U.S. patent application number 14/230766 was filed with the patent office on 2014-08-21 for method for producing monatin using an l-amino acid aminotransferase.
This patent application is currently assigned to Ajinomoto Co., Inc.. The applicant listed for this patent is Ajinomoto Co., Inc.. Invention is credited to Hidemi Fujii, Koki Ishikawa, Kenichi Mori, Hiroomi Ogino, Masakazu Sugiyama, Eri Tabuchi, Uno Tagami, Yasuaki Takakura.
Application Number | 20140234916 14/230766 |
Document ID | / |
Family ID | 47021630 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234916 |
Kind Code |
A1 |
Takakura; Yasuaki ; et
al. |
August 21, 2014 |
METHOD FOR PRODUCING MONATIN USING AN L-AMINO ACID
AMINOTRANSFERASE
Abstract
The present invention provides a methodology for improving a
yield of 2R,4R-Monatin. Specifically, the present invention
provides a method for producing 2S,4R-Monatin or a salt thereof,
comprising contacting 4R-IHOG with an L-amino acid aminotransferase
in the presence of an L-amino acid to form the 2S,4R-Monatin; a
method for producing 2R,4R-Monatin or a salt thereof, comprising
isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin; and the
like. These production methods may further comprise condensing
indole-3-pyruvate and pyruvate to form the 4R-IHOG, and deaminating
a tryptophan to form the indole-3-pyruvate.
Inventors: |
Takakura; Yasuaki;
(Kawasaki-shi, JP) ; Ogino; Hiroomi;
(Kawasaki-shi, JP) ; Sugiyama; Masakazu;
(Kawasaki-shi, JP) ; Mori; Kenichi; (Kawasaki-shi,
JP) ; Tabuchi; Eri; (Kawasaki-shi, JP) ;
Ishikawa; Koki; (Kawasaki-shi, JP) ; Tagami; Uno;
(Kawasaki-shi, JP) ; Fujii; Hidemi; (Kawasaki-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ajinomoto Co., Inc. |
Kawasaki-shi |
|
JP |
|
|
Assignee: |
Ajinomoto Co., Inc.
Kawasaki-shi
JP
|
Family ID: |
47021630 |
Appl. No.: |
14/230766 |
Filed: |
March 31, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13271280 |
Oct 12, 2011 |
8771997 |
|
|
14230766 |
|
|
|
|
61477402 |
Apr 20, 2011 |
|
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Current U.S.
Class: |
435/106 |
Current CPC
Class: |
C12P 17/10 20130101;
C12N 9/1096 20130101; C12P 13/04 20130101 |
Class at
Publication: |
435/106 |
International
Class: |
C12P 13/04 20060101
C12P013/04 |
Claims
1.-30. (canceled)
31. A method for producing 2R,4R-Monatin or a salt thereof,
comprising the following (I) and (II): (I) contacting 4R form of
4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid (4R-IHOG) with
an L-amino acid aminotransferase in the presence of an L-amino acid
to form the 2S,4R-Monatin, wherein the L-amino acid
aminotransferase consists of an amino acid sequence having 95% or
more identity to the amino acid sequence of SEQ ID NO:2; and (II)
isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin.
32. The production method of claim 31, wherein a keto acid formed
from the L-amino acid due to the action of the L-amino acid
aminotransferase is contacted with a decarboxylase to degrade said
keto acid.
33. The production method of claim 31, wherein the L-amino acid is
L-aspartate.
34. The production method of claim 33, wherein oxaloacetate formed
from the L-aspartate due to the action of the L-amino acid
aminotransferase is contacted with an oxaloacetate decarboxylase to
degrade said oxaloacetate to irreversibly form pyruvate.
35. The production method of claim 31, wherein the L-amino acid
aminotransferase is derived from a microorganism belonging to the
genus Arthrobacter, Bacillus, Candida, Corynebacterium,
Lodderomyces, Micrococcus, Microbacterium, Nocardia, Pseudomonas,
Rhizobium, Stenotrophomonas, Dietzia, Ochrobactrum, Brevundimonas,
Burkholderia, Carnimonas, Yarrowia, Clostridium, Deinococcus,
Eubacterium, Lactobacillus, Methanothermobacter, Phormidium,
Pyrococcus, Rhodococcus, Saccharomyces, Saccharophagus,
Sinorhizobium, Thermoanaerobacter, Thermotoga or Thermus.
36. The production method of claim 35, wherein the L-amino acid
aminotransferase is derived from Arthrobacter sp., Bacillus
altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, Bacillus
sp., Candida norvegensis, Candida inconspicua, Corynebacterium
ammoniagenes, Corynebacterium glutamicum, Lodderomyces
elongisporus, Micrococcus luteus, Microbacterium sp., Nocardia
globerula, Pseudomonas chlororaphis, Pseudomonas citronocllolis,
Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha,
Pseudomonas taetrolens, Pseudomonas sp., Rhizobium radiobacter,
Rhizobium sp., Stenotrophomonas sp., Dietzia maxis, Ochrobactrum
pseudogrignonense, Brevundimonas diminuta, Burkholderia sp.,
Carnimonas sp., Yarrowia lypolytica, Clostridium cellulolyticum,
Deinococcus geothermalis, Eubacterium rectale, Lactobacillus
acidophilus, Methanothermobacter thermautotrophicus, Phormidium
lapideum, Pyrococcus horikoshii, Rhodococcus erythropolis,
Saccharomyces cerevisiae, Saccharophagus degradans, Sinorhizobium
meliloti, Thermoanaerobacter tengcongensis, Thermotoga maritima, or
Thermus thermophilus.
37. The production method of claim 31, wherein the L-amino acid
aminotransferase comprises one or more mutations of amino acid
residues selected from the group consisting of the amino acid
residues at position 39, position 109, position 128, position 150,
position 258, position 287, position 288, position 289, position
303, position 358 and position 431 in the amino acid sequence of
SEQ ID NO:2.
38. The production method of claim 37, wherein the one or more
mutations of amino acid residues are selected from the group
consisting of: i) substitution of the lysine at position 39 with an
arginine; ii) substitution of the serine at position 258 with a
glycine; iii) substitution of the glutamine at position 287 with a
glutamic acid; iv) substitution of the threonine at position 288
with a glycine; v) substitution of the isoleucine at position 289
with an alanine; vi) substitution of the aspartic acid at position
109 with a glycine; vii) substitution of the histidine at position
150 with a tyrosine; viii) substitution of the phenylalanine at
position 303 with a leucine; ix) substitution of the aspartic acid
at position 358 with a tyrosine; x) substitution of the serine at
position 431 with a threonine; and xi) substitution of the glutamic
acid at position 128 with a glycine.
39. The production method of claim 31, wherein the 4R-IHOG is
contacted with the L-amino acid aminotransferase using a
transformant that expresses the L-amino acid aminotransferase.
40. The production method of claim 31, further comprising
condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG.
41. The production method of claim 40, wherein the
indole-3-pyruvate and the pyruvate are condensed by contacting the
indole-3-pyruvate and the pyruvate with an aldolase.
42. The production method of claim 40, wherein at least part of the
pyruvate used in the formation of the 4R-IHOG is from pyruvate
formed from the oxaloacetate due to the action of an oxaloacetate
decarboxylase.
43. The production method of claim 40, further comprising
deaminating a tryptophan to form the indole-3-pyruvate.
44. The production method of claim 43, wherein the tryptophan is
deaminated by contacting the tryptophan with a deamination
enzyme.
45. The production method of claim 41, wherein the production of
2S,4R-Monatin or the salt thereof is carried out in one
reactor.
46. The production method of claim 40, wherein the production of
the 2S,4R-Monatin or the salt thereof is carried out in one
reactor.
47. The production method of claim 41, wherein the 2S,4R-Monatin is
isomerized in the presence of an aromatic aldehyde.
48. The production method of claim 41, wherein the salt is a sodium
salt or a potassium salt.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
provisional Patent Application No. 61/477,402, filed on Apr. 20,
2011, the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing
Monatin using an L-amino acid aminotransferase, and the like.
BACKGROUND ART
[0003] Monatin [4-(indole-3-yl-methyl)-4-hydroxy-glutamic acid] is
a compound that is one of amino acids contained in roots of
Schlerochitom ilicifolius that is a shrub in South Africa and is
particularly expected as a low calorie sweetener because of having
sweetness one thousand and several hundreds times sweeter than
sucrose (see Patent Document 1). The Monatin has asymmetric carbon
atoms at positions 2 and 4, and a naturally occurring stereoisomer
of Monatin is a 2S,4S-isomer. Naturally non-occurring three
stereoisomers have been synthesized by organic chemistry processes.
All of these stereoisomers are excellent in sweetness, and expected
to be used as the sweeteners.
[0004] Several methods have been reported as the methods for
producing the Monatin (e.g., see Patent Document 2). However, all
of the reported methods require a step of multiple stages, and
thus, it is required to improve a synthetic yield of the
Monatin.
[0005] Specifically, for the method for producing the Monatin, the
following method for producing 2R,4R-Monatin by synthesizing
indole-3-pyruvate (hereinafter referred to as "IPA" as needed) from
L-tryptophan (L-Trp), synthesizing 4R form of
4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid (hereinafter
referred to as "4R-IHOG" as needed) from the resulting IPA and
pyruvate, and subsequently subjecting the obtained 4R-IHOG to an
oximation reaction, a reduction reaction and an
epimerization-crystallization method has been known (conventional
method (1)) (see Patent Document 2).
[0006] However, an aldolase step (second step) is an equilibrium
reaction, and thus, a satisfactory yield is not always obtained in
this reaction.
##STR00001##
[0007] In order to improve the yield of the 2R,4R-Monatin, the
method for producing the 2R,4R-Monatin by a one-pot enzymatic
reaction has been invented (conventional method (2)) (see Patent
Documents 3 to 6). [0008] Patent Document 1: JP Sho-64-25757-A
[0009] Patent Document 2: International Publication WO2003/059865
[0010] Patent Document 3: International Publication WO2007/133184
[0011] Patent Document 4: International Publication WO2005/042756
[0012] Patent Document 5: US Patent Application Publication No.
2006/0252135 Specification [0013] Patent Document 6: US Patent
Application Publication No. 2008/020434 Specification
SUMMARY OF INVENTION
Problem to be Solved by the Invention
[0014] The object of the present invention is to provide a method
for producing Monatin with a good yield.
Means for Solving Problem
[0015] As a result of an extensive study, the present inventors
have found that the above problem can be solved by using an L-amino
acid aminotransferase, and completed the present invention. No
L-amino acid aminotransferase that acts upon 4R-IHOG has been known
so far.
[0016] Accordingly, the present invention is as follows.
[1] A method for producing 2S,4R-Monatin or a salt thereof,
comprising contacting 4R-IHOG with an L-amino acid aminotransferase
in the presence of an L-amino acid to form the 2S,4R-Monatin. [2]
The production method of [1], further comprising contacting a keto
acid with a decarboxylase to degrade the keto acid, wherein the
keto acid is formed from the L-amino acid due to action of the
L-amino acid aminotransferase. [3] The production method of [1],
wherein the L-amino acid is L-aspartate. [4] The production method
of [3], further comprising contacting oxaloacetate with an
oxaloacetate decarboxylase to irreversibly form pyruvate, wherein
the oxaloacetate is formed from the L-aspartate by action of the
L-amino acid aminotransferase. [5] The production method of [1],
wherein the L-amino acid aminotransferase is derived from a
microorganism belonging to genus Arthrobacter, genus Bacillus,
genus Candida, genus Corynebacterium, genus Lodderomyces, genus
Micrococcus, genus Microbacterium, genus Nocardia, genus
Pseudomonas, genus Rhizobium, genus Stenotrophomonas, genus
Dietzia, genus Ochrobactrum, genus Brevundimonas, genus
Burkholderia, genus Carnimonas, genus Yarrowia, genus Clostridium,
genus Deinococcus, genus Eubacterium, genus Lactobacillus, genus
Methanothermobacter, genus Phormidium, genus Pyrococcus, genus
Rhodococcus, genus Saccharomyces, genus Saccharophagus, genus
Sinorhizobium, genus Thermoanaerobacter, genus Thermotoga or genus
Thermus. [6] The production method of [5], wherein the L-amino acid
aminotransferase is derived from a microorganism belonging to
Arthrobacter sp., Bacillus altitudinis, Bacillus cellulosilyticus,
Bacillus pumilus, Bacillus sp., Candida norvegensis, Candida
inconspicua, Corynebacterium ammoniagenes, Corynebacterium
glutamicum, Lodderomyces elongisporus, Micrococcus luteus,
Microbacterium sp., Nocardia globerula, Pseudomonas chlororaphis,
Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida,
Pseudomonas synxantha, Pseudomonas taetrolens, Pseudomonas sp.,
Rhizobium radiobacter, Rhizobium sp., Stenotrophomonas sp., Dietzia
marls, Ochrobactrum pseudogrignonense, Brevundimonas diminuta,
Burkholderia sp., Carnimonas sp., Yarrowia lypolytica, Clostridium
cellulolyticum, Deinococcus geothermalis, Eubacterium rectale,
Lactobacillus acidophilus, Methanothermobacter thermautotrophicus,
Phormidium lapideum, Pyrococcus horikoshii, Rhodococcus
erythropolis, Saccharomyces cerevisiae, Saccharophagus degradans,
Sinorhizobium meliloti, Thermoanaerobacter tengcongensis,
Thermotoga maritima, or Thermus thermophilus. [7] The production
method of [1], wherein the L-amino acid aminotransferase consists
of an amino acid sequence showing 90% or more identity to the amino
acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID
NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ
ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ ID NO:85,
SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,
SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111. [8]
The production method of [7], wherein the L-amino acid
aminotransferase comprises one or more mutations of amino acid
residues selected from the group consisting of the amino acid
residues at position 39, position 109, position 128, position 150,
position 258, position 287, position 288, position 289, position
303, position 358 and position 431 in the amino acid sequence
represented by SEQ ID NO:2. [9] The production method of [8],
wherein the one or more mutations of amino acid residues are
selected from the group consisting of: i) substitution of the
lysine at position 39 with an arginine; ii) substitution of the
serine at position 258 with a glycine; iii) substitution of the
glutamine at position 287 with a glutamic acid; iv) substitution of
the threonine at position 288 with a glycine; v) substitution of
the isoleucine at position 289 with an alanine; vi) substitution of
the aspartic acid at position 109 with a glycine; vii) substitution
of the histidine at position 150 with a tyrosine; viii)
substitution of the phenylalanine at position 303 with a leucine;
ix) substitution of the aspartic acid at position 358 with a
tyrosine; x) substitution of the serine at position 431 with a
threonine; and xi) substitution of the glutamic acid at position
128 with a glycine. [10] The production method of [1], wherein the
4R-IHOG is contacted with the L-amino acid aminotransferase using a
transformant that expresses the L-amino acid aminotransferase. [11]
The production method of [1], further comprising condensing
indole-3-pyruvate and pyruvate to form the 4R-IHOG. [12] The
production method of [11], the indole-3-pyruvate and the pyruvate
are condensed by contacting the indole-3-pyruvate and the pyruvate
with an aldolase. [13] The production method of [11], wherein at
least part of the pyruvate used in the formation of the 4R-IHOG is
from pyruvate formed from the oxaloacetate due to action of the
oxaloacetate decarboxylase. [14] The production method of [11],
further comprising deaminating a tryptophan to form the
indole-3-pyruvate. [15] The production method of [14], wherein the
tryptophan is deaminated by contacting the tryptophan with a
deamination enzyme. [16] The production method of [11] or [14],
wherein the production of the 2S,4R-Monatin or the salt thereof is
carried out in one reactor. [17] A method for producing
2R,4R-Monatin or a salt thereof, comprising the following (I) and
(II): (I) performing the method of [1] to form the 2S,4R-Monatin;
and (II) isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin.
[18] The production method of [17], wherein the 2S,4R-Monatin is
isomerized in the presence of an aromatic aldehyde. [19] The
production method of [17], wherein the salt is a sodium salt or a
potassium salt. [20] An L-amino acid aminotransferase that is a
protein selected form the group consisting of the following
(A)-(D): (A) a protein consisting of the amino acid sequence
represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID
NO:61; (B) a protein comprising the amino acid sequence represented
by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID NO:61; (C) a
protein consisting of an amino acid sequence showing 90% or more
identity to the amino acid sequence represented by SEQ ID NO:2, SEQ
ID NO:48, SEQ ID NO:53, or SEQ ID NO:61, and having an L-amino acid
aminotransferase activity; and (D) a protein consisting of an amino
acid sequence comprising mutation of one or several amino acid
residues, which is selected from the group consisting of deletion,
substitution, addition and insertion of the amino acid residues in
the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48,
SEQ ID NO:53, or SEQ ID NO:61, and having an L-amino acid
aminotransferase activity. [21] The L-amino acid aminotransferase
of [20], wherein the L-amino acid aminotransferase comprises one or
more mutations of amino acid residues selected from the group
consisting of the amino acid residues at position 39, position 109,
position 128, position 150, position 258, position 287, position
288 and position 289, position 303, position 358 and position 431
in the amino acid sequence represented by SEQ ID NO:2. [22] The
L-amino acid aminotransferase of [21], wherein the one or more
mutations of amino acid residues are selected from the group
consisting of: i) substitution of the lysine at position 39 with an
arginine; ii) substitution of the serine at position 258 with a
glycine; iii) substitution of the glutamine at position 287 with a
glutamic acid; iv) substitution of the threonine at position 288
with a glycine; v) substitution of the isoleucine at position 289
with an alanine; vi) substitution of the aspartic acid at position
109 with a glycine; vii) substitution of the histidine at position
150 with a tyrosine; viii) substitution of the phenylalanine at
position 303 with a leucine; ix) substitution of the aspartic acid
at position 358 with a tyrosine; x) substitution of the serine at
position 431 with a threonine; and xi) substitution of the glutamic
acid at position 128 with a glycine. [23] A polynucleotide selected
from the group consisting of the following (a)-(e): (a) a
polynucleotide consisting of the nucleotide sequence represented by
SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60; (b) a
polynucleotide comprising the nucleotide sequence represented by
SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60; (c) a
polynucleotide consisting of a nucleotide sequence showing 90% or
more identity to the amino acid sequence represented by SEQ ID
NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60, and encoding a
protein having an L-amino acid aminotransferase activity; (d) a
polynucleotide that hybridizes under a stringent condition with a
polynucleotide consisting of the nucleotide sequence complementary
to the nucleotide sequence represented by SEQ ID NO:1, SEQ ID
NO:47, SEQ ID NO:52, or SEQ ID NO:60, and encodes a protein having
an L-amino acid aminotransferase activity; and (e) a polynucleotide
encoding the L-amino acid aminotransferase of [20]. [24] An
expression vector comprising the polynucleotide of [23]. [25] A
transformant introduced with the expression vector of [24]. [26] A
method for producing an L-aminotransfearase, comprising culturing
the transformant of [25] in a medium to obtain the L-amino acid
aminotransferase. [27] A method of producing 2S,4R-Monatin or a
salt thereof, comprising contacting 4R-IHOG with the L-amino acid
aminotransferase of [20] in the presence of an L-amino acid to form
the 2S,4R-Monatin. [28] A method for producing 2R,4R-Monatin or a
salt thereof, comprising the following (I') and (II'): (I')
performing the method of [27] to form the 2S,4R-Monatin; and (II')
isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin. [29] The
production method of [28], wherein the 2S,4R-Monatin is isomerized
in the presence of an aromatic aldehyde. [30] The production method
of [28], wherein the salt is a sodium salt or a potassium salt.
Effect of the Invention
[0017] The method of the present invention can contribute to
improvement of the yield of the Monatin by producing the
2S,4R-Monatin with a good yield from 4R-IHOG using the L-amino acid
aminotransferase. The method of the present invention has an
advantage that it is not necessary to use an expensive D-amino acid
(D-Asp and the like) as a substrate when the 2S,4R-Monatin is
formed from IHOG or that it is not necessary to add an enzyme such
as racemase to form the D-amino acid from an L-amino acid. In the
method of the present invention, when performing not only the
reaction to form the 2S,4R-Monatin from 4R-IHOG (third step) but
also the reaction to form IPA from L-Trp (first step) and the
reaction to form 4R-IHOG from IPA (second step), whole reaction
equilibrium can be defined in the third step and the reaction
equilibrium in the second step can be largely shifted to a
direction to form 4R-IHOG. In this case, the method of the present
invention makes it possible to produce the 2S,4R-Monatin with a
very good yield by avoiding a by-product of L-Trp (progress of a
reverse reaction of the first step).
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a view showing one example of the production
method of the present invention. Trp: tryptophan; IPA:
indole-3-pyruvate; IHOG:
4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid; Monatin:
4-(indole-3-yl-methyl)-4-hydroxy-glutamic acid.
[0019] FIG. 2 is a view showing one example of the production
method of the present invention. Abbreviations are the same as in
FIG. 1; and
[0020] FIG. 3 is a view showing a preferable example of the
production method of the present invention. L-Trp: L-tryptophan;
L-Asp: L-aspartic acid; OAA: oxaloacetate; PA: pyruvate; and the
other abbreviations are the same as in FIG. 1.
[0021] FIG. 4 is a graph showing a reaction of forming
2S,4R-Monatin from L-Trp in 400 ml scale using the L-amino acid
aminotransferase mutant (ID166). SR-Monatin: 2S,4R-Monatin;
SS-Monatin: 2S,4S-Monatin; IHOG: 4R-IHOG; Trp: L-Trp.
[0022] FIG. 5 is a graph showing a reaction of forming
2S,4R-Monatin from L-Trp in 80 ml scale using the L-amino acid
aminotransferase mutant (ID189). The abbreviations are similar to
those of FIG. 4.
[0023] FIG. 6 is a graph showing a reaction of forming
2S,4R-Monatin from L-Trp in 80 ml scale using the L-amino acid
aminotransferase mutant (ID296). The abbreviations are similar to
those of FIG. 4.
BEST MODES FOR CARRYING OUT THE INVENTION
(1) Method for Producing 2S,4R-Monatin or a Salt Thereof
[0024] The present invention provides a method (1) for producing
2S,4R-Monatin or a salt thereof. The production method of the
present invention can be classified into (1-1) a method for
producing the 2S,4R-Monatin from 4R-IHOG, (1-2) a method for
producing the 2S,4R-Monatin from IPA and pyruvate, and (1-3) a
method for producing the 2S,4R-Monatin from tryptophan. The methods
(1-1), (1-2) and (1-3) are common in contacting 4R-IHOG with an
L-amino acid aminotransferase in the presence of the L-amino acid
to form the 2S,4R-Monatin.
(1-1) Method for Producing 2S,4R-Monatin from 4R-IHOG
[0025] This method comprises contacting 4R-IHOG with the L-amino
acid aminotransferase in the presence of the L-amino acid to form
the 2S,4R-Monatin (reaction 1). By contacting 4R-IHOG with the
L-amino acid aminotransferase in the presence of the L-amino acid,
an amino group in the L-amino acid can be transferred to 4R-IHOG to
form the 2S,4R-Monatin.
##STR00002##
[0026] The kinds of the L-amino acid is not particularly limited as
long as the amino group in the L-amino acid can be transferred to
4R-IHOG that is an objective substrate by the L-amino acid
aminotransferase. Various L-amino acids such as L-.alpha.-amino
acids are known as such an L-amino acid. Specifically, such an
L-amino acid includes L-aspartic acid, L-alanine, L-lysine,
L-arginine, L-histidine, L-glutamic acid, L-asparagine,
L-glutamine, L-serine, L-threonine, L-tyrosine, L-cysteine,
L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine,
L-methionine and L-tryptophan. A salt form of the L-amino acid may
be added to a reaction solution. The concentration of the L-amino
acid in a reaction solution is, for example, 1 mM to 3 M,
preferably 20 mM to 1 M, more preferably 100 mM to 500 mM.
[0027] In one embodiment, the L-amino acid aminotransferase may be
a protein derived from a microorganism such as a bacterium,
actinomycete or yeast. The classification of the microorganisms can
be carried out by a classification method well-known in the art,
e.g., a classification method used in the database of NCBI
(National Center for Biotechnology Information)
(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347).
Examples of the microorganisms from which the L-amino acid
aminotransferase is derived include microorganisms belonging to
genus Arthrobacter, genus Bacillus, genus Candida, genus
Corynebacterium, genus Lodderomyces, genus Micrococcus, genus
Microbacterium, genus Nocardia, genus Pseudomonas, genus Rhizobium,
genus Stenotrophomonas, genus Dietzia, genus Ochrobactrum, genus
Brevundimonas, genus Burkholderia, genus Carnimonas, genus
Yarrowia, genus Clostridium, genus Deinococcus, genus Eubacterium,
genus Lactobacillus, genus Methanococcus, genus
Methanothermobacter, genus Phormidium, genus Pyrococcus, genus
Rhodococcus, genus Saccharomyces, genus Saccharophagus, genus
Sinorhizobium, genus Thermoanaerobacter, genus Thermotoga, and
genus Thermus.
[0028] Specifically, examples of the microorganisms belonging to
genus Arthrobacter include Arthrobacter sp.
[0029] Examples of the microorganisms belonging to genus Bacillus
include Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus
pumilus, and Bacillus sp. Examples of the microorganisms belonging
to genus Candida include Candida norvegensis and Candida
inconspicua. Examples of the microorganisms belonging to genus
Corynebacterium include Corynebacterium ammonia genes, and
Corynebacterium glutamicum. Examples of the microorganisms
belonging to genus Lodderomyces include Lodderomyces elongisporus.
Examples of the microorganisms belonging to genus Micrococcus
include Micrococcus luteus. Examples of the microorganisms
belonging to genus Microbacterium include Microbacterium sp.
Examples of the microorganisms belonging to genus Nocardia include
Nocardia globerula.
[0030] Examples of the microorganisms belonging to genus
Pseudomonas include Pseudomonas chlororaphis (e.g., Pseudomonas
chlororaphis subsp. chlororaphis), Pseudomonas citronocllolis,
Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha,
Pseudomonas taetrolens, and Pseudomonas sp.
[0031] Examples of the microorganisms belonging to genus Rhizobium
include Rhizobium radiobacter and Rhizobium sp. Examples of the
microorganisms belonging to genus Stenotrophomonas include
Stenotrophomonas sp. Examples of the microorganisms belonging to
genus Dietzia include Dietzia marls. Examples of the microorganisms
belonging to genus Ochrobactrum include Ochrobactrum
pseudogrignonense. Examples of the microorganisms belonging to
genus Brevundimonas include Brevundimonas diminuta. Examples of the
microorganisms belonging to genus Burkholderia include Burkholderia
sp. Examples of the microorganisms belonging to genus Carnimonas
include Carnimonas sp. Examples of the microorganisms belonging to
genus Yarrowia include Yarrowia lypolytica.
[0032] Examples of the microorganisms belonging to genus
Clostridium include Clostridium cellulolyticum. Examples of the
microorganisms belonging to genus Deinococcus include Deinococcus
geothermalis. Examples of the microorganisms belonging to genus
Eubacterium include Eubacterium rectale. Examples of the
microorganisms belonging to genus Lactobacillus include
Lactobacillus acidophilus. Examples of the microorganisms belonging
to genus Methanococcus include Methanococcus jannaschii. Examples
of the microorganisms belonging to genus Methanothermobacter
include Methanothermobacter thermautotrophicus. Examples of the
microorganisms belonging to genus Phormidium include Phormidium
lapideum. Examples of the microorganisms belonging to genus
Pyrococcus include Pyrococcus horikoshii. Examples of the
microorganisms belonging to genus Rhodococcus include Rhodococcus
erythropolis. Examples of the microorganisms belonging to genus
Saccharomyces include Saccharomyces cerevisiae. Examples of the
microorganisms belonging to genus Saccharophagus include
Saccharophagus degradans. Examples of the microorganisms belonging
to genus Sinorhizobium include Sinorhizobium meliloti. Examples of
the microorganisms belonging to genus Thermoanaerobacter include
Thermoanaerobacter tengcongensis. Examples of the microorganisms
belonging to genus Thermotoga include Thermotoga maritima. Examples
of the microorganisms belonging to genus Thermus include Thermus
thermophilus.
[0033] In another embodiment, the L-amino acid aminotransferase may
be a naturally occurring protein or an artificial mutant protein.
Such an L-amino acid aminotransferase includes those consisting of
an amino acid sequence having high homology (e.g., similarity,
identity) to an amino acid sequence represented by SEQ ID NO:2, SEQ
ID NO:48, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67,
SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID
NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ
ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,
SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ
ID NO:111, and having an L-amino acid aminotransferase activity.
The term "L-amino acid aminotransferase activity" refers to an
activity of transferring the amino group in the L-amino acid to
4R-IHOG that is the objective substrate for forming the 2S,4R
Monatin that is an objective compound having the amino group.
Specifically, the L-amino acid aminotransferase includes a protein
consisting of the amino acid sequence showing 80% or more,
preferably 90% or more, more preferably 95% or more and
particularly preferably 98% or more or 99% or more homology (e.g.,
similarity, identity) to the amino acid sequence represented by SEQ
ID NO:2, and having the L-amino acid aminotransferase activity.
[0034] The homology of the amino acid sequences and nucleotide
sequences can be determined using algorithm BLAST by Karlin and
Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA by
Pearson (Methods Enzymol., 183, 63 (1990)). Programs referred to as
BLASTP and BLASTN (see http://www.ncbi.nlm.nih.gov) have been
developed based on this algorithm BLAST. Thus, the homology of the
amino acid sequences and the nucleotide sequences may be calculated
using these programs with default setting. A numerical value
obtained when matching count is calculated as a percentage by using
GENETYX Ver. 7.0.9 that is software from GENETYX Corporation and
using full length polypeptide chains encoded in ORF with setting of
Unit Size to Compare=2 may be used as the homology of the amino
acid sequences. The lowest value among the values derived from
these calculations may be employed as the homology of the amino
acid sequences and the nucleotide sequences.
[0035] In further another embodiment, the L-amino acid
aminotransferase may be a protein consisting of an amino acid
sequence comprising mutation (e.g., deletion, substitution,
addition and insertion) of one or several amino acid residues in
the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48,
SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID
NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ
ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,
SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID
NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID
NO:111, and having the L-amino acid aminotransferase activity. The
mutation of one or several amino acid residues may be introduced
into one region or multiple different regions in the amino acid
sequence. The term "one or several amino acid residues" indicate a
range in which a three dimensional structure and the activity of
the protein are not largely impaired. The term "one or several
amino acid residues" in the case of the protein denote, for
example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1
to 30, 1 to 20, 1 to 10 or 1 to 5 amino acid residues. Such
mutation may be attributed to naturally occurring mutation (mutant
or variant) based on individual difference, species difference and
the like of the microorganism carrying a gene encoding the L-amino
acid aminotransferase.
[0036] A position of the amino acid residue to be mutated in the
amino acid sequence is apparent to those skilled in the art.
Specifically, a person skilled in the art can recognize the
correlation between the structure and the function by 1) comparing
the amino acid sequences of the multiple proteins having the same
kind of activity (e.g., the amino acid sequence represented by SEQ
ID NO:2, and amino acid sequences of other L-amino acid
aminotransferase), 2) clarifying relatively conserved regions and
relatively non-conserved regions, and then 3) predicting a region
capable of playing an important role for its function and a region
incapable of playing the important role for its function from the
relatively conserved regions and the relatively non-conserved
regions, respectively. Therefore, a person skilled in the art can
specify the position of the amino acid residue to be mutated in the
amino acid sequence of the L-amino acid aminotransferase.
[0037] When an amino acid residue is mutated by the substitution,
the substitution of the amino acid may be conservative
substitution. As used herein, the term "conservative substitution"
means that a certain amino acid residue is substituted with an
amino acid residue having an analogous side chain. Families of the
amino acid residues having the analogous side chain are well-known
in the art. Examples of such families include an amino acid having
a basic side chain (e.g., lysine, arginine or histidine), an amino
acid having an acidic side chain (e.g., aspartic acid or glutamic
acid), an amino acid having a non-charged polar side chain (e.g.,
asparagine, glutamine, serine, threonine, tyrosine or cysteine), an
amino acid having a non-polar side chain (e.g., glycine, alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine or
tryptophan), an amino acid having a .beta.-position branched side
chain (e.g., threonine, valine or isoleucine), an amino acid having
an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan
or histidine), an amino acid having a hydroxyl group (e.g.,
alcoholic or phenolic)-containing side chain (e.g., serine,
threonine or tyrosine), and an amino acid having a
sulfur-containing side chain (e.g., cysteine or methionine).
Preferably, the conservative substitution of the amino acids may be
the substitution between aspartic acid and glutamic acid, the
substitution among arginine, lysine and histidine, the substitution
between tryptophan and phenylalanine, the substitution between
phenylalanine and valine, the substitution among leucine,
isoleucine and alanine, and the substitution between glycine and
alanine.
[0038] In further another embodiment, the L-amino acid
aminotransferase may be a protein encoded by DNA that hybridizes
under a stringent condition with a nucleotide sequence
complementary to a nucleotide sequence represented by SEQ ID NO:2,
SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ
ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108,
or SEQ ID NO:110, and having the L-amino acid aminotransferase
activity. The "stringent condition" refers to the condition where a
so-called specific hybrid is formed whereas no non-specific hybrid
is formed. Although it is difficult to clearly quantify this
condition, one example of this condition is the condition where a
pair of polynucleotides with high homology (e.g., identity), for
example, a pair of polynucleotides having the homology of 80% or
more, preferably 90% or more, more preferably 95% or more, and
particularly preferably 90% or more are hybridized whereas a pair
of polynucleotides with lower homology than that are not
hybridized. Specifically, such a condition includes hybridization
in 6.times.SSC (sodium chloride/sodium citrate) at about 45.degree.
C. followed by one or two or more washings in 0.2.times.SSC and
0.1% SDS at 50 to 65.degree. C.
[0039] In a preferred embodiment, the L-amino acid aminotransferase
may be L-amino acid aminotransferase mutant in which one or more
(e.g., one or two) of any amino acid residues selected from the
group consisting of the amino acid residues at position 39,
position 109, position 128, position 150, position 258, position
287, position 288, position 289, position 303, position 358, and
position 431 in the amino acid sequence represented by SEQ ID NO:2
are mutated (e.g., substituted). Preferred examples of the L-amino
acid aminotransferase mutant comprise one or more (e.g., one or
two) substitutions selected from the group consisting of:
i) substitution of the lysine at position 39 with an arginine; ii)
substitution of the serine at position 258 with a glycine; iii)
substitution of the glutamine at position 287 with a glutamic acid;
iv) substitution of the threonine at position 288 with a glycine;
v) substitution of the isoleucine at position 289 with an alanine;
vi) substitution of the aspartic acid at position 109 with a
glycine; vii) substitution of the histidine at position 150 with a
tyrosine; viii) substitution of the phenylalanine at position 303
with a leucine; ix) substitution of the aspartic acid at position
358 with a tyrosine; x) substitution of the serine at position 431
with a threonine; and xi) substitution of the glutamic acid at
position 128 with a glycine.
[0040] For the combination of the substitution of one or more
(e.g., one or two) of any amino acid residues selected from the
group consisting of the amino acid residues at position 39,
position 109, position 128, position 150, position 258, position
287, position 288, position 289, position 303, position 358 and
position 431 in the amino acid sequence represented by SEQ ID NO:2,
the combined mutations as shown below can be introduced although
the combination of the amino acid substitutions which can be
utilized in the present invention is not limited to the
following:
a) T288G
b) S258G/I289A
c) K39R/T288G
d) Q287E/T288G
e) K39R/D109R/T288G/S431T
f) K39R/D109R/T288G/F303L
g) D109R/Q287E/T288G/F303L
h) D109R/S258G/I289A/F303L
i) D109R/Q287E/T288G/S431T
j) D109R/S258G/I289A/S431T
k) K39R/D109R/E128G/T288G/F303L
l) K39R/D109G/E128G/I288G/F303L
m) D109R/E128G/Q287E/T288G/F303L
n) D109R/E128G/S258G/I289A/S431T
o) D109G/E128G/Q287E/T288G/F303L
p) D109G/E128G/S258G/I289A/F303L
q) K39R/D109G/H150Y/T288G/F303L/D358Y/S431T
r) K39R/D109G/E128G/H150Y/T288G/F303L/D358Y
s) D109G/H150Y/Q287E/T288G/F303L/D35BY/S431T
t) D109G/H150Y/S258G/I289A/F303L/D358Y/S431T
u) D109G/E128G/H150Y/Q287E/T288G/F303L/D358Y or
v) D109G/E128G/H150Y/S258G/I289A/F303L/D358Y
[0041] In one embodiment, the contact of 4R-IHOG with the L-amino
acid aminotransferase can be accomplished by allowing 4R-IHOG and
the L-amino acid aminotransferase extracted from an L-amino acid
aminotransferase-producing microorganism (extracted enzyme) to
coexist in a reaction solution. Examples of the L-amino acid
aminotransferase-producing microorganism include the microorganisms
that naturally produce the L-amino acid aminotransferase (e.g., the
aforementioned microorganisms), and transformants that express the
L-amino acid aminotransferase. Specifically, examples of the
extracted enzyme include a purified enzyme, a crude enzyme, an
immorbilized enzyme, a cuture broth, and a treated product of the
culture broth (e.g., an L-amino acid aminotransferase-containing
fraction prepared from the above enzyme-producing microorganism,
and a disrupted product of and a lysate of the above
enzyme-producing microorganism). Examples of the treatment for
obtaining the treated product of the culture broth from the culture
broth include a heat treatment (42.degree. C. to 80.degree. C., pH
3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene,
toluene, ethanol, isopropylalcohol), a surfactant (e.g., Tween 20,
Triton X-100), and a treatment with a bacteriolytic enzyme (e.g.,
lysozyme treatment). Alternatively, the culture broth is subjected
to a reaction after retaining it with adjusting temperature, pH and
the like to enhance an enzymatic activity detected in the broth. In
this case, the temperature may be set at 4.degree. C. to 60.degree.
C., preferably 20.degree. C. to 37.degree. C. In addition, the pH
may be set at 3 to 12, preferably 7 to 9. The time may be set for
about 5 minutes to 20 days, preferably about 1 hour to 7 days.
During retaining the broth, aeration and agitation may be or may
not be carried out.
[0042] In another embodiment, the contact of 4R-IHOG with the
L-amino acid aminotransferase can be accomplished by allowing
4R-IHOG and the L-amino acid aminotransferase-producing
microorganism to coexist in the reaction solution (e.g., culture
medium).
[0043] The reaction solution used in the production method (1) of
the present invention is not particularly limited as long as the
objective reaction progresses, and for example, water and buffer
are used. Examples of the reaction solution include Tris buffer,
phosphate buffer (e.g., KH.sub.2PO.sub.4), carbonate buffer, borate
buffer and acetate buffer. The concentration of the buffer may be,
for example, 0.1 mM to 10 M, preferably 1 mM to 1 M. When the
L-amino acid aminotransferase-producing microorganism is used in
the production method of the present invention, the culture medium
may be used as the reaction solution. Such a culture medium can be
prepared using a medium described later. The reaction solution used
in the production method of the present invention may further
comprise pyridoxal phosphate (PLP) as a coenzyme. A salt form of
PLP may be added to the reaction solution. The concentration of PLP
in the reaction solution may be, for example, 1 .mu.M to 100 mM,
preferably 10 .mu.M to 1 mM. When the reaction solution comprises
PLP, an effect to form 2R,4R-Monatin from the 2S,4R-Monatin can be
expected by an isomerization reaction which can be catalyzed by PLP
(e.g., see Example 11).
[0044] A pH value of the reaction solution used in the production
method (1) of the present invention is not particularly limited as
long as the objective reaction progresses, and is, for example, pH
5 to 10, is preferably pH 6 to 9 and is more preferably pH 7 to
8.
[0045] A reaction temperature in the production method (1) of the
present invention is not particularly limited as long as the
objective reaction progresses, and is, for example, 10 to
50.degree. C., is preferably 20 to 40.degree. C. and is more
preferably 25 to 35.degree. C.
[0046] A reaction time period in the production method (1) of the
present invention is not particularly limited as long as the time
period is sufficient to form the 2S,4R-Monatin, and is, for
example, 2 to 100 hours, is preferably 4 to 50 hours and is more
preferably 8 to 25 hours.
[0047] When a transformant that expresses the L-amino acid
aminotransferase is used as the L-amino acid
aminotransferase-producing microorganism, this transformant can be
made by, for example, making an expression vector of the L-amino
acid aminotransferase, and then introducing this expression vector
into a host. For example, the transformant that expresses the
L-amino acid aminotransferase can be obtained by making the
expression vector incorporating DNA having the nucleotide sequence
represented by SEQ ID NO:1, and introducing it into an appropriate
host. For example, various prokaryotic cells including bacteria
belonging to genus Escherichia such as Escherichia coli, genus
Corynebacterium (e.g., Corynebacterium glutamicum) and genes
Bacillus (e.g., Bacillus subtilis), and various eukaryotic cells
including genus Saccharomyces (e.g., Saccharomyces cerevisiae),
genus Pichia (e.g., Pichia stipitis) and genus Aspergillus (e.g.,
Aspergillus oryzae) can be used as the host for expressing the
L-amino acid aminotransferase. For the host, a strain having
deletion of a certain gene may be used. Examples of such a gene
which may be deleted include AspC, an L-amino acid aminotransferase
derived from a host, an aldolase derived from a host, a deamination
enzyme derived from a host. Examples of the transformants include a
transformant carrying a vector in its cytoplasm, and a transformant
introduced with a gene of interest into its genome.
[0048] An L-amino acid aminotransferase-producing microorganism can
be cultured using certain culture apparatus (e.g., a test tube, a
flask, or a jar fermenter) in a medium having the composition
mentioned below. The culture condition can be set appropriately.
Specifically, the culture temperature may be 25.degree. C. to
37.degree. C., pH may be 6.5 to 7.5, the culture time may be 1 hour
to 100 hours. The cultivation may be carried out with controlling
the concentration of dissolved oxygen. In this case, the
concentration of dissolved oxygen (DO value) in the culture
solution may be utilized as an indicator of the controlling.
[0049] The condition on aeration and agitation can be controlled
such that relative concentration of dissolved oxygen (DO value) in
the case of the concentration of oxygen in air being considered 21%
is not less than 1% to 10%, preferably 3% to 8%. The cultivation
may be batch cultivation or fed-batch cultivation. In the case of
the fed-batch cultivation, a sugar source solution and a solution
containing phosphate can be continuously or discontinuously added
in a sequential manner to continue the cultivation.
[0050] The hosts to be transformed are as described above.
Describing Escherichia coli in detail, the host can be selected
from Escherichia coli K12 strain subspecies, Escherichia coli
JM109, DH5.alpha., HB101, BL21 (DE3) strains and the like. Methods
for performing the transformation and methods for selecting the
transformant are described in Molecular Cloning: A Laboratory
Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the
like. A method for making transformed Escherichia coli and
producing a certain enzyme by the use thereof will be specifically
described below as one example.
[0051] As a promoter for expressing DNA encoding the L-amino acid
aminotransferase, the promoter typically used for producing a
heterogeneous protein in E. coli can be used, and includes potent
promoters such as PhoA, PhoC, T7 promoter, lac promoter, trp
promoter, trc promoter, tac promoter, PR and PL promoters of lambda
phage, and T5 promoter. PhoA, PhoC and lac are preferred. As the
vector, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG
(e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010),
pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118,
pMW219, pMW218), pQE (e.g., pQE30) and derivatives thereof, and the
like may be used. The vectors of phage DNA may also be utilized as
the other vectors. Further, the expression vector containing the
promoter and capable of expressing the inserted DNA sequence may be
used. Preferably, the vector may be pUC, pSTV or pMW.
[0052] A terminator that is a transcription termination sequence
may be ligated to downstream of an L-amino acid aminotransferase
gene. Examples of such a terminator include T7 terminator, fd phage
terminator, T4 terminator, a terminator of a tetracycline resistant
gene, and a terminator of an E. coli trpA gene.
[0053] So-called multiple copy types are preferable as the vector
for introducing the L-amino acid aminotransferase gene into E.
coli, and include plasmids having a replication origin derived from
ColE1, such as pUC type plasmids, pBR322 type plasmids or
derivatives thereof. Here, the "derivatives" means those in which
modification is given to the plasmids by substitution, deletion,
insertion, addition and/or inversion of nucleotides. The
"modification" as referred to here also includes the modification
by mutagenic treatments by mutagenic agents and UV irradiation, or
natural mutation, or the like.
[0054] For selecting the transformant, it is preferable that the
vector has a marker such as an ampicillin resistant gene. As such a
plasmid, the expression vectors carrying the strong promoter are
commercially available (e.g., pUC types (supplied from TAKARA BIO
Inc.), pPROK types (supplied from Clontech), pKK233-2 (supplied
from Clontech)).
[0055] The L-amino acid aminotransferase is expressed by
transforming E. coli with the obtained expression vector and
culturing this E. coli.
[0056] A medium such as M9-casamino acid medium and LB medium
typically used for culturing E. coli may be used as the medium. The
medium may contain a certain carbon source, a nitrogen source, and
a coenzyme (e.g., pyridoxine hydrochloride). Specifically, peptone,
yeast extract, NaCl, glucose, MgSO.sub.4, ammonium sulfate,
potassium dihydrogen phosphate, ferric sulfate, manganese sulfate,
thiamine, hydrolysate of soy with hydrochloric acid, Disfoam
GD113-K (NOF Corporation) and the like may be used. Culture
conditions and production induction conditions are appropriately
selected depending on types of the marker and the promoter in the
used vector, the host bacterium and the like.
[0057] The following methods and the like are available for
recovering the L-amino acid aminotransferase. The L-amino acid
aminotransferase can be obtained as a disrupted product or a lysate
by collecting the L-amino acid aminotransferase-producing
microorganism followed by disrupting (e.g., sonication,
homogenization) or lysing (e.g., lysozyme treatment) the microbial
cells. Also, the purified enzyme, the crude enzyme, the L-amino
acid aminotransferase-containing fraction, or the like can be
obtained by subjecting such a disrupted product or lysate to
techniques such as extraction, precipitation, filtration and column
chromatography.
[0058] In a preferred embodiment, the production method of the
present invention further comprises contacting a keto acid
(R--COCOOH) formed from the L-amino acid (e.g., L-.alpha.-amino
acid) by action of the L-amino acid aminotransferase with a
decarboxylase to degrade the keto acid (see the reaction 1'). By
promoting the degradation of the keto acid formed from the L-amino
acid by an amino group transfer reaction, it is possible to shift
the equilibrium of the reaction to form the 2S,4R-Monatin from
4R-IHOG so that the 2S,4R-Monatin is formed in a larger amount.
##STR00003##
[0059] The decarboxylase used in the present invention is the
enzyme that catalyzes a decarboxylation reaction of the keto acid.
The decarboxylation reaction by the decarboxylase can be
irreversible. Various enzymes are known as the decarboxylase used
for the irreversible decarboxylation reaction of the keto acid, and
examples thereof include an oxaloacetate decarboxylase derived from
Pseudomonas stutzeri (Arch Biochem Biophys., 365, 17-24, 1999) and
a pyruvate decarboxylase derived from Zymomonas mobilis (Applied
Microbiology and Biotechnology, 17, 152-157, 1983).
[0060] In a particularly preferred embodiment, the production
method of the present invention comprises contacting oxaloacetate
(CAA) formed from L-aspartic acid (L-Asp) by action of the L-amino
acid aminotransferase with the oxaloacetate decarboxylase to form
the pyruvate (PA) (see the reaction 1''). By promoting the
irreversible formation of the pyruvate from the oxaloacetate, it is
possible to shift the equilibrium of the reaction to form the
2S,4R-Monatin from 4R-IHOG so that the 2S,4R-Monatin is formed in a
larger amount. A salt form of L-aspartic acid may be added to the
reaction solution. The concentration of L-aspartate in the reaction
solution is 1 mM to 3 M, preferably 20 mM to 1 M, more preferably
100 mM to 500 mM.
##STR00004##
[0061] The oxaloacetate decarboxylase used in the present invention
is the enzyme that catalyzes the decarboxylation reaction of the
oxaloacetate to form the pyruvate. The decarboxylation reaction by
the oxaloacetate decarboxylase can be irreversible. Various enzymes
are known as the oxaloacetate decarboxylase used for the
irreversible decarboxylation reaction of the oxaloacetate. Examples
of such an oxaloacetate decarboxylase include the oxaloacetate
decarboxylase derived from Pseudomonas stutzeri (Arch Biochem
Biophys., 365, 17-24, 1999), the oxaloacetate decarboxylase derived
from Klebsiella aerogenes (FEBS Lett., 141, 59-62, 1982), and the
oxaloacetate decarboxylase derived from Sulfolobus solfataricus
(Biochim Biophys Acta., 957, 301-311, 1988).
[0062] When the decarboxylase is used in the production of the
2S,4R-Monatin from 4R-IHOG, the contact of the keto acid formed
from the L-amino acid with the decarboxylase can be accomplished by
allowing the keto acid and the decarboxylase extracted from a
decarboxylase-producing microorganism (extracted enzyme) or the
decarboxylase-producing microorganism to coexist in the reaction
solution (e.g., culture medium). Examples of the
decarboxylase-producing microorganism include microorganisms that
naturally produce the decarboxylase and transformants that express
the decarboxylase. Examples of the extracted enzyme include a
purified enzyme, a crude enzyme, an immobilized enzyme, a culture
broth, and a treated product of the culture broth (e.g., a
decarboxylase-containing fraction prepared from the above
decarboxylase-producing microorganism, and a disrupted product of
and a lysate of the above decarboxylase-producing microorganism).
Examples of the treatment for obtaining the treated product of the
culture broth from the culture broth include a heat treatment
(42.degree. C. to 80.degree. C., pH 3 to 12, 1 minute to 24 hours),
a solvent treatment (e.g, xylene, toluene, ethanol,
isopropylalcohol), a surfactant (e.g., Tween 20, Triton X-100), and
a treatment with a bacteriolytic enzyme (e.g., lysozyme treatment).
Alternatively, the culture broth is subjected to a reaction after
retaining it with adjusting temperature, pH and the like to enhance
an enzymatic activity detected in the broth. In this case, the
temperature may be set at 4.degree. C. to 60.degree. C., preferably
20.degree. C. to 37.degree. C. The pH may be set at 3 to 12,
preferably 7 to 9. The time may be set for about 5 minutes to 20
days, preferably about 1 hour to 7 days. During retaining the
broth, aeration and agitation may be or may not be carried out.
[0063] When both the L-amino acid aminotransferase and the
decarboxylase are used in the production of the 2S,4R-Monatin from
4R-IHOG, the L-amino acid aminotransferase and the decarboxylase
may be provided in the reaction solution in the following
manner:
[0064] L-amino acid aminotransferase (extracted enzyme) and
decarboxylase (extracted enzyme);
[0065] L-amino acid aminotransferase-producing microorganism and
decarboxylase (extracted enzyme);
[0066] L-amino acid aminotransferase (extracted enzyme) and
decarboxylase-producing microorganism;
[0067] L-amino acid aminotransferase-producing microorganism and
decarboxylase-producing microorganism; and
[0068] L-amino acid aminotransferase- and decarboxylase-producing
microorganism.
[0069] Preferably, the L-amino acid aminotransferase- and
decarboxylase-producing microorganism may be a transformant. Such a
transformant can be made by i) introducing an expression vector of
the L-amino acid aminotransferase into the decarboxylase-producing
microorganism, ii) introducing an expression vector of the
decarboxylase into the L-amino acid aminotransferase-producing
microorganism, (iii) introducing a first expression vector of the
L-amino acid aminotransferase and a second expression vector of the
decarboxylase into a host microorganism, and (iv) introducing an
expression vector of the L-amino acid aminotransferase and the
decarboxylase into the host microorganism. Examples of the
expression vector of the L-amino acid aminotransferase and the
decarboxylase include i') an expression vector containing a first
expression unit composed of a first polynucleotide encoding the
L-amino acid aminotransferase and a first promoter operatively
linked to the first polynucleotide, and a second expression unit
composed of a second polynucleotide encoding the decarboxylase and
a second promoter operatively linked to the second polynucleotide;
and ii') an expression vector containing a first polynucleotide
encoding the L-amino acid aminotransferase, a second polynucleotide
encoding the decarboxylase and a promoter operatively linked to the
first polynucleotide and the second polynucleotide (vector capable
of expressing polycistronic mRNA). The first polynucleotide
encoding the L-amino acid aminotransferase may be located upstream
or downstream the second polynucleotide encoding the
decarboxylase.
(1-2) Method for Producing 2S,4R-Monatin from IPA and Pyruvate
[0070] The production method of the present invention may further
comprise condensing IPA and the pyruvate to form 4R-IHOG in order
to prepare 4R-IHOG. The condensation of IPA and the pyruvate can be
carried out by the organic chemistry process, or an enzymatic
method using an aldolase. The method for forming 4R-IHOG by
condensing IPA and the pyruvate by the organic chemistry process is
disclosed in, for example, International Publication WO2003/059865
and US Patent Application Publication No. 2008/0207920. The method
for forming 4R-IHOG by condensing IPA and the pyruvate by the
enzymatic method using the aldolase is disclosed in, for example,
International Publication WO2003/056026, JP 2006-204285-A, US
Patent Application Publication No. 2005/0244939 and International
Publication WO2007/103989. Therefore, in the present invention,
these methods can be used in order to prepare 4R-IHOG from IPA and
the pyruvate.
[0071] IPA used for the preparation of 4R-IHOG is an unstable
compound. Therefore, the condensation of IPA and the pyruvate may
be carried out in the presence of a stabilizing factor for IPA.
Examples of the stabilizing factor for IPA include superoxide
dismutase (e.g., see International Publication WO2009/028338) and
mercaptoethanol (e.g., see International Publication
WO2009/028338). For example, the transformant expressing the
superoxide dismutase is disclosed in International Publication
WO2009/028338. Thus, such a transformant may be used in the method
of the present invention.
[0072] The reaction to form 4R-IHOG from IPA and the pyruvate and
the reaction to form the 2S,4R-Monatin from 4R-IHOG may be
progressed separately or in parallel. These reactions may be
carried out in one reactor. When these reactions are carried out in
one reactor, these reactions can be carried out by adding the
substrates and the enzymes sequentially or simultaneously.
Specifically, when the reaction to form 4R-IHOG from IPA and the
pyruvate by the enzymatic method using the aldolase and the
reaction to form the 2S,4R-Monatin from 4R-IHOG by the L-amino acid
aminotransferase are carried out, (1) IPA, the pyruvate and the
aldolase, and (2) the L-amino acid and the L-amino acid
aminotransferase may be added in one reactor sequentially or
simultaneously. A salt form of pyruvate (e.g., sodium salt) may be
added to the reaction solution. Pyruvate may be added to the
reaction solution in any manner (e.g., batch method, or feed
method). The concentration of pyruvate in the reaction solution may
be, for example, 0.1 mM to 10 M, preferably 1 mM to 1 M.
[0073] In a preferred embodiment, the production method of the
present invention is combined with the above reaction 1'' as
follows. In this case, the pyruvate irreversibly formed from the
oxaloacetate is utilized for the preparation of 4R-IHOG. In other
words, at least a part of the pyruvate used for the formation of
4R-IHOG can be from the pyruvate formed from the oxaloacetate by
action of the oxaloacetate decarboxylase. In this case, it should
be noted that an initial amount of the pyruvate in the reaction
system is not necessarily important if an amount of the L-amino
acid present in the reaction system is sufficient because the
pyruvate is formed from the oxaloacetate in conjunction with the
formation of the 2S,4R-Monatin. Therefore, the larger amount of the
L-amino acid may be added to the reaction system compared with the
pyruvate.
##STR00005##
[0074] When the aldolase is used in the production of 4R-IHOG from
IPA and the pyruvate, the contact of IPA and the pyruvate with the
aldolase can be accomplished by allowing IPA, the pyruvate and the
aldolase extracted from an aldolase-producing microorganism
(extracted enzyme) or the aldolase-producing microorganism to
coexist in the reaction solution (e.g., culture medium). Examples
of the aldolase-producing microorganism include microorganisms that
naturally produce the aldolase and transformants that express the
aldolase. Examples of the extracted enzyme include a purified
enzyme, a crude enzyme, an immobilized enzyme, a culture broth, and
a treated product of the culture broth (e.g., an
aldolase-containing fraction prepared from the above
aldolase-producing microorganism, a disrupted product of and a
lysate of the above aldolase-producing microorganism). Examples of
the treatment for obtaining the treated product of the culture
broth from the culture broth include a heat treatment (42.degree.
C. to 80.degree. C., pH 3 to 12, 1 minute to 24 hours), a solvent
treatment (e.g, xylene, toluene), a surfactant treatment. The
culture broth may be used under a condition of 4.degree. C. to
60.degree. C., pH 3 to 12, and 5 minutes to 20 days (with or
without aeration and agitation). The aldolase-producing
microorganism may further express other enzyme(s) (e.g., superoxide
dismutase, L-amino acid aminotransferase, decarboxylase).
Alternatively, a microorganism that produces the other enzyme in
addition to the aldolase-producing microorganism may be allowed to
coexist in the reaction solution. Those described in the production
method (1-1) of the present invention can be used as the reaction
solution.
[0075] Preferably, the aldolase-, L-amino acid aminotransferase-
and decarboxylase-producing microorganism may be a transformant.
The expression of the aldolase, the L-amino acid aminotransferase
and the decarboxylase may be carried out using the same
transformant, or it may be carried out with a combination of two
transformants, or the three enzymes may be expressed in separate
transformants. the aldolase, L-amino acid aminotransferase and
decarboxylase genes are expressed in the same transformant, these
genes may be integrated into its chromosome, or the aldolase,
L-amino acid aminotransferase and decarboxylase genes are inserted
to one vector. Alternatively, an expression vector of the L-amino
acid aminotransferase may be introduced to a microorganism which
produces the decarboxylase and aldolase, or a first expression
vector of the L-amino acid aminotransferase and a second expression
vector of the decarboxylase and the aldolase may be introduced to a
host microorganism. Examples of the expression vector of the
aldolase, the L-amino acid aminotransferase and the decarboxylase
include i') an expression vector containing a first expression unit
composed of a first polynucleotide encoding the L-amino acid
aminotransferase and a first promoter operatively linked to the
first polynucleotide, a second expression unit composed of a second
polynucleotide encoding the decarboxylase and a second promoter
operatively linked to the second polynucleotide, and a third
expression unit composed of a third polynucleotide encoding the
decarboxylase and a third promoter operatively linked to the third
polynucleotide; and ii') an expression vector containing a first
expression unit composed of a first polynucleotide encoding the
L-amino acid aminotransferase, a second polynucleotide encoding the
decarboxylase and a promoter operatively linked to the first
polynucleotide and the second polynucleotide, and a second
expression unit composed of a third polynucleotide encoding the
aldolase and a promoter operatively linked to the third
polynucleotide (a vector capable of expressing a polycistronic
mRNA). The positions of genes encoding the L-amino acid
aminotransferase, the decarboxylase and the aldolase on a plasmid
are not particularly limited.
[0076] Various conditions such as the temperature, the pH value and
the time period in the reaction can be appropriately established as
long as the objective reaction can progress. For example, the
conditions of the enzymatic method using the aldolase may be the
same as those described in the production method (1-1) of the
present invention.
(1-3) Method for Producing 2S,4R-Monatin or a Salt Thereof from
Tryptophan or a Salt Thereof
[0077] The production method of the present invention may further
comprise deaminating a tryptophan (Trp) in order to prepare IPA.
Trp includes L-Trp, D-Trp and a mixture of L-Trp and D-Trp. The
deamination of Trp can be performed by the organic chemistry
technique and the enzymatic method using a deamination enzyme.
[0078] Various methods are known as the method for deaminating Trp
to form IPA by the organic chemistry technique. Examples of such a
method include the method in which the tryptophan is used as a
starting material and reacted with pyridine aldehyde in the
presence of a base for dehydration of a proton acceptor (e.g., see
JP Sho-62-501912 and International Publication WO1987/000169), and
the method of subjecting to acid hydrolysis after a condensation
reaction using indole and ethyl-3-bromopyruvate ester oxime as raw
materials (e.g., European Patent Application Publication No.
421946).
[0079] As used herein, the term "deamination enzyme" refers to the
enzyme capable of forming IPA from Trp. The formation of IPA from
Trp is essentially conversion of the amino group (--NH.sub.2) in
Trp to an oxy group (.dbd.O). Therefore, the enzymes that catalyze
this reaction are sometimes termed as other names such as an amino
acid deaminase, an aminotransferase and an amino acid oxidase.
Therefore, the term "deamination enzyme" means any enzyme that can
form IPA from Trp, and the enzymes having the other name (e.g.,
amino acid deaminase, aminotransferase, amino acid oxidase) which
catalyze the reaction to form IPA from Trp are also included in the
"deamination enzyme."
[0080] Examples of the method for forming IPA from Trp using the
amino acid deaminase or an amino acid deaminase-producing
microorganism include the method disclosed in International
Publication WO2009/028338. A general formula of the reaction
catalyzed by the amino acid deaminase includes the following
formula: Amino acid+H.sub.2O.fwdarw.2-oxo acid+NH.sub.3.
[0081] Examples of the method for forming IPA from Trp using the
aminotransferase or an aminotransferase-producing microorganism
include the methods disclosed in East Germany Patent DD 297190, JP
Sho-59-95894-A, International Publication WO2003/091396 and US
Patent Application Publication No. 2005/028226.
[0082] Examples of the method for forming IPA from Trp using the
L-amino acid oxidase or an L-amino acid oxidase-producing
microorganism include the methods disclosed in U.S. Pat. No.
5,002,963, John A. Duerre et al. (Journal of Bacteriology 1975,
vol. 121, No. 2, p 656-663), JP Sho-57-146573, International
Publication WO2003/056026 and International Publication
WO2009/028338. The general formula of the reaction catalyzed by the
amino acid oxidase includes the following formula: Amino
acid+O.sub.2+H.sub.2O.fwdarw.2-Oxo acid+H.sub.2O.sub.2+NH.sub.3.
For the purpose of suppressing the degradation of the compound by
hydrogen peroxide as the by-product produced at that time, a
hydrogen peroxide-degrading enzyme such as a catalase may be added
in the reaction solution.
[0083] The reaction to form IPA from Trp, the reaction to form
4R-IHOG from IPA and the pyruvate and the reaction to form
2S,4R-Monatin from 4R-IHOG may be progressed separately or in
parallel. These reactions may be carried out in one reactor. When
these reactions are carried out in one reactor, these reactions can
be carried out by adding the substrates and the enzymes
sequentially or simultaneously. Specifically, when the reaction to
deaminate Trp by the enzymatic method using the deamination enzyme
to form IPA, the reaction to form 4R-IHOG from IPA and the pyruvate
by the enzymatic method using the aldolase, and the reaction to
form 2S,4R-Monatin from 4R-IHOG by the L-amino acid
aminotransferase are carried out, (1) Trp and the deamination
enzyme, (2) the pyruvate and the aldolase, and (3) the L-amino acid
and the L-amino acid aminotransferase may be added in one reactor
sequentially or simultaneously.
[0084] When the deamination enzyme is used in the production of IPA
from Trp, the contact of Trp with the deamination enzyme can be
accomplished by allowing Trp and the deamination enzyme extracted
from a deamination enzyme-producing microorganism (extracted
enzyme) or the deamination enzyme-producing microorganism to
coexist in the reaction solution. Examples of the deamination
enzyme-producing microorganism include microorganisms that
naturally produce the deamination enzyme and transformants that
express the deamination enzyme. For example, the pTB2 strain
described in Example 2 of WO 2009/028338 (the modified strain of E.
coli introduced with the amino acid deaminase gene derived from the
strain of Providencia rettgeri) may be used. An operative promoter
(e.g., phoA, phoC, trp, lac, or tac promoter) may be linked to the
deaminase gene in the plasmid. When E. coli is used as a host, a
plasmid capable of expressing a deaminase may be introduced to a
host having a deletion of a certain gene such as aspC gene.
Examples of the extracted enzyme include a purified enzyme, a crude
enzyme, an immobilized enzyme, a cuture broth, and a treated
product of the culture broth (e.g., a deamination enzyme-containing
fraction prepared from the above deamination enzyme-producing
microorganism, a disrupted product of and a lysate of the above
deamination enzyme-producing microorganism). Examples of the
treatment for obtaining the treated product of the culture broth
from the culture broth include a heat treatment (42.degree. C. to
80.degree. C., pH 3 to 12, 1 minute to 24 hours), a solvent
treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a
surfactant (e.g., Tween 20, Triton X-100), and a treatment with a
bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the
culture broth is subjected to a reaction after retaining it with
adjusting temperature, pH and the like to enhance an enzymatic
activity detected in the broth. In this case, the temperature may
be set at 4.degree. C. to 60.degree. C., preferably 20.degree. C.
to 37.degree. C. In addition, the pH may be set at 3 to 12,
preferably 7 to 9. The time may be set for about 5 minutes to 20
days, preferably about 1 hour to 7 days. During retaining the
broth, aeration and agitation may be or may not be carried out. The
deamination enzyme-producing microorganism may further express the
other enzyme(s) (e.g., aldolase, superoxide dismutase, L-amino acid
aminotransferase, decarboxylase). Alternatively, the other
enzyme-producing microorganism in addition to the deamination
enzyme-producing microorganism may be allowed to coexist in the
reaction solution. Those described in the production method (1-1)
of the present invention can be used as the reaction solution. Trp
is preferably L-trp. A salt form of Trp may be added to the
reaction solution. The concentration of Trp in the reaction
solution is, for example, 1 mM to 3 M, preferably 20 mM to 1 M,
more preferably 20 mM to 300 mM.
[0085] Various conditions such as the temperature, the pH value and
the time period in the reaction can be appropriately established as
long as the objective reaction can progress. For example, the
conditions of the enzymatic method using the deamination enzyme may
be the same as those described in the production method (1-1) of
the present invention.
[0086] In a preferred embodiment, when the production method (1-3)
of the present invention is carried out in one reactor, the
deaminase, the aldolase, the L-amino acid aminotransferase, and the
oxaloacetate decarboxylase, and/or one or more transformants
expressing them are used. The superoxide dimustase, and/or a
transformant expressing it may be further used. These enzymes may
be mutants. For an expression system of the enzymes, the
aforementioned transformants can be used. Specifically, a
transformant carrying the expression vector of a gene of interest
in its cytoplasm, a transformant introduced with a gene of interest
on its genome, and a transformant which carries the expression
vector of a gene of interest in its cytoplasm, and which is
introduced with a gene of interest on its genome. For an expression
vector used in the preparation of the transformant, the
aforementioned expression vector can be used.
[0087] In a preferred embodiment, when the production method (1-3)
of the present invention is carried out in one reactor, a reaction
solution containing a certain concentrations of L-Trp, L-Asp, PA, a
buffer (e.g., phosphate buffer, Tris buffer) and PLP can be used.
The concentration of L-Trp is, for example, 1 mM to 3 M, preferably
10 mM to 1 M, more preferably 50 mM to 300 mM. The concentration of
L-Asp is, for example, 1 mM to 3 M, preferably 100 mM to 1 M, more
preferably 200 mM to 400 mM. L-Asp may be a salt form (e.g., sodium
salt, potassium salt) or a free form. When L-Asp is used in a free
form, pH may be appropriately adjusted after supplying it in the
reaction solution. In this case, an alkaline solution (e.g., NaOH
aqueous solution, KOH aqueous solution) may be used for the
adjustment of pH. The concentration of PA is, for example, 1 mM to
3 M, preferably 10 mM to 100 mM. PA may be a salt form (e.g.,
sodium salt, potassium salt) or a free form. When PA is used in a
free form, pH may be adjusted after supplying it in the reaction
solution. The concentration of PLP is, for example, 1 .mu.M to 100
mM, preferably 10 .mu.M to 1 mM. The reaction solution may further
contain magnesium, phosphate, and antifoaming agent. When magnesium
is used as a salt, the salt form of magnesium is not particularly
limited, and examples of the salt form include magnesium chloride
and magnesium sulfate. The concentration of magnesium is, for
example, 0.1 mM to 100 mM, preferably 0.5 mM to 5 mM. In addition,
the phosphate is used as a salt, the salt form of the phosphate is
not particularly limited, and examples of the salt form include a
potassium salt (e.g., monopotassium salt, dipotassium salt,
tripotassium salt) and a sodium salt (e.g., monosodium salt,
disodium salt, trisodium salt). The concentration of the phosphate
is, for example, 1 mM to 100 mM, preferably 10 mM to 50 mM. The
antifoaming agent is not particularly limited, and examples of the
antifoaming agent include GD113K. The concentration of the
antifoaming agent is not particularly limited, and is 0.0001% to 1%
(v/v), preferably 0.001% to 0.1% (v/v). The reaction condition such
as pH, temperature, aeration, agitation and time can be
appropriately set. The pH of the reaction solution is, for example,
5 to 10, preferably 6 to 9, more preferably 7 to 8. The control of
pH during the reaction may be achieved by adding an acid or
alkaline appropriately. The acid or alkaline used in this case is
not particularly limited, and examples of the acid or alkaline
include hydrochloric acid, phosphoric acid, sulfuric acid, ammonium
gas, ammonium aqueous solution, NaOH aqueous solution, and KOH
aqueous solution. The concentration of the acid or alkaline used in
the adjustment of pH is not particularly limited. It is, for
example, 0.1 N to 20 N, preferably 3 N to 12 N, when a solution of
the acid or alkaline is used. The reaction temperature is, for
example, 10.degree. C. to 50.degree. C., preferably 20.degree. C.
to 40.degree. C., more preferably 25.degree. C. to 35.degree. C.
When a container capable of controlling aeration and agitation
(e.g., jar fermenter) is used for the reaction, the concentration
of dissolved oxygen in the reaction solution can be set by
controlling the conditions on aeration and agitation. A person
skilled in the art can set the conditions on aeration and agitation
according to the used container. For example, when a jar fermenter
with a volume of 1 litter is used, the condition on aeration is,
for example, 1/200 to 1 vvm, preferably 1/100 to 1/10 vvm. The
condition on agitation is, for example, 100 rpm to 1000 rpm,
preferably 400 rpm to 700 rpm. Examples of the enzyme to be added
to the reaction include a purified enzyme, a microorganism
expressing an enzyme, a treated product of a microorganism
expressing an enzyme, a cuture broth containing a microorganism
expressing an enzyme, and a treated product of a culture broth
containing a microorganism expressing an enzyme. Examples of the
treatment for obtaining the treated product of the culture broth
from the culture broth include a heat treatment (42.degree. C. to
80.degree. C., pH 3 to 12, 1 minute to 24 hours), a solvent
treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a
surfactant (e.g., Tween 20, Triton X-100), and a treatment with a
bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the
culture broth is subjected to a reaction after retaining it with
adjusting temperature, pH and the like to enhance an enzymatic
activity detected in the broth. In this case, the temperature of
the culture broth may be 4.degree. C. to 60.degree. C., preferably
20.degree. C. to 37.degree. C. The pH of the culture broth may be 3
to 12, preferably 7 to 9. The retaining time may be about 5 minutes
to 20 days, preferably about 1 hour to 7 days. During retaining the
broth, aeration and agitation may be or may not be carried out.
[0088] Each enzyme to be added to the reaction solutions can be
appropriately determined by measuring an activity of each enzyme
previously. The deaminease activity, the aldolase activity, the
L-amino acid aminotransferase activity, and the oxaloacetate
decarboxylase activity can be measured by the following
methods.
[0089] Deaminase activity: 10 mM L-Phe, 100 mM NH.sub.4Cl, 100 mM
Tris-HCl (pH 8.0), 0.25 mM NADH and phenylalanine dehydrogenase
(manufactured by UNITIKA, derived from Thermoactinomyces
intermedius) at 25.degree. C. The activity is calculated from the
reduction of the absorbance measured at 340 nm.
[0090] L-amino acid aminotransferase activity (L-Asp/.alpha.-KG
activity): 100 mM L-Asp-Na-laq, 10 mM .alpha.-KG-2Na, 50 .mu.M PLP,
100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 2 U/mL of MDH at
25.degree. C. The activity is calculated from the reduction of the
absorbance at 340 nm. Malic dehydrogenase from porcine heart
(Sigma) was used as MDH.
[0091] Aldolase activity: 2 mM 4-phenyl-4-hydroxy-2-oxo glutarate
(PHOG), 100 mM Tris-HCl (pH 7.0), 1 mM MgCl.sub.2, 0.25 mM NADH, 10
U/ml lactate dehydrogenase (manufactured by ORIENTAL YEAST Co.,
Ltd., derived from Leuconostoc mesenteroides) at 25.degree. C. The
activity is calculated from the reduction of the absorbance at 340
nm.
[0092] Oxaloacetate decarboxylase activity: 1 mM oxaloacetate, 100
mM Tris-HCl (pH 8.0), 0.25 mM NADH, 10 U/ml lactate dehydrogenase
(manufactured by ORIENTAL YEAST Co., Ltd., derived from Leuconostoc
mesenteroides) at 25.degree. C. The activity is calculated from the
reduction of the absorbance at 340 nm.
[0093] Based on the enzymatic activities determined as mentioned
above, the amounts of enzymes to be added to the reaction solution
may be as follows. The amount of the deaminase to be added to the
reaction solution is, for example, 0.1 to 20 U/ml, preferably 0.5
to 2 U/ml. The amount of the aldolase to be added to the reaction
solution is, for example, 1 to 1000 U/ml, preferably 10 to 100
U/ml. The amount of the L-amino acid aminotransferase to be added
to the reaction solution is, for example, 1 to 1000 U/ml,
preferably 10 to 100 U/ml. The amount of the oxaloacetate
decarboxylase to be added to the reaction solution is, for example,
0.01 U/ml or more, preferably 0.1 U/ml or more. Each substrate may
be added to a reaction system by a batch method or a feed method.
The enzyme, the microorganism expressing the enzyme, the treated
product of the microorganism expressing the enzyme, the cuture
broth containing the microorganism expressing the enzyme, and the
treated product of the culture broth containing the microorganism
expressing the enzyme may also be added to the reaction system by a
batch method or a feed method. The reaction time is, for example, 2
to 100 hours, preferably 4 to 50 hours, more preferably 8 to 25
hours. The reaction solution may be sterilized under an appropriate
condition (e.g., temperature, pH, time).
[0094] When the production method (1-2) of the present invention is
carried out in one reactor, such a production method can be carried
out similar to the production method (1-3) of the present
invention.
[0095] The purified 2S,4R-Monatin can be obtained by taking
advantage of known purification methods such as column treatment,
crystallization treatment and extraction treatment for a
2S,4R-Monatin-containing reaction solution obtained by any of the
production methods (1-1), (1-2) and (1-3) of the present invention.
The purified 2S,4R-Monatin can be provided to a method (2) for
producing 2R,4R-Monatin or a salt thereof. The
2S,4R-Monatin-containing reaction solution obtained by any of the
production methods (1-1), (1-2) and (1-3) of the present invention
can also be directly provided to the method (2) for producing the
2R,4R-Monatin or the salt thereof.
(2) Method for Producing 2R,4R-Monatin or a Salt Thereof
[0096] The present invention provides a method (2) for producing
2R,4R-Monatin or the salt thereof. The production method of the
present invention comprises performing the production method (1) of
the present invention to form the 2S,4R-Monatin or a salt thereof,
and isomerizing the 2S,4R-Monatin or the salt thereof to form
2R,4R-Monatin or a salt thereof.
[0097] The isomerization of the 2S,4R-monatin to the 2R,4R-Monatin
can be performed by any method that enables the isomerization
(e.g., see International Publication WO2005/082850 and
International Publication WO03/059865). However, in terms of
enhancing a yield of the 2R,4R-Monatin, the isomerization of the
2S,4R-Monatin is preferably performed by
epimerization-crystallization (e.g., see International Publication
WO2005/082580). The epimerization-crystallization is a method in
which the isomerization reaction and the crystallization are
performed simultaneously. In this case, the isomerization reaction
at position 2 to convert the 2S,4R-Monatin into the 2R,4R-Monatin
and the crystallization of the converted 2R,4R-Monatin are
performed simultaneously by the epimerization-crystallization.
[0098] In the epimerization-crystallization, the isomerization
reaction may be performed in the presence of an aldehyde. The
aldehyde includes an aliphatic aldehyde and an aromatic aldehyde,
and the aromatic aldehyde is preferred. A purified 2S,4R-Monatin or
a 2S,4R-Monatin-containing reaction solution may be used as the
2S,4R-Monatin used for the isomerization reaction.
[0099] For the aliphatic aldehyde, for example, a saturated or
unsaturated aldehyde having 1 to 7 carbon atoms, such as
formaldehyde, acetaldehyde, propionaldehyde, n-butyl aldehyde,
1-butyl aldehyde, n-valeraldehyde, capronaldehyde,
n-heptylaldehyde, acrolein or methacrolein can be used.
[0100] For the aromatic aldehyde, the aromatic aldehyde such as
benzaldehyde, salicylaldehyde, m-hydroxybenzaldehyde,
p-hydroxybenzaldehyde, o-nitrobenzaldehyde, p-nitrobenzaldehyde,
5-nitrosalicylaldehyde, 3,5-dichlorosalicylaldehyde, anisaldehyde,
o-vanillin, vanillin, furfural, pyridoxal or 5-phosphate pyridoxal
can be used. Particularly, pyridoxal, 5-nitrosalicylaldehyde, or
3,5-dichlorosalicylaldehyde is preferred as the aromatic
aldehyde.
[0101] The aldehyde can be used in the range of 0.01 to 1 mol
equivalent and more preferably 0.05 to 0.5 mol equivalent to the
Monatin present in the system.
[0102] The epimerization-crystallization is performed in the
presence of the aldehyde, and a mixed solvent of water and an
organic solvent is used as a solvent. The organic solvent miscible
with the water is used as the organic solvent, and particularly,
alcohol such as methanol, ethanol, propanol or isopropanol is
preferred. Two or more different kinds of organic solvents may be
used in mixture. A volume ratio of the organic solvent to the water
is set in the range of preferably 1:0.01 to 1:1 and more preferably
1:0.1 to 1:0.5 (organic solvent:water).
[0103] The temperature in the epimerization-crystallization is set
in the range of preferably 0 to 100.degree. C. and more preferably
40 to 80.degree. C. The time period for performing the
epimerization-crystallization is set in the range of preferably 10
hours to one week and more preferably 15 hours to 96 hours.
[0104] The pH value is set in the range of 4 to 13, preferably 4.5
to 10 and more preferably 5 to 9. The pH value can be adjusted
using an acid or an alkali. The acid to be used is not particularly
limited, and an organic acid such as acetic acid, or an inorganic
acid such as hydrochloric acid or sulfuric acid can be used. The
alkali is not also particularly limited, and an alkali metal
hydroxide such as sodium hydroxide or potassium hydroxide, or an
organic base such as ammonia or amine can be used.
[0105] Each compound obtained by the above method can be isolated
and purified by optionally combining known separation and
purification procedures such as concentration, reduced pressure
concentration, solvent extraction, crystallization,
recrystallization, solvent transfer, a treatment with activated
charcoal, and treatments with chromatography and the like using ion
exchange resin or synthetic adsorption resin. The salts of the
compound used in the method of the present invention and the
compound (objective compound) produced by the method of the present
invention can be produced, for example, by adding the inorganic
acid or the organic acid to the objective compound according to the
method publicly known per se. The objective compound and the salt
thereof may be hydrate, and both hydrate and non-hydrate are
included in the scope of the present invention. The compounds
(e.g., Trp, IPA, 4R-IHOG, 2S,4R-Monatin) used for the production
methods of the present invention may be the forms of various salts
such as sodium salts, potassium salts and ammonium salts. The
compounds (e.g., IPA, 4R-IHOG, 2S,4R-Monatin, 2R,4R-Monatin)
obtained by the production method of the present invention may also
be the forms of various salts.
[0106] The present invention will be described in detail by the
following Examples, but the present invention is not limited by
these Examples.
EXAMPLES
Analytical Condition of HPLC
[0107] In Examples 1 to 7, if HPLC analysis was performed, the HPLC
analysis was performed under the condition shown in the
Example.
[0108] In Examples 8 to 15, the HPLC analysis was performed under
the condition shown below.
[0109] Detector: Ultraviolet absorption spectrometer (measured
wavelength: 210 nm)
[0110] Column temperature: 40.degree. C.
[0111] Column: CAPCELLPAK C18 Type MGII, inner diameter: 3 mm,
length: 25 cm, and particle diameter: 5 .mu.m, Shiseido Co.,
Ltd.
[0112] Mobile phase: Solution A (aqueous solution of 20 mM
potassium dihydrogen phosphate:acetonitrile=95:5) and solution B
(aqueous solution of 20 mM potassium dihydrogen
phosphate:acetonitrile=60:40)
[0113] Gradient program: See the following Table 1
TABLE-US-00001 TABLE 1 Gradient program Time (min) Mobile phase A
(%) Mobile phase B (%) 0.0 100 0 15.0 100 0 40.0 0 100 45.0 0 100
45.1 100 0
[0114] Flow: 0.45 mL/minute
[0115] Injection amount: 20 IAL
[0116] Analysis time period: 60 minutes
Example 1
Formation of 2S,4R-Monatin from 4R-IHOG Using Extraction Solution
from Bacillus altitudinis AJ1616 Microbial Cells
[0117] Bacillus altitudinis AJ1616 was streaked on CM2G agar medium
(10 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of glucose,
5 g/L of sodium chloride, 15 g/L of agar, pH 7.0), and cultured at
30.degree. C. for 2 days.
[0118] One loopful of the resulting microbial cells was inoculated
to 3 mL of an enzyme production medium (10 g/L of yeast extract, 10
g/L of polypeptone, 1 g/L of glucose, 3 g/L of dipotassium hydrogen
phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of
magnesium sulfate heptahydrate, 5 g/L of ammonium sulfate) in a
test tube, which was then cultured with shaking at 30.degree. C.
for 16 hours. The microbial cells were collected from 2 mL of the
cultured medium by centrifugation, washed with and suspended in 20
mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell
suspension.
[0119] 1 g of glass beads (0.1 mm) was added to 1 mL of this
microbial cell suspension, and the microbial cells were disrupted
using a multi beads shocker (Yasui Kikai Co., Ltd.). The resulting
disrupted cell solution was centrifuged to use a supernatant as a
microbial cell extract.
[0120] A 2S,4R-Monatin synthesis reaction solution (0.1 mL) (9.5 mM
4R-IHOG, 0.5 mM 4S-IHOG, 100 mM L-Asp, 50 .mu.M PLP, 100 mM
Tris-HCl, pH 8.0) was prepared so that 0.05 mL of the Bacillus
altitudinis AJ1616 microbial cell extract was contained. The
reaction solution was reacted at 30.degree. C. for 20 hours. After
termination of the reaction, the formed 2S,4R-Monatin was
quantified, and its concentration was 0.21 mM.
[0121] The 2S,4R-Monatin was quantified using UPLC (Waters). The
analytical condition is as follows.
[0122] Mobile phase: 20 mM KH.sub.2PO.sub.4/asetonitrile=100/5
[0123] Flow rate: 0.15 mL/minute
[0124] Column temperature: 40.degree. C.
[0125] Detection: UV 210 nm
[0126] Column: ACQUITY UPLC BEH C18, 2.1.times.50 mm, 1.7 .mu.m
(Waters).
Example 2
Purification of Aminotransferase Derived from Bacillus altitudinis
AJ1616
[0127] An aminotransferase for forming the 2S,4R-Monatin was
purified from a soluble fraction of Bacillus altitudinis AJ1616 as
follows. The reaction for synthesizing 2S,4R-Monatin and the
quantification of 2S,4R-Monatin were performed in the same manner
as in Example 1.
(1) Preparation of Soluble Fraction
[0128] Bacillus altitudinis AJ1616 was streaked on CM2G agar medium
(10 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of glucose,
5 g/L of sodium chloride, 15 g/L of agar, pH 7.0), and cultured at
30.degree. C. for 2 days.
[0129] One loopful of the resulting microbial cells was inoculated
to 160 mL of TB (Terrific Broth) medium in a 500 mL Sakaguchi
flask, which was then cultured with shaking at 30.degree. C. for 16
hours. The microbial cells were collected from about 2000 mL of the
cultured medium by centrifugation, washed with and suspended in 20
mM Tris-HCl (pH 7.6), 100 mM NaCl, and then disrupted by sonication
at 4.degree. C. for 30 minutes. Microbial cell debris was removed
from the disrupted solution by centrifugation, and the resulting
supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0130] The above soluble fraction was applied onto an anion
exchange chromatography column HiLoad 26/10 Q Sepharose HP
(supplied from GE Health Care Bioscience, CV=53 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and adsorbed to the
carrier. Proteins that had not been adsorbed to the carrier
(unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6),
100 mM NaCl, and subsequently the adsorbed proteins were eluted by
linearly changing the concentration of NaCl from 100 mM to 500 mM
at a flow rate of 8 mL/minute. A 2S,4R-Monatin forming activity was
measured in each fraction, and detected in the fractions
corresponding to about 200 mM NaCl.
(3) Hydrophobic Chromatography
[0131] The fractions in which the 2S,4R-Monatin forming activity
had been detected were combined, and ammonium sulfate and Tris-HCl
(pH 7.6) were added thereto at final concentrations of 1.4 M and 20
mM, respectively. This solution was applied to a hydrophobic
chromatography column HiLoad 16/10 Phenyl Sepharose HP (supplied
from GE Health Care Bioscience, CV=20 mL) equilibrated with 1.4 M
ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and adsorbed to the
carrier. Unadsorbed proteins that had not been adsorbed to the
carrier were washed out with 1.4 M ammonium sulfate, 20 mM Tris-HCl
(pH 7.6), and subsequently, a 2S,4R-Monatin forming enzyme was
eluted by linearly changing the concentration of ammonium sulfate
from 1.4 M to 0 M at a flow rate of 3 mL/minute. The 2S,4R-Monatin
forming activity was measured in each fraction, and detected in the
fractions corresponding to about 1.0 M ammonium sulfate.
(4) Gel Filtration Chromatography
[0132] The fractions in which the 2S,4R-Monatin forming activity
had been detected were combined and concentrated using Amicon
Ultra-15 30K (Millipore). The resulting concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6), 150 mM NaCl. This solution
was applied to a gel filtration column HiLoad 16/60 Superdex 200 pg
(supplied from GE Health Care Bioscience, CV=120 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and eluted at a flow
rate of 1 mL/minute. This manipulation confirmed the 2S,4R-Monatin
forming activity in a location estimated as a molecular weight of
about 120 kDa.
(5) Anion Exchange Chromatography
[0133] The fractions in which the 2S,4R-Monatin forming activity
had been detected were combined and applied to an anion exchange
chromatography column Mono Q 5/5 (supplied from Pharmacia (GE
Health Care Bioscience), CV=1 mL) equilibrated with 20 mM Tris-HCl,
100 mM NaCl (pH 7.6), and adsorbed to the carrier. Proteins that
had not been adsorbed to the carrier (unadsorbed proteins) were
washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and
subsequently the adsorbed proteins were eluted by linearly changing
the concentration of NaCl from 100 mM to 500 mM at a flow rate of
0.5 mL/minute. The 2S,4R-Monatin forming activity was measured in
each fraction, and detected in the fractions corresponding to about
200 mM NaCl.
(6) SDS-PAGE
[0134] The obtained fractions were subjected to SDS-PAGE, and a
band around 45 kDa was observed in the active fraction. This band
was subjected to analysis of an N-terminal amino acid sequence as a
candidate for the aminotransferase for forming the 2S,4R-Monatin.
The band was also subjected to the analysis of an internal amino
acid sequence.
Example 3
Determination of N-Terminal and Internal Amino Acid Sequences of
Aminotransferase Derived from Bacillus altitudinis AJ1616
[0135] The purified enzyme solution obtained in Example 2 was
subjected to the analysis of the N-terminal amino acid sequence,
and the sequence SGFTALSEAELNDLY (SEQ ID NO:4) was obtained as the
N-terminal amino acid sequence. The sample in SDS-PAGE gel was
treated with trypsin (pH 8.0, 35.degree. C., 20 hours), and
subsequently subjected to reverse phase HPLC to separate peptide
fragments. The amino acid sequences in the fractionated fractions
were analyzed, and the sequence QLDLSMGMLDVV (SEQ ID NO:5) was
obtained as the internal amino acid sequence. Both the N-terminal
amino acid sequence and the internal amino acid sequence exhibited
high homology to the aminotransferase derived from Bacillus pumilus
SAFR-032 (YP001487343).
Example 4
Cloning of Aminotransferase Gene Derived from Bacillus altitudinis
AJ1616
[0136] Bacillus altitudinis AJ1616 was cultured in the same manner
as in Example 1. The microbial cells were collected from the
cultured medium by centrifugation, and genomic DNA was
extracted.
[0137] A DNA fragment including an aminotransferase gene was
amplified by PCR using the obtained genomic DNA as a template. For
primers, the primer Bp-u300-f (5'-ctcaggaagcaggcgcaaaaagattaattt-3'
(SEQ ID NO:6) and the primer Bp-d200-r
(5'-ggatgctgtctttgtcatcccaaagtggat-3' (SEQ ID NO:7) were used,
which were designed from DNA sequences of upstream 300 bp and
downstream 200 bp in the aminotransferase gene with reference to
the genomic DNA sequence of Bacillus pumilus SAFR-032 (CP000813).
PCR was performed using KOD-plus-ver. 2 (Toyobo) under the
following condition.
TABLE-US-00002 1 cycle 94.degree. C., 2 min 25 cycles 98.degree.
C., 10 sec 55.degree. C., 10 sec 68.degree. C., 60 sec 1 cycle
68.degree. C., 60 sec 4.degree. C.
[0138] A nucleotide sequence of about 1800 bp of the amplified DNA
fragment was determined, and the nucleotide sequence was shown to
include 1308 bp of ORF that had the high homology to the
aminotransferase gene derived from Bacillus pumilus SAFR-032 (NC
009848). The homology was 89% in the DNA sequences and 93% in the
amino acid sequences.
[0139] The N-terminal amino acid sequence and the internal amino
acid sequence obtained in Example 3 were found in this sequence.
Thus, it was thought that the aminotransferase gene having the
2S,4R-Monatin forming activity could have been acquired.
Example 5
Expression of Aminotransferase Derived from Bacillus altitudinis
AJ1616 in E. coli
[0140] (1) Construction of Plasmid Expressing Aminotransferase
Derived from Bacillus altitudinis AJ1616
[0141] A DNA fragment including the aminotransferase gene derived
from Bacillus altitudinis AJ1616 was amplified by PCR using the
genomic DNA of Bacillus altitudinis AJ1616 as the template. The
primer 1616AT-Nde-f (5'-ggaattccatATGAGCGGTTTTACAGCGTT-3': SEQ ID
NO:8) and the primer 1616-xho-r
(5'-gtcaaggagtttttctcgagTACCGTTGGTGCTGATTGAC-3': SEQ ID NO:9) were
used as the primers. A NdeI sequence in the aminotransferase gene
was converted using the primer 1616-delNde-f
(5'-GGATTGAAGGAACAcATGAAAAAGCATGC-3': SEQ ID NO:10) and the primer
1616-delNde-r (5'-GCATGCTTTTTCATgTGTTCCTTCAATCC-3': SEQ ID NO:11).
PCR was performed using KOD-plus-ver. 2 (Toyobo) under the
following condition.
TABLE-US-00003 1 cycle 94.degree. C., 2 min 25 cycles 98.degree.
C., 10 sec 55.degree. C., 10 sec 68.degree. C., 60 sec 1 cycle
68.degree. C., 60 sec 4.degree. C.
[0142] The resulting DNA fragment of about 1300 bp was treated with
restriction enzymes NdeI and XhoI, and then ligated to pET-22b
(Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was
transformed with this solution containing the ligated product, the
objective plasmid was extracted from ampicillin resistant colonies,
and this plasmid was designated as pET-22-1616AT-His. This plasmid
expresses the aminotransferase derived from Bacillus altitudinis
AJ1616 which has the His-tag to C-terminus end (1616AT-His).
(2) Purification of 1616AT-His from E. coli Expression Strain
[0143] The constructed expression plasmid pET-22-1616AT-His was
introduced into E. coli BL21 (DE3). One loopful of the resulting
transformant was inoculated to 160 mL of Overnight Express Instant
TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and cultured with shaking at 37.degree. C. for 16
hours. After the termination of the cultivation, microbial cells
were collected from about 1000 mL of the resulting cultured medium
by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH
7.6), 100 mM NaCl and 20 mM imidazole, and disrupted by sonication
at 4.degree. C. for 30 minutes. Microbial cell debris was removed
from the disrupted solution by centrifugation, and the resulting
supernatant was used as a soluble fraction.
[0144] The obtained soluble fraction was applied to a His-tag
protein purification column HisPrep FF 16/10 (supplied from
Pharmacia (GE Health Care Bioscience), CV=20 mL) equilibrated with
20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and
adsorbed to the carrier. Proteins that had not been adsorbed to the
carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl
(pH 7.6), 100 mM NaCl and 20 mM imidazole, and subsequently the
adsorbed proteins were eluted by linearly changing the
concentration of imidazole from 20 mM to 250 mM at a flow rate of 3
mL/minute.
[0145] The obtained fractions were combined and concentrated using
Amicon Ultra-15 30K (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and applied to
the anion exchange chromatography column HiLoad 16/10 Q Sepharose
HP (supplied from GE health Care Bioscience, CV=20 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and adsorbed to the
carrier. The proteins that had not been adsorbed to the carrier
(unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6),
100 mM NaCl, and subsequently the adsorbed proteins were eluted by
linearly changing the concentration of NaCl from 100 mM to 500 mM
at a flow rate of 3 mL/minute.
[0146] The 2S,4R-Monatin forming activity was measured in each
eluted fraction, and the fractions in which the 2S,4R-Monatin
forming activity had been confirmed were combined and concentrated
using Amicon Ultra-15 30K (Millipore). The concentrated solution
was diluted with 20 mM Tris-HCl (pH 7.6) to use as a 1616AT-His
solution.
Example 6
Synthesis Reaction of 2S,4R-Monatin, Using 1616AT-His
[0147] The 2S,4R-Monatin was quantified by HPLC analysis. The
analytical condition was as follows.
[0148] Mobile phase: 20 mM KH.sub.2PO.sub.4/acetonitrile-100/5
[0149] Flow rate: 1.0 mL/minute
[0150] Column temperature: 40.degree. C.
[0151] Detection: UV 280 nm
[0152] Column: CAPCELL PAK MGII, 4.6.times.150 mm, 3 .mu.m,
(Shiseido Co., Ltd.)
(1) Synthesis of 2S,4R-Monatin from 4R-IHOG
[0153] The 1616AT-His solution prepared so as to contain 0.5 mg of
1616AT-His (Example 5) was added to 0.1 mL of the reaction solution
(9.5 mM 4R-IHOG, 0.5 mM 4S-IHOG, 80 mM L-Asp, 50 .mu.M PLP, 100 mM
Tris-HCl, pH 8.0), and then reacted at 25.degree. C. for 12 hours.
After the termination of the reaction, the formed 2S,4R-Monatin was
quantified, and its concentration was 8.6 mM.
(2) Synthesis of 2S,4R-Monatin from Indole Pyruvate (IPA) and
Pyruvate (PA)
[0154] A reaction mixture was prepared so as to contain 0.5 mg of
1616AT-His (the 1616AT-His solution in Example 5 was used), 0.01 mg
of SpAld (a solution having an aldolase activity, the preparation
method of the solution is explained in detail below, see also JP
2006-204285-A) and 1 U of oxaloacetate decarboxylase (Sigma, 04878)
in 0.1 mL of a reaction solution (50 mM IPA, 100 mM PA, 100 mM
L-Asp, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 100 mM
potassium phosphate buffer, pH 8.0), and reacted at 25.degree. C.
for 2 hours. After the termination of the reaction, the formed
2S,4R-Monatin was quantified, and its concentration was 5.0 mM.
(3) Synthesis of 2S,4R-Monatin from L-Trp
[0155] A reaction mixture was prepared so as to contain 5 mg of
1616AT-His (the 1616AT-His solution in Example 5 was used), 0.2 mg
of SpAld, 0.4 mL of the cultured medium (TB medium) of pTB2 strain
(a bacterial strain capable of expressing a deamination enzyme, the
preparation method of the bacterial strain is explained in detail
below, see also WO2009/028338) in the Sakaguchi flask, 200 U of
superoxide dismutase (Sigma, S8160) and 10 U of oxaloacetate
decarboxylase (Sigma, 04878) in 1.0 mL of a reaction solution (50
mM L-Trp, 100 mM PA, 400 mM L-Asp, 1 mM MgCl.sub.2, 50 .mu.M PLP,
100 mM Tris-HCl, 100 mM potassium phosphate buffer, pH 6.5), and
reacted at 25.degree. C. for 12 hours. The reaction was performed
using a test tube with shaking at 140 rpm. After the termination of
the reaction, the formed 2S,4R-Monatin was quantified, and its
concentration was 22 mM (44% of yield).
[0156] SpAld was prepared by the following method.
[0157] A DNA fragment including a SpAld gene was amplified by PCR
using plasmid DNA, ptrpSpALD described in Example 5 in JP
2006-204285-A as the template. The primer SpAld-f-NdeI
(5'-GGAATTCCATATGACCCAGACGCGCCTCAA-3': SEQ ID NO:12) and the primer
SpAld-r-HindIII (5'-GCCCAAGCTTTCAGTACCCCGCCAGTTCGC-3': SEQ ID
NO:13) were used. E. coli rare codons (6L-ctc, 13L-ctc, 18P-ccc,
38P-ccc, 50P-ccc, 77P-ccc, 81P-ccc and 84R-cga) in an aldolase gene
were converted to 6L-ctg, 13L-ctg, 18P-ccg, 38P-ccg, 50P-ccg,
77P-ccg, 81P-ccg and 84R-cgc, respectively. When 6L was converted,
the primer 6L-f (5'-ACCCAGACGCGCCTGAACGGCATCATCCG-3': SEQ ID NO:14)
and the primer 6L-r (5'-CGGATGATGCCGTTCAGGCGCGTCTGGGT-3': SEQ ID
NO:15) were used. When 13L was converted, the primer 13L-f
(5'-ATCATCCGCGCTCTGGAAGCCGGCAAGCC-3': SEQ ID NO:16) and the primer
13L-r (5'-GGCTTGCCGGCTTCCAGAGCGCGGATGAT-3': SEQ ID NO:17) were
used. When 18P was converted, the primer 18P-f
(5'-GAAGCCGGCAAGCCGGCTTTCACCTGCTT-3': SEQ ID NO:18) and the primer
18P-r (5'-AAGCAGGTGAAAGCCGGCTTGCCGGCTTC-3': SEQ ID NO:19) were
used. When 38P was converted, the primer 38P-f
(5'-CTGACCGATGCCCCGTATGACGGCGTGGT-3': SEQ ID NO:20) and the primer
38P-r (5'-ACCACGCCGTCATACGGGGCATCGGTCAG-3': SEQ ID NO:21) were
used. When 50P was converted, the primer 50P-f
(5'-ATGGAGCACAACCCGTACGATGTCGCGGC-3': SEQ ID NO:22) and the primer
50P-r (5'-GCCGCGACATCGTACGGGTTGTGCTCCAT-3': SEQ ID NO:23) were
used. When 77P, 81P and 84P were converted, the primer
77P-81P-84R-f (5'-CGGTCGCGCCGTCGGTCACCCCGATCGCGCGCATCCCGGCCA-3':
SEQ ID NO:24) and the primer 77P-81P-84R-r
(5'-TGGCCGGGATGCGCGCGATCGGGGTGACCGACGGCGCGACCG-3': SEQ ID NO:25)
were used. PCR was performed using KOD-plus (Toyobo) under the
following condition.
TABLE-US-00004 1 cycle 94.degree. C., 2 min 25 cycles 94.degree.
C., 15 sec 55.degree. C., 15 sec 68.degree. C., 60 sec 1 cycle
68.degree. C., 60 sec 4.degree. C.
[0158] The resulting DNA fragment of about 900 bp was treated with
the restriction enzymes NdeI and HindIII, and ligated to pSFN
Sm_Aet (Examples 1, 6 and 12 in International Publication
WO2006/075486) likewise treated with NdeI and HindIII. E. coli
JM109 was transformed with this solution containing the ligated
product. The objective plasmid was extracted from ampicillin
resistant strains, and this plasmid was designated as
pSFN-SpAld.
[0159] One loopful of E. coli JM 109/pSFN-SpAld that was the
bacterial strain carrying the constructed plasmid pSFN-SpAld was
inoculated to 50 mL of LB liquid medium containing 100 mg/L of
ampicillin in a 500 mL Sakaguchi flask, and cultured with shaking
at 36.degree. C. for 8 hours. After the termination of the culture,
0.0006 mL of the obtained cultured medium was added to 300 mL of a
seed liquid medium (10 g of glucose, 5 g of ammonium sulfate, 1.4 g
of potassium dihydrogen phosphate, 0.45 g of hydrolyzed soybeans as
a nitrogen amount, 1 g of magnesium sulfate heptahydrate, 0.02 g of
iron (II) sulfate heptahydrate, 0.02 g of manganese (II) sulfate
pentahydrate, 1 mg of thiamin hydrochloride, 0.1 mL of Disfoam
GD-113K (NOF Corporation), pH 6.3, made to one liter with water)
containing 100 mg/L of ampicillin in a 1000 mL volume of jar
fermenter, and seed cultivation was started. The seed cultivation
was performed at 33.degree. C. with ventilation at 1/1 vvm with
stirring at 700 rpm and controlling pH at 6.3 with ammonia until
glucose was consumed. Then, 15 mL of the cultured medium obtained
as above was added to 285 mL of a main liquid medium (15 g of
glucose, 5 g of ammonium sulfate, 3.5 g of phosphoric acid, 0.45 g
of hydrolyzed soybeans as the nitrogen amount, 1 g of magnesium
sulfate heptahydrate, 0.05 g of iron (II) sulfate heptahydrate,
0.05 g of manganese (II) sulfate pentahydrate, 1 mg of thiamin
hydrochloride, 0.1 mL of Disfoam GD-113K (NOF Corporation), pH 6.3,
made to 0.95 L with water) containing 100 mg/L of ampicillin in a
1000 mL volume of jar fermenter, and main cultivation was started.
The main cultivation was performed at 36.degree. C. with
ventilation at 1/1 vvm, pH was controlled to 6.3 with ammonia, and
stirring was controlled at 700 rpm or more so that the
concentration of dissolved oxygen was 5% or more. After glucose
contained in the main medium was consumed, the cultivation was
continued with dropping a glucose solution at 500 g/L for total 50
hours.
[0160] Microbial cells were collected by centrifugation from 100 mL
of the obtained cultured medium, washed with and suspended in 20 mM
Tris-HCl (pH 7.6), and disrupted by sonication at 4.degree. C. for
30 minutes. Microbial cell debris was removed from the disrupted
solution by centrifugation, and the obtained supernatant was used
as a soluble fraction.
[0161] The above soluble fraction was applied to the anion exchange
chromatography column HiLoad 26/10 Q Sepharose HP (supplied from GE
health Care Bioscience, CV=53 mL) equilibrated with 20 mM Tris-HCl
(pH 7.6), and adsorbed to the carrier. The proteins that had not
been adsorbed to the carrier (unadsorbed proteins) were washed out
with 20 mM Tris-HCl (pH 7.6), and subsequently, the adsorbed
proteins were eluted by linearly changing the concentration of NaCl
from 0 mM to 500 mM at a flow rate of 8 mL/minute. Fractions having
an aldolase activity were combined, and ammonium sulfate and
Tris-HCl (pH 7.6) were added thereto at final concentrations of 1 M
and 20 mM, respectively.
[0162] The resulting solution was applied to the hydrophobic
chromatography column HiLoad 16/10 Phenyl Sepharose HP (supplied
from GE health Care Bioscience, CV=20 mL) equilibrated with 1 M
ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and adsorbed to the
carrier. The proteins that had not been adsorbed to the carrier
were washed out with 1 M ammonium sulfate, 20 mM Tris-HCl (pH 7.6),
and subsequently, the adsorbed proteins were eluted by linearly
changing the concentration of ammonium sulfate from 1 M to 0 M at a
flow rate of 3 mL/minute. The fractions having the aldolase
activity were combined and concentrated using Amicon Ultra-15 10K
(Millipore). The obtained concentrated solution was diluted with 20
mM Tris-HCl (pH 7.6), and used as a SpAld solution. The aldolase
activity was measured as an aldol degradation activity using PHOG
as the substrate under the following condition.
[0163] Reaction condition: 50 mM Phosphate buffer (pH 7.0), 2 mM
PHOG, 0.25 mM NADH, 1 mM MgCl.sub.2, 16 U/mL lactate dehydrogenase,
an absorbance at 340 nm was measured at 25.degree. C.
[0164] pTB2 strain was prepared by the following method.
[0165] One loopful of pTB2 strain described in Example 2 in
International Publication WO2009/028338 was inoculated to 50 mL of
the TB liquid medium containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and cultured with shaking at 37.degree. C. for 16
hours. The obtained cultured medium was used as the cultured medium
of pTB2 strain in the Sakaguchi flask (TB medium).
Example 7
Synthesis of 2S,4R-Monatin by Microorganisms Having 2S,4R-Monatin
Forming Activity
(1) Synthesis of 2S,4R-Monatin by Bacteria
[0166] Rhizobium radiobacter LAT1, Rhizobium radiobacter AJ11568,
Dietzia maris AJ2788, Stenotrophomonas sp. AJ3447, Stenotrophomonas
sp. AJ13127, Pseudomonas chlororaphis subsp. chlororaphis NBRC3904,
Micrococcus luteus NBRC3067, Stenotrophomonas sp. AJ11634,
Pseudomonas putida NBRC12668, Ochrobactrum pseudogrignonense
AJ3735, Stenotrophomonas sp. AJ1591, Stenotrophomonas sp. AJ3839,
Brevundimonas diminuta AJ3958, Pseudomonas citronocllolis
ATCC13674, Arthrobacter sp. AJ1436, Rhizobium sp. AJ12469,
Rhizobium radiobacter AJ2777, Burkholderia sp. AJ3084,
Microbacterium sp. AJ2787, Pseudomonas taetrolens ATCC4683,
Rhizobium radiobacter ATCC4452, Rhizobium radiobacter AJ2557,
Carnimonas sp. AJ3230, Rhizobium radiobacter NBRC12667, Pseudomonas
fragi NBRC3458, Rhizobium radiobacter NBRC12664, Corynebacterium
ammoniagenes NBRC12072, Pseudomonas sp. AJ1594, Rhizobium
radiobacter ATCC6466, Pseudomonas synxantha NBRC3912, Rhizobium
radiobacter ATCC4720, or Pseudomonas sp. AJ2438 was applied onto a
nutrient broth (NB) agar medium or the CM2G agar medium (10 g/L of
yeast extract, 10 g/L of polypeptone, 5 g/L of glucose, 5 g/L of
NaCl, 15 g/L of agar, pH 7.0), and cultured at 30.degree. C. for 2
days.
[0167] One loopful of the obtained microbial cells was inoculated
to 3 mL of an enzyme production medium (10 g/L of yeast extract, 10
g/L of polypeptone, 1 g/L of glucose, 3 g/L of dipotassium hydrogen
phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of
magnesium sulfate heptahydrate, 5 g/L of ammonium sulfate) in a
test tube, which was then cultured with shaking at 30.degree. C.
for 16 hours. The microbial cells were collected from 2 mL of the
cultured medium by centrifugation, washed with and suspended in 20
mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell
suspension.
[0168] Then, 1 g of glass beads (0.1 mm) was added to 1 mL of this
microbial cell suspension, and the microbial cells were disrupted
using the multi beads shocker (Yasui Kikai Co., Ltd.). The
resulting disrupted cell solution was centrifuged to use a
supernatant as a microbial cell extract.
[0169] The reaction of synthesizing 2S,4R-Monatin and the
quantification of 2S,4R-Monatin were performed in the same manner
as in Example 1, and amounts of the 2S,4R-Monatin which was formed
were as follows (Table 2)
TABLE-US-00005 TABLE 2 Amount of 2S,4R-Monatin which was produced
Amount of 2S,4R-Monatin Microorganism which was formed Rhizobium
radiobacter LAT1 3.8 mM Rhizobium radiobacter AJ11568 3.5 mM
Dietzia maris AJ2788 3.2 mM Stenotrophomonas sp. AJ3447 2.7 mM
Stenotrophomonas sp. AJ13127 2.7 mM Pseudomonas chlororaphis subsp
2.6 mM chlororaphis NBRC3904 Micrococcus luteus NBRC3067 2.3 mM
Stenotrophomonas sp. AJ11634 2.2 mM Pseudomonas putida NBRC12668
2.2 mM Ochrobactrum pseudogrignonense AJ3735 2.2 mM
Stenotrophomonas sp. AJ1591 2.1 mM Stenotrophomonas sp. AJ3839 2.1
mM Brevundimonas diminuta AJ3958 2.0 mM Pseudomonas citronocllolis
ATCC13674 1.9 mM Arthrobacter sp. AJ1436 1.7 mM Rhizobium sp.
AJ12469 1.6 mM Rhizobium radiobacter AJ2777 1.5 mM Burkholderia sp.
AJ3084 1.5 mM Microbacterium sp. AJ2787 1.5 mM Pseudomonas
taetrolens ATCC4683 1.4 mM Rhizobium radiobacter ATCC4452 1.4 mM
Rhizobium radiobacter AJ2557 1.4 mM Carnimonas sp. AJ3230 1.4 mM
Rhizobium radiobacter NBRC12667 1.3 mM Pseudomonas fragi NBRC3458
1.3 mM Rhizobium radiobacter NBRC12664 1.3 mM Corynebacterium
ammoniagenes 1.2 mM NBRC12072 Pseudomonas sp. AJ1594 1.2 mM
Rhizobium radiobacter ATCC6466 1.2 mM Pseudomonas synxantha
NBRC3912 1.1 mM Rhizobium radiobacter ATCC4720 1.1 mM Pseudomonas
sp. AJ2438 1.0 mM
(2) Synthesis of 2S,4R-Monatin by Actinomycete
[0170] Nocardia globerula ATCC21022 was applied onto a YMPG agar
medium (3 g/L of yeast extract, 3 g/L of malt extract, 5 g/L of
polypeptone, 10 g/L of glucose, 15 g/L of agar, pH 7.0), and
cultured at 30.degree. C. for 2 days.
[0171] One loopful of the obtained microbial cells was inoculated
to 3 mL of a YMPG medium (3 g/L of yeast extract, 3 g/L of malt
extract, 5 g/L of polypeptone, 10 g/L of glucose, pH 7.0) in a test
tube, and cultured with shaking at 30.degree. C. for 16 hours. The
microbial cells were collected from 2 mL of the cultured medium by
centrifugation, washed with and suspended in 20 mM Tris-HCl (pH
7.6) to prepare 1 mL of a microbial cell suspension.
[0172] Then, 1 g of glass beads (0.1 mm) was added to 1 mL of this
microbial cell suspension, and the microbial cells were disrupted
using the multi beads shocker (Yasui Kikai Co., Ltd.). The
resulting disrupted cell solution was centrifuged to use a
supernatant as a microbial cell extract.
[0173] The reaction of synthesizing 2S,4R-Monatin and the
quantification of 2S,4R-Monatin were performed in the same manner
as in Example 1, and amount of the 2S,4R-Monatin which was formed
was as follows (Table 3)
TABLE-US-00006 TABLE 3 Amount of 2S,4R-Monatin which was formed
Amount of 2S4R-Monatin Microoganism which was formed Nocardia
globerula ATCC21022 0.57 mM
(3) Synthesis of 2S,4R-Monatin by Yeast
[0174] Lodderomyces elongisporus CBS2605, Candida norvegensis
NBRC0970, Candida inconspicua NBRC0621 or Yarrowia lypolytica
NBRC0746 was applied onto a YPD agar medium (10 g/L of yeast
extract, 20 g/L of polypeptone, 20 g/L of glucose, 15 g/L of agar),
and cultured at 30.degree. C. for 2 days.
[0175] One loopful of the obtained microbial cells was inoculated
to 3 mL of a YPD medium (10 g/L of yeast extract, 20 g/L of
polypeptone, 20 g/L of glucose) in a test tube, and cultured with
shaking at 30.degree. C. for 16 hours. The microbial cells were
collected from 2 mL of the cultured medium by centrifugation,
washed with and suspended in 20 mM Tris-HCl (pH 7.6) to prepare 1
mL of a microbial cell suspension.
[0176] Then, 1 g of glass beads (0.5 mm) was added to 1 mL of this
microbial cell suspension, and the microbial cells were disrupted
using the multi beads shocker (Yasui Kikai Co., Ltd.). The
resulting disrupted cell solution was centrifuged to use a
supernatant as a microbial cell extract.
[0177] The reaction of synthesizing 2S,4R-Monatin and the
quantification of 2S,4R-Monatin were performed in the same manner
as in Example 1, and amount of the 2S,4R-Monatin which was formed
were as follows (Table 4)
TABLE-US-00007 TABLE 4 Amount of 2S,4R-Monatin which was formed
Amount of 2S4R-Monatin Microorganism which was formed Lodderomyces
elongisporus CBS2605 0.57 mM Candida norvegensis NBRC0970 0.55 mM
Candida inconspicua NBRC0621 0.52 mM Yarrowia lypolytica NBRC0746
0.52 mM
Example 8
Production of 2S,4R-Monatin Potassium Salt Dihydrate
[0178] After 149.00 g of ethanol was added to a reduction reaction
concentrated solution (containing 36.62 g (125.28 mmol) of Monatin,
(2S,4R):(2R,4R)=32:68), 0.25 g of 2R,4R-Monatin potassium salt
monohydrate was added as seed crystals, and the mixture was stirred
at 56.degree. C. for 4 hours to perform preferential
crystallization of the 2R,4R-Monatin potassium salt monohydrate.
The crystallized crystals were separated by filtration (wet
crystals 31.27 g) to obtain 225.80 g of a mother solution
(containing 22.41 g (76.68 mmol) of Monatin,
(2S,4R):(2R,4R)=53:47). This mother solution was cooled to
10.degree. C. and stirred for 5 hours to crystallize 2S,4R-Monatin
potassium salt dihydrate. The crystals were separated by filtration
(wet crystals 32.74 g), and dried under reduced pressure to yield
9.88 g (15.68 mmol) of the objective 2S,4R-Monatin potassium salt
dihydrate (HPLC purity: 55.5%). Then, 9.35 g of the crude crystals
were dissolved in 25.37 g of water, and 58.99 g of ethanol was
added to this dissolved solution, which was stirred at 25.degree.
C. for 5 hours to refine the 2S,4R-Monatin potassium salt dehydrate
by crystallization. The crystals were separated by filtration (wet
crystals 4.49 g), and dried under reduced pressure to yield 3.75 g
(9.62 mmol) of the objective 2S,4R-Monatin potassium salt dihydrate
(HPLC purity: 96.0%).
[0179] A water content and a potassium content of the obtained
crystals (2S,4R-Monatin potassium salt dihydrate) were analyzed by
a water measurement method and a cation analysis method using ion
chromatography. Details of the performed water measurement method
and cation analysis method are shown below.
(Water Measurement Method)
[0180] Measurement apparatus: Hiranuma Automatic Water Measurement
Apparatus AQV-2000 (supplied from Hiranuma Sangyo Corporation)
[0181] Measurement condition: Titration solution=Hydranal Composite
5K (supplied from Riedel de Haen)
(Cation Analysis Method)
[0182] Apparatus: Tosoh IC2001
[0183] Column: TSKgel SuperIC-Cation (4.6.times.150 mm)
[0184] Guard column: TSKgel SuperIC-Cation (1 cm)
[0185] Suppress gel: TSKgel TSKsuppressIC-C
[0186] Column temperature: 40.degree. C.
[0187] Eluant flow: 0.7 mL/minute
[0188] Sample injection amount: 30 .mu.L
[0189] Detection: Electric conductivity
[0190] Eluant composition: 2.2 mM methanesulfonic acid+1.0 mM
18-crown-6-ether+0.5 mM histidine mixed aqueous solution
[0191] .sup.1H NMR (400 MHz, D.sub.2O) .delta.:2.11 (dd, J=19.0,
27.0 Hz, 1H), 2.39 (dd, J=5.0, 27.0 Hz, 1H), 3.14 (s, 2H), 3.90
(dd, J=5.0, 19.0 Hz, 1H), 7.06 (m, 1H), 7.13 (m, 1H), 7.15 (s, 1H),
7.40 (d, 8.5 Hz, 1H), 7.6 (d, 8.5 Hz, 1H)
[0192] ESI-MS Calculated value:
C.sub.14H.sub.16N.sub.2O.sub.5=292.11
[0193] ESI-MS Analyzed value: C.sub.14H.sub.16N.sub.2O.sub.5=290.9
[M-H].sup.-
Example 9
Isomerization Reaction Using 5-Nitrosalicylaldehyde
[0194] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of an aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 7.6 mg (0.045
mmol) of 5-nitrosalicylaldehyde and 7.5 .mu.L (0.13 mmol) of acetic
acid were added to that dissolved solution, and stirred at
60.degree. C. for 48 hours. The reaction solution was analyzed and
quantified by HPLC, and a molar ratio of 2S,4R-Monatin and
2R,4R-Monatin in the reaction solution was 1:2.1.
Example 10
Isomerization Reaction Using Pyridoxal Hydrochloride Salt
[0195] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of the aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 9.1 mg (0.045
mmol) of pyridoxal hydrochloride and 7.5 .mu.L (0.13 mmol) of
acetic acid were added to that dissolved solution, and stirred at
60.degree. C. for 48 hours. The reaction solution was analyzed and
quantified by HPLC, and the molar ratio of 2S,4R-Monatin and
2R,4R-Monatin in the reaction solution was 1:1.3.
Example 11
Isomerization Reaction Using Pyridoxal 5-Phosphate Monohydrate
[0196] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of the aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 12.8 mg (0.048
mmol) of pyridoxal 5-phosphate monohydrate and 7.5 .mu.L (0.13
mmol) of acetic acid were added to that dissolved solution, and
stirred at 60.degree. C. for 48 hours. The reaction solution was
analyzed and quantified by HPLC, and the molar ratio of
2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was
1:1.1.
Example 12
Isomerization Reaction Using Salicylaldehyde
[0197] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of the aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 5.3 mg (4.6
.mu.L, 0.043 mmol) of salicylaldehyde and 7.5 .mu.L (0.13 mmol) of
acetic acid were added to that dissolved solution, and stirred at
60.degree. C. for 48 hours. The reaction solution was analyzed and
quantified by HPLC, and the molar ratio of 2S,4R-Monatin and
2R,4R-Monatin in the reaction solution was 1:0.6.
Example 13
Isomerization Reaction Using 3,5-Dichlorosalicylaldehyde
[0198] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of the aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 8.1 mg (0.042
mmol) of 3,5-dichlorosalicylaldehyde and 7.5 .mu.L (0.13 mmol) of
acetic acid were added to that dissolved solution, and stirred at
60.degree. C. for 48 hours. The reaction solution was analyzed and
quantified by HPLC, and the molar ratio of 2S,4R-Monatin and
2R,4R-Monatin in the reaction solution was 1:1.5.
Example 14
Production of 2R,4R-Monatin Potassium Salt Monohydrate by
Isomerization-Crystallization Using 2S,4R-Monatin Potassium Salt
Dihydrate as Starting Material
[0199] The 2S,4R-Monatin potassium salt dihydrate is added to an
aqueous solution of 20% ethanol and completely dissolved at
60.degree. C. 5 molar percent 5-Nitrosalicylaldehyde relative to
the 2S,4R-Monatin, and 30 molar percent acetic acid relative to the
2S,4R-Monatin are added to this dissolved solution, and stirred for
48 hours. Ethanol at a final concentration of 70% is added to this
reaction solution (2S,4R-Monatin:2R,4R-Monatin=1:2.1), subsequently
one percent 2R,4R-Monatin potassium salt monohydrate relative to
the 2R,4R-Monatin in the reaction solution is added as the seed
crystals thereto, and the mixture is stirred at 60.degree. C. for
48 hours to perform the isomerization-crystallization. The
crystallized crystals are separated by filtration, and dried under
reduced pressure to yield the objective 2R,4R-Monatin potassium
salt monohydrate.
Example 15
Isomerization Reaction Using Glyoxylic Acid
[0200] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt
dihydrate was added to 10.0 g of the aqueous solution of 70%
ethanol, and completely dissolved at 60.degree. C. 5.1 mg (0.069
mmol) of glyoxylic acid and 7.5 .mu.L (0.13 mmol) of acetic acid
were added to that dissolved solution, and stirred at 60.degree. C.
for 48 hours. The reaction solution was analyzed and quantified by
HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the
reaction solution was 1:0.07
Example 16
Production of L-Amino Acid Aminotransferase (LAT) Mutants Derived
from AJ1616 Strain and Measurement of Specific Activity for Various
Keto Acids
(1) Production of Mutated LAT-Expressing Plasmid by Site-Directed
Mutagenesis
[0201] Plasmids expressing a mutated LAT derived from AJ1616 strain
were produced by site-directed mutagenesis in accordance with
protocols of QuickChange Site-Directed Mutagenesis Kit supplied
from Stratagene. One set of primers designed so that a mutation
(substitution) was introduced into a target nucleotide residue and
became complementary in respective chains of double stranded DNA
was synthesized. The produced mutants and the nucleotide sequences
of the primers used for the production of the mutants are shown in
Tables 5 and 6, respectively. The mutant plasmids were produced
using pET22-AJ1616LAT-His(C) as the template under the following
PCR condition:
TABLE-US-00008 1 cycle 95.degree. C., 1 min 18 cycles 95.degree.
C., 30 sec 55.degree. C., 1 min 68.degree. C., 8 min after
completion of the cycles 4.degree. C.
[0202] The template pET22-AJ1616LAT-His(C) was cleaved by treating
with the restriction enzyme Dpn I (37.degree. C., one hour)
cleaving by recognition of methylated DNA, and subsequently E. coli
JM109 was transformed with the resulting reaction solution. The
plasmid was collected from the transformant, and it was confirmed
by sequencing the nucleotides that the mutation (substitution) of
the target nucleotide residue had been introduced. ID136 that was a
double mutant of S258C/I289A was constructed by making an S258G
mutant plasmid followed by repeating the same manipulation using
the primers for introducing an I289A mutation. ID189 that was a
double mutant of K39R/T288G was constructed by making an ID166
(T288G) mutant plasmid followed by repeating the same manipulation
using the primers for introducing a K39R mutation. ID296 that was a
double mutant of Q287E/T288G was constructed by making a T288G
mutant plasmid followed by repeating the same manipulation using
the primers for introducing a Q287E/T288G mutation.
TABLE-US-00009 TABLE 5 Mutants which were prepared ID Mutants ID136
S258G/I289A ID166 T288G ID189 K39R/T288G ID296 Q287E/T288G
TABLE-US-00010 TABLE 6 Nucleotide sequences of primers used for
introducing mutation Nucleotide sequences Mutants Primer names (SEQ
ID NOs) K39R K39R_FW gacatgtctagagggcgtccttcaccaaaacag (SEQ ID NO:
26) K39R_RV ctgttttggtgaaggacgccctctagacatgtc (SEQ ID NO: 27) S258G
S258G_FW gttcgcctctactggtaaaattacgttccc (SEQ ID NO: 28) S258G_RV
gggaacgtaattttaccagtagaggcgaac (SEQ ID NO: 29) T288G T288G_FW
cagctatcagttcaaggcattgggccagataaaatc (SEQ ID NO: 30) T288G_RV
gattttatctggcccaatgccttgaactgatagctg (SEQ ID NO: 31) I289A I289A_FW
ctatcagttcaaaccgctgggccagataaaatc (SEQ ID NO: 32) I289A_RV
gattttatctggcccagcggtttgaactgatag (SEQ ID NO: 33) Q287E/
Q287E_T288G_FW Cagctatcagttgaaggcattgggccag T288G (SEQ ID NO: 34)
Q287E_T288G_RV ctggcccaatgccttcaactgatagctg (SEQ ID NO: 35)
(2) Expression and Purification of Mutated LAT
[0203] E. coli JM109 (DE3) was transformed with the obtained mutant
AJ1616 LAT-expressing plasmid to produce a mutant AJ1616
LAT-expressing strain. Microbial cells of the mutant AJ1616
LAT-expressing strain pET22-AJ1616LATmut-His(C)/E. coli JM109 (DE3)
that was grown on an LB-amp (100 mg/L) plate were inoculated to 100
mL of Overnight Express Instant TB Medium (Novagen) containing 100
mg/L of ampicillin, and cultured with shaking at 37.degree. C. for
16 hours using a Sakaguchi flask. After completion of the
cultivation, the microbial cells were collected from the resulting
medium by centrifugation, washed with and suspended in 20 mM
Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, followed by
being sonicated. Microbial cell debris was removed from the
disrupted suspension by centrifugation, and the resulting
supernatant was used as a soluble fraction. The resulting soluble
fraction was applied onto a His-tagged protein purification column,
His TALON superflow 5 ml Cartridge (Clontech) equilibrated with 20
mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and absorbed
to the carrier. Proteins that had not been absorbed to the carrier
(unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6),
300 mM NaCl and 10 mM imidazole, and subsequently, the absorbed
proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and
150 mM imidazole at a flow rate of 5 mL/minute. Resulting fractions
were combined, and the combined fraction was concentrated using
Amicon Ultra-15 30K (Millipore). The concentrated fraction was
diluted with 20 mM Tris-HCl (pH 7.6) to use as a mutant AJ1616 LAT
solution. If necessary, the purification was performed by
increasing the amount of the medium and the number of the His TALON
columns to be connected.
(3) Measurement of Protein Concentration
[0204] A protein concentration was measured using a protein assay
CBB solution (diluted to 5 folds for the use) supplied from Nacalai
Tesque. The protein concentration was calculated by preparing a
standard curve using solutions containing 0.05, 0.1, 0.25 and 0.5
mL/mL BSA as the standards.
(4) Measurement of Activity for L-Asp/.alpha.-KG, L-Asp/PA and
L-Asp/.+-.MHOG by colorimetric assay
[0205] The activity of AJ1616 LAT for various substrates was
measured. 100 mM L-Asp was used as an amino donor substrate in a
transamination reaction, and a specific activity for 10 mM various
keto acids was measured by a colorimetric assay.
[0206] Activity for L-Asp/.alpha.-KG (.alpha.-ketoglutaric acid):
measured in 100 mM L-Asp-Na, 10 mM .alpha.-KG-2Na, 50 .mu.M PLP,
100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL MDH at
25.degree. C. The activity was calculated from the reduction of
absorbance at 340 nm. Malic dehydrogenase from porcine heart
(Sigma) was used as MDH. The activity for L-Asp/.alpha.-KG is shown
in the column ".alpha.-KG" of the aminotransferase activity in
Table 9.
[0207] Activity for L-Asp/PA: measured in 100 mM L-Asp-Na, 10 mM
PA-2Na, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2
U/mL MDH (same as above) at 25.degree. C. The activity was
calculated from the reduction of the absorbance at 340 nm. The
activity for L-Asp/PA is shown in the column "PA" of the
aminotransferase activity in Table 9.
[0208] Activity for L-Asp/(.+-.)-MHOG
(4-hydroxy-4-methyl-2-ketoglutarate): measured in 100 mM L-Asp-Na,
10 mM (.+-.)-MHOG, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM
NADH, 2 U/mL MDH and 10 U/mL LDH at 25.degree. C. The activity was
calculated from the reduction of the absorbance at 340 nm.
D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental
Yeast) was used as LDH. LDH was added in order to remove PA in a
trace amount contaminated in (.+-.)-MHOG. The activity for
L-Asp/(.+-.)-MHOG is shown in the column "(.+-.)-MHOG" of the
aminotransferase activity in Table 9.
(5) Measurement of Activity for L-Asp/4R-IHOG and L-Asp/IPA
[0209] The activity of forming 2S,4R-Monatin from 4R-IHOG, which
was an objective activity, and the activity of forming a byproduct
L-Trp from IPA were measured. 100 mM L-Asp was used as the amino
donor substrate in the transamination reaction, the transamination
reaction to 10 mM keto acid was performed. The amount of formed
amino acid was quantified by UPLC or HPLC, and the specific
activity was calculated.
[0210] Activity for L-Asp/4R-IHOG (10 mM): measured in 100 mM
L-Asp-Na, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50
.mu.M PLP, and 100 mM Tris-HCl (pH 8.0) at 25.degree. C. Formed
2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis.
The reaction was stopped using a 200 mM citrate Na solution (pH
4.5). The activity for L-Asp/4R-IHOG is shown in the column
"4R-IHOG" of the aminotransferase activity in Table 9.
[0211] Activity for L-Asp/IPA: measured in 100 mM L-Asp-Na, 10 mM
IPA, 50 .mu.M PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to
8.0 with 1 N NaOH after preparing the reaction solution) at
25.degree. C. Formed Trp was quantified by the UPLC analysis. The
reaction was stopped using the 200 mM citrate Na solution (pH 4.5).
The activity for L-Asp/IPA is shown in the column "IPA" of the
aminotransferase activity in Table 9.
[0212] Formed Monatin and Trp were quantified using ACQUITY UPLC
system supplied from Waters. A measurement condition is shown
below. The reaction in 0.2 mL was performed for 15 minutes, and
then stopped. The reaction solution after stopping the reaction was
centrifuged, and then about 0.2 mL of the supernatant was subjected
to the UPLC analysis. Results obtained by measurement using serial
dilutions in which the concentrations of the samples and a blank
fell into the range of 0.01 to 0.05 mM were employed as activity
values.
TABLE-US-00011 TABLE 7 UPLC Column: ACQUITY UPLC HSS T3 2.1 .times.
50 mm Column Temp.: 40.degree. C. Sample Temp.: 4.degree. C.
Detection: UV 210 nm Injection vol.: 5 .mu.l Mobile Phase A: 20 mM
KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20
mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40
2.2 60 40 2.3 96 4 3.0 96 4
[0213] 2S,4R-Monatin, 2S,4S-Monatin and Trp can be quantified
separately at 1.1 minutes, 1.5 minutes and 1.3 minutes,
respectively.
[0214] The quantification using HPLC under the following analysis
condition was also performed in conjunction with the above.
[0215] HPLC condition (quantification condition for Monatin, Trp,
IPA, IAA (indole acetate), IAD (indole aldehyde))
[0216] Column: CAPCELL PAK C18 TYPE MGII 3 .mu.m, 4.6 mm.times.150
mm (Shiseido)
[0217] Column temperature: 40.degree. C.
[0218] Detection wavelength: 280 nm
[0219] Flow rate: 1.0 mL/minute
[0220] Mobile phase: A: 20 mM KH.sub.2PO.sub.4/CH.sub.3CN=100/5, B:
CH.sub.3CN
TABLE-US-00012 TABLE 8 Time (min) A (%) B (%) 0 100 0 6 100 0 11 90
10 25 90 10 26 100 0 30 100 0
(6) Results of Measuring Specific Activity of AJ1616 Strain LAT
Mutants Against Various Keto Acids
[0221] The results of the specific activity against 10 mM keto acid
are shown in Table 9, which were measured with the produced mutant
and L-Asp as the amino donor. The objective activity of forming
2S,4R-Monatin using 4R-IHOG as the substrate was enhanced in any of
the produced mutants. Concerning relative values of side reaction
relative to the objective activity, the activity of producing the
byproduct L-Trp, the activity of producing the byproduct MHG
(4-hydroxy-4-methyl glutamate), and the activity of producing the
byproduct L-Ala, relative to the objective activity (activity of
forming 2S,4R-Monatin) were reduced in any of the mutants.
TABLE-US-00013 TABLE 9 Specific activities of mutants relative to
various keto acids. Relative values of side reaction relative to
Aminotransferase activity of forming activity (U/mg) 2S,4R-Monatin
(SR) ID Mutants .alpha.-KG PA .+-.MHOG 4R-IHOG IPA Trp/SR MHG/SR
Ala/SR WT WT 235 0.45 1.3 0.92 0.11 0.12 1.45 0.49 136 S258G/I289A
14 0.06 0.09 6.8 0.54 0.08 0.01 0.01 166 T288G 184 0.23 2.1 6.7
0.28 0.04 0.31 0.03 189 K39R/T288G 90 0.23 1.6 9.7 0.31 0.03 0.17
0.02 296 Q287E/T288G 50 0.18 1.5 11.1 0.24 0.02 0.14 0.02
Example 17
Construction of E. coli JM109 .DELTA.aspC Strain and Production of
Broth Containing Expressed Deaminase
[0222] E. coli JM109 .DELTA.aspC was constructed by following
methods. E. coli JM109/pKD46 was cultured at 30.degree. C.
overnight on the LB-amp (100 mg/L) plate. Obtained microbial cells
were inoculated to 50 mL of LB (containing 100 mg/L of Amp and 10
mM L-arabinose). This was cultured with shaking at 30.degree. C.
using the Sakaguchi flask. When OD.sub.610 became about 0.6, a
cultivation temperature was changed to 37.degree. C. and the
cultivation was continued with shaking for additional one hour. The
microbial cells were collected from the resulting medium by
centrifugation, washed with 10% glycerol, and collected again by
centrifugation. These were suspended in 10% glycerol to use as
competent cells.
[0223] Amplification by PCR was performed with pMW118-attL-cat-attR
as the template using the primer aspC-L1
(5'-TTTGAGAACATTACCGCCGCTCCTGCCGACCCGATTCTGGGCtgaagcctgcttttttat-3':
SEQ ID NO:36) and the primer aspC-R1
(5'-CAGCACTGCCACAATCGCTTCGCACAGCGGAGCCATGTTATCcgctcaagttagtataaa-3:
SEQ ID NO:37). The resulting PCR product was extracted from agarose
to use as a DNA fragment for aspC gene disruption. PCR was
performed using KOD-plus-ver. 2 (Toyobo).
[0224] The competent cells were transformed with the purified DNA
fragment, and an objective transformant was selected on an LB-Cm
(20 mg/L) plate at 37.degree. C. It was confirmed by colony PCR
that attL-cat-attR was inserted into the aspC gene region of the
transformant. The primers used are the primer aspC-up
(5'-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3': SEQ ID NO: 38), the primer
attL-1 (5'-TAGTGACCTGTTCGTTGC-3': SEQ ID NO:39), the primer
aspC-down (5'-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5': SEQ ID NO:40), and
the primer attR-1 (5'-TTACGTTTCTCGTTCAGC-3': SEQ ID NO:41). Z-taq
(TAKARA) was used for PCR.
[0225] The obtained transformant was inoculated to 3 mL of LB (Cm
20 mg/L), and cultured with shaking at 37.degree. C. for 6 hours.
Microbial cells were collected from the resulting medium by
centrifugation, washed with 10% glycerol, and the microbial cells
were collected again by centrifugation. These were suspended in 10%
glycerol to use as competent cells.
[0226] The competent cells were transformed with pMW-intxis-ts in
order to remove the Cm resistant gene sequence inserted in the
genomic DNA. An objective transformant was selected on the LB-amp
(100 mg/L) plate at 30.degree. C. The obtained transformant was
cultured on the LB plate at 42.degree. C. overnight, and the
microbial cells were streaked on the LB-amp (100 mg/L) plate and on
the LB-Cm (20 mg/L) plate, respectively and cultured at 37.degree.
C. The transformant was confirmed not to grow on both the plate
containing Amp and the plate containing Cm. Further the removal of
the Cm resistant gene was confirmed by colony PCR using the primer
aspC-up (5'-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3': SEQ ID NO:38) and
the primer aspC-down (5'-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5': SEQ ID
NO:40). Z-tag (TAKARA) was used for PCR.
[0227] The obtained strain was designated as an aspC-deficient
strain, E. coli JM109.DELTA.aspC. A deaminase-expressing strain
pTB2/E. coli JM109.DELTA.aspC was constructed by transforming E.
coli JM109.DELTA.aspC with a deaminase-expressing plasmid, pTB2.
This bacterial strain was cultured on the LB-amp (100 mg/L) at
37.degree. C. overnight. The obtained microbial cells were
inoculated to 100 mL of TB-amp (100 mg/L) and cultured with shaking
at 37.degree. C. for 16 hours using the Sakaguchi flask. The
resulting medium was used as Ps_aad broth.
Example 18
Construction of Oxaloacetate Decarboxylase-Expressing Strain
[0228] Synthesis of an OAA decarboxylase gene derived from
Pseudomonas putida KT2440 strain was asked GenScript, and a plasmid
DNA in which a DNA fragment including the OAA decarboxylase gene
had been inserted in pUC57 was obtained. A frequency of codon usage
was optimized for expression in E. coli (see SEQ ID NOS:42 and 43).
This plasmid was cleaved with NdeI and XhoI, inserted into pET22b
cleaved with NdeI and XhoI, and the resulting plasmid was
designated as pET22-PpODC-His(C). E. coli BL21 (DE3) was
transformed with the resulting plasmid to obtain a
PpODC-His(C)-expressing strain, pET22-PpODC-His(C)/E. coli BL21
(DE3). Microbial cells of the PpODC-His(C)-expressing strain,
pET22-PpODC-His(C)/E. coli BL21 (DE3) grown on the LB-amp (100
mg/L) plate were inoculated to 100 mL of Overnight Express Instant
TB Medium (Novagen), and cultured with shaking at 30.degree. C. for
16 hours using the Sakaguchi flask. After the termination of
cultivation, microbial cells were collected from the resulting
medium, and washed with and suspended in 20 mM Tris-HCl (pH 7.6),
300 mM NaCl and 10 mM imidazole, followed by being sonicated.
Microbial cell debris was removed from the disrupted solution by
centrifugation, and the resulting supernatant was used as a soluble
fraction. The resulting soluble fraction was applied onto a
His-tagged protein purification column, His TALON superflow 5 ml
Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300
mM NaCl and 10 mM imidazole, and absorbed to the carrier. Proteins
that had not been absorbed to the carrier (unabsorbed proteins)
were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM
imidazole, and subsequently, the absorbed proteins were eluted
using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at
a flow rate of 5 mL/minute. Resulting fractions were combined, and
the obtained solution was concentrated using Amicon Ultra-15 10K
(Millipore). The obtained solution was diluted with 20 mM Tris-HCl
(pH 7.6) to use as a PpODC solution.
[0229] An ODC activity was measured under the condition shown
below.
[0230] The measurement of the ODC activity was performed under the
following condition.
[0231] 10 mM OAA, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 10
U/mL of LDH at 25.degree. C. The activity was calculated from the
reduction of the absorbance at 340 nm. D-Lactate dehydrogenase from
Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. The
reaction and the analysis on a scale of 1 mL were performed, and
activity values in serial dilutions in which a measured value
[(sample .DELTA.340 nm/min)-(blank .DELTA.340 nm/min)] fell onto
the range of 0.05 to 0.15 were employed. The enzyme was diluted
with 20 mM Tris-HCl (pH 7.6) and 0.01% BSA.
Example 19
One-Pot Synthesis Reaction of 2S,4R-Monatin from 100 mM L-Trp (WT,
ID136, ID166)
[0232] A reaction was performed for 22 hours using the purified
mutant AJ1616 LAT under the following condition. The reaction was
performed in a volume of 1 mL using a test tube. Sampling was
performed after 14, 18 and 22 hours. The sample was diluted with TE
buffer, which was then ultrafiltrated using an Amicon Ultra-0.5 mL
centrifugation type filter 10 kDa, and a filtrate was analyzed.
HPLC was used for the analysis.
[0233] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM
L-Asp-Na, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 20 mM
KPB, pH 7.0, 40% Ps_aad broth, 0.2 mg/mL of purified SpAld enzyme,
10 U/mL of commercially available OAA DCase enzyme, 2 U/mL of
purified mutant AJ1616 LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL
of commercially available SOD enzyme at 25.degree. C. at 140
rpm.
[0234] Methods for preparing the enzyme subjected to the reaction
are described below.
[0235] Ps_aad broth: Prepared according to the method described in
Example 17.
[0236] Purified SpAld enzyme: A jar cultivation of the
SpAld-expressing strain was performed according to the method
described in Example 6, and the thermal treatment at 60.degree. C.
was further performed for one hour. Microbial cells were collected
from 100 mL of the resulting medium after the thermal treatment by
centrifugation, and washed with and suspended in 20 mM Tris-HCl (pH
7.6), followed by being sonicated. Microbial cell debris was
removed from the disrupted solution by centrifugation. The
resulting supernatant was used as a soluble fraction. Ammonium
sulfate and Tris-HCl (pH 7.6) were added so that this soluble
fraction contained 1 M ammonium sulfate and 20 mM Tris-HCl (pH
7.6). This solution was applied onto a hydrophobic chromatography
column HiLoad 26/10 Phenyl Sepharose HP (supplied from GE
Healthcare Bioscience, CV=53 mL) equilibrated with 1 M ammonium
sulfate and 20 mM Tris-HCl (pH 7.6), and absorbed to the carrier.
Unabsorbed proteins which had not been absorbed to the carrier were
washed out with 1 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6).
Subsequently, the absorbed proteins were eluted by linearly
changing the concentration of ammonium sulfate from 1 M to 0 M at a
flow rate of 8 mL/minute. Fractions in which the activity had been
detected were combined, and the obtained solution was concentrated
using Amicon Ultra-15 10k (Millipore). The resulting concentrated
solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as an
SpAld solution. A PHOG degrading activity measurement method was
used for measuring the aldolase activity (measured in 2 mM PHOG, 50
mM KPB, 1 mM MgCl.sub.2, 0.25 mM NADH, and 16 U/mL of LDH at
25.degree. C. (pH 7.0). The activity was calculated from the
reduction of the absorbance at 340 nm). D-Lactate dehydrogenase
from Leuconostoc mesenteroides (Oriental Yeast) was used as
LDH.
[0237] Mutant AJ1616 LAT: Microbial cells of the mutant AJ1616
LAT-expressing strain, pET22-AJ1616LATmut-His(C)/E. coli JM109
(DE3) grown on the LB-amp (100 mg/L) plate were inoculated to 100
mL of Overnight Express Instant TB Medium (Novagen) containing 100
mg/L of ampicillin, and cultured with shaking at 37.degree. C. for
16 hours using the Sakaguchi flask. After the termination of
cultivation, the microbial cells were collected from the resulting
medium, and washed with and suspended in 20 mM Tris-HCl (pH 7.6),
300 mM NaCl and 10 mL imidazole, followed by being sonicated.
Microbial cell debris was removed from the disrupted solution by
centrifugation, and the resulting supernatant was used as a soluble
fraction. The resulting soluble fraction was applied onto a
His-tagged protein purification column, His TALON superflow 5 ml
Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300
mM NaCl and 10 mM imidazole, and absorbed to the carrier. Proteins
that had not been absorbed to the carrier (unabsorbed proteins)
were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM
imidazole, and subsequently, the absorbed proteins were eluted
using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at
a flow rate of 5 mL/minute. Resulting fractions were combined, and
the obtained solution was concentrated using Amicon Ultra-15 30K
(Millipore). The concentrated solution was diluted with 20 mM
Tris-HCl (pH 7.6) to use as a mutant AJ1616 LAT solution. If
necessary, the purification was performed by increasing the amount
of the culture medium and the number of the His TALON columns to be
connected.
[0238] OAA DCase: Oxaloacetate decarboxylase from Pseudomonas sp.
(Sigma) was used. The value described by the manufacturer was used
as the amount of the enzyme (U).
[0239] SOD: Superoxide dismutase from bovine liver (Sigma) was
used. The value described by the manufacturer was used as the
amount of the enzyme (U).
[0240] As a result of the one-pot reaction, the yield of
2S,4R-Monatin was enhanced in cases of using the produced 10136 and
ID166 mutant enzymes compared with the wild enzyme (Table 10).
TABLE-US-00014 TABLE 10 Yield of 2S,4R-Monatin in one-pot reaction
using 100 mM Trp as substrate Yield of 2S,4R-Monatin in one-pot
reaction (vs. yield of Trp (%)) ID Mutants 14 hr 18 hr 22 hr WT WT
23 30 30 136 S259G/I289A 68 77 72 166 T288G 84 83 85
Example 20
One-Pot Synthesis Reaction of 2S,4R-Monatin from 100 mM Trp (10166
on Scale of 400 mL)
[0241] A reaction was performed for 6 hours using purified AJ1616
LAT-ID166 under the following condition. The reaction was performed
in a volume of 400 mL using a 1 liter volume jar. Sampling was
appropriately performed, the sample was diluted with TE buffer,
which was then ultrafiltrated using an Amicon Ultra-0.5 mL
centrifugation type filter 10 kDa, and a filtrate was analyzed.
HPLC and capillary electrophoresis were used for the analysis.
[0242] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM
L-Asp-Na, 1 mM MgCl.sub.2, 50 .mu.M PLP, 20 mM KPB (pH 7.6),
pH<7.6 (1 M H.sub.2SO.sub.4), 40% Ps_aad broth, 10% SpAld broth,
5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID166 (vs 10 mM 4R-IHOG) and
100 U/mL of SOD at 25.degree. C. at 500 rpm, and with air at 20
mL/min (1/20 vvm).
[0243] pTB2/E. coli JM109.DELTA.aspC broth was used as Ps_aad
broth. The thermally treated broth described in Example 19 was used
as SpAld broth. The purified enzyme described in Example 18 was
used as PpODC. Superoxide dismutase from bovine liver (Sigma) was
used as SOD.
[0244] As a result, the accumulation of 86 mM 2S,4R-Monatin was
confirmed after 6 hours (FIG. 4). The yield relative to L-Trp
calculated after calibrating the solution amount was 89%.
Example 21
One-Pot Synthesis Reaction of 2S,4R-Motatin from 150 mM L-Trp
(ID189 on Scale of 80 mL)
[0245] A reaction was performed for 27 hours using purified AJ1616
LAT-ID189 under the following condition. The reaction was performed
in a volume of 80 mL using a 250 mL volume jar. Sampling was
appropriately performed, the sample was diluted with TE buffer,
which was then ultrafiltrated using the Amicon Ultra-0.5 mL
centrifugation type filter 10 kDa, and a filtrate was analyzed.
HPLC and capillary electrophoresis were used for the analysis.
[0246] Reaction condition: 150 mM L-Trp, 50 mM PA-Na, 400 mM
L-Asp-Na, 1 mM MgCl.sub.2, 50 .mu.M PLP, 20 mM KPB (pH 7.6),
pH<7.6 (1 M H.sub.2SO.sub.4), 40% Ps_aad broth, 10% SpAld broth,
5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID189 (vs 10 mM 4R-IHOG) and
100 U/mL of SOD at 25.degree. C. (380 rpm), and with air at 4
mL/min (1/20 vvm).
[0247] pTB2/E. coli JM109.DELTA.aspC broth was used as the Ps_aad
broth. The thermally treated broth described in Example 19 was used
as the SpAld broth. The purified enzyme described in Example 18 was
used as PpODC. Superoxide dismutase from bovine liver (Sigma) was
used as SOD.
[0248] As a result, the accumulation of 105 mM 2S,4R-Monatin was
confirmed after 27 hours (FIG. 5). The yield relative to L-Trp
calculated after calibrating the solution amount was 78% (FIG.
5).
Example 22
Isolation of 2S,4R-Monatin
[0249] 2.59 g Of ZN charcoal was added to 435.66 g of a permeated
solution obtained by treating 435.45 g of the enzyme reaction
solution in Example 20 (lot 101213 J4) with UF (MWCO: 3000), and
the mixture was stirred at room temperature (about 26.degree. C.)
for one hour. The activated charcoal was filtrated with a Kiriyama
filter (5C), and the resulting filtrate was transferred to a 1
liter four-necked flask. The flask was immersed in an incubator at
5.degree. C., the solution was neutralized with 35% hydrochloric
acid to adjust pH to 3.5, and stirred using a mechanical stirrer
(120 rpm). Then, 48 mg of seed crystals were added, and 1 N
hydrochloric acid was sequentially added using a pH controller and
a peristaltic pump to keep a target pH because the pH value
elevated when the crystals began to precipitate. A slurry solution
obtained by stirring for 24 hours was filtrated, the crystals were
washed with 10 mL of water, and the wet crystals were dried under
reduced pressure at 40.degree. C. to yield 6.81 g of 2S,4R-Monatin.
The quality of the obtained crystals was confirmed by HPLC and
.sup.1H-NMR analysis.
[0250] HPLC area purity (210 nm): 98.4%
[0251] .sup.1H-NMR (in D.sub.2O+K.sub.2CO.sub.3) 2.08-2.14 (1H,
dd), 2.35-2.39 (1H, dd), 3.09-3.17 (2H, dd), 3.85-3.88 (1H, dd),
7.04-7.15 (3H, m), 7.39-7.41 (1H, m), 7.64-7.66 (1H, d).
Example 23
Synthesis of 2R,4R-Monatin
[0252] 3.10 g (10.4 mmol) Of 2S,4R-Monatin obtained in Example 22
and 1.165 g (10.4 mmol) of 50% KOH were dissolved in 3.27 g of
water, and further 1.3 g of EtOH, 0.0869 g (0.052 mmol) of
5-nitrosalicylaldehyde, and 0.187 g (3.12 mmol) of acetic acid were
added thereto. After 25 hours, 20.5 g of EtOH and 10 mg of seed
crystals (2R,4R-Monatin) were added, and the mixture was stirred
for additional 46.5 hours. The resulting slurry solution was cooled
to room temperature, and then filtrated. The crystals were washed
with 4 g of 85% EtOH-water, and the wet crystals were dried under
reduced pressure at 40.degree. C. to yield 2.3 g of crude
2R,4R-Monatin. 2.1 g of the resulting crude 2R,4R-Monatin was
dissolved in 6 mL of water, 0.2 g of BA charcoal was added, and the
mixture was stirred at room temperature (around 25.degree. C.) for
one hour and then filtrated with a 0.45 .mu.m membrane filter. The
filtrate was concentrated to 6.38 g under reduced pressure. 12 g Of
EtOH was dripped to the concentrated filtrate at 45.degree. C.,
which was then stirred for one hour. Further, 13.5 g of EtOH was
quantitatively dripped over one hour, which was then stirred at
45.degree. C. for 16 hours and subsequently cooled to 25.degree. C.
The resulting slurry solution was filtrated, the crystals were
washed with 3 g of 85% EtOH-water, and the wet crystals were dried
under reduced pressure at 40.degree. C. to yield 1.9 g (5.46 mmol)
of 2R,4R-Monatin. The obtained crystals, the mother solution, and
the washing solution were analyzed by HPLC to analyze yield and
quality.
[0253] HPLC area purity (210 nm): 99.9%
[0254] .sup.1H-NMR (in D.sub.2O) 1.93-2.00 (1H, dd), 2.57-2.61 (1H,
dd), 2.99-3.02 (1H, d), 3.19-3.22 (1H, d), 3.55-3.56 (1H, dd),
7.04-7.15 (3H, m), 7.39-7.41 (1H, m), 7.64-7.66 (1H, d).
TABLE-US-00015 TABLE 11 HPLC analysis condition DEGASSER
DGU-20A.sub.3 [SHIMAZU] PUMP LC-20AD [SHIMAZU, Two units] Column
oven CTO-20AC [SHIMAZU] DIODE ARRAY SPD-M20A [SHIMAZU] DETECTOR
Auto sampler SIL-20AC.sub.HT [SHIMAZU] COMMUNICATION CBM-20A BUS
MODULE System LC solution [SHIMAZU] Column CAPCELL PAC C18 Type MG
II 5 .mu.m 3.0 mm .PHI. .times. 250 mm [supplied from Shiseido]
Column temperature 40.degree. C. Detection wavelength 210 nm Flow
rate 0.35 ml/min Composition of mobile Solution A: 20 mM
KH.sub.2PO.sub.4/CH.sub.3CN = 100/5 solution Solution B: 20 mM
KH.sub.2PO.sub.4/CH.sub.3CN = 30/70 Injection amount 5 .mu.l
Autosampler solution CH.sub.3CN/H.sub.2O = 30/70 Solution A
Solution B Time (min) (%) (%) Gradient pattern 0 100 0 15 100 0 40
46 54 45 46 54 45.1 100 0 60 100 0
Example 24
One-Pot Synthesis Reaction of 2S,4R-Motatin from 150 mM L-Trp
(ID296 on Scale of 80 mL)
[0255] A reaction was performed for 51 hours using purified AJ1616
LAT-ID296 under the following condition. The reaction was performed
in a volume of 80 mL using a 250 mL volume jar. Sampling was
appropriately performed, the sample was diluted with TE buffer,
which was then ultrafiltrated using the Amicon Ultra-0.5 mL
centrifugation type filter 10 kDa, and a filtrate was analyzed.
HPLC was used for the analysis.
[0256] Reaction condition: 150 mM L-Trp, 50 mM PA-Na, 400 mM
L-Asp-Na, 1 mM MgCl.sub.2, 50 .mu.M PLP, 20 mM KPB (pH 7.6),
pH<7.6 (1 M H.sub.2SO.sub.4), 40% Ps_aad broth, 10% SpAld broth,
5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID296 (vs 10 mM 4R-IHOG) and
100 U/mL of SOD at 25.degree. C. (380 rpm), and with air at 4
mL/min (1/20 vvm).
[0257] pTB2/E. coli JM109.DELTA.aspC broth was used as the Ps_aad
broth. The thermally treated broth described in Example 19 was used
as the SpAld broth. The purified enzyme described in Example 18 was
used as PpODC. Superoxide dismutase from bovine liver (Sigma) was
used as SOD.
[0258] As a result, the accumulation of 113 mM 2S,4R-Monatin was
confirmed after 39 hours (FIG. 6). The yield relative to L-Trp
calculated after calibrating the solution amount was 86% (FIG.
6).
Example 25
Purification of Aminotransferase Derived from Rhizobium radiobacter
AJ3976
[0259] An aminotransferase that forms 2S,4R-Monatin was purified
from a soluble fraction of Rhizobium radiobacter AJ3976 as follows.
The reaction was performed in 100 mM L-Asp-Na-laq, 10 mM 4R-IHOG
(containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and 100 mM
Tris-HCl (pH 8.0) at 25.degree. C. The formed 2S,4R-Monatin was
quantified by UPLC analysis.
TABLE-US-00016 TABLE 12-1 UPLC Column: ACQUITY UPLC HSS T3 2.1
.times. 50 mm Column Temp.: 40.degree. C. Sample Temp.: 4.degree.
C. Detection: UV 210 nm Injection vol.: 5 .mu.l Mobile Phase A: 20
mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method:
20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60
40 2.2 60 40 2.3 96 4 3.0 96 4
(1) Preparation of Soluble Fraction
[0260] Microbial cells of Rhizobium radiobacter AJ3976 were spread
on an LB agar medium and cultured at 30.degree. C. for two
days.
[0261] One loopful of the obtained microbial cells was inoculated
to 160 mL of an enzyme production medium (10 g/L of yeast extract,
10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen
phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of
magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a
500 mL Sakaguchi flask, and cultured at 30.degree. C. for 20 hours
with shaking. The microbial cells were collected from about 1920 mL
of the resulting cultured medium by centrifugation, washed with and
suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4.degree. C.
for 30 minutes. Microbial cell debris was removed from the
sonicated cell suspension by the centrifugation, and the resulting
supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0262] The above soluble fraction was applied onto an anion
exchange chromatographic column HiLoad 26/10 Q Sepharose HP
(supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6) and absorbed to the carrier. Proteins
that had not been absorbed to the carrier (unabsorbed protein) were
washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, proteins
that had been absorbed to the carrier were eluted by linearly
changing a concentration of NaCl from 0 mM to 500 mM at a flow rate
of 2 mL/minute. A 2S,4R Monatin-forming activity was measured in
each eluted fraction, and the 2S,4R-Monatin-forming activity was
detected in fractions corresponding to about 250 mM NaCl.
(3) Hydrophobic Chromatography
[0263] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and ammonium sulfate and Tris-HCl
(pH 7.6) were added thereto so that the concentrations of ammonium
sulfate and Tris-HCl (pH 7.6) were 1.0 M and 20 mM, respectively.
The resulting solution was applied onto a hydrophobic
chromatographic column HiLoad 16/10 Phenyl Sepharose HP (supplied
from GE Healthcare Bioscience, CV=20 mL) equilibrated with 1.0 M
ammonium sulfate and 20 mM Tris-HCl (pH 7.6), and absorbed to the
carrier. Unabsorbed proteins that had not been absorbed to the
carrier were washed out using 1.0 M ammonium sulfate and 20 mM
Tris-HCl (pH 7.6). Subsequently, a 2S,4R-Monatin-forming enzyme was
eluted by linearly changing the concentration of ammonium sulfate
from 1.0 M to 0 M at a flow rate of 3 mL/minute. The
2S,4R-Monatin-forming activity was measured in each obtained
fraction, and the 2S,4R-Monatin-forming activity was detected in
fractions corresponding to about 0.9 M of ammonium sulfate.
(4) Gel Filtration Chromatography
[0264] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and concentrated using Amicon
Ultra-15 10k (Millipore). The resulting concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting
solution was applied onto a gel filtration column HiLoad 16/60
Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL)
equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and
proteins were eluted at a flow rate of 1 mL/minute. This
manipulation confirmed the 2S,4R-Monatin-forming activity at a
position in which a molecular weight was estimated to be about 100
kDa.
(5) SDS-PAGE
[0265] The resulting fraction was subjected to SDS-PAGE, and a
single band derived from the active fraction was detected near 47
kDa. This band was subjected to analysis of an N-terminal amino
acid sequence as a candidate of the aminotransferase that forms
2S,4R-Monatin.
Example 26
Determination of N-Terminal Amino Acid Sequence of Aminotransferase
Derived from Rhizobium radiobacter AJ3976
[0266] The purified enzyme solution obtained in Example 25 was
subjected to the analysis of the N-terminal amino acid sequence,
and the N-terminal amino acid sequence of AFLADILSRVKPSATIAVTQ (SEQ
ID NO:44) was obtained. The N-terminal amino acid sequence showed a
high homology to that of aspartate aminotransferase (AAK87940)
derived from Agrobacterium tumefaciens str. C58.
Example 27
Cloning of Aminotransferase Gene Derived from Rhizobium radiobacter
AJ3976
[0267] The microbial cells of Rhizobium radiobacter AJ3976 were
cultured in the same manner as in Example 25. The microbial cells
were collected from the cultured medium by centrifugation, and
genomic DNA was extracted therefrom.
[0268] A DNA fragment including the aminotransferase gene was
amplified by PCR using the obtained genomic DNA as a template.
Primers were designed from DNA sequences of upstream 100 bp and
downstream 100 bp of the aminotransferase gene with reference to
the genomic DNA sequence of Agrobacterium tumefaciens str. C58. The
primer Ag-u100-f (5'-ctggtgcagataagccggcttttgacc-3': SEQ ID NO:45)
and the primer Ag-d100-r (5'-ccaccttcatcatgctgctgtttctcg-3': SEQ ID
NO:46) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo)
under the following condition.
TABLE-US-00017 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0269] The nucleotide sequence of the amplified DNA fragment of
about 1400 bp was determined, and was shown to be the nucleotide
sequence including 1203 bp of ORF (SEQ ID NOs: 47 and 48), which
had the high homology to the aspartate aminotransferase gene
(Atu2196) derived from Agrobacterium tumefaciens str. C58. The
homology was 92% in their DNA sequences and 97% in their amino acid
sequences.
[0270] This amino acid sequence was consistent with the N-terminal
amino acid sequence obtained in Example 26. Thus, it has been
thought that the aminotransferase gene having the
2S,4R-Monatin-forming activity could be acquired.
Example 28
Expression of Aminotransferase Derived from Rhizobium radiobacter
AJ3976 in E. coli
[0271] (1) Construction of Expression Plasmid for Aminotransferase
Derived from Rhizobium radiobacter AJ3976
[0272] A DNA fragment including the aminotransferase gene derived
from Rhizobium radiobacter AJ3976 was amplified by PCR with the
genomic DNA of Rhizobium radiobacter AJ3976 as the template. The
primer 3976AT-Nde-f (5'-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3': SEQ
ID NO:49) and the primer 3976-xho-r
(5'-actccgctcgagACGGCAATCGGCGCAGAAACGCTGA-3': SEQ ID NO:50) were
used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the
following condition.
TABLE-US-00018 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0273] The resulting DNA fragment was treated with restriction
enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise
treated with NdeI and XhoI. E. coli JM109 was transformed with this
ligation solution, an objective plasmid was selected from
ampicillin resistant colonies, and this plasmid was designated as
pET-22-3976AT-His. In this plasmid, the aminotransferase derived
from Rhizobium radiobacter AJ3976 which having a His-tag added to a
C-terminus end (3976AT-His) is expressed.
(2) Purification of 3976AT-His from E. coli Strain Expressing
3976AT-His
[0274] The constructed expression plasmid pET-22-3976AT-His was
introduced into E. coli BL21 (DE3), and one loopful of the
transformant was inoculated to 160 mL of Overnight Express Instant
TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and the Sakaguchi flask was shaken at 37.degree.
C. for 16 hours. After completion of the cultivation, microbial
cells were collected from about 1000 mL of the cultured medium by
centrifugation, washed with and suspended in 20 mM Tris-HCl (pH
7.6), 100 mM NaCl and 20 mM imidazole, and sonicated at 4.degree.
C. for 30 minutes. Microbial cell debris was removed from the
sonicated cell suspension by centrifugation, and the resulting
supernatant was used as a soluble fraction.
[0275] The obtained soluble fraction was applied onto a His-tag
protein purification column HisPrep FF 16/10 (supplied from
Pharmacia (GE Healthcare Bioscience), CV=20 mL) equilibrated with
20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole to absorb
proteins to the carrier. Proteins that had not been absorbed to the
carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl
(pH 7.6), 100 mM NaCl and 20 mM imidazole. Subsequently, the
absorbed proteins were eluted by linearly changing the
concentration of imidazole from 20 mM to 250 mM at a flow rate of 3
mL/minute.
[0276] The obtained fractions were combined and concentrated using
Amicon Ultra-15 30k (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto an
anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP
(supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier.
Proteins that had not been absorbed to the carrier (unabsorbed
protein) were washed out with 20 mM Tris-HCl (pH 7.6).
Subsequently, the proteins that had been absorbed to the carrier
were eluted by linearly changing the concentration of NaCl from 0
mM to 500 mM at a flow rate of 3 mL/minute.
[0277] The 2S,4R Monatin-forming activity was measured in each
eluted fraction, and the fractions in which the 2S,4R
Monatin-forming activity had been detected were combined, and
concentrated using Amicon Ultra-15 30k (Millipore).
[0278] The concentrated solution was diluted with 20 mM Tris-HCl
(pH 7.6), and used as a 3976AT-His solution.
Example 29
Results of Measuring Specific Activity of AJ3976LAT for Various
Keto Acids
(1) Measurement of Activity for L-Asp/.alpha.-KG, L-Asp/PA and
L-Asp/(.+-.)-MHOG by Colorimetric Method
[0279] The activity of AJ3976LAT for various substrates was
measured. The specific activities for 10 mM various keto acids were
measured by a colorimetric method, using 100 mM L-Asp as an amino
donor substrate for a transamination reaction.
[0280] Activity for L-Asp/.alpha.-KG: 100 mM L-Asp-Na-laq, 10 mM
.alpha.-KG-2Na, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM
NADH and 2 U/mL of MDH at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. Malic
dehydrogenase from porcine heart (Sigma) was used as MDH. The
activity for L-Asp/.alpha.-KG is shown in the column ".alpha.-KG"
of the aminotransferase activity in Table 13.
[0281] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50
.mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of
MDH (same as above) at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. The
activity for L-Asp/PA is shown in the column "PA" of the
aminotransferase activity in Table 13.
[0282] Activity for L-ASP/(.+-.)-MHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-MHOG, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH,
2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25.degree. C.
The activity was calculated from the reduction of the absorbance
measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc
mesenteroides (Oriental Yeast) was used as LDH. LDH was added in
order to remove PA in a trace amount existed in (.+-.)-MHOG. The
activity for L-Asp/(.+-.)-MHOG is shown in the column "(.+-.)-MHOG"
of the aminotransferase activity in Table 13.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(.+-.)-IHOG
and L-Asp/IPA
[0283] The activity to form the 2S,4R-Monatin from 4R-IHOG, the
activity to form the 2S,4R-Monatin and 2S,4S-Monatin from
(.+-.)-IHOG, which are objective activities, and the activity to
form L-Trp as a by-product from IPA were measured individually. The
transamination reaction to 10 mM keto acid was performed using 100
mM L-Asp as the amino donor substrate, and the amount of the formed
amino acid was quantified by UPLC to calculate the specific
activity.
[0284] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM
4R-IHOG (containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and
100 mM Tris-HCl (pH 8.0) at 25.degree. C. The formed 2S,4R-Monatin
and 2S,4S-Monatin were quantified by UPLC analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/4R-IHOG is shown in the column
"4R-IHOG" of the aminotransferase activity in Table 13.
[0285] Activity for L-Asp/(.+-.)-IHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-IHOG, 50 .mu.M PLP, and 100 mM Tris-HCl (pH 8.0) at
25.degree. C. The formed 2S,4R-Monatin and 2S,4S-Monatin were
quantified by the UPLC analysis. A solution of 200 mM sodium
citrate (pH 4.5) was used as a solution for stopping the reaction.
The activity for L-Asp/4R-IHOG is shown in the column "(.+-.)-IHOG"
of the aminotransferase activity in Table 13.
[0286] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50
.mu.M PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0
with 1 N NaOH after preparing the reaction solution) at 25.degree.
C. Formed Trp was quantified by the UPCL analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/IPA is shown in the column
"IPA" of the aminotransferase activity in Table 13.
[0287] The formed Monatin and Trp were quantified using ACQUITY
UPLC system supplied from Waters. The condition for the measurement
is shown below. 0.2 mL of the reaction solution was reacted for 15
minutes, then the reaction was stopped. The reaction solution after
stopping the reaction was centrifuged, and about 0.2 mL of the
supernatant was subjected to the UPLC analysis.
TABLE-US-00019 TABLE 12-2 UPLC Column: ACQUITY UPLC HSS T3 2.1
.times. 50 mm Column Temp.: 40.degree. C. Sample Temp.: 4.degree.
C. Detection: UV 210 nm Injection vol.: 5 .mu.l Mobile Phase A: 20
mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method:
20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60
40 2.2 60 40 2.3 96 4 3.0 96 4
[0288] The 2S,4R-Monatin, the 2S,4S-Monatin and Trp can be
quantified distinctively at 1.1 minutes, 1.5 minutes and 1.3
minutes, respectively.
(3) Results of Measuring Specific Activity of AJ3976LAT for Various
Keto Acids
[0289] The results of measuring the specific activity for 10 mM
keto acid when 3976-AT-His was used and L-Asp was used as the amino
donor are shown in Table 13.
TABLE-US-00020 TABLE 13 Specific activity of AJ3976LAT for various
keto acids Aminotransferase activity (U/mg) 4R-IHOG .+-.IHOG
.alpha.-KG PA .+-.MHOG SR SS SR SS IPA 106 4.0 48 0.58 1.5 0.052
3.7 0.012
Example 30
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-3976AT-His/E.
coli BL21 (DE3)
[0290] One loopful of microbial cells of pET-22-3976AT-His/E. coli
BL21 (DE3) prepared in Example 28 was inoculated to 3 mL of
Overnight Express Instant TB medium (Novagen) containing 100 mg/L
of ampicillin in a test tube, and the test tube was then shaken at
37.degree. C. for 16 hours. After the completion of the
cultivation, the microbial cells were collected from 1 mL of the
cultured medium by centrifugation, and suspended in 1 mL of
BugBuster Master Mix (Novagen). The resulting suspension was
incubated at room temperature for 15 minutes to lyse the microbial
cells. Microbial cell debris was removed by centrifugation, and the
resulting supernatant was used as a soluble fraction.
[0291] The reaction for the synthesis of the 2S,4R-Monatin from
4R-IHOG was carried out using the obtained soluble fraction. To 0.1
mL of a reaction solution [100 mM L-Asp-Na-laq, 10 mM 4R-IHOG
(containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and 100 mM
Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was
added, and the mixture was reacted at 25.degree. C. for one hour.
After the completion of the reaction, the formed 2S,4R-Monatin was
quantified to be 0.84 mM. The 2S,4R-Monatin was quantified by the
UPLC analysis. The condition for the analysis is the same in
Example 29.
Example 31
Purification of Aminotransferase Derived from Rhizobium sp.
AJ12469
[0292] Aminotransferase that forms 2S,4R-Monatin was purified from
the soluble fraction of Rhizobium sp. AJ12469 as follows. The
synthetic reaction and quantification of 2S,4R-Monatin was carried
out in the same manner as in Example 25.
(1) Preparation of Soluble Fraction
[0293] Microbial cells of Rhizobium sp. AJ12469 were spread on the
LB agar medium, and cultured at 30.degree. C. for two days.
[0294] One loopful of the resulting microbial cells was inoculated
to 160 mL of an enzyme production medium (10 g/L of yeast extract,
10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen
phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of
magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a
500 mL Sakaguchi flask, and cultured at 30.degree. C. for 16 hours
with shaking. The microbial cells were collected from about 1920 mL
of the resulting cultured medium by centrifugation, washed with and
suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4.degree. C.
for 30 minutes. The microbial cell debris was removed from the
sonicated cell suspension by centrifugation, and the resulting
supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0295] The above soluble fraction was applied onto an anion
exchange chromatographic column HiLoad 26/10 Q Sepharose HP
(supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6) to be absorbed to the carrier.
Proteins that had not been absorbed to the carrier (unabsorbed
protein) were washed out with 20 mM Tris-HCl (pH 7.6).
Subsequently, proteins that had been absorbed to the carrier were
eluted by linearly changing the concentration of NaCl from 0 mM to
500 mM at a flow rate of 8 mL/minute. The 2S,4R Monatin-forming
activity was measured in each eluted fraction, and the
2S,4R-Monatin-forming activity was detected in fractions
corresponding to about 200 mM NaCl.
(3) Hydrophobic Chromatography
[0296] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and ammonium sulfate and Tris-HCl
(pH 7.6) were added thereto so that the concentrations of ammonium
sulfate and Tris-HCl (pH 7.6) were 1.5 M and 20 mM, respectively.
The resulting solution was applied onto the hydrophobic
chromatographic column HiLoad 16/10 Phenyl Sepharose HP (supplied
from GE Healthcare Bioscience, CV=20 mL) equilibrated with 1.5 M
ammonium sulfate and 20 mM Tris-HCl (pH 7.6) to be absorbed to the
carrier. Unabsorbed proteins that had not been absorbed to the
carrier were washed out using 1.5 M ammonium sulfate and 20 mM
Tris-HCl (pH 7.6). Subsequently, the 2S,4R-Monatin-forming enzyme
was eluted by linearly changing the concentration of ammonium
sulfate from 1.5 M to 0 M at a flow rate of 3 mL/minute. The
2S,4R-Monatin-forming activity was measured in obtained each
fraction, and the 2S,4R-Monatin-forming activity was detected in
fractions corresponding to about 0.8 M ammonium sulfate.
(4) Gel Filtration Chromatography
[0297] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and concentrated using Amicon
Ultra-15 10k (Millipore). The resulting concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting
solution was applied onto a gel filtration column HiLoad 16/60
Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL)
equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and
proteins were eluted at a flow rate of 1 mL/minute. This
manipulation confirmed the 2S,4R-Monatin-forming activity at a
position at which the molecular weight was estimated to be about
100 kDa.
(5) Anion Exchange Chromatography
[0298] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and the resulting solution was
applied onto an anion exchange chromatographic column Mono Q 5/5
(supplied from Pharmacia (GE Healthcare Bioscience, CV=1 mL) to
absorb proteins to the carrier. Proteins that had not been absorbed
to the carrier (unabsorbed proteins) were washed out with 20 mM
Tris-HCl (pH 7.6). Subsequently, the absorbed proteins were eluted
by linearly changing the concentration of NaCl from 0 mM to 500 mM
at a flow rate of 0.5 mL/minute. The 2S,4R-Monatin-forming activity
was measured in each fraction, and the 2S,4R-Monatin-forming
activity was detected in the fractions corresponding to about 300
mM NaCl.
(6) SDS-PAGE
[0299] The obtained fractions were subjected to SDS-PAGE, and a
band derived from the active fraction was detected near 47 kDa.
This band was subjected to the analysis of the N-terminal amino
acid sequence as the candidate for the aminotransferase that forms
2S,4R-Monatin.
Example 32
Determination of N-Terminal Amino Acid Sequence of Aminotransferase
Derived from Rhizobium sp. AJ12469
[0300] The purified enzyme solution obtained in Example 31 was
subjected to the analysis of the N-terminal amino acid sequence,
and an N-terminal amino acid sequence of AFLADILSRVKPSATIAVTQ (SEQ
ID NO:51) was obtained. The N-terminal amino acid sequence showed
the high homology to aspartate aminotransferase (AAK87940) derived
from Agrobacterium tumefaciens str. C58.
Example 33
Cloning of Aminotransferase Gene Derived from Rhizobium sp.
AJ12469
[0301] The microbial cells of Rhizobium radiobacter AJ3976 were
cultured in the same manner as in Example 31. The microbial cells
were collected from the cultured medium by centrifugation, and
genomic DNA was extracted therefrom.
[0302] A DNA fragment containing the aminotransferase gene was
amplified by PCR with the obtained genomic DNA as the template.
Primers were designed from DNA sequences of upstream 100 bp and
downstream 100 bp of the aminotransferase gene with reference to
the genomic DNA sequence of Agrobacterium tumefaciens str. C58. The
primer Ag-u100-f (5'-ctggtgcagataagccggcttttgacc-3': SEQ ID NO:45)
and the primer Ag-d100-r (5'-ccaccttcatcatgctgctgtttctcg-3': SEQ ID
NO:46) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo)
under the following condition.
TABLE-US-00021 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0303] A nucleotide sequence of the amplified DNA fragment of about
1400 bp was determined, and was shown to be the nucleotide sequence
including 1203 bp of ORF (SEQ ID NOs:52 and 53), which had the high
homology to the aspartate aminotransferase gene (Atu2196) derived
from Agrobacterium tumefaciens str. C58. The homology was 97% in
their DNA sequences and 99% in their amino acid sequences.
[0304] This amino acid sequence was consistent with the N-terminal
amino acid sequence obtained in Example 32. Thus, it has been
thought that the aminotransferase gene having the
2S,4R-Monatin-forming activity could be acquired.
Example 34
Expression of Aminotransferase Derived from Rhizobium sp. AJ12469
in E. coli
[0305] (1) Construction of Expression Plasmid for Aminotransferase
Derived from Rhizobium sp. AJ12469
[0306] A DNA fragment containing an aminotransferase gene derived
from Rhizobium sp. AJ12469 was amplified by PCR with the genomic
DNA of Rhizobium sp. AJ12469 as the template. The primer
12469AT-Nde-f (5'-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3': SEQ ID
NO:54) and the primer 12469-xho-r
(5'-actccgctcgagGCGGCAATCGGCGCAGAAACGCTGA-3': SEQ ID NO:55) were
used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the
following condition.
TABLE-US-00022 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0307] The resulting DNA fragment was treated with restriction
enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise
treated with NdeI and XhoI. E. coli JM109 was transformed with this
ligation solution, an objective plasmid was selected from
ampicillin resistant colonies, and this plasmid was designated as
pET-22-12469AT-His. In this plasmid, the aminotransferase derived
from Rhizobium sp. AJ12469 which has a His-tag added to the
C-terminus end (12469AT-His) is expressed.
(2) Purification of 12469AT-His from E. coli Strain Expressing
12469AT-His
[0308] The constructed expression plasmid pET-22-12469AT-His was
introduced into E. coli BL21 (DE3), and one loopful of the
transformant was inoculated to 160 mL of Overnight Express Instant
TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and the Sakaguchi flask was shaken at 37.degree.
C. for 16 hours. After the completion of the cultivation, microbial
cells were collected from about 1000 mL of the cultured medium by
centrifugation, washed with and suspended in 20 mM Tris-HCl (pH
7.6), 100 mM NaCl and 20 mM imidazole, and sonicated at 4.degree.
C. for 30 minutes. The microbial cell debris was removed from the
sonicated cell suspension by centrifugation, and the resulting
supernatant was used as a soluble fraction.
[0309] The obtained soluble fraction was applied onto the His-tag
protein purification column HisPrep FF 16/10 (supplied from
Pharmacia (GE Healthcare Bioscience), CV=20 mL) equilibrated with
20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole to absorb
proteins to the carrier. Proteins that had not been absorbed to the
carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl
(pH 7.6), 100 mM NaCl and 20 mM imidazole. Subsequently, absorbed
proteins were eluted by linearly changing the concentration of
imidazole from 20 mM to 250 mM at a flow rate of 3 mL/minute.
[0310] The obtained fractions were combined and concentrated using
Amicon Ultra-15 30k (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto the
anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP
(supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier.
Proteins that had not been absorbed to the carrier (unabsorbed
protein) were washed out with 20 mM Tris-HCl (pH 7.6).
Subsequently, absorbed proteins were eluted by linearly changing
the concentration of NaCl from 0 mM to 500 mM at a flow rate of 3
mL/minute.
[0311] The 2S,4R-Monatin-forming activity was measured in each
eluted fraction. The fractions in which the 2S,4R-Monatin-forming
activity had been detected were combined, and concentrated using
Amicon Ultra-15 30k (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6) to use as a 12469AT-His
solution.
Example 35
Results of Measuring Specific Activity of AJ12469LAT for Various
Keto Acids
(1) Measurement of Activity for L-Asp/.alpha.-KG, L-Asp/PA and
L-Asp/(.+-.)-MHOG by Colorimetric Method
[0312] The activity of AJ12469LAT for various substrates was
measured. The specific activity for 10 mM keto acid was measured by
the colorimetric method, using 100 mM L-Asp as the amino donor
substrate for the transamination reaction.
[0313] Activity for L-Asp/.alpha.-KG: 100 mM L-Asp-Na-laq, 10 mM
.alpha.-KG-2Na, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM
NADH and 2 U/mL of MDH at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. Malic
dehydrogenase from porcine heart (Sigma) was used as MDH. The
activity for L-Asp/.alpha.-KG is shown in the column ".alpha.-KG"
of the aminotransferase activity in Table 15.
[0314] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50
.mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of
MDH (same as above) at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. The
activity for L-Asp/PA is shown in the column "PA" of the
aminotransferase activity in Table 15.
[0315] Activity for L-ASP/(.+-.)-MHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-MHOG, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH,
2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25.degree. C.
The activity was calculated from the reduction of the absorbance
measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc
mesenteroides (Oriental Yeast) was used as LDH. LDH was added in
order to remove PA in a trace amount contaminated in (.+-.)-MHOG.
The activity for L-Asp/(.+-.)-MHOG is shown in the column
"(.+-.)-MHOG" of the aminotransferase activity in Table 15.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(.+-.)-IHOG
and L-Asp/IPA
[0316] The activity to form 2S,4R-Monatin from 4R-IHOG, the
activity to form 2S,4R-Monatin and 2S,4S-Monatin from (.+-.)-IHOG,
which are the objective activities, and the activity to form L-Trp
as a by-product from IPA were measured individually. The
transamination reaction to 10 mM keto acid was performed using 100
mM L-Asp as the amino donor substrate for the transamination
reaction, and the amount of the formed amino acid was quantified by
UPLC to calculate the specific activity.
[0317] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM
4R-IHOG (containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and
100 mM Tris-HCl (pH 8.0) at 25.degree. C. Formed 2S,4R-Monatin and
2S,4S-Monatin were quantified by the UPLC analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/4R-IHOG is shown in the column
"4R-IHOG" of the aminotransferase activity in Table 15.
[0318] Activity for L-Asp/(.+-.)-IHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-IHOG, 50 .mu.M PLP, and 100 mM Tris-HCl (pH 8.0) at
25.degree. C. The formed 2S,4R-Monatin and 2S,4S-Monatin were
quantified by the UPLC analysis. A solution of 200 mM sodium
citrate (pH 4.5) was used as a solution for stopping the reaction.
The activity for L-Asp/(.+-.)-IHOG is shown in the column
"(.+-.)-IHOG" of the aminotransferase activity in Table 13.
[0319] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50
.mu.M PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0
with 1 N NaOH after preparing the reaction solution) at 25.degree.
C. Formed Trp was quantified by the UPCL analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/IPA is shown in the column
"IPA" of the aminotransferase activity in Table 15.
[0320] The formed Monatin and Trp were quantified using ACQUITY
UPLC system supplied from Waters. The condition for the measurement
is shown below. 0.2 mL of the reaction solution was reacted for 15
minutes, then the reaction was stopped. The reaction solution after
stopping the reaction was centrifuged, and about 0.2 mL of the
supernatant was subjected to the UPLC analysis.
TABLE-US-00023 TABLE 14 UPLC Column: ACQUITY UPLC HSS T3 2.1
.times. 50 mm Column Temp.: 40.degree. C. Sample Temp.: 4.degree.
C. Detection: UV 210 nm Injection vol.: 5 .mu.l Mobile Phase A: 20
mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method:
20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60
40 2.2 60 40 2.3 96 4 3.0 96 4
[0321] The 2S,4R-Monatin, the 2S,4S-Monatin and Trp can be
quantified distinctively at 1.1 minutes, 1.5 minutes and 1.3
minutes, respectively.
(3) Results of Measuring Specific Activity of AJ12469LAT for
Various Keto Acids
[0322] The results of measuring the specific activity for 10 mM
keto acid when 12469-AT-His was used and 100 mM L-Asp was used as
the amino donor are shown in Table 15.
TABLE-US-00024 TABLE 15 Specific activity of AJ12469LAT for various
keto acids Aminotransferase acitivity (U/mg) 4R-IHOG .+-.IHOG
.alpha.-KG PA .+-.MHOG SR SS SR SS IPA 96 4.8 44 0.56 1.6 0.066 3.3
0.016
Example 36
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-12469AT-His/E.
coli BL21 (DE3)
[0323] One loopful of microbial cells of pET-22-12469AT-His/E. coli
BL21 (DE3) prepared in Example 34 was inoculated to 3 mL of
Overnight Express Instant TB medium (Novagen) containing 100 mg/L
of ampicillin in a test tube, and the test tube was then shaken at
37.degree. C. for 16 hours. After the completion of the
cultivation, the microbial cells were collected from 1 mL of the
cultured medium by centrifugation, and suspended in 1 mL of
BugBuster Master Mix (Novagen). The resulting suspension was
incubated at room temperature for 15 minutes to lyse the microbial
cells. The microbial cell debris was removed by centrifugation, and
the resulting supernatant was used as a soluble fraction.
[0324] The reaction for the synthesis of the 2S,4R-Monatin from
4R-IHOG was carried out using the obtained soluble fraction. To 0.1
mL of a reaction solution [100 mM L-Asp-Na-lag, 10 mM 4R-IHOG
(containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and 100 mM
Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was
added, and the mixture was reacted at 25.degree. C. for one hour.
After the completion of the reaction, the amount of the formed
2S,4R-Monatin was quantified to be 0.87 mM. The 2S,4R-Monatin was
quantified by the UPLC analysis. The condition for the analysis is
the same as that in Example 29.
Example 37
Purification of Aminotransferase Derived from Corynebacterium
ammoniagenes AJ1444
[0325] Aminotransferase that formed the 2S,4R-Monatin was purified
from a soluble fraction from Corynebacterium ammoniagenes AJ1444 as
follows. The synthetic reaction and quantification of the
2S,4R-Monatin were carried out in the same manner as in Example
25.
(1) Preparation of Soluble Fraction
[0326] Microbial cells of Corynebacterium ammonia genes AJ1444 were
spread on the LB agar medium and cultured at 30.degree. C. for two
days.
[0327] One loopful of the obtained microbial cells was inoculated
to 160 mL of the enzyme production medium (10 g/L of yeast extract,
10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen
phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of
magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a
500 mL Sakaguchi flask, and cultured at 30.degree. C. for 16 hours
with shaking. The microbial cells were collected from about 1760 mL
of the cultured medium by centrifugation, washed with and suspended
in 20 mM Tris-HCl (pH 7.6), and disrupted by adding glass beads and
using a multibead shocker (Yasui Kikai Corporation). The microbial
cell debris was removed from the disrupted cell suspension by
centrifugation, and the resulting supernatant was used as a soluble
fraction.
(2) Ammonium Sulfate Precipitation
[0328] Ammonium sulfate was added to the above soluble fraction so
that a final concentration of ammonium sulfate was 90% (w/w), and
an ammonium sulfate precipitate was obtained by centrifugation.
(3) Hydrophobic Chromatography
[0329] The above ammonium sulfate precipitate was dissolved in 1.0
M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). This solution was
applied onto the hydrophobic chromatographic column HiLoad 26/10
Phenyl Sepharose HP (supplied from GE Healthcare Bioscience, CV=53
mL) equilibrated with 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH
7.6) to absorb proteins to the carrier. Unabsorbed proteins that
had not been absorbed to the carrier were washed out with 1.0 M
ammonium sulfate and 20 mM Tris-HCl (pH 7.6). Subsequently, the
2S,4R-Monatin-forming enzyme was eluted by linearly changing the
concentration of ammonium sulfate from 1.0 M to 0 M at a flow rate
of 3 mL/minute. The 2S,4R-Monatin-forming activity was measured in
each eluted fraction, and detected in fractions corresponding to
about 0.2 M ammonium sulfate.
(4) Anion Exchange Chromatography
[0330] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and dialyzed against 20 mM
Tris-HCl (pH 7.6) overnight. The resulting solution was applied
onto the anion exchange chromatographic column HiLoad 16/10 Q
Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL)
equilibrated with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the
carrier. Proteins that had not been absorbed to the carrier
(unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6).
Subsequently, absorbed proteins were eluted by linearly changing
the concentration of NaCl from 0 mM to 500 mM at a flow rate of
2.25 mL/minute. The 2S,4R-Monatin-forming activity was measured in
each eluted fraction, and detected in the fractions corresponding
to about 400 mM NaCl.
(5) Gel Filtration Chromatography
[0331] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and concentrated using Amicon
Ultra-15 10k (Millipore). The resulting concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting
solution was applied onto the gel filtration column HiLoad 16/60
Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL)
equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and
proteins were eluted at a flow rate of 1.2 mL/minute. This
manipulation confirmed the 2S,4R-Monatin-forming activity at a
position at which the molecular weight was estimated to be about 85
kDa.
(6) Anion Exchange Chromatography
[0332] The fractions in which the 2S,4R-Monatin-forming activity
had been detected were combined, and the resulting solution was
applied onto the anion exchange chromatographic column Mono Q 5/5
(supplied from Pharmacia (GE Healthcare Bioscience), CV=1 mL) to
absorb proteins to the carrier. Proteins that had not been absorbed
to the carrier (unabsorbed proteins) were washed out with 20 mM
Tris-HCl (pH 7.6). Subsequently, absorbed proteins were eluted by
linearly changing the concentration of NaCl from 0 mM to 500 mM at
a flow rate of 1 mL/minute. The 2S,4R-Monatin-forming activity was
measured in each fraction, and the 2S,4R-Monatin-forming activity
was detected in the fractions corresponding to about 400 mM
NaCl.
(7) SDS-PAGE
[0333] The obtained fractions were subjected to SDS-PAGE, and a
band derived from the active fraction was detected near 43 kDa.
This band was subjected to the analysis of the N-terminal amino
acid sequence as the candidate for the aminotransferase that forms
the 2S,4R-Monatin.
Example 38
Determination of N-Terminal Amino Acid Sequence of Aminotransferase
Derived from Corynebacterium ammoniagenes AJ1444
[0334] The purified enzyme solution obtained in Example 37 was
subjected to the analysis of the N-terminal amino acid sequence,
and the N-terminal amino acid sequence of MSXIAQXILDQ (SEQ ID
NO:112) was obtained. This N-terminal amino acid sequence showed
the high homology to aspartate aminotransferase (ZP.sub.--03935516)
derived from Corynebacterium striatum ATCC6940 and aspartate
aminotransferase (ZP.sub.--06838515) derived from Corynebacterium
ammoniagenes DSM20306.
Example 39
Cloning of Aminotransferase Gene Derived from Corynebacterium
ammoniagenes AJ1444
[0335] Microbial cells of Corynebacterium ammoniagenes AJ1444 were
cultured in the same manner as in Example 37. The microbial cells
were collected from the resulting cultured medium by
centrifugation, and genomic DNA was extracted therefrom.
[0336] A DNA fragment including the aminotransferase gene was
amplified by PCR with the obtained genomic DNA as the template. The
primer Co-d50-r (5'-cttccttggaacaagtcgaggaagac-3': SEQ ID NO:56)
designed from the DNA sequence of downstream 50 bp of the
aminotransferase gene with reference to the genomic DNA sequence of
Corynebacterium ammoniagenes DSM20306, and the primer Co-800-f
(5'-gctatcgcacaattccaccgcacctt-3': SEQ ID NO:57) designed with
reference to partial sequences that had the high homology between
the aspartate aminotransferase (ZP.sub.--03935516) derived from
Corynebacterium striatum ATCC6940 and the aspartate
aminotransferase (ZP.sub.--06838515) derived from Corynebacterium
ammoniagenes DSM20306 were used. PCR was performed using
KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00025 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0337] A nucleotide sequence of about 400 bp of the amplified DNA
fragment was determined, and the primer Co-890-r
(5'-acatcgttaagcaagcgaaccaccag-3': SEQ ID NO:58) and the primer
Co-1060-r (5'-gaaagacaagcgaatgtggtgctcg-3': SEQ ID NO:59 were
designed based on that nucleotide sequence. PCR was performed using
LA PCR in vitro Cloning Kit (Takara). PCR was performed using
KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00026 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0338] As a result, the nucleotide sequence including 1134 bp of
ORF (SEQ ID NOs: 60 and 61), which has the high homology to the
aspartate aminotransferase gene (HMPREF0281.sub.--02480) derived
from Corynebacterium ammoniagenes DSM20306 was determined. The
homology was 76% in their DNA sequences and 82% in their amino acid
sequences.
[0339] This amino acid sequence was consistent with the N-terminal
amino acid sequence obtained in Example 38. Thus, it has been
thought that the aminotransferase gene having the
2S,4R-Monatin-forming activity could be acquired.
Example 40
Expression of Aminotransferase Derived from Corynebacterium
ammoniagenes AJ1444 in E. coli
[0340] (1) Construction of Expression Vector for Aminotransferase
Derived from Corynebacterium ammoniagenes AJ1444
[0341] A DNA fragment including the aminotransferase gene derived
from Corynebacterium ammoniagenes AJ1444 was amplified by PCR with
the genomic DNA of Corynebacterium ammoniagenes AJ1444 as the
template. The primer 1444AT-Nde-f
(5'-ggaattccatATGAGCCACATCGCTCAACGCATCC-3': SEQ ID NO:62) and a
primer 1444-xho-r (5'-actccgctcgagGGACTTTTCGAAGTATTGGCGAATG-3': SEQ
ID NO:63) were used. PCR was performed using KOD-plus-ver. 2
(Toyobo) under the following condition.
TABLE-US-00027 1 cycle at 94.degree. C. for 2 minutes 25 cycles at
98.degree. C. for 10 seconds 55.degree. C. for 10 seconds and
68.degree. C. for 60 seconds 1 cycle at 68.degree. C. for 60
seconds, and 4.degree. C.
[0342] The resulting DNA fragment was treated with the restriction
enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise
treated with NdeI and XhoI. E. coli JM109 was transformed with this
ligation solution, an objective plasmid was selected from
ampicillin resistant E. coli colonies, and this plasmid was
designated as pET-22-1444AT-His. In this plasmid, the
aminotransferase derived from Corynebacterium ammoniagenes AJ1444
which has the His-tag added to the C-terminus end (1444AT-His) is
expressed.
(2) Purification of 1444AT-His from E. coli Strain Expressing
1444AT-His
[0343] The constructed expression plasmid pET-22-1444AT-His was
introduced into E. coli BL21 (DE3), and one loopful of the
transformant was inoculated to 160 mL of Overnight Express Instant
TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and the Sakaguchi flask was shaken at 37.degree.
C. for 16 hours. After completion of the cultivation, microbial
cells were collected from about 1000 mL of the cultured medium by
centrifugation, washed with and suspended in 20 mM Tris-HCl (pH
7.6), 300 mM NaCl and 10 mM imidazole, and sonicated at 4.degree.
C. for 30 minutes. The microbial cell debris was removed from the
sonicated cell suspension by centrifugation, and the resulting
supernatant was used as a soluble fraction.
[0344] The obtained soluble fraction was applied onto a His-tag
protein purification column His TALON superflow 5 mL Centrifuge
(Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl
and 10 mM imidazole to absorb proteins to the carrier. Proteins
that had not been absorbed to the carrier (unabsorbed protein) were
washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM
imidazole. Subsequently, absorbed proteins were eluted using 20 mM
Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate
of 5 mL/minute.
[0345] The obtained fractions were combined and concentrated using
Amicon Ultra-15 30k (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto the
anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP
(supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated
with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier.
Proteins that had not been absorbed to the carrier (unabsorbed
protein) were washed out with 20 mM Tris-HCl (pH 7.6).
Subsequently, unabsorbed proteins were eluted by linearly changing
the concentration of NaCl from 0 mM to 500 mM at a flow rate of 3
mL/minute.
[0346] The 2S,4R-Monatin-forming activity was measured in each
eluted fraction. The fractions in which the 2S,4R-Monatin-forming
activity had been detected were combined, and concentrated using
Amicon Ultra-15 30k (Millipore). The concentrated solution was
diluted with 20 mM Tris-HCl (pH 7.6) to use as a 1444AT-His
solution.
Example 41
Results of Measuring Specific Activity of AJ1444LAT for Various
Keto Acids
(1) Measurement of Activity for L-Asp/.alpha.-KG, L-Asp/PA,
L-Asp/(.+-.)-MHOG, L-Glu/PA and L-Glu/(.+-.)-MHOG by Colorimetric
Method
[0347] The activity of AJ1444LAT for various substrates was
measured. The specific activity for 10 mM keto acid was measured by
colorimetric method, using 100 mM L-Asp or L-Glu as the amino donor
substrate for the transamination reaction.
[0348] Activity for L-Asp/.alpha.-KG: 100 mM L-Asp-Na-laq, 10 mM
.alpha.-KG-2Na, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM
NADH and 2 U/mL of MDH at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. Malic
dehydrogenase from porcine heart (Sigma) was used as MDH. The
activity for L-Asp/.alpha.-KG is shown in the column ".alpha.-KG"
of the aminotransferase activity in Table 17.
[0349] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50
.mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of
MDH (same as above) at 25.degree. C. The activity was calculated
from the reduction of the absorbance measured at 340 nm. The
activity for L-Asp/PA is shown in the column "PA" of the
aminotransferase activity in Table 17.
[0350] Activity for L-Asp/(.+-.)-MHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-MHOG, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH,
2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25.degree. C.
The activity was calculated from the reduction of the absorbance
measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc
mesenteroides (Oriental Yeast) was used as LDH. LDH was added in
order to remove PA in a trace amount contaminated in (.+-.)-MHOG.
The activity for L-Asp/(.+-.)-MHOG is shown in the column
"(.+-.)-MHOG" of the aminotransferase activity in Table 17.
[0351] Activity for L-Glu/PA: 100 mM L-Glu-Na, 10 mM PA, 50 .mu.M
PLP, 100 mM Tris-HCl (pH 8.0), 100 mM NH.sub.4Cl, 0.25 mM NADH and
10 U/mL of GDH at 25.degree. C. The activity was calculated from
the reduction of the absorbance measured at 340 nm. L-Glutamic
dehydrogenase from bovine liver (Sigma) was used as GDH. The
activity for L-Glu/PA is shown in the column "PA" of the
aminotransferase activity in Table 17.
[0352] Activity for L-Glu/(.+-.)-MHOG: 100 mM L-Glu-Na, 10 mM
(.+-.)-MHOG, 50 .mu.M PLP, 100 mM Tris-HCl (pH 8.0), 100 mM
NH.sub.4Cl, 0.25 mM NADH, and 10 U/mL of GDH at 25.degree. C. The
activity was calculated from the reduction of the absorbance
measured at 340 nm. The activity for L-Glu/(.+-.)-MHOG is shown in
the column "(.+-.)-MHOG" of the aminotransferase activity in Table
17.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(.+-.)-IHOG,
L-Asp/IPA, L-Glu/4R-IHOG and L-Glu/IPA
[0353] The activity to form 2S,4R-Monatin from 4R-IHOG, the
activity to form 2S,4R-Monatin and 2S,4S-Monatin from (.+-.)-IHOG,
which are the objective activities, and the activity to form L-Trp
as the by-product from IPA were measured individually. The
transamination reaction to 10 mM keto acid was performed using 100
mM L-Asp or L-Glu as the amino donor substrate of the
transamination reaction, and the amount of the formed amino acid
was quantified by UPLC to calculate the specific activity.
[0354] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM
4R-IHOG (containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and
100 mM Tris-HCl (pH 8.0) at 25.degree. C. The formed 2S,4R-Monatin
and 2S,4S-Monatin were quantified by UPLC analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/4R-IHOG is shown in the column
"4R-IHOG" of the aminotransferase activity in Table 17.
[0355] Activity for L-Asp/(.+-.)-IHOG: 100 mM L-Asp-Na-laq, 10 mM
(.+-.)-IHOG, 50 .mu.M PLP, and 100 mM Tris-HCl (pH 8.0) at
25.degree. C. The formed 2S,4R-Monatin and 2S,4S-Monatin were
quantified by UPLC analysis. A solution of 200 mM sodium citrate
(pH 4.5) was used as a solution for stopping the reaction. The
activity for L-Asp/(.+-.)-IHOG is shown in the column "(.+-.)-IHOG"
of the aminotransferase activity in Table 17.
[0356] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50
.mu.M PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0
with 1 N NaOH after preparing the reaction solution) at 25.degree.
C. The formed Trp was quantified by UPCL analysis. A solution of
200 mM sodium citrate (pH 4.5) was used as a solution for stopping
the reaction. The activity for L-Asp/IPA is shown in the column
"IPA" of the aminotransferase activity in Table 17.
[0357] Activity for L-Glu/4R-IHOG: 100 mM L-Glu-Na, 10 mM 4R-IHOG
(containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and 100 mM
Tris-HCl (pH 8.0) at 25.degree. C. The formed 2S,4R-Monatin and
2S,4S-Monatin were quantified by UPLC analysis. A solution of 200
mM sodium citrate (pH 4.5) was used as a solution for stopping the
reaction. The activity for L-Glu/4R-IHOG is shown in the column
"4R-IHOG" of the aminotransferase activity in Table 17.
[0358] Activity for L-Glu/IPA: 100 mM L-Glu-Na, 10 mM IPA, 50 PLP,
and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH
after preparing the reaction solution) at 25.degree. C. The formed
Trp was quantified by UPCL analysis. A solution of 200 mM sodium
citrate (pH 4.5) was used as a solution for stopping the reaction.
The activity for L-Glu/IPA is shown in the column "IPA" of the
aminotransferase activity in Table 17.
[0359] The formed Monatin and Trp were quantified using ACQUITY
UPLC system supplied from Waters. The condition for the measurement
is shown below. 0.2 mL of the reaction solution was reacted for 15
minutes, then the reaction was stopped. The reaction solution after
stopping the reaction was centrifuged, and about 0.2 mL of the
supernatant was subjected to UPLC analysis.
TABLE-US-00028 TABLE 16 UPLC Column: ACQUITY UPLC HSS T3 2.1
.times. 50 mm Column Temp.: 40.degree. C. Sample Temp.: 4.degree.
C. Detection: UV 210 nm Injection vol.: 5 .mu.l Mobile Phase A: 20
mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method:
20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60
40 2.2 60 40 2.3 96 4 3.0 96 4
[0360] The 2S,4R-Monatin, 2S,4S-Monatin and Trp can be quantified
distinctively at 1.1 minutes, 1.5 minutes and 1.3 minutes,
respectively.
(3) Results of Measuring Specific Activity of AJ1444LAT for Various
Keto Acids
[0361] The results of measuring the specific activity for 10 mM
keto acid when 1444-AT-His was used and L-Asp was used as the amino
donor are shown in Table 17.
TABLE-US-00029 TABLE 17 Specific activity of AJ1444LAT for various
keto acids Aminotransferase activity (U/mg) 4R-IHOG .+-.IHOG
.alpha.-KG PA .+-.MHOG SR SS SR SS IPA L-Asp 4.0 1.7 2.7 2.4 0.26
0.91 1.7 0.085 L-Glu -- 8.7 145 200 15 -- -- 0.21
Example 42
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-1444AT-His/E.
coli BL21 (DE3)
[0362] One loopful of microbial cells of pET-22-1444AT-His/E. coli
BL21 (DE3) prepared in Example 40 was inoculated to 3 mL of
Overnight Express Instant TB medium (Novagen) containing 100 mg/L
of ampicillin in a test tube, and the test tube was then shaken at
37.degree. C. for 16 hours. After the completion of the
cultivation, the microbial cells were collected from 1 mL of the
cultured medium by centrifugation, and suspended in 1 mL of
BugBuster Master Mix (Novagen). The resulting suspension was left
stand at room temperature for 15 minutes to lyse the microbial
cells. The microbial cell debris was removed by centrifugation, and
the resulting supernatant was used as a soluble fraction.
[0363] The reaction for the synthesis of 2S,4R-Monatin from 4R-IHOG
was carried out using the obtained soluble fraction. To 0.1 mL of
the reaction solution [100 mM L-Asp-Na-laq, 10 mM 4R-IHOG
(containing 4S-IHOG in a trace amount), 50 .mu.M PLP, and 100 mM
Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was
added, and the mixture was reacted at 25.degree. C. for one hour.
After the completion of the reaction, the amount of the formed
2S,4R-Monatin was quantified to be 0.13 mM. The 2S,4R-Monatin was
quantified by the UPLC analysis. The condition for the analysis is
the same as that in Example 29.
Example 43
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 20 mM L-Trp
(AJ3976LAT, AJ12469LAT, AJ1444LAT)
[0364] A reaction was performed under the following condition for
12 hours using purified 3976AT-His, 12469AT-His and 1444AT-His. The
reaction was performed in 1 mL using a test tube. The reaction
solution was appropriately sampled, the sample was diluted with TE
buffer, ultrafiltrated using an Amicon Ultra-0.5 mL centrifugal
filter 10 kDa (Millipore), and the resulting filtrate was analyzed.
HPLC and capillary electrophoresis were used for the analysis.
[0365] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM
L-Asp-Na-laq, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 20 mM
KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme,
10 U/mL of commercially available OAA DCase enzyme, 2 U/mL of
purified LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL of
commercially available SOD enzyme at 25.degree. C. at 120 rpm.
[0366] The methods for preparing the enzymes subjected to the
reaction are shown below.
[0367] Ps_aad broth: it was prepared according to the method
described in Example 17.
[0368] Purified SpAld enzyme: it was prepared according to the
method described in Example 19.
[0369] AJ3976LAT, AJ12469LAT and AJ1444LAT: they are prepared
according to the methods described in Examples 28, 34 and 40.
[0370] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp.
(Sigma) was used. A value described by the manufacturer was used as
an enzyme amount (U).
[0371] SOD: superoxide dismutase from bovine liver (Sigma) was
used. A value described by the manufacturer was used as an enzyme
amount (U).
[0372] As a result of the one-pot reactions, 12 mM, 11 mM and 13 mM
2S,4R-Monatin were formed after 4 hours using AJ3976LAT, AJ12469LAT
and AJ1444LAT, respectively, and their yields from L-Trp were 58%,
53% and 64%, respectively.
Example 44
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 50 mM Trp
(AJ3976 on Scale of 80 mL)
[0373] A reaction was performed for 12 hours using purified
3976AT-HIs under the following condition. The reaction was
performed in a volume of 80 mL using a 250 mL volume mini-jar. The
reaction solution was appropriately sampled, the sample was diluted
with TE buffer, which was then ultrafiltrated using the Amicon
Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the
resulting filtrate was analyzed. HPLC and capillary electrophoresis
were used for the analysis.
[0374] Reaction condition: 50 mM L-Trp, 50 mM PA-Na, 200 mM
L-Asp-Na-laq, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl (pH
7.6), 20 mM KPB (pH 7.6), 0.0025% GD113K, pH<7.6 (1 M
H.sub.2SO.sub.4), 20% Ps_aad broth, 30 U/mL of purified SpAld
enzyme, 10 U/mL of commercially available OAA DCase enzyme, 2 U/mL
of purified LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL of
commercially available SOD enzyme at 25.degree. C. at 350-400 rpm
with air at 8 mL/minute (1/10 vvm).
[0375] The methods for preparing the enzymes subjected to the
reaction are shown below.
[0376] Ps_aad broth: it was prepared according to the method
described in Example 17.
[0377] Purified SpAld enzyme: it was prepared according to the
method described in Example 19.
[0378] AJ3976LAT: it was prepared according to the methods
described in Examples 2B.
[0379] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp.
(Sigma) was used. The value described by the manufacturer was used
as the enzyme amount (U).
[0380] SOD: superoxide dismutase from bovine liver (Sigma) was
used. The value described by the manufacturer was used as the
enzyme amount (U).
[0381] As a result of the one-pot reaction, 27 mM 2S,4R-Monatin was
confirmed to be accumulated after 8 hours, and the yield from L-Trp
which was calculated after calibrating the solution amounts was
56%.
Example 45
Expression of in Silico Selected Aminotransferase in E. coli
(1) Construction of Expression Plasmid for in Silico Selected
Aminotransferase
[0382] A DNA sequence obtained by conferring a NdeI recognition
sequence and a XhoI recognition sequence to the 5'-end and 3'-end
of the genetic sequence of the aminotransferase selected in silico
was subjected to Optimum Gene Codon Optimization Analysis supplied
by GenScript to obtain synthesized DNA, an expression efficiency of
which had been optimized in E. coli. Types of the aminotransferase
are as follows.
[0383] Putative aminotransferase derived from Deinococcus
Geothermalis DSM 11300 (Dge, ABF45244) (SEQ ID NOs: 64 and 65),
hypothetical protein derived from Corynebacterium glutamicum R
(Cgl, BAF53276) (SEQ ID NOs: 66 and 67), Lysn, alpha-aminoadipate
aminotransferase derived from Thermus thermophilus HB27 (TtHB,
AAS80391) (SEQ ID NOs: 68 and 69), aminotransferase (Putative)
derived from Thermotoga Maritima (Tma1, AAD36207) (SEQ ID NOs: 70
and 71), human kynurenine aminotransferase II Homologue derived
from Pyrococcus Horikoshii Ot3 (PhoH, 1X0M) (SEQ ID NOs: 72 and
73), aspartate aminotransferase derived from Phormidium Lapideum
(Pla, BAB86290) (SEQ ID NOs: 74 and 75), aspartate aminotransferase
derived from Thermus Thermophilus (Tth, BAD69869) (SEQ ID NOs: 76
and 77), aromatic aminotransferase derived from Pyrococcus
Horikoshii Ot3 (PhoA, 1DJU) (SEQ ID NOs: 78 and 79), Mj0684 derived
from Methanococcus jannaschii (Mja, AAB98679) (SEQ ID NOs: 80 and
81), aspartate aminotransferase derived from Thermotoga Maritima
(Tma2, AAD36764) (SEQ ID NOs: 82 and 83), aspartate
aminotransferase derived from Saccharomyces cerevisiae (Sce,
CAY81265) (SEQ ID NOs: 84 and 85), aspartate aminotransferase
derived from Eubacterium rectale (Ere, ACR74350) (SEQ ID NOs: 86
and 87), aspartate aminotransferase derived from Bacillus pumilus
SAFR-032 (Bpu, ABV62783) (SEQ ID NOs: 88 and 89), putative
transcriptional regulator (GntR family) derived from Bacillus
cellulosilyticus DSM 2522 (Bce, ADU30616) (SEQ ID NOs: 90 and 91),
aspartate aminotransferase aspC derived from Bacillus species
(strain YM-2) (Bsp, AAA22250) (SEQ ID NOs: 92 and 93), aspartate
aminotransferase aatB derived from Sinorhizobium meliloti 1021
(SmeB, CAC47870) (SEQ ID NOs: 94 and 95), branched-chain amino-acid
aminotransferase derived from Methanothermobacter
thermautotrophicus str. Delta H (Mth, AAB85907) (SEQ ID NOs: 96 and
97), aspartate aminotransferase derived from Lactobacillus
acidophilus (Lba, AAV43507) (SEQ ID NOs: 98 and 99), aspartate
aminotransferase aatA derived from Sinorhizobium meliloti 1021
(SmeA, CAC46904) (SEQ ID NOs: 100 and 101), hypothetical serine
aminotransferase derived from Pyrococcus horikoshi OT3 (PhoS,
BAA30413) (SEQ ID NOs: 102 and 103), PLP-dependent
aminotransferases derived from Thermoanaerobacter tengcongensis MB4
(Tte, AAM24436) (SEQ ID NOs: 104 and 105), putative transcriptional
regulator (GntR family) derived from Clostridium cellulolyticum H10
(Cce, ACL75101) (SEQ ID NOs: 106 and 107), aspartate
aminotransferase AspT derived from Rhodococcus erythropolis PR4
(Rer, BAH31070) (SEQ ID NOs: 108 and 109), and transcriptional
regulator derived from Saccharophagus degradans 2-40 (Sde,
ABD82545) (SEQ ID NOs: 110 and 111).
TABLE-US-00030 TABLE 18 Comparison of percent identities of amino
acid sequences Amino acid Amino acid sequence identity sequence
identity ID Abbreviation (%) to AJ1616LAT (%) to AJ3976LAT 1 Dge 46
23 2 Cgl 46 30 3 TtHB 20 22 4 Tmal 21 20 5 PhoH 20 22 6 Pla 18 45 7
Tth 17 47 8 PhoA 16 39 9 Mja 17 33 10 Tma2 15 27 11 Sce 20 19 12
Ere 30 26 13 Bpu 93 23 14 Bce 67 22 15 Bsp 17 45 16 SmeB 20 58 17
Mth 17 16 18 Lba 20 24 19 SmeA 21 89 20 PhoS 19 15 21 Tte 17 48 22
Cce 61 24 23 Rer 49 16 24 Sde 49 26
[0384] The synthesized DNA was treated with the restriction enzymes
NdeI and XhoI, and ligated to pET-22b (Novagen) likewise treated
with NdeI and XhoI. E. coli JM109 was transformed with this
ligation solution, the objective plasmids were selected from
ampicillin resistant colonies, and these plasmid were designated as
pET-22-AT-His. In these plasmids, the aminotransferases having the
His-tag added to the C terminus end (AT-His) are expressed.
(2) Purification of AT-His from E. coli Strains Expressing
AT-His
[0385] Each of the constructed plasmids pET-22-AT-His was
introduced into E. coli BL21 (DE3), and one loopful of the
transformant was inoculated to 100 mL of Overnight Express Instant
TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL
Sakaguchi flask, and the Sakaguchi flask was shaken for 16 hours.
The shaking was performed for Lba at 25.degree. C., for Dge, Pla,
Tth, Tma2, Sce, Ere, Bpu, Bce, Bsp, SmeA, PhoS, Rer and Sde at
30.degree. C., for Cgl, TtHB, PhoH, PhoA, SmeB, Tte and Cce at
37.degree. C., and for Tma1, Mja and Mth at 42.degree. C. After the
completion of the cultivation, microbial cells were collected from
the cultured medium by the centrifugation, washed with and
suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM
imidazole, and sonicated. The microbial cell debris was removed
from the sonicated cell suspension by the centrifugation, and the
resulting supernatant was used as a soluble fraction.
[0386] The obtained soluble fraction was applied onto the His-tag
protein purification column His TALON superflow 5 mL Centrifuge
(Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl
and 10 mM imidazole to absorb proteins to the carrier. Proteins
that had not been absorbed to the carrier (unabsorbed protein) were
washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM
imidazole. Subsequently, absorbed proteins were eluted using 20 mM
Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate
of 5 mL/minute. The obtained fractions were combined and
concentrated using Amicon Ultra-15 10k (Millipore). The
concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to
use as a LAT solution. If necessary, further purification was
carried out by increasing the amount of the medium to be cultured
and the number of His TALON columns to be linked.
Example 46
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 20 mM
L-Trp
[0387] Each reaction was performed under the following condition
for 15 hours using purified various AT-His. The reaction was
performed in a volume of 1 mL using a test tube. After the
completion of the reaction, each sample was diluted with TE buffer,
ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal filter 10
kDa (Millipore), and the resulting filtrate was analyzed. HPLC and
capillary electrophoresis were used for the analysis.
[0388] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM
L-Asp-Na-laq, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 20 mM
KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme,
10 U/mL of commercially available OAA DCase enzyme, 1 mg/mL of
purified LAT enzyme, and 200 U/mL of commercially available SOD
enzyme at 25.degree. C. at 120 rpm.
[0389] The methods for preparing the enzymes subjected to the
reaction were shown below.
[0390] Ps_aad broth: it was prepared according to the method
described in Example 17.
[0391] Purified SpAld enzyme: it was prepared according to the
method described in Example 19.
[0392] Various LAT: they were prepared according to the method
described in Example 45.
[0393] OAA DCase: oxaloacetate decarboxylase from Pseudomonas
Example 47
One-pot reaction for synthesis of 2S,4R-Monatin from 20 mM L-Trp
(Tth, Bpu, SmeA and Sde)
[0394] Reactions were performed under the following condition for
15 hours using purified various AT-His. The reaction was performed
in a volume of 1 mL using a test tube. After the completion of the
reaction, the sample was diluted with TE buffer, ultrafiltrated
using the Amicon Ultra-0.5 mL centrifugal filter 10 kDa
(Millipore), and the resulting filtrate was analyzed. HPLC and
capillary electrophoresis were used for the analysis.
[0395] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM
L-Asp-Na-laq, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 20 mM
KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme,
10 U/mL of commercially available OAA DCase enzyme, 3 mg/mL of
purified LAT enzyme (12 mg/mL of Tth, 1 mg/mL of Bpu), and 200 U/mL
of commercially available SOD enzyme at 25.degree. C. at 120
rpm.
[0396] The methods for preparing the enzymes subjected to the
reaction are shown below.
[0397] Ps_aad broth: it was prepared according to the method
described in Example 17.
[0398] Purified SpAld enzyme: it was prepared according to the
method described in Example 19.
[0399] Various LAT: they were prepared according to the method
described in Example 45.
[0400] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp.
(Sigma) was used. The value described by the manufacturer was used
as the enzyme amount (U).
[0401] SOD: superoxide dismutase from bovine liver (Sigma) was
used. The value described by the manufacturer was used as the
enzyme amount (U).
[0402] The results of the one-pot reactions are shown in Table 20.
The 2S,4R-Monatin at 18 mM, 17 mM, 11 mM and 12 mM were formed
using Tth, Bpu, SmeA and Sde, respectively, and their yields from
L-Trp were 92%, 87%, 54% and 61%, respectively.
TABLE-US-00031 TABLE 20 Yields of 2S,4R-Monatin in one-pot reaction
using 20 mM Trp as substrate Abbreviation Yield from Trp (%) Tth 92
Bpu 87 SmeA 54 Sde 61
Example 48
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 100 mM L-Trp
(Tth, Bpu, SmeA and Sde)
[0403] Reactions were performed under the following condition for
18 hours using purified various AT-His, Tth, Bpu, SmeA and Sde. The
reaction was performed in a volume of 1 mL using a test tube. After
the completion of the reaction, the sample was diluted with TE
buffer, ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal
filter 10 kDa (Millipore), and the resulting filtrate was analyzed.
HPLC and capillary electrophoresis were used for the analysis.
[0404] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM
L-Asp-Na-laq, 1 mM MgCl.sub.2, 50 .mu.M PLP, 100 mM Tris-HCl, 20 mM
KPB (pH 7.0), 40% Ps_aad broth, 60 U/mL of purified SpAld enzyme,
10 U/mL of commercially available OAA DCase enzyme, 3 mg/mL of
purified LAT enzyme (12 mg/mL for Tth), and 200 U/mL of
commercially available SOD enzyme at 25.degree. C. at 150 rpm.
[0405] The methods for preparing the enzymes subjected to the
reaction are shown below.
[0406] Ps_aad broth: it was prepared according to the method
described in Example 17.
[0407] Purified SpAld enzyme: it was prepared according to the
method described in Example 19.
[0408] Various LAT: they were prepared according to the method
described in Example 45.
[0409] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp.
(Sigma) was used. The value described by the manufacturer was used
as the enzyme amount (U).
[0410] SOD: superoxide dismutase from bovine liver (Sigma) was
used. The value described by the manufacturer was used as the
enzyme amount (U).
[0411] The results of the one-pot reactions are shown in Table 21.
The 2S,4R-Monatin at 72 mM, 46 mM, 6.4 mM and 20 mM were formed
using Tth, Bpu, SmeA and Sde, respectively, and their yields from
L-Trp were 72%, 46%, 6.4% and 20%, respectively.
TABLE-US-00032 TABLE 21 Yields of 2S,4R-MOnatin in one pot reaction
using 100 mM Trp as substrate Yield from Abbreviation Trp (%) Tth
72 Bpu 46 SmeA 6.4 Sde 20
(Information on Microorganisms)
[0412] The microorganisms specified by deposit numbers which are
described herein can be available from certain deposit authority.
The microorganisms described in Table 22 have been deposited to
National Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary (1-1-1 Central No. 6
Higashi, Tsukuba-shi, Ibaraki Prefecture, Japan) on the following
dates, and the following deposit numbers have been conferred to
them. As described in Table 22, these microorganisms are currently
classified in the following ways as a result of reidentification,
although different names were previously conferred to them.
TABLE-US-00033 TABLE 22 Current names for microorganims (Previous
names for Deposit microorganims) number Deposited date Bacillus
altitudinis AJ1616 FERM-BP 11429 Oct. 4, (Bacillus sp. AJ1616) 2011
Stenotrophomonas sp. AJ3447 FERM-BP 11422 Sep. 30, (Xanthomonas
oryzae AJ3447) 2011 Stenotrophomonas sp. AJ11634 FERM-BP 11423 Sep.
30, (Xanthomonas albilineans 2011 AJ11634) Ochrobactrum
pseudogrignonense FERM-BP 11432 Oct. 4, AJ3735 2011 (Pseudomonas
betainovorans AJ3735) Stenotrophomonas sp. AJ1591 FERM-BP 11419
Sep. 27, (Pseudomonas putrefaciens 2011 AJ1591) Stenotrophomonas
sp. AJ3839 FERM-BP 11416 Sep. 15, (Pseudomonas peptidolytica 2011
AJ3839) Brevundimonas diminuta AJ3958 FERM-BP 11425 Sep. 30,
(Pseudomonas hydrogenovora 2011 AJ3958) Rhizobium sp. AJ12469
FERM-BP 11430 Oct. 4, (Alcaligenes faecalis AJ12469) 2011
Carnimonas sp. AJ3230 FERM-BP 11431 Oct. 4, (Achromobacter
brunificans 2011 AJ3230) Pseudomonas sp. AJ1594 FERM-BP 11424 Sep.
30, (Pseudomonas ovalis AJ1594) 2011
[0413] In addition, the microorganisms described in Table 23 are
currently classified in the following ways as a result of
reidentification, although different names were previously
conferred to them. The bacterial strain, Stenotrophomonas sp.
AJ13127 is identical to the known bacterial strain specified by the
deposit number FERM-BP 5568.
TABLE-US-00034 TABLE 23 Current names for microorganims (Previous
names for microorganims) Rhizobium radiobacter LAT1 (Rhizobium sp.
LAT1) Rhizobium radiobacter AJ11568 (Pseudomonas umorosa AJ11568)
Dietzia maris AJ2788 (Pseudomonas tabaci AJ2788) Stenotrophomonas
sp. AJ13127 (Stenotrophomonas sp. AJ13127) Arthrobacter sp. IAM1390
(Arthrobacter ureafaciens IAM1390) Burkholderia sp. AJ3084
(Pseudomonas multivorans AJ3084) Rhizobium radiobacter AJ2557
(Alcaligenes metalcaligenes AJ2557) Pseudomonas sp. LMG2833
(Achromobacter butyri LMG2833)
INDUSTRIAL APPLICABILITY
[0414] As described above, the methods of the present invention are
useful for producing the Monatin which can be used as the
sweetener.
Sequence Listing Free Text
[0415] SEQ ID NO:1: Nucleotide sequence of aminotransferase gene
derived from Bacillus altitudinis SEQ ID NO:2: Amino acid sequence
of aminotransferase derived from Bacillus altitudinis SEQ ID NO:3:
Nucleotide sequence of aminotransferase gene (nucleotide numbers
231-1538) and the upstream and downstream regions thereof which are
derived from Bacillus altitudinis SEQ ID NO:4: Amino acid sequence
of a fragment of aminotransferase derived from Bacillus altitudinis
SEQ ID NO:5: Amino acid sequence of a fragment of aminotransferase
derived from Bacillus altitudinis SEQ ID NO:6: Forward primer for
amplifying DNA fragment containing aminotransferase gene derived
from Bacillus altitudinis (Bp-u200-f) SEQ ID NO:7: Reverse primer
for amplifying DNA fragment containing aminotransferase gene
derived from Bacillus altitudinis (Bp-d200-r) SEQ ID NO:8: Forward
primer for amplifying DNA fragment containing aminotransferase gene
derived from Bacillus altitudinis (1616AT-Nde-f) SEQ ID NO:9:
Reverse primer for amplifying DNA fragment containing
aminotransferase gene derived from Bacillus altitudinis
(1616-xho-r) SEQ ID NO:10: Forward primer for converting DNA
sequence recognized by NdeI, which is found on aminotransferase
gene derived from Bacillus altitudinis (1616-delNde-f) SEQ ID
NO:11: Reverse primer for converting DNA sequence recognized by
NdeI, which is found on aminotransferase gene derived from Bacillus
altitudinis (1616-delNde-r) SEQ ID NO:12: Forward primer for
amplifying DNA fragment containing SpAld gene (SpAld-f-NdeI) SEQ ID
NO:13: Reverse primer for amplifying DNA fragment containing SpAld
gene (SpAld-r-HindIII) SEQ ID NO:14: Forward primer for converting
rare codon 6L in SpAld gene (6L-f) SEQ ID NO:15: Reverse primer for
converting rare codon 6L in SpAld gene (6L-r) SEQ ID NO:16: Forward
primer for converting rare codon 13L in SpAld gene (13L-f) SEQ ID
NO:17: Reverse primer for converting rare codon 13L in SpAld gene
(13L-r) SEQ ID NO:18: Forward primer for converting rare codon 18P
in SpAld gene (18P-f) SEQ ID NO:19: Reverse primer for converting
rare codon 18P in SpAld gene (18P-r) SEQ ID NO:20: Forward primer
for converting rare codon 38P in SpAld gene (38P-f) SEQ ID NO:21:
Reverse primer for converting rare codon 38P in SpAld gene (38P-r)
SEQ ID NO:22: Forward primer for converting rare codon 50P in SpAld
gene (50P-f) SEQ ID NO:23: Reverse primer for converting rare codon
50P in SpAld gene (50P-r) SEQ ID NO:24: Forward primer for
converting rare codons 77P, 81P and 84R in SpAld gene
(77P-81P-84R-f) SEQ ID NO:25: Reverse primer for converting rare
codons 77P, 81P and B4R in SpAld gene (77P-81P-84R-r) SEQ ID NO:26:
Forward primer for preparing the aminotransferase mutant K39R
derived from Bacillus altitudinis AJ1616 (K39R_FW) SEQ ID NO:27:
Reverse primer for preparing the aminotransferase mutant K39R
derived from Bacillus altitudinis AJ1616 (K39R_RV) SEQ ID NO:28:
Forward primer for preparing the aminotransferase mutant S258G
derived from Bacillus altitudinis AJ1616 (S258G_FW) SEQ ID NO:29:
Reverse primer for preparing the aminotransferase mutant S258G
derived from Bacillus altitudinis AJ1616 (S258G_RV) SEQ ID NO:30:
Forward primer for preparing the aminotransferase mutant T288G
derived from Bacillus altitudinis AJ1616 (T288G_FW) SEQ ID NO:31:
Reverse primer for preparing the aminotransferase mutant T288G
derived from Bacillus altitudinis AJ1616 (T288G_RV) SEQ ID NO:32:
Forward primer for preparing the aminotransferase mutant I289A
derived from Bacillus altitudinis AJ1616 (I289A_FW) SEQ ID NO:33:
Reverse primer for preparing the aminotransferase mutant I289A
derived from Bacillus altitudinis AJ1616 (I289A_RV) SEQ ID NO:34:
Forward primer for preparing the aminotransferase mutant
Q287E/T288G derived from Bacillus altitudinis AJ1616
(Q287E/T288G_FW) SEQ ID NO:35: Reverse primer for preparing the
aminotransferase mutant Q287E/T288G derived from Bacillus
altitudinis AJ1616 (Q287E/T288G_RV) SEQ ID NO:36: Primer for
preparing a DNA fragment for destroying aspC gene (aspC-L1) SEQ ID
NO:37: Primer for preparing a DNA fragment for destroying aspC gene
(aspC-R1) SEQ ID NO:38: Primer for confirming the insertion of
attL-cat-attR in the region of aspC gene (aspC-up) SEQ ID NO:39:
Primer for confirming the insertion of attL-cat-attR in the region
of aspC gene (attL-1) SEQ ID NO:40: Primer for confirming the
insertion of attL-cat-attR in the region of aspC gene (aspC-down)
SEQ ID NO:41: Primer for confirming the insertion of attL-cat-attR
in the region of aspC gene (attR-1) SEQ ID NO:42: Nucleotide
sequence of oxaloacetate decarboxylase gene derived from
Pseudomonas putida SEQ ID NO:43: Amino acid sequence of
oxaloacetate decarboxylase derived from Pseudomonas putida SEQ ID
NO:44: Amino acid sequence of a fragment of aminotransferase
derived from Rhizobium radiobacter SEQ ID NO:45: Forward primer
which is designed based on the genomic DNA sequence from
Agrobacterium tumefaciens str. C58 (Ag-u100-f) SEQ ID NO:46:
Reverse primer which is designed based on the genomic DNA sequence
from Agrobacterium tumefaciens str. C58 (Ag-d100-r) SEQ ID NO:47:
Nucleotide sequence of aminotransferase gene derived from Rhizobium
radiobacter SEQ ID NO:48: Amino acid sequence of aminotransferase
derived from Rhizobium radiobacter SEQ ID NO:49: Forward primer for
amplifying DNA fragment containing aminotransferase gene derived
from Rhizobium radiobacter (3976AT-Nde-f) SEQ ID NO:50: Reverse
primer for amplifying DNA fragment containing aminotransferase gene
derived from Rhizobium radiobacter (3976-xho-r) SEQ ID NO:51: Amino
acid sequence of a fragment of aminotransferase derived from
Rhizobium sp. SEQ ID NO:52: Nucleotide sequence of aminotransferase
gene derived from Rhizobium sp. SEQ ID NO:53: Amino acid sequence
of aminotransferase derived from Rhizobium sp. SEQ ID NO:54:
Forward primer for amplifying DNA fragment containing
aminotransferase gene derived from Rhizobium sp. (12469AT-Nde-f)
SEQ ID NO:55: Reverse primer for amplifying DNA fragment containing
aminotransferase gene derived from Rhizobium sp. (12469-xho-r) SEQ
ID NO:56: Forward primer which is designed based on the genomic DNA
sequence from Corynebacterium ammonia genes DSM20306 (Co-d50-r) SEQ
ID NO:57: Reverse primer which is designed based on a homologus
region between the genomic DNA sequences corresponding to the
aspartate aminotransferases from Corynebacterium striatum ATCC6940
(ZP.sub.--03935516) and from Corynebacterium ammonia genes DSM20306
SEQ ID NO:58: Forward primer for amplifying DNA fragment containing
aminotransferase gene derived from Corynebacterium ammoniagenes
(Co-890-r) SEQ ID NO:59: Reverse primer for amplifying DNA fragment
containing aminotransferase gene derived from Corynebacterium
ammoniagenes (Co-1060-r) SEQ ID NO:60: Nucleotide sequence of
aminotransferase gene derived from Corynebacterium ammoniagenes SEQ
ID NO:61: Amino acid sequence of aminotransferase derived from
Corynebacterium ammoniagenes SEQ ID NO:62: Forward primer for
amplifying DNA fragment containing aminotransferase gene derived
from Corynebacterium ammoniagenes (1444AT-Nde-f) SEQ ID NO:63:
Reverse primer for amplifying DNA fragment containing
aminotransferase gene derived from Corynebacterium ammoniagenes
(1444-xho-r) SEQ ID NO:64: Nucleotide sequence of aminotransferase
gene derived from Deinococcus geothermalis SEQ ID NO:65: Amino acid
sequence of aminotransferase derived from Deinococcus geothermalis
SEQ ID NO:66: Nucleotide sequence of aminotransferase gene derived
from Corynebacterium glutamicum SEQ ID NO:67: Amino acid sequence
of aminotransferase derived from Corynebacterium glutamicum SEQ ID
NO:68: Nucleotide sequence of aminotransferase gene derived from
Thermus thermophilus SEQ ID NO:69: Amino acid sequence of
aminotransferase derived from Thermus thermophilus SEQ ID NO:70:
Nucleotide sequence of aminotransferase gene derived from
Thermotoga maritima SEQ ID NO:71: Amino acid sequence of
aminotransferase derived from Thermotoga maritima SEQ ID NO:72:
Nucleotide sequence of aminotransferase gene derived from
Pyrococcus horikoshii SEQ ID NO:73: Amino acid sequence of
aminotransferase derived from Pyrococcus horikoshii SEQ ID NO:74:
Nucleotide sequence of aminotransferase gene derived from
Phormidium lapideum SEQ ID NO:75: Amino acid sequence of
aminotransferase derived from Phormidium lapideum SEQ ID NO:76:
Nucleotide sequence of aminotransferase gene derived from Thermos
thermophilus SEQ ID NO:77: Amino acid sequence of aminotransferase
derived from Thermos thermophilus SEQ ID NO:78: Nucleotide sequence
of aminotransferase gene derived from Pyrococcus horikoshii SEQ ID
NO:79: Amino acid sequence of aminotransferase derived from
Pyrococcus horikoshii SEQ ID NO:80: Nucleotide sequence of
aminotransferase gene derived from Methanococcus jannaschii SEQ ID
NO:81: Amino acid sequence of aminotransferase derived from
Methanococcus jannaschii SEQ ID NO:82: Nucleotide sequence of
aminotransferase gene derived from Thermotoga maritima SEQ ID
NO:83: Amino acid sequence of aminotransferase derived from
Thermotoga maritima SEQ ID NO:84: Nucleotide sequence of
aminotransferase gene derived from Saccharomyces cerevisiae SEQ ID
NO:85: Amino acid sequence of aminotransferase derived from
Saccharomyces cerevisiae SEQ ID NO:86: Nucleotide sequence of
aminotransferase gene derived from Eubacterium rectale SEQ ID
NO:87: Amino acid sequence of aminotransferase derived from
Eubacterium rectale SEQ ID NO:88: Nucleotide sequence of
aminotransferase gene derived from Bacillus pumilus SEQ ID NO:89:
Amino acid sequence of aminotransferase derived from Bacillus
pumilus SEQ ID NO:90: Nucleotide sequence of aminotransferase gene
derived from Bacillus cellulosilyticus SEQ ID NO:91: Amino acid
sequence of aminotransferase derived from Bacillus cellulosilyticus
SEQ ID NO:92: Nucleotide sequence of aminotransferase gene derived
from Bacillus sp. SEQ ID NO:93: Amino acid sequence of
aminotransferase derived from Bacillus sp. SEQ ID NO:94: Nucleotide
sequence of aminotransferase gene derived from Sinorhizobium
meliloti SEQ ID NO:95: Amino acid sequence of aminotransferase
derived from Sinorhizobium meliloti SEQ ID NO:96: Nucleotide
sequence of aminotransferase gene derived from Methanothermobacter
thermautotrophicus SEQ ID NO:97: Amino acid sequence of
aminotransferase derived from Methanothermobacter
thermautotrophicus SEQ ID NO:98: Nucleotide sequence of
aminotransferase gene derived from Lactobacillus acidophilus SEQ ID
NO:99: Amino acid sequence of aminotransferase derived from
Lactobacillus acidophilus SEQ ID NO:100: Nucleotide sequence of
aminotransferase gene derived from Sinorhizobium meliloti SEQ ID
NO:101: Amino acid sequence of aminotransferase derived from
Sinorhizobium meliloti SEQ ID NO:102: Nucleotide sequence of
aminotransferase gene derived from Pyrococcus horikoshii SEQ ID
NO:103: Amino acid sequence of aminotransferase derived from
Pyrococcus horikoshii SEQ ID NO:104: Nucleotide sequence of
aminotransferase gene derived from Thermoanaerobacter tengcongensis
SEQ ID NO:105: Amino acid sequence of aminotransferase derived from
Thermoanaerobacter tengcongensis SEQ ID NO:106: Nucleotide sequence
of aminotransferase gene derived from Clostridium cellulolyticum
SEQ ID NO:107: Amino acid sequence of aminotransferase derived from
Clostridium cellulolyticum SEQ ID NO:108: Nucleotide sequence of
aminotransferase gene derived from Rhodococcus erythropolis SEQ ID
NO:109: Amino acid sequence of aminotransferase derived from
Rhodococcus erythropolis SEQ ID NO:110: Nucleotide sequence of
aminotransferase gene derived from Saccharophagus degradans SEQ ID
NO:111: Amino acid sequence of aminotransferase derived from
Saccharophagus degradans SEQ ID NO:112: Amino acid sequence of a
fragment of aminotransferase derived from Corynebacterium
ammoniagenes
Sequence CWU 1
1
11211308DNABacillus altitudinisCDS(1)..(1308) 1atg agc ggt ttt aca
gcg tta agt gaa gca gaa tta aat gac cta tat 48Met Ser Gly Phe Thr
Ala Leu Ser Glu Ala Glu Leu Asn Asp Leu Tyr 1 5 10 15 gca gca cga
caa aaa gag tat gaa acg tac aaa agt aaa aac tta cac 96Ala Ala Arg
Gln Lys Glu Tyr Glu Thr Tyr Lys Ser Lys Asn Leu His 20 25 30 tta
gac atg tct aga ggg aaa cct tca cca aaa cag ctc gat tta tct 144Leu
Asp Met Ser Arg Gly Lys Pro Ser Pro Lys Gln Leu Asp Leu Ser 35 40
45 atg ggc atg ctc gat gtc gtg aca tca aag gat gca atg aca gct gag
192Met Gly Met Leu Asp Val Val Thr Ser Lys Asp Ala Met Thr Ala Glu
50 55 60 gat ggt aca gat gtg cga aac tat ggc ggc ttg aca ggc ctt
cct gaa 240Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Thr Gly Leu
Pro Glu 65 70 75 80 aca aag aaa ttt ttt gca agt gtg ctc aat ctg aag
cca gaa caa atc 288Thr Lys Lys Phe Phe Ala Ser Val Leu Asn Leu Lys
Pro Glu Gln Ile 85 90 95 atc att ggc ggt aat tct agc cta aat atg
atg cat gac aca att gcc 336Ile Ile Gly Gly Asn Ser Ser Leu Asn Met
Met His Asp Thr Ile Ala 100 105 110 cgt gct atg act cac ggc gta tat
ggc agc aaa aca cct tgg gga gag 384Arg Ala Met Thr His Gly Val Tyr
Gly Ser Lys Thr Pro Trp Gly Glu 115 120 125 ctt cca aag gta aaa ttc
ctt gca cca agc cca ggg tat gat cgt cat 432Leu Pro Lys Val Lys Phe
Leu Ala Pro Ser Pro Gly Tyr Asp Arg His 130 135 140 ttt gcc att tgt
gag cat ttt aac ata gag atg att acg gta gat atg 480Phe Ala Ile Cys
Glu His Phe Asn Ile Glu Met Ile Thr Val Asp Met 145 150 155 160 aag
tcg gat gga cct gac atg gat cag gtg gaa aaa ttg gtt gca gaa 528Lys
Ser Asp Gly Pro Asp Met Asp Gln Val Glu Lys Leu Val Ala Glu 165 170
175 gat gaa gcc atc aaa ggg att tgg tgt gta cca aaa tat agc aac cct
576Asp Glu Ala Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro
180 185 190 gac ggc att acg tat tca gat gag gtt gtc gac cgt ctt gct
tcc atg 624Asp Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Leu Ala
Ser Met 195 200 205 cag aca aaa gca gac gac ttc cgt att ttt tgg gat
gat gcc tat gca 672Gln Thr Lys Ala Asp Asp Phe Arg Ile Phe Trp Asp
Asp Ala Tyr Ala 210 215 220 gtc cac cat cta aca gat acg cct gat acg
tta aaa gat att ttt caa 720Val His His Leu Thr Asp Thr Pro Asp Thr
Leu Lys Asp Ile Phe Gln 225 230 235 240 gca gta gac aaa gca ggg cat
gca aac cgt gtg ttt atg ttc gcc tct 768Ala Val Asp Lys Ala Gly His
Ala Asn Arg Val Phe Met Phe Ala Ser 245 250 255 act tct aaa att acg
ttc cca ggc tca ggt gtt gca ctg atg gca tct 816Thr Ser Lys Ile Thr
Phe Pro Gly Ser Gly Val Ala Leu Met Ala Ser 260 265 270 agt cag gac
aac gtc agc ttt att caa aaa cag cta tca gtt caa acc 864Ser Gln Asp
Asn Val Ser Phe Ile Gln Lys Gln Leu Ser Val Gln Thr 275 280 285 att
ggg cca gat aaa atc aat caa tta aga cac ctt cgt ttc ttc aag 912Ile
Gly Pro Asp Lys Ile Asn Gln Leu Arg His Leu Arg Phe Phe Lys 290 295
300 aat cca gaa gga ttg aag gaa cat atg aaa aag cat gca gcg att att
960Asn Pro Glu Gly Leu Lys Glu His Met Lys Lys His Ala Ala Ile Ile
305 310 315 320 aag ccg aaa ttt gac ctc gtt ctt tcg atc ctt gat gaa
aag ctt ggt 1008Lys Pro Lys Phe Asp Leu Val Leu Ser Ile Leu Asp Glu
Lys Leu Gly 325 330 335 gga aca ggc atc gct gag tgg cac aaa cca aat
ggc gga tat ttt att 1056Gly Thr Gly Ile Ala Glu Trp His Lys Pro Asn
Gly Gly Tyr Phe Ile 340 345 350 agc tta aat aca ctc gat cat tgt gca
aaa gct gtt gtg caa aaa gcg 1104Ser Leu Asn Thr Leu Asp His Cys Ala
Lys Ala Val Val Gln Lys Ala 355 360 365 aaa gaa gcc ggt gtg aca cta
aca ggt gca ggg gcg aca tat cct tat 1152Lys Glu Ala Gly Val Thr Leu
Thr Gly Ala Gly Ala Thr Tyr Pro Tyr 370 375 380 gga aac gac ccg ctt
gat cgt aac atc cgt att gcg cca acg ttc cca 1200Gly Asn Asp Pro Leu
Asp Arg Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390 395 400 acg ctt
gaa gaa cta gag cag gcg att gat atc ttt acg tta tgc gtt 1248Thr Leu
Glu Glu Leu Glu Gln Ala Ile Asp Ile Phe Thr Leu Cys Val 405 410 415
cag ctt gtc agc att gaa aag ctg ctg tct gag aaa agt caa tca gca
1296Gln Leu Val Ser Ile Glu Lys Leu Leu Ser Glu Lys Ser Gln Ser Ala
420 425 430 cca acg gta taa 1308Pro Thr Val 435 2435PRTBacillus
altitudinis 2Met Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu Leu Asn
Asp Leu Tyr 1 5 10 15 Ala Ala Arg Gln Lys Glu Tyr Glu Thr Tyr Lys
Ser Lys Asn Leu His 20 25 30 Leu Asp Met Ser Arg Gly Lys Pro Ser
Pro Lys Gln Leu Asp Leu Ser 35 40 45 Met Gly Met Leu Asp Val Val
Thr Ser Lys Asp Ala Met Thr Ala Glu 50 55 60 Asp Gly Thr Asp Val
Arg Asn Tyr Gly Gly Leu Thr Gly Leu Pro Glu 65 70 75 80 Thr Lys Lys
Phe Phe Ala Ser Val Leu Asn Leu Lys Pro Glu Gln Ile 85 90 95 Ile
Ile Gly Gly Asn Ser Ser Leu Asn Met Met His Asp Thr Ile Ala 100 105
110 Arg Ala Met Thr His Gly Val Tyr Gly Ser Lys Thr Pro Trp Gly Glu
115 120 125 Leu Pro Lys Val Lys Phe Leu Ala Pro Ser Pro Gly Tyr Asp
Arg His 130 135 140 Phe Ala Ile Cys Glu His Phe Asn Ile Glu Met Ile
Thr Val Asp Met 145 150 155 160 Lys Ser Asp Gly Pro Asp Met Asp Gln
Val Glu Lys Leu Val Ala Glu 165 170 175 Asp Glu Ala Ile Lys Gly Ile
Trp Cys Val Pro Lys Tyr Ser Asn Pro 180 185 190 Asp Gly Ile Thr Tyr
Ser Asp Glu Val Val Asp Arg Leu Ala Ser Met 195 200 205 Gln Thr Lys
Ala Asp Asp Phe Arg Ile Phe Trp Asp Asp Ala Tyr Ala 210 215 220 Val
His His Leu Thr Asp Thr Pro Asp Thr Leu Lys Asp Ile Phe Gln 225 230
235 240 Ala Val Asp Lys Ala Gly His Ala Asn Arg Val Phe Met Phe Ala
Ser 245 250 255 Thr Ser Lys Ile Thr Phe Pro Gly Ser Gly Val Ala Leu
Met Ala Ser 260 265 270 Ser Gln Asp Asn Val Ser Phe Ile Gln Lys Gln
Leu Ser Val Gln Thr 275 280 285 Ile Gly Pro Asp Lys Ile Asn Gln Leu
Arg His Leu Arg Phe Phe Lys 290 295 300 Asn Pro Glu Gly Leu Lys Glu
His Met Lys Lys His Ala Ala Ile Ile 305 310 315 320 Lys Pro Lys Phe
Asp Leu Val Leu Ser Ile Leu Asp Glu Lys Leu Gly 325 330 335 Gly Thr
Gly Ile Ala Glu Trp His Lys Pro Asn Gly Gly Tyr Phe Ile 340 345 350
Ser Leu Asn Thr Leu Asp His Cys Ala Lys Ala Val Val Gln Lys Ala 355
360 365 Lys Glu Ala Gly Val Thr Leu Thr Gly Ala Gly Ala Thr Tyr Pro
Tyr 370 375 380 Gly Asn Asp Pro Leu Asp Arg Asn Ile Arg Ile Ala Pro
Thr Phe Pro 385 390 395 400 Thr Leu Glu Glu Leu Glu Gln Ala Ile Asp
Ile Phe Thr Leu Cys Val 405 410 415 Gln Leu Val Ser Ile Glu Lys Leu
Leu Ser Glu Lys Ser Gln Ser Ala 420 425 430 Pro Thr Val 435 3
1678DNABacillus altitudinis 3gaaacaatta ctcaaagaag cccgcgatat
attcccgctg actgaagctg cacatgattt 60cttttctttt cctattgacc gtaccctgta
aaaattggat gaatcgtcaa aaatagttgt 120gtgatttttt tgatatttat
gatgacgctc tttttcaggt agtggtaaaa tggtgaagaa 180aaaacaaatg
acgaattaca ctatgaagaa tacgggaggc acaatcaaag atgagcggtt
240ttacagcgtt aagtgaagca gaattaaatg acctatatgc agcacgacaa
aaagagtatg 300aaacgtacaa aagtaaaaac ttacacttag acatgtctag
agggaaacct tcaccaaaac 360agctcgattt atctatgggc atgctcgatg
tcgtgacatc aaaggatgca atgacagctg 420aggatggtac agatgtgcga
aactatggcg gcttgacagg ccttcctgaa acaaagaaat 480tttttgcaag
tgtgctcaat ctgaagccag aacaaatcat cattggcggt aattctagcc
540taaatatgat gcatgacaca attgcccgtg ctatgactca cggcgtatat
ggcagcaaaa 600caccttgggg agagcttcca aaggtaaaat tccttgcacc
aagcccaggg tatgatcgtc 660attttgccat ttgtgagcat tttaacatag
agatgattac ggtagatatg aagtcggatg 720gacctgacat ggatcaggtg
gaaaaattgg ttgcagaaga tgaagccatc aaagggattt 780ggtgtgtacc
aaaatatagc aaccctgacg gcattacgta ttcagatgag gttgtcgacc
840gtcttgcttc catgcagaca aaagcagacg acttccgtat tttttgggat
gatgcctatg 900cagtccacca tctaacagat acgcctgata cgttaaaaga
tatttttcaa gcagtagaca 960aagcagggca tgcaaaccgt gtgtttatgt
tcgcctctac ttctaaaatt acgttcccag 1020gctcaggtgt tgcactgatg
gcatctagtc aggacaacgt cagctttatt caaaaacagc 1080tatcagttca
aaccattggg ccagataaaa tcaatcaatt aagacacctt cgtttcttca
1140agaatccaga aggattgaag gaacatatga aaaagcatgc agcgattatt
aagccgaaat 1200ttgacctcgt tctttcgatc cttgatgaaa agcttggtgg
aacaggcatc gctgagtggc 1260acaaaccaaa tggcggatat tttattagct
taaatacact cgatcattgt gcaaaagctg 1320ttgtgcaaaa agcgaaagaa
gccggtgtga cactaacagg tgcaggggcg acatatcctt 1380atggaaacga
cccgcttgat cgtaacatcc gtattgcgcc aacgttccca acgcttgaag
1440aactagagca ggcgattgat atctttacgt tatgcgttca gcttgtcagc
attgaaaagc 1500tgctgtctga gaaaagtcaa tcagcaccaa cggtataacg
aaaaaactcc ttgactgatg 1560tccggtcaag gagtttttgt ttttagttag
ctgtttgata ataagtggca ggctgttttg 1620ctgcacacca tacatcataa
atcgcaagct gcacaatatg tggttcatcg tgatgaat 1678415PRTBacillus
altitudinis 4Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu Leu Asn Asp
Leu Tyr 1 5 10 15 512PRTBacillus altitudinis 5Gln Leu Asp Leu Ser
Met Gly Met Leu Asp Val Val 1 5 10 630DNAArtificial SequenceForward
primer for amplifying DNA fragment containing aminotransferase gene
derived from Bacillus altitudinis (Bp-u200-f) 6ctcaggaagc
aggcgcaaaa agattaattt 30730DNAArtificial SequenceReverse primer for
amplifying DNA fragment containing aminotransferase gene derived
from Bacillus altitudinis (Bp-d200-r) 7ggatgctgtc tttgtcatcc
caaagtggat 30830DNAArtificial SequenceForward primer for amplifying
DNA fragment containing aminotransferase gene derived from Bacillus
altitudinis (1616AT-Nde-f) 8ggaattccat atgagcggtt ttacagcgtt
30940DNAArtificial SequenceReverse primer for amplifying DNA
fragment containing aminotransferase gene derived from Bacillus
altitudinis (1616-xho-r) 9gtcaaggagt ttttctcgag taccgttggt
gctgattgac 401029DNAArtificial SequenceForward primer for
converting DNA sequence recognized by NdeI, which is found on
aminotransferase gene derived from Bacillus altitudinis
(1616-delNde-f) 10ggattgaagg aacacatgaa aaagcatgc
291129DNAArtificial SequenceReverse primer for converting DNA
sequence recognized by NdeI, which is found on aminotransferase
gene derived from Bacillus altitudinis (1616-delNde-r) 11gcatgctttt
tcatgtgttc cttcaatcc 291230DNAArtificial SequenceForward primer for
amplifying DNA fragment containing SpAld gene (SpAld-f-NdeI)
12ggaattccat atgacccaga cgcgcctcaa 301330DNAArtificial
SequenceReverse primer for amplifying DNA fragment containing SpAld
gene (SpAld-r-HindIII) 13gcccaagctt tcagtacccc gccagttcgc
301429DNAArtificial SequenceForward primer for converting rare
codon 6L in SpAld gene (6L-f) 14acccagacgc gcctgaacgg catcatccg
291529DNAArtificial SequenceReverse primer for converting rare
codon 6L in SpAld gene (6L-r) 15cggatgatgc cgttcaggcg cgtctgggt
291629DNAArtificial SequenceForward primer for converting rare
codon 13L in SpAld gene (13L-f) 16atcatccgcg ctctggaagc cggcaagcc
291729DNAArtificial SequenceReverse primer for converting rare
codon 13L in SpAld gene (13L-r) 17ggcttgccgg cttccagagc gcggatgat
291829DNAArtificial SequenceForward primer for converting rare
codon 18P in SpAld gene (18P-f) 18gaagccggca agccggcttt cacctgctt
291929DNAArtificial SequenceReverse primer for converting rare
codon 18P in SpAld gene (18P-r) 19aagcaggtga aagccggctt gccggcttc
292029DNAArtificial SequenceForward primer for converting rare
codon 38P in SpAld gene (38P-f) 20ctgaccgatg ccccgtatga cggcgtggt
292129DNAArtificial SequenceReverse primer for converting rare
codon 38P in SpAld gene (38P-r) 21accacgccgt catacggggc atcggtcag
292229DNAArtificial SequenceForward primer for converting rare
codon 50P in SpAld gene (50P-f) 22atggagcaca acccgtacga tgtcgcggc
292329DNAArtificial SequenceReverse primer for converting rare
codon 50P in SpAld gene (50P-r) 23gccgcgacat cgtacgggtt gtgctccat
292442DNAArtificial SequenceForward primer for converting rare
codons 77P, 81P and 84R in SpAld gene (77P-81P-84R-f) 24cggtcgcgcc
gtcggtcacc ccgatcgcgc gcatcccggc ca 422542DNAArtificial
SequenceReverse primer for converting rare codons 77P, 81P and 84R
in SpAld gene (77P-81P-84R-r) 25tggccgggat gcgcgcgatc ggggtgaccg
acggcgcgac cg 422633DNAArtificial SequenceForward primer for
preparing the aminotransferase mutant K39R derived from Bacillus
altitudinis AJ1616 (K39R_FW) 26gacatgtcta gagggcgtcc ttcaccaaaa cag
332733DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant K39R derived from Bacillus altitudinis
AJ1616 (K39R_RV) 27ctgttttggt gaaggacgcc ctctagacat gtc
332830DNAArtificial SequenceForward primer for preparing the
aminotransferase mutant S258G derived from Bacillus altitudinis
AJ1616 (S258G_FW) 28gttcgcctct actggtaaaa ttacgttccc
302930DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant S258G derived from Bacillus altitudinis
AJ1616 (S258G_RV) 29gggaacgtaa ttttaccagt agaggcgaac
303036DNAArtificial SequenceForward primer for preparing the
aminotransferase mutant T288G derived from Bacillus altitudinis
AJ1616 (T288G_FW) 30cagctatcag ttcaaggcat tgggccagat aaaatc
363136DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant T288G derived from Bacillus altitudinis
AJ1616 (T288G_RV) 31gattttatct ggcccaatgc cttgaactga tagctg
363233DNAArtificial SequenceForward primer for preparing the
aminotransferase mutant I289A derived from Bacillus altitudinis
AJ1616 (I289A_FW) 32ctatcagttc aaaccgctgg gccagataaa atc
333333DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant I289A derived from Bacillus altitudinis
AJ1616 (I289A_RV) 33gattttatct ggcccagcgg tttgaactga tag
333428DNAArtificial SequenceForward primer for preparing the
aminotransferase mutant Q287E/T288G derived from Bacillus
altitudinis AJ1616 (Q287E/T288G_FW) 34cagctatcag ttgaaggcat
tgggccag 283528DNAArtificial SequenceReverse primer for preparing
the
aminotransferase mutant Q287E/T288G derived from Bacillus
altitudinis AJ1616 (Q287E/T288G_RV) 35ctggcccaat gccttcaact
gatagctg 283660DNAArtificial SequencePrimer for preparing a DNA
fragment for destroying aspC gene (aspC-L1) 36tttgagaaca ttaccgccgc
tcctgccgac ccgattctgg gctgaagcct gcttttttat 603760DNAArtificial
SequencePrimer for preparing a DNA fragment for destroying aspC
gene (aspC-R1) 37cagcactgcc acaatcgctt cgcacagcgg agccatgtta
tccgctcaag ttagtataaa 603830DNAArtificial SequencePrimer for
confirming the insertion of attL-cat-attR in the region of aspC
gene (aspC-up) 38aacctcttgg caacggtaaa aaagctgaac
303918DNAArtificial SequencePrimer for confirming the insertion of
attL-cat-attR in the region of aspC gene (attL-1) 39tagtgacctg
ttcgttgc 184030DNAArtificial SequencePrimer for confirming the
insertion of attL-cat-attR in the region of aspC gene (aspC-down)
40gcctgcgcaa agtcgtatgt ttggtctgga 304118DNAArtificial
SequencePrimer for confirming the insertion of attL-cat-attR in the
region of aspC gene (attR-1) 41ttacgtttct cgttcagc
1842882DNAPseudomonas putidaCDS(1)..(882) 42atg att atg ccg aaa gcc
tcc cat cag gat ctg cgt ttt gcg ttc cgt 48Met Ile Met Pro Lys Ala
Ser His Gln Asp Leu Arg Phe Ala Phe Arg 1 5 10 15 gaa ctg ctg gcc
tct ggt agc tgt ttc cac acc gcg tca gtt ttt gat 96Glu Leu Leu Ala
Ser Gly Ser Cys Phe His Thr Ala Ser Val Phe Asp 20 25 30 ccg atg
agc gca cgt att gcg gcc gac ctg ggc ttc gaa gtc ggt atc 144Pro Met
Ser Ala Arg Ile Ala Ala Asp Leu Gly Phe Glu Val Gly Ile 35 40 45
ctg ggc ggt agt gtc gcg tcc ctg caa gtg ctg gca gct ccg gat ttt
192Leu Gly Gly Ser Val Ala Ser Leu Gln Val Leu Ala Ala Pro Asp Phe
50 55 60 gcc ctg att acg ctg tct gaa ttc gtg gaa cag gca acc cgt
atc ggt 240Ala Leu Ile Thr Leu Ser Glu Phe Val Glu Gln Ala Thr Arg
Ile Gly 65 70 75 80 cgt gtt gct caa ctg ccg gtc ctg gca gat gca gac
cat ggt tat ggt 288Arg Val Ala Gln Leu Pro Val Leu Ala Asp Ala Asp
His Gly Tyr Gly 85 90 95 aac gca ctg aat gtt atg cgt acc gtc att
gaa ctg gaa cgt gct ggt 336Asn Ala Leu Asn Val Met Arg Thr Val Ile
Glu Leu Glu Arg Ala Gly 100 105 110 gtg gca gca ctg acc atc gaa gat
acg ctg ctg ccg gcg cag ttt ggt 384Val Ala Ala Leu Thr Ile Glu Asp
Thr Leu Leu Pro Ala Gln Phe Gly 115 120 125 cgc aaa agt acc gac ctg
att ccg gtg gaa gaa ggc gtt ggt aaa atc 432Arg Lys Ser Thr Asp Leu
Ile Pro Val Glu Glu Gly Val Gly Lys Ile 130 135 140 cgt gca gct ctg
gaa gcc cgc gtt gat agc tct ctg tcc att atc gcg 480Arg Ala Ala Leu
Glu Ala Arg Val Asp Ser Ser Leu Ser Ile Ile Ala 145 150 155 160 cgt
acc aac gcc ggt gtc ctg agc acg gaa gaa att atc gtg cgc acc 528Arg
Thr Asn Ala Gly Val Leu Ser Thr Glu Glu Ile Ile Val Arg Thr 165 170
175 cag tct tat caa aaa gca ggc gct gat ggt att tgc atg gtc ggc gtg
576Gln Ser Tyr Gln Lys Ala Gly Ala Asp Gly Ile Cys Met Val Gly Val
180 185 190 aaa gac ttt gaa cag ctg gaa caa atc gcg gaa cat ctg acg
gtg ccg 624Lys Asp Phe Glu Gln Leu Glu Gln Ile Ala Glu His Leu Thr
Val Pro 195 200 205 ctg atg ctg gtt acc tac ggt aac ccg aat ctg cgt
gat gac gaa cgt 672Leu Met Leu Val Thr Tyr Gly Asn Pro Asn Leu Arg
Asp Asp Glu Arg 210 215 220 ctg gca cgt ctg ggt gtt cgt att gtg gtt
gat ggt cac gcg gcc tat 720Leu Ala Arg Leu Gly Val Arg Ile Val Val
Asp Gly His Ala Ala Tyr 225 230 235 240 ttc gca gct atc aaa gcc acg
tac gac tgt ctg cgt ctg caa cgt ggc 768Phe Ala Ala Ile Lys Ala Thr
Tyr Asp Cys Leu Arg Leu Gln Arg Gly 245 250 255 cgc caa aac aaa tca
gaa aat ctg tcg gca acc gaa ctg agc cac acc 816Arg Gln Asn Lys Ser
Glu Asn Leu Ser Ala Thr Glu Leu Ser His Thr 260 265 270 tac acc cag
ccg gaa gac tac att cgt tgg gca aaa gaa tac atg agc 864Tyr Thr Gln
Pro Glu Asp Tyr Ile Arg Trp Ala Lys Glu Tyr Met Ser 275 280 285 gtt
gaa gaa ctc gag tga 882Val Glu Glu Leu Glu 290 43293PRTPseudomonas
putida 43Met Ile Met Pro Lys Ala Ser His Gln Asp Leu Arg Phe Ala
Phe Arg 1 5 10 15 Glu Leu Leu Ala Ser Gly Ser Cys Phe His Thr Ala
Ser Val Phe Asp 20 25 30 Pro Met Ser Ala Arg Ile Ala Ala Asp Leu
Gly Phe Glu Val Gly Ile 35 40 45 Leu Gly Gly Ser Val Ala Ser Leu
Gln Val Leu Ala Ala Pro Asp Phe 50 55 60 Ala Leu Ile Thr Leu Ser
Glu Phe Val Glu Gln Ala Thr Arg Ile Gly 65 70 75 80 Arg Val Ala Gln
Leu Pro Val Leu Ala Asp Ala Asp His Gly Tyr Gly 85 90 95 Asn Ala
Leu Asn Val Met Arg Thr Val Ile Glu Leu Glu Arg Ala Gly 100 105 110
Val Ala Ala Leu Thr Ile Glu Asp Thr Leu Leu Pro Ala Gln Phe Gly 115
120 125 Arg Lys Ser Thr Asp Leu Ile Pro Val Glu Glu Gly Val Gly Lys
Ile 130 135 140 Arg Ala Ala Leu Glu Ala Arg Val Asp Ser Ser Leu Ser
Ile Ile Ala 145 150 155 160 Arg Thr Asn Ala Gly Val Leu Ser Thr Glu
Glu Ile Ile Val Arg Thr 165 170 175 Gln Ser Tyr Gln Lys Ala Gly Ala
Asp Gly Ile Cys Met Val Gly Val 180 185 190 Lys Asp Phe Glu Gln Leu
Glu Gln Ile Ala Glu His Leu Thr Val Pro 195 200 205 Leu Met Leu Val
Thr Tyr Gly Asn Pro Asn Leu Arg Asp Asp Glu Arg 210 215 220 Leu Ala
Arg Leu Gly Val Arg Ile Val Val Asp Gly His Ala Ala Tyr 225 230 235
240 Phe Ala Ala Ile Lys Ala Thr Tyr Asp Cys Leu Arg Leu Gln Arg Gly
245 250 255 Arg Gln Asn Lys Ser Glu Asn Leu Ser Ala Thr Glu Leu Ser
His Thr 260 265 270 Tyr Thr Gln Pro Glu Asp Tyr Ile Arg Trp Ala Lys
Glu Tyr Met Ser 275 280 285 Val Glu Glu Leu Glu 290
4420PRTRhizobium radiobacter 44Ala Phe Leu Ala Asp Ile Leu Ser Arg
Val Lys Pro Ser Ala Thr Ile 1 5 10 15 Ala Val Thr Gln 20
4527DNAArtificial SequenceForward primer which is designed based on
the genomic DNA sequence from Agrobacterium tumefaciens str. C58
(Ag-u100-f) 45ctggtgcaga taagccggct tttgacc 274627DNAArtificial
SequenceReverse primer which is designed based on the genomic DNA
sequence from Agrobacterium tumefaciens str. C58 (Ag-d100-r)
46ccaccttcat catgctgctg tttctcg 27471203DNARhizobium
radiobacterCDS(1)..(1203) 47atg gcc ttc ctt gcc gac att ctc tcc cgc
gta aag cca tcc gcc acc 48Met Ala Phe Leu Ala Asp Ile Leu Ser Arg
Val Lys Pro Ser Ala Thr 1 5 10 15 atc gcc gtt acc cag aaa gcc cgt
gag ctg aaa gcg aag ggc cgc gat 96Ile Ala Val Thr Gln Lys Ala Arg
Glu Leu Lys Ala Lys Gly Arg Asp 20 25 30 gtg atc agc ctt ggc gcc
ggc gag ccg gat ttc gat acg ccc gat aat 144Val Ile Ser Leu Gly Ala
Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35 40 45 atc aag gaa gcg
gcc atc gac gcc atc aag cgc ggc gaa acg aaa tac 192Ile Lys Glu Ala
Ala Ile Asp Ala Ile Lys Arg Gly Glu Thr Lys Tyr 50 55 60 acg ccc
gtt tcc ggc att ccg gaa ctg cgc aag gcc att gct gac aag 240Thr Pro
Val Ser Gly Ile Pro Glu Leu Arg Lys Ala Ile Ala Asp Lys 65 70 75 80
ttc aag cgc gaa aac ggc ctc gac tac aag ccg gag cag acg att gtc
288Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro Glu Gln Thr Ile Val
85 90 95 ggc acc ggc ggc aag cag ata ctt ttc aac gcc ttc atg gcc
acc ctc 336Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe Met Ala
Thr Leu 100 105 110 aac ccg ggt gac gaa gtc gtc att ccc gcg cct tac
tgg gtc agc tac 384Asn Pro Gly Asp Glu Val Val Ile Pro Ala Pro Tyr
Trp Val Ser Tyr 115 120 125 ccg gaa atg gtg gcg atc tgc ggc ggc acg
cct gta ttc gtc gac acc 432Pro Glu Met Val Ala Ile Cys Gly Gly Thr
Pro Val Phe Val Asp Thr 130 135 140 acg ctt gaa gac aat ttc aag ctg
acg ccg gaa gcg ctg gaa aag gcg 480Thr Leu Glu Asp Asn Phe Lys Leu
Thr Pro Glu Ala Leu Glu Lys Ala 145 150 155 160 atc aca ccg aag aca
aag tgg ttc gtc ttc aac tcg cct tca aac ccc 528Ile Thr Pro Lys Thr
Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165 170 175 tcg ggt gcc
gcc tat tcg cat gac gaa ctg aag gcg ctg acg gac gtg 576Ser Gly Ala
Ala Tyr Ser His Asp Glu Leu Lys Ala Leu Thr Asp Val 180 185 190 ctg
gtc aag cat ccg caa gtc tgg gtg ctg acg gac gac atg tac gag 624Leu
Val Lys His Pro Gln Val Trp Val Leu Thr Asp Asp Met Tyr Glu 195 200
205 cac ctc acc tat ggc gat ttc aaa ttc gtc acc ccg gtt gag gtt gag
672His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr Pro Val Glu Val Glu
210 215 220 cct gcg ctc tat gat cgc acg ctg acg atg aac ggc gtc tcc
aag gcc 720Pro Ala Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly Val Ser
Lys Ala 225 230 235 240 tat gcc atg acc ggc tgg cgt atc ggt tac gcg
gcc ggc ccg ctg ccg 768Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala
Ala Gly Pro Leu Pro 245 250 255 ctg atc aag gcc atg gac atg atc cag
ggc cag cag acc tcg ggc gcc 816Leu Ile Lys Ala Met Asp Met Ile Gln
Gly Gln Gln Thr Ser Gly Ala 260 265 270 agc tcg atc gcg caa tgg gcg
gct gtt gaa gcg ctg aac ggc acg cag 864Ser Ser Ile Ala Gln Trp Ala
Ala Val Glu Ala Leu Asn Gly Thr Gln 275 280 285 gat ttc att ccg acc
aac aag aaa atc ttc gaa ggt cgc cgt gat ctc 912Asp Phe Ile Pro Thr
Asn Lys Lys Ile Phe Glu Gly Arg Arg Asp Leu 290 295 300 gtc gtc tcc
atg ctc aac cag gcc aag ggc atc aat tgc ccg tca ccg 960Val Val Ser
Met Leu Asn Gln Ala Lys Gly Ile Asn Cys Pro Ser Pro 305 310 315 320
gaa ggc gca ttc tac gtc tac ccg tcc tgc gcc ggc ctg att ggc aag
1008Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys
325 330 335 acc gcg cca tct ggc aag gtc atc gag tcg gat gtg gac ttc
gtc tcc 1056Thr Ala Pro Ser Gly Lys Val Ile Glu Ser Asp Val Asp Phe
Val Ser 340 345 350 gag ctt ctg gaa gcc gaa ggc gtc gcc gtc gtg cag
gga tcg gct ttc 1104Glu Leu Leu Glu Ala Glu Gly Val Ala Val Val Gln
Gly Ser Ala Phe 355 360 365 ggc ctc ggc ccg aac ttc cgc att tcc tac
gcc acg tcg gaa agc ctg 1152Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr
Ala Thr Ser Glu Ser Leu 370 375 380 ctg gaa gaa gcc tgc aag cgc att
cag cgt ttc tgc gcc gat tgc cgt 1200Leu Glu Glu Ala Cys Lys Arg Ile
Gln Arg Phe Cys Ala Asp Cys Arg 385 390 395 400 tga
120348400PRTRhizobium radiobacter 48Met Ala Phe Leu Ala Asp Ile Leu
Ser Arg Val Lys Pro Ser Ala Thr 1 5 10 15 Ile Ala Val Thr Gln Lys
Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp 20 25 30 Val Ile Ser Leu
Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35 40 45 Ile Lys
Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly Glu Thr Lys Tyr 50 55 60
Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Lys Ala Ile Ala Asp Lys 65
70 75 80 Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro Glu Gln Thr
Ile Val 85 90 95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe
Met Ala Thr Leu 100 105 110 Asn Pro Gly Asp Glu Val Val Ile Pro Ala
Pro Tyr Trp Val Ser Tyr 115 120 125 Pro Glu Met Val Ala Ile Cys Gly
Gly Thr Pro Val Phe Val Asp Thr 130 135 140 Thr Leu Glu Asp Asn Phe
Lys Leu Thr Pro Glu Ala Leu Glu Lys Ala 145 150 155 160 Ile Thr Pro
Lys Thr Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165 170 175 Ser
Gly Ala Ala Tyr Ser His Asp Glu Leu Lys Ala Leu Thr Asp Val 180 185
190 Leu Val Lys His Pro Gln Val Trp Val Leu Thr Asp Asp Met Tyr Glu
195 200 205 His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr Pro Val Glu
Val Glu 210 215 220 Pro Ala Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly
Val Ser Lys Ala 225 230 235 240 Tyr Ala Met Thr Gly Trp Arg Ile Gly
Tyr Ala Ala Gly Pro Leu Pro 245 250 255 Leu Ile Lys Ala Met Asp Met
Ile Gln Gly Gln Gln Thr Ser Gly Ala 260 265 270 Ser Ser Ile Ala Gln
Trp Ala Ala Val Glu Ala Leu Asn Gly Thr Gln 275 280 285 Asp Phe Ile
Pro Thr Asn Lys Lys Ile Phe Glu Gly Arg Arg Asp Leu 290 295 300 Val
Val Ser Met Leu Asn Gln Ala Lys Gly Ile Asn Cys Pro Ser Pro 305 310
315 320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly
Lys 325 330 335 Thr Ala Pro Ser Gly Lys Val Ile Glu Ser Asp Val Asp
Phe Val Ser 340 345 350 Glu Leu Leu Glu Ala Glu Gly Val Ala Val Val
Gln Gly Ser Ala Phe 355 360 365 Gly Leu Gly Pro Asn Phe Arg Ile Ser
Tyr Ala Thr Ser Glu Ser Leu 370 375 380 Leu Glu Glu Ala Cys Lys Arg
Ile Gln Arg Phe Cys Ala Asp Cys Arg 385 390 395 400
4935DNAArtificial SequenceForward primer for amplifying DNA
fragment containing aminotransferase gene derived from Rhizobium
radiobacter (3976AT-Nde-f) 49ggaattccat atggccttcc ttgccgacat tctct
355037DNAArtificial SequenceReverse primer for amplifying DNA
fragment containing aminotransferase gene derived from Rhizobium
radiobacter (3976-xho-r) 50actccgctcg agacggcaat cggcgcagaa acgctga
375120PRTRhizobium sp. 51Ala Phe Leu Ala Asp Ile Leu Ser Arg Val
Lys Pro Ser Ala Thr Ile 1 5 10 15 Ala Val Thr Gln 20
521203DNARhizobium sp.CDS(1)..(1203) 52atg gcc ttc ctt gcc gac att
ctc tcc cgc gta aag cca tcc gcc acc 48Met Ala Phe Leu Ala Asp Ile
Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5 10
15 atc gcc gtt acc cag aaa gcc cgt gag ctg aaa gcc aag ggt cgc gat
96Ile Ala Val Thr Gln Lys Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp
20 25 30 gtg att agc ctt ggc gcc ggc gag ccg gat ttc gat acg ccc
gat aat 144Val Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro
Asp Asn 35 40 45 atc aag gaa gcg gcc att gac gcc atc aag cgc ggc
gaa acc aaa tac 192Ile Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly
Glu Thr Lys Tyr 50 55 60 acg ccg gtt tcc ggc att cct gaa ctg cgc
aag gcg att gcc gac aag 240Thr Pro Val Ser Gly Ile Pro Glu Leu Arg
Lys Ala Ile Ala Asp Lys 65 70 75 80 ttc aag cgt gaa aac ggc ctc gac
tac aag ccg gaa cag acc atc gtc 288Phe Lys Arg Glu Asn Gly Leu Asp
Tyr Lys Pro Glu Gln Thr Ile Val 85 90 95 ggc acc ggc ggc aag cag
atc ctc ttc aac gcc ttc atg gcg acg ctg 336Gly Thr Gly Gly Lys Gln
Ile Leu Phe Asn Ala Phe Met Ala Thr Leu 100 105 110 aac ccc ggt gat
gag gtc gtc att ccc gcg cct tac tgg gtc agc tac 384Asn Pro Gly Asp
Glu Val Val Ile Pro Ala Pro Tyr Trp Val Ser Tyr 115 120 125 ccg gaa
atg gtg gcg atc tgc ggc ggt acg ccg gtt ttc gtc aac gcc 432Pro Glu
Met Val Ala Ile Cys Gly Gly Thr Pro Val Phe Val Asn Ala 130 135 140
acg ctc gaa gac aat ttc aag ctg aag ccg gaa gcg ctg gaa aag gct
480Thr Leu Glu Asp Asn Phe Lys Leu Lys Pro Glu Ala Leu Glu Lys Ala
145 150 155 160 atc acg ccg aag aca aag tgg ttc gtc ttc aac tcg cct
tcc aac ccc 528Ile Thr Pro Lys Thr Lys Trp Phe Val Phe Asn Ser Pro
Ser Asn Pro 165 170 175 tcg ggt gcg gcc tat tcg cat gag gag ttg aag
gcg ctg acg gac gtg 576Ser Gly Ala Ala Tyr Ser His Glu Glu Leu Lys
Ala Leu Thr Asp Val 180 185 190 ctg gtc aag cat ccg cat gtc tgg gtg
ctg acg gac gac atg tat gag 624Leu Val Lys His Pro His Val Trp Val
Leu Thr Asp Asp Met Tyr Glu 195 200 205 cac ctg acc tat ggc gat ttc
aaa ttc gtc acc cct gtg gaa gtc gag 672His Leu Thr Tyr Gly Asp Phe
Lys Phe Val Thr Pro Val Glu Val Glu 210 215 220 cct tcg ctc tat gac
cgg acg ttg acg atg aac ggc gtc tcc aag gcc 720Pro Ser Leu Tyr Asp
Arg Thr Leu Thr Met Asn Gly Val Ser Lys Ala 225 230 235 240 tat gcc
atg acc ggc tgg cgt atc ggt tac gct gcc ggc ccg ctg ccg 768Tyr Ala
Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Pro Leu Pro 245 250 255
ctg atc aag gcc atg gac atg atc cag ggc cag cag acc tcg ggc gca
816Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln Gln Thr Ser Gly Ala
260 265 270 agc tcg atc gca cag tgg gcc gct gtc gaa gct ctg aac ggc
acg cag 864Ser Ser Ile Ala Gln Trp Ala Ala Val Glu Ala Leu Asn Gly
Thr Gln 275 280 285 gat ttc att ccg gcg aac aag aag atc ttc gaa ggc
cgt cgc gat ctc 912Asp Phe Ile Pro Ala Asn Lys Lys Ile Phe Glu Gly
Arg Arg Asp Leu 290 295 300 gtc gtt tcc atg ctc aac cag gcc aag ggc
atc agc tgc ccg tca ccg 960Val Val Ser Met Leu Asn Gln Ala Lys Gly
Ile Ser Cys Pro Ser Pro 305 310 315 320 gaa ggt gca ttc tac gtc tac
ccg tcc tgc gcc ggc ttg atc ggc aag 1008Glu Gly Ala Phe Tyr Val Tyr
Pro Ser Cys Ala Gly Leu Ile Gly Lys 325 330 335 acc gcg cct tcg ggc
aag gtc atc gag acg gat acg gat ttc gtt tcc 1056Thr Ala Pro Ser Gly
Lys Val Ile Glu Thr Asp Thr Asp Phe Val Ser 340 345 350 gag ctt ctg
gaa gcc gaa ggc gtt gcc gtc gtg cag gga tcg gct ttc 1104Glu Leu Leu
Glu Ala Glu Gly Val Ala Val Val Gln Gly Ser Ala Phe 355 360 365 ggc
ctt ggc ccg aac ttc cgc atc tcc tac gcc acg tcg gaa act ctt 1152Gly
Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr Ser Glu Thr Leu 370 375
380 ctc gaa gag gcc tgc aag cgc att cag cgt ttc tgc gcc gat tgc cgc
1200Leu Glu Glu Ala Cys Lys Arg Ile Gln Arg Phe Cys Ala Asp Cys Arg
385 390 395 400 taa 120353400PRTRhizobium sp. 53Met Ala Phe Leu Ala
Asp Ile Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5 10 15 Ile Ala Val
Thr Gln Lys Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp 20 25 30 Val
Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35 40
45 Ile Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly Glu Thr Lys Tyr
50 55 60 Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Lys Ala Ile Ala
Asp Lys 65 70 75 80 Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro Glu
Gln Thr Ile Val 85 90 95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn
Ala Phe Met Ala Thr Leu 100 105 110 Asn Pro Gly Asp Glu Val Val Ile
Pro Ala Pro Tyr Trp Val Ser Tyr 115 120 125 Pro Glu Met Val Ala Ile
Cys Gly Gly Thr Pro Val Phe Val Asn Ala 130 135 140 Thr Leu Glu Asp
Asn Phe Lys Leu Lys Pro Glu Ala Leu Glu Lys Ala 145 150 155 160 Ile
Thr Pro Lys Thr Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165 170
175 Ser Gly Ala Ala Tyr Ser His Glu Glu Leu Lys Ala Leu Thr Asp Val
180 185 190 Leu Val Lys His Pro His Val Trp Val Leu Thr Asp Asp Met
Tyr Glu 195 200 205 His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr Pro
Val Glu Val Glu 210 215 220 Pro Ser Leu Tyr Asp Arg Thr Leu Thr Met
Asn Gly Val Ser Lys Ala 225 230 235 240 Tyr Ala Met Thr Gly Trp Arg
Ile Gly Tyr Ala Ala Gly Pro Leu Pro 245 250 255 Leu Ile Lys Ala Met
Asp Met Ile Gln Gly Gln Gln Thr Ser Gly Ala 260 265 270 Ser Ser Ile
Ala Gln Trp Ala Ala Val Glu Ala Leu Asn Gly Thr Gln 275 280 285 Asp
Phe Ile Pro Ala Asn Lys Lys Ile Phe Glu Gly Arg Arg Asp Leu 290 295
300 Val Val Ser Met Leu Asn Gln Ala Lys Gly Ile Ser Cys Pro Ser Pro
305 310 315 320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu
Ile Gly Lys 325 330 335 Thr Ala Pro Ser Gly Lys Val Ile Glu Thr Asp
Thr Asp Phe Val Ser 340 345 350 Glu Leu Leu Glu Ala Glu Gly Val Ala
Val Val Gln Gly Ser Ala Phe 355 360 365 Gly Leu Gly Pro Asn Phe Arg
Ile Ser Tyr Ala Thr Ser Glu Thr Leu 370 375 380 Leu Glu Glu Ala Cys
Lys Arg Ile Gln Arg Phe Cys Ala Asp Cys Arg 385 390 395 400
5435DNAArtificial SequenceForward primer for amplifying DNA
fragment containing aminotransferase gene derived from Rhizobium
sp. (12469AT-Nde-f) 54ggaattccat atggccttcc ttgccgacat tctct
355537DNAArtificial SequenceReverse primer for amplifying DNA
fragment containing aminotransferase gene derived from Rhizobium
sp. (12469-xho-r) 55actccgctcg aggcggcaat cggcgcagaa acgctga
375626DNAArtificial SequenceForward primer which is designed based
on the genomic DNA sequence from Corynebacterium ammoniagenes
DSM20306 (Co-d50-r) 56cttccttgga acaagtcgag gaagac
265726DNAArtificial SequenceReverse primer which is designed based
on a homologus region between the genomic DNA sequences
corresponding to the aspartate aminotransferases from
Corynebacterium striatum ATCC6940 (ZP_03935516) and from
Corynebacterium ammoniagenes DSM20306 57gctatcgcac aattccaccg
cacctt 265826DNAArtificial SequenceForward primer for amplifying
DNA fragment containing aminotransferase gene derived from
Corynebacterium ammoniagenes (Co-890-r) 58acatcgttaa gcaagcgaac
caccag 265925DNAArtificial SequenceReverse primer for amplifying
DNA fragment containing aminotransferase gene derived from
Corynebacterium ammoniagenes (Co-1060-r) 59gaaagacaag cgaatgtggt
gctcg 25601134DNACorynebacterium ammoniagenesCDS(1)..(1134) 60atg
agc cac atc gct caa cgc atc ctt gac cag cgt caa gca tct ctt 48Met
Ser His Ile Ala Gln Arg Ile Leu Asp Gln Arg Gln Ala Ser Leu 1 5 10
15 cgc ccg ccg ctt ggg gtg gtg ccg ccc ggc gcg gtg tcg ttg gcg ttg
96Arg Pro Pro Leu Gly Val Val Pro Pro Gly Ala Val Ser Leu Ala Leu
20 25 30 ggc gag ccg gac ttt gcc cca ccg cag gcg gtt atc gat gcc
acc acg 144Gly Glu Pro Asp Phe Ala Pro Pro Gln Ala Val Ile Asp Ala
Thr Thr 35 40 45 caa gca gtc gcc caa ggc cgc acc aac tac acg gat
cag cac ggt atc 192Gln Ala Val Ala Gln Gly Arg Thr Asn Tyr Thr Asp
Gln His Gly Ile 50 55 60 gct gag ctt cgc gat gcc ctc ctc gcc gcc
ctt ccc acc cgc ccc tct 240Ala Glu Leu Arg Asp Ala Leu Leu Ala Ala
Leu Pro Thr Arg Pro Ser 65 70 75 80 aac tgg gac cgc gac aat att gtg
gtg aca cac ggt gca acc gcg gga 288Asn Trp Asp Arg Asp Asn Ile Val
Val Thr His Gly Ala Thr Ala Gly 85 90 95 ctg ggt gcg ctg ttt ttc
gcg ctg att gaa ccc ggt gac aag gtc gtt 336Leu Gly Ala Leu Phe Phe
Ala Leu Ile Glu Pro Gly Asp Lys Val Val 100 105 110 atc cca cag cct
gcg tat tct ttg tac gcc gac cag gtg gtt tta gcc 384Ile Pro Gln Pro
Ala Tyr Ser Leu Tyr Ala Asp Gln Val Val Leu Ala 115 120 125 ggc ggc
acc gtg gaa ttt gtt ccc atg ggc aag gac ctc cac ttt gat 432Gly Gly
Thr Val Glu Phe Val Pro Met Gly Lys Asp Leu His Phe Asp 130 135 140
ttt gac cag ctt gcc acg gcg ctt gac ggc gca aag atg gtg gtc ttt
480Phe Asp Gln Leu Ala Thr Ala Leu Asp Gly Ala Lys Met Val Val Phe
145 150 155 160 tct aat cct tct aat ccc aat gga atc gtg cat acc cgc
gat gag ctg 528Ser Asn Pro Ser Asn Pro Asn Gly Ile Val His Thr Arg
Asp Glu Leu 165 170 175 gaa agg ctt gca cag ctt ctc gat gcc acc gat
acc ctc gtc gtt tcc 576Glu Arg Leu Ala Gln Leu Leu Asp Ala Thr Asp
Thr Leu Val Val Ser 180 185 190 gac gaa gcc tac tcg gcc ctc acc tat
acc gcc gaa cca ttc acc tcc 624Asp Glu Ala Tyr Ser Ala Leu Thr Tyr
Thr Ala Glu Pro Phe Thr Ser 195 200 205 gct tta gag gtt ccc ggc ttg
cag gag cgc acg tta tac gtg cag acc 672Ala Leu Glu Val Pro Gly Leu
Gln Glu Arg Thr Leu Tyr Val Gln Thr 210 215 220 ttt tcc aag aaa tac
tgc atg acg ggc ttc cgc gtc ggc tac gtc gcg 720Phe Ser Lys Lys Tyr
Cys Met Thr Gly Phe Arg Val Gly Tyr Val Ala 225 230 235 240 ggc gcg
aga gat ttg atc gct gcg att gcg cag atg cac cgc acc ttc 768Gly Ala
Arg Asp Leu Ile Ala Ala Ile Ala Gln Met His Arg Thr Phe 245 250 255
aac ggc tca gtg tcg gag cag gcg cag ctg gca gcg ctc gcc gcg gta
816Asn Gly Ser Val Ser Glu Gln Ala Gln Leu Ala Ala Leu Ala Ala Val
260 265 270 tct ctg ccg gag tct gtg gtc aca ccg atg ttg gaa gaa tac
gcc cag 864Ser Leu Pro Glu Ser Val Val Thr Pro Met Leu Glu Glu Tyr
Ala Gln 275 280 285 cgc cgc gac ctg gtg gtt cgc ttg ctt aac gat gtc
ccc cac gtc caa 912Arg Arg Asp Leu Val Val Arg Leu Leu Asn Asp Val
Pro His Val Gln 290 295 300 ctc ttc gag ccc gaa ggc gcg ttt tat gca
ttc ttc tct tat gac ttg 960Leu Phe Glu Pro Glu Gly Ala Phe Tyr Ala
Phe Phe Ser Tyr Asp Leu 305 310 315 320 gac aag ccc tct tcc cag gta
gca gcc gag ctg gcc gaa cgc ggt gta 1008Asp Lys Pro Ser Ser Gln Val
Ala Ala Glu Leu Ala Glu Arg Gly Val 325 330 335 cta gtg cgc gct ggt
gcc gaa tat ggc ccc gcc gcc gag cac cac att 1056Leu Val Arg Ala Gly
Ala Glu Tyr Gly Pro Ala Ala Glu His His Ile 340 345 350 cgc ttg tct
ttc gca gcc tcg caa gca gat atc gaa cgt ggc att gga 1104Arg Leu Ser
Phe Ala Ala Ser Gln Ala Asp Ile Glu Arg Gly Ile Gly 355 360 365 atc
att cgc caa tac ttc gaa aag tcc tag 1134Ile Ile Arg Gln Tyr Phe Glu
Lys Ser 370 375 61377PRTCorynebacterium ammoniagenes 61Met Ser His
Ile Ala Gln Arg Ile Leu Asp Gln Arg Gln Ala Ser Leu 1 5 10 15 Arg
Pro Pro Leu Gly Val Val Pro Pro Gly Ala Val Ser Leu Ala Leu 20 25
30 Gly Glu Pro Asp Phe Ala Pro Pro Gln Ala Val Ile Asp Ala Thr Thr
35 40 45 Gln Ala Val Ala Gln Gly Arg Thr Asn Tyr Thr Asp Gln His
Gly Ile 50 55 60 Ala Glu Leu Arg Asp Ala Leu Leu Ala Ala Leu Pro
Thr Arg Pro Ser 65 70 75 80 Asn Trp Asp Arg Asp Asn Ile Val Val Thr
His Gly Ala Thr Ala Gly 85 90 95 Leu Gly Ala Leu Phe Phe Ala Leu
Ile Glu Pro Gly Asp Lys Val Val 100 105 110 Ile Pro Gln Pro Ala Tyr
Ser Leu Tyr Ala Asp Gln Val Val Leu Ala 115 120 125 Gly Gly Thr Val
Glu Phe Val Pro Met Gly Lys Asp Leu His Phe Asp 130 135 140 Phe Asp
Gln Leu Ala Thr Ala Leu Asp Gly Ala Lys Met Val Val Phe 145 150 155
160 Ser Asn Pro Ser Asn Pro Asn Gly Ile Val His Thr Arg Asp Glu Leu
165 170 175 Glu Arg Leu Ala Gln Leu Leu Asp Ala Thr Asp Thr Leu Val
Val Ser 180 185 190 Asp Glu Ala Tyr Ser Ala Leu Thr Tyr Thr Ala Glu
Pro Phe Thr Ser 195 200 205 Ala Leu Glu Val Pro Gly Leu Gln Glu Arg
Thr Leu Tyr Val Gln Thr 210 215 220 Phe Ser Lys Lys Tyr Cys Met Thr
Gly Phe Arg Val Gly Tyr Val Ala 225 230 235 240 Gly Ala Arg Asp Leu
Ile Ala Ala Ile Ala Gln Met His Arg Thr Phe 245 250 255 Asn Gly Ser
Val Ser Glu Gln Ala Gln Leu Ala Ala Leu Ala Ala Val 260 265 270 Ser
Leu Pro Glu Ser Val Val Thr Pro Met Leu Glu Glu Tyr Ala Gln 275 280
285 Arg Arg Asp Leu Val Val Arg Leu Leu Asn Asp Val Pro His Val Gln
290 295 300 Leu Phe Glu Pro Glu Gly Ala Phe Tyr Ala Phe Phe Ser Tyr
Asp Leu 305 310 315 320 Asp Lys Pro Ser Ser Gln Val Ala Ala Glu Leu
Ala Glu Arg Gly Val 325 330 335 Leu Val Arg Ala Gly Ala Glu Tyr Gly
Pro Ala Ala Glu His His Ile 340 345 350 Arg Leu
Ser Phe Ala Ala Ser Gln Ala Asp Ile Glu Arg Gly Ile Gly 355 360 365
Ile Ile Arg Gln Tyr Phe Glu Lys Ser 370 375 6235DNAArtificial
SequenceForward primer for amplifying DNA fragment containing
aminotransferase gene derived from Corynebacterium ammoniagenes
(1444AT-Nde-f) 62ggaattccat atgagccaca tcgctcaacg catcc
356337DNAArtificial SequenceReverse primer for amplifying DNA
fragment containing aminotransferase gene derived from
Corynebacterium ammoniagenes (1444-xho-r) 63actccgctcg agggactttt
cgaagtattg gcgaatg 37641275DNADeinococcus
geothermalisCDS(1)..(1275) 64atg acc aaa gaa gca tcc cgc ccg gca
ctg gac ctg gct cgt caa gcg 48Met Thr Lys Glu Ala Ser Arg Pro Ala
Leu Asp Leu Ala Arg Gln Ala 1 5 10 15 tat gaa gca ttt aaa gct cgt
ggc ctg aat ctg aat atg cag cgt ggt 96Tyr Glu Ala Phe Lys Ala Arg
Gly Leu Asn Leu Asn Met Gln Arg Gly 20 25 30 caa ccg gct gat gcg
gac ttt gat ctg tct aac ggc ctg ctg acc gtt 144Gln Pro Ala Asp Ala
Asp Phe Asp Leu Ser Asn Gly Leu Leu Thr Val 35 40 45 ctg ggt gcc
gaa gac gtc cgt atg gac ggc ctg gat ctg cgc aat tat 192Leu Gly Ala
Glu Asp Val Arg Met Asp Gly Leu Asp Leu Arg Asn Tyr 50 55 60 ccg
ggc ggt gtg gca ggt ctg ccg agc gcc cgc gca ctg ttt gcc ggt 240Pro
Gly Gly Val Ala Gly Leu Pro Ser Ala Arg Ala Leu Phe Ala Gly 65 70
75 80 tac ctg gat gtt aaa gca gaa aac gtt ctg gtc tgg aac aat agc
tct 288Tyr Leu Asp Val Lys Ala Glu Asn Val Leu Val Trp Asn Asn Ser
Ser 85 90 95 ctg gaa ctg caa ggt ctg gtt ctg acc ttc gcc ctg ctg
cat ggt gtc 336Leu Glu Leu Gln Gly Leu Val Leu Thr Phe Ala Leu Leu
His Gly Val 100 105 110 cgt ggt agc acg ggt ccg tgg ctg tct caa acc
ccg aaa atg att gtg 384Arg Gly Ser Thr Gly Pro Trp Leu Ser Gln Thr
Pro Lys Met Ile Val 115 120 125 acg gtt ccg ggc tat gat cgc cac ttt
ctg ctg ctg caa acc ctg ggt 432Thr Val Pro Gly Tyr Asp Arg His Phe
Leu Leu Leu Gln Thr Leu Gly 130 135 140 ttc gaa ctg ctg acg gtg gac
atg caa agc gat ggc ccg gac gtc gat 480Phe Glu Leu Leu Thr Val Asp
Met Gln Ser Asp Gly Pro Asp Val Asp 145 150 155 160 gcc gtg gaa cgt
ctg gca ggc acc gat ccg tct gtg aaa ggt att ctg 528Ala Val Glu Arg
Leu Ala Gly Thr Asp Pro Ser Val Lys Gly Ile Leu 165 170 175 ttt gtt
ccg acc tac tca aac ccg ggc ggt gaa acg atc tcg ctg gaa 576Phe Val
Pro Thr Tyr Ser Asn Pro Gly Gly Glu Thr Ile Ser Leu Glu 180 185 190
aaa gct cgt cgc ctg gca ggt ctg caa gcg gcc gca ccg gac ttt acg
624Lys Ala Arg Arg Leu Ala Gly Leu Gln Ala Ala Ala Pro Asp Phe Thr
195 200 205 att ttc gct gat gac gcg tat cgt gtc cat cac ctg gtg gaa
gaa gat 672Ile Phe Ala Asp Asp Ala Tyr Arg Val His His Leu Val Glu
Glu Asp 210 215 220 cgc gcc gaa ccg gtg aat ttc gtg gtt ctg gcc cgt
gac gca ggt tac 720Arg Ala Glu Pro Val Asn Phe Val Val Leu Ala Arg
Asp Ala Gly Tyr 225 230 235 240 ccg gat cgt gcc ttt gtt ttc gca tca
acc tcg aaa atc acg ttt gct 768Pro Asp Arg Ala Phe Val Phe Ala Ser
Thr Ser Lys Ile Thr Phe Ala 245 250 255 ggt gca ggt ctg ggt ttc gtg
gcg agt tcc gaa gat aac att cgt tgg 816Gly Ala Gly Leu Gly Phe Val
Ala Ser Ser Glu Asp Asn Ile Arg Trp 260 265 270 ctg agt aaa tat ctg
ggc gcg cag tcc atc ggt ccg aat aaa gtc gaa 864Leu Ser Lys Tyr Leu
Gly Ala Gln Ser Ile Gly Pro Asn Lys Val Glu 275 280 285 caa gcc cgt
cat gtg aaa ttt ctg acc gaa tac ccg ggc ggt ctg gaa 912Gln Ala Arg
His Val Lys Phe Leu Thr Glu Tyr Pro Gly Gly Leu Glu 290 295 300 ggt
ctg atg cgc gac cac gct gcg att atc gct ccg aaa ttc cgt gcg 960Gly
Leu Met Arg Asp His Ala Ala Ile Ile Ala Pro Lys Phe Arg Ala 305 310
315 320 gtt gat gaa gtc ctg cgc gct gaa ctg ggc gaa ggc ggt gaa tat
gca 1008Val Asp Glu Val Leu Arg Ala Glu Leu Gly Glu Gly Gly Glu Tyr
Ala 325 330 335 acc tgg acg ctg ccg aaa ggc ggt tac ttt atc agt ctg
gac acc gct 1056Thr Trp Thr Leu Pro Lys Gly Gly Tyr Phe Ile Ser Leu
Asp Thr Ala 340 345 350 gaa ccg gtg gcg gat cgc gtc gtg aaa ctg gcg
gaa gcc gca ggc gtt 1104Glu Pro Val Ala Asp Arg Val Val Lys Leu Ala
Glu Ala Ala Gly Val 355 360 365 agc ctg acc ccg gcg ggt gca acg tat
ccg gca ggt caa gat ccg cat 1152Ser Leu Thr Pro Ala Gly Ala Thr Tyr
Pro Ala Gly Gln Asp Pro His 370 375 380 aac cgt aat ctg cgt ctg gca
ccg acc cgt ccg ccg gtg gaa gaa gtt 1200Asn Arg Asn Leu Arg Leu Ala
Pro Thr Arg Pro Pro Val Glu Glu Val 385 390 395 400 cgc acg gca atg
caa gtg gtc gcc gcg tgt atc cgc ctg gca acc gaa 1248Arg Thr Ala Met
Gln Val Val Ala Ala Cys Ile Arg Leu Ala Thr Glu 405 410 415 gaa tat
cgt gct ggt cat ctc gag tga 1275Glu Tyr Arg Ala Gly His Leu Glu 420
65424PRTDeinococcus geothermalis 65Met Thr Lys Glu Ala Ser Arg Pro
Ala Leu Asp Leu Ala Arg Gln Ala 1 5 10 15 Tyr Glu Ala Phe Lys Ala
Arg Gly Leu Asn Leu Asn Met Gln Arg Gly 20 25 30 Gln Pro Ala Asp
Ala Asp Phe Asp Leu Ser Asn Gly Leu Leu Thr Val 35 40 45 Leu Gly
Ala Glu Asp Val Arg Met Asp Gly Leu Asp Leu Arg Asn Tyr 50 55 60
Pro Gly Gly Val Ala Gly Leu Pro Ser Ala Arg Ala Leu Phe Ala Gly 65
70 75 80 Tyr Leu Asp Val Lys Ala Glu Asn Val Leu Val Trp Asn Asn
Ser Ser 85 90 95 Leu Glu Leu Gln Gly Leu Val Leu Thr Phe Ala Leu
Leu His Gly Val 100 105 110 Arg Gly Ser Thr Gly Pro Trp Leu Ser Gln
Thr Pro Lys Met Ile Val 115 120 125 Thr Val Pro Gly Tyr Asp Arg His
Phe Leu Leu Leu Gln Thr Leu Gly 130 135 140 Phe Glu Leu Leu Thr Val
Asp Met Gln Ser Asp Gly Pro Asp Val Asp 145 150 155 160 Ala Val Glu
Arg Leu Ala Gly Thr Asp Pro Ser Val Lys Gly Ile Leu 165 170 175 Phe
Val Pro Thr Tyr Ser Asn Pro Gly Gly Glu Thr Ile Ser Leu Glu 180 185
190 Lys Ala Arg Arg Leu Ala Gly Leu Gln Ala Ala Ala Pro Asp Phe Thr
195 200 205 Ile Phe Ala Asp Asp Ala Tyr Arg Val His His Leu Val Glu
Glu Asp 210 215 220 Arg Ala Glu Pro Val Asn Phe Val Val Leu Ala Arg
Asp Ala Gly Tyr 225 230 235 240 Pro Asp Arg Ala Phe Val Phe Ala Ser
Thr Ser Lys Ile Thr Phe Ala 245 250 255 Gly Ala Gly Leu Gly Phe Val
Ala Ser Ser Glu Asp Asn Ile Arg Trp 260 265 270 Leu Ser Lys Tyr Leu
Gly Ala Gln Ser Ile Gly Pro Asn Lys Val Glu 275 280 285 Gln Ala Arg
His Val Lys Phe Leu Thr Glu Tyr Pro Gly Gly Leu Glu 290 295 300 Gly
Leu Met Arg Asp His Ala Ala Ile Ile Ala Pro Lys Phe Arg Ala 305 310
315 320 Val Asp Glu Val Leu Arg Ala Glu Leu Gly Glu Gly Gly Glu Tyr
Ala 325 330 335 Thr Trp Thr Leu Pro Lys Gly Gly Tyr Phe Ile Ser Leu
Asp Thr Ala 340 345 350 Glu Pro Val Ala Asp Arg Val Val Lys Leu Ala
Glu Ala Ala Gly Val 355 360 365 Ser Leu Thr Pro Ala Gly Ala Thr Tyr
Pro Ala Gly Gln Asp Pro His 370 375 380 Asn Arg Asn Leu Arg Leu Ala
Pro Thr Arg Pro Pro Val Glu Glu Val 385 390 395 400 Arg Thr Ala Met
Gln Val Val Ala Ala Cys Ile Arg Leu Ala Thr Glu 405 410 415 Glu Tyr
Arg Ala Gly His Leu Glu 420 661287DNACorynebacterium
glutamicumCDS(1)..(1287) 66atg agc tcg gtg tcg ctg caa gac ttt gac
gct gaa cgc att ggc ctg 48Met Ser Ser Val Ser Leu Gln Asp Phe Asp
Ala Glu Arg Ile Gly Leu 1 5 10 15 ttc cac gaa gat att aaa cgt aaa
ttt gat gaa ctg aaa tct aaa aac 96Phe His Glu Asp Ile Lys Arg Lys
Phe Asp Glu Leu Lys Ser Lys Asn 20 25 30 ctg aaa ctg gat ctg acc
cgt ggt aaa ccg agc tct gaa cag ctg gat 144Leu Lys Leu Asp Leu Thr
Arg Gly Lys Pro Ser Ser Glu Gln Leu Asp 35 40 45 ttt gcg gac gaa
ctg ctg gcc ctg ccg ggc aag ggt gat ttc aaa gcg 192Phe Ala Asp Glu
Leu Leu Ala Leu Pro Gly Lys Gly Asp Phe Lys Ala 50 55 60 gcc gat
ggc acc gac gtt cgt aac tat ggc ggt ctg gat ggt att gtc 240Ala Asp
Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Asp Gly Ile Val 65 70 75 80
gac atc cgc cag att tgg gcg gat ctg ctg ggc gtg ccg gtt gaa caa
288Asp Ile Arg Gln Ile Trp Ala Asp Leu Leu Gly Val Pro Val Glu Gln
85 90 95 gtg ctg gca ggt gat gct agt tcc ctg aat atc atg ttt gac
gtg att 336Val Leu Ala Gly Asp Ala Ser Ser Leu Asn Ile Met Phe Asp
Val Ile 100 105 110 agc tgg tct tat atc ttc ggc aac aat gat tca gtt
cag ccg tgg tcg 384Ser Trp Ser Tyr Ile Phe Gly Asn Asn Asp Ser Val
Gln Pro Trp Ser 115 120 125 aaa gaa gaa acg gtg aaa tgg att tgc ccg
gtt ccg ggc tac gac cgt 432Lys Glu Glu Thr Val Lys Trp Ile Cys Pro
Val Pro Gly Tyr Asp Arg 130 135 140 cat ttt tct att acc gaa cgc ttt
ggt ttc gaa atg atc agt gtt ccg 480His Phe Ser Ile Thr Glu Arg Phe
Gly Phe Glu Met Ile Ser Val Pro 145 150 155 160 atg aac gaa gat ggc
ccg gat atg gac gca gtt gaa gaa ctg gtc aaa 528Met Asn Glu Asp Gly
Pro Asp Met Asp Ala Val Glu Glu Leu Val Lys 165 170 175 gac ccg caa
gtc aaa ggc atg tgg gtg gtt ccg gtg ttt agt aat ccg 576Asp Pro Gln
Val Lys Gly Met Trp Val Val Pro Val Phe Ser Asn Pro 180 185 190 acc
ggc ttc acg gtg tcc gaa gat gtt gcc aaa cgt ctg tca acc atg 624Thr
Gly Phe Thr Val Ser Glu Asp Val Ala Lys Arg Leu Ser Thr Met 195 200
205 gaa acg gca gct ccg gat ttt cgc gtc gtg tgg gac aat gcg tac gcc
672Glu Thr Ala Ala Pro Asp Phe Arg Val Val Trp Asp Asn Ala Tyr Ala
210 215 220 gtg cac acc ctg acg gat gaa ttc ccg gaa gtc att gac atc
gtg ggt 720Val His Thr Leu Thr Asp Glu Phe Pro Glu Val Ile Asp Ile
Val Gly 225 230 235 240 ctg ggt gaa gcg gcc ggt aac ccg aat cgt ttt
tgg gcg ttc acc agt 768Leu Gly Glu Ala Ala Gly Asn Pro Asn Arg Phe
Trp Ala Phe Thr Ser 245 250 255 acg tcc aaa att acc ctg gca ggc gct
ggt gtc agc ttt ttc atg acg 816Thr Ser Lys Ile Thr Leu Ala Gly Ala
Gly Val Ser Phe Phe Met Thr 260 265 270 agc gcg gaa aac cgt aaa tgg
tat agc ggc cat gct ggt atc cgc ggc 864Ser Ala Glu Asn Arg Lys Trp
Tyr Ser Gly His Ala Gly Ile Arg Gly 275 280 285 att ggt ccg aac aaa
gtt aat cag ctg gcg cac gcc cgc tac ttt ggc 912Ile Gly Pro Asn Lys
Val Asn Gln Leu Ala His Ala Arg Tyr Phe Gly 290 295 300 gat gca gaa
ggt gtc cgt gct gtg atg cgc aaa cat gca gct tcc ctg 960Asp Ala Glu
Gly Val Arg Ala Val Met Arg Lys His Ala Ala Ser Leu 305 310 315 320
gcg ccg aaa ttc aat aaa gtg ctg gaa atc ctg gat agt cgt ctg gcg
1008Ala Pro Lys Phe Asn Lys Val Leu Glu Ile Leu Asp Ser Arg Leu Ala
325 330 335 gaa tat ggt gtt gca cag tgg acc gtc ccg gcc ggc ggt tac
ttt att 1056Glu Tyr Gly Val Ala Gln Trp Thr Val Pro Ala Gly Gly Tyr
Phe Ile 340 345 350 tcg ctg gac gtt gtc ccg ggt acg gca agc cgc gtt
gcg gaa ctg gcc 1104Ser Leu Asp Val Val Pro Gly Thr Ala Ser Arg Val
Ala Glu Leu Ala 355 360 365 aaa gaa gca ggc atc gct ctg acc ggc gcg
ggt tca tcg tat ccg ctg 1152Lys Glu Ala Gly Ile Ala Leu Thr Gly Ala
Gly Ser Ser Tyr Pro Leu 370 375 380 cgt caa gat ccg gaa aac aaa aat
ctg cgt ctg gca ccg agc ctg ccg 1200Arg Gln Asp Pro Glu Asn Lys Asn
Leu Arg Leu Ala Pro Ser Leu Pro 385 390 395 400 ccg gtg gaa gaa ctg
gaa gtt gcg atg gat ggt gtg gct acc tgc gtg 1248Pro Val Glu Glu Leu
Glu Val Ala Met Asp Gly Val Ala Thr Cys Val 405 410 415 ctg ctg gct
gcc gcc gaa cat tat gct tct ctc gag tga 1287Leu Leu Ala Ala Ala Glu
His Tyr Ala Ser Leu Glu 420 425 67428PRTCorynebacterium glutamicum
67Met Ser Ser Val Ser Leu Gln Asp Phe Asp Ala Glu Arg Ile Gly Leu 1
5 10 15 Phe His Glu Asp Ile Lys Arg Lys Phe Asp Glu Leu Lys Ser Lys
Asn 20 25 30 Leu Lys Leu Asp Leu Thr Arg Gly Lys Pro Ser Ser Glu
Gln Leu Asp 35 40 45 Phe Ala Asp Glu Leu Leu Ala Leu Pro Gly Lys
Gly Asp Phe Lys Ala 50 55 60 Ala Asp Gly Thr Asp Val Arg Asn Tyr
Gly Gly Leu Asp Gly Ile Val 65 70 75 80 Asp Ile Arg Gln Ile Trp Ala
Asp Leu Leu Gly Val Pro Val Glu Gln 85 90 95 Val Leu Ala Gly Asp
Ala Ser Ser Leu Asn Ile Met Phe Asp Val Ile 100 105 110 Ser Trp Ser
Tyr Ile Phe Gly Asn Asn Asp Ser Val Gln Pro Trp Ser 115 120 125 Lys
Glu Glu Thr Val Lys Trp Ile Cys Pro Val Pro Gly Tyr Asp Arg 130 135
140 His Phe Ser Ile Thr Glu Arg Phe Gly Phe Glu Met Ile Ser Val Pro
145 150 155 160 Met Asn Glu Asp Gly Pro Asp Met Asp Ala Val Glu Glu
Leu Val Lys 165 170 175 Asp Pro Gln Val Lys Gly Met Trp Val Val Pro
Val Phe Ser Asn Pro 180 185 190 Thr Gly Phe Thr Val Ser Glu Asp Val
Ala Lys Arg Leu Ser Thr Met 195 200 205 Glu Thr Ala Ala Pro Asp Phe
Arg Val Val Trp Asp Asn Ala Tyr Ala 210 215 220 Val His Thr Leu Thr
Asp Glu Phe Pro Glu Val Ile Asp Ile Val Gly 225 230 235 240 Leu Gly
Glu Ala Ala Gly Asn Pro Asn Arg Phe Trp Ala Phe Thr Ser 245 250 255
Thr Ser Lys Ile Thr Leu Ala Gly Ala Gly Val Ser Phe Phe Met Thr 260
265 270 Ser Ala Glu Asn Arg Lys Trp Tyr Ser Gly His Ala Gly Ile
Arg
Gly 275 280 285 Ile Gly Pro Asn Lys Val Asn Gln Leu Ala His Ala Arg
Tyr Phe Gly 290 295 300 Asp Ala Glu Gly Val Arg Ala Val Met Arg Lys
His Ala Ala Ser Leu 305 310 315 320 Ala Pro Lys Phe Asn Lys Val Leu
Glu Ile Leu Asp Ser Arg Leu Ala 325 330 335 Glu Tyr Gly Val Ala Gln
Trp Thr Val Pro Ala Gly Gly Tyr Phe Ile 340 345 350 Ser Leu Asp Val
Val Pro Gly Thr Ala Ser Arg Val Ala Glu Leu Ala 355 360 365 Lys Glu
Ala Gly Ile Ala Leu Thr Gly Ala Gly Ser Ser Tyr Pro Leu 370 375 380
Arg Gln Asp Pro Glu Asn Lys Asn Leu Arg Leu Ala Pro Ser Leu Pro 385
390 395 400 Pro Val Glu Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr
Cys Val 405 410 415 Leu Leu Ala Ala Ala Glu His Tyr Ala Ser Leu Glu
420 425 681200DNAThermus thermophilusCDS(1)..(1200) 68atg aaa ccg
ctg tcc tgg tct gaa gcg ttc ggc aaa ggt gct ggt cgt 48Met Lys Pro
Leu Ser Trp Ser Glu Ala Phe Gly Lys Gly Ala Gly Arg 1 5 10 15 atc
caa gcc tct acc att cgt gaa ctg ctg aaa ctg acg cag cgc ccg 96Ile
Gln Ala Ser Thr Ile Arg Glu Leu Leu Lys Leu Thr Gln Arg Pro 20 25
30 ggt att ctg agc ttt gca ggc ggt ctg ccg gct ccg gaa ctg ttc ccg
144Gly Ile Leu Ser Phe Ala Gly Gly Leu Pro Ala Pro Glu Leu Phe Pro
35 40 45 aaa gaa gaa gcg gcc gaa gca gct gcg cgt atc ctg cgt gaa
aaa ggt 192Lys Glu Glu Ala Ala Glu Ala Ala Ala Arg Ile Leu Arg Glu
Lys Gly 50 55 60 gaa gtt gca ctg caa tat agc ccg acc gaa ggt tac
gct ccg ctg cgt 240Glu Val Ala Leu Gln Tyr Ser Pro Thr Glu Gly Tyr
Ala Pro Leu Arg 65 70 75 80 gca ttt gtc gct gaa tgg att ggt gtt cgt
ccg gaa gaa gtc ctg atc 288Ala Phe Val Ala Glu Trp Ile Gly Val Arg
Pro Glu Glu Val Leu Ile 85 90 95 acc acg ggc tct cag caa gcg ctg
gat ctg gtg ggt aaa gtt ttc ctg 336Thr Thr Gly Ser Gln Gln Ala Leu
Asp Leu Val Gly Lys Val Phe Leu 100 105 110 gac gaa ggc agt ccg gtt
ctg ctg gaa gcc ccg tcc tat atg ggt gcg 384Asp Glu Gly Ser Pro Val
Leu Leu Glu Ala Pro Ser Tyr Met Gly Ala 115 120 125 att cag gcc ttt
cgc ctg caa ggt ccg cgt ttc ctg acc gtc ccg gca 432Ile Gln Ala Phe
Arg Leu Gln Gly Pro Arg Phe Leu Thr Val Pro Ala 130 135 140 ggt gaa
gaa ggc ccg gat ctg gac gct ctg gaa gaa gtg ctg aaa cgt 480Gly Glu
Glu Gly Pro Asp Leu Asp Ala Leu Glu Glu Val Leu Lys Arg 145 150 155
160 gaa cgc ccg cgt ttt ctg tac ctg atc ccg agc ttc cag aac ccg acc
528Glu Arg Pro Arg Phe Leu Tyr Leu Ile Pro Ser Phe Gln Asn Pro Thr
165 170 175 ggc ggt ctg acg ccg ctg ccg gca cgt aaa cgt ctg ctg caa
atg gtg 576Gly Gly Leu Thr Pro Leu Pro Ala Arg Lys Arg Leu Leu Gln
Met Val 180 185 190 atg gaa cgt ggt ctg gtg gtt gtc gaa gat gac gcg
tat cgc gaa ctg 624Met Glu Arg Gly Leu Val Val Val Glu Asp Asp Ala
Tyr Arg Glu Leu 195 200 205 tac ttt ggc gaa gcc cgt ctg ccg tca ctg
ttc gaa ctg gca cgc gaa 672Tyr Phe Gly Glu Ala Arg Leu Pro Ser Leu
Phe Glu Leu Ala Arg Glu 210 215 220 gct ggt tat ccg ggc gtg att tac
ctg ggt agc ttt tct aaa gtt ctg 720Ala Gly Tyr Pro Gly Val Ile Tyr
Leu Gly Ser Phe Ser Lys Val Leu 225 230 235 240 tcg ccg ggt ctg cgt
gtg gca ttc gca gtt gct cat ccg gaa gcg ctg 768Ser Pro Gly Leu Arg
Val Ala Phe Ala Val Ala His Pro Glu Ala Leu 245 250 255 caa aaa ctg
gtt cag gcg aaa caa ggt gcc gat ctg cat acc ccg atg 816Gln Lys Leu
Val Gln Ala Lys Gln Gly Ala Asp Leu His Thr Pro Met 260 265 270 ctg
aac caa atg ctg gtg cac gaa ctg ctg aaa gaa ggc ttt tct gaa 864Leu
Asn Gln Met Leu Val His Glu Leu Leu Lys Glu Gly Phe Ser Glu 275 280
285 cgt ctg gaa cgt gtc cgt cgc gtg tat cgc gaa aaa gcg cag gcc atg
912Arg Leu Glu Arg Val Arg Arg Val Tyr Arg Glu Lys Ala Gln Ala Met
290 295 300 ctg cac gca ctg gac cgt gaa gtc ccg aaa gaa gtg cgc tac
acg cgt 960Leu His Ala Leu Asp Arg Glu Val Pro Lys Glu Val Arg Tyr
Thr Arg 305 310 315 320 ccg aaa ggc ggt atg ttt gtg tgg atg gaa ctg
ccg aaa ggt ctg agt 1008Pro Lys Gly Gly Met Phe Val Trp Met Glu Leu
Pro Lys Gly Leu Ser 325 330 335 gcc gaa ggc ctg ttt cgt cgc gcg ctg
gaa gaa aat gtt gcc ttc gtc 1056Ala Glu Gly Leu Phe Arg Arg Ala Leu
Glu Glu Asn Val Ala Phe Val 340 345 350 ccg ggc ggt ccg ttt ttc gca
aac ggc ggt ggc gaa aat acc ctg cgc 1104Pro Gly Gly Pro Phe Phe Ala
Asn Gly Gly Gly Glu Asn Thr Leu Arg 355 360 365 ctg tcc tat gca acg
ctg gat cgt gaa ggc atc gcc gaa ggt gtc cgc 1152Leu Ser Tyr Ala Thr
Leu Asp Arg Glu Gly Ile Ala Glu Gly Val Arg 370 375 380 cgc ctg ggt
cgt gct ctg aaa ggt ctg ctg gcc ctg gtt ctc gag tga 1200Arg Leu Gly
Arg Ala Leu Lys Gly Leu Leu Ala Leu Val Leu Glu 385 390 395
69399PRTThermus thermophilus 69Met Lys Pro Leu Ser Trp Ser Glu Ala
Phe Gly Lys Gly Ala Gly Arg 1 5 10 15 Ile Gln Ala Ser Thr Ile Arg
Glu Leu Leu Lys Leu Thr Gln Arg Pro 20 25 30 Gly Ile Leu Ser Phe
Ala Gly Gly Leu Pro Ala Pro Glu Leu Phe Pro 35 40 45 Lys Glu Glu
Ala Ala Glu Ala Ala Ala Arg Ile Leu Arg Glu Lys Gly 50 55 60 Glu
Val Ala Leu Gln Tyr Ser Pro Thr Glu Gly Tyr Ala Pro Leu Arg 65 70
75 80 Ala Phe Val Ala Glu Trp Ile Gly Val Arg Pro Glu Glu Val Leu
Ile 85 90 95 Thr Thr Gly Ser Gln Gln Ala Leu Asp Leu Val Gly Lys
Val Phe Leu 100 105 110 Asp Glu Gly Ser Pro Val Leu Leu Glu Ala Pro
Ser Tyr Met Gly Ala 115 120 125 Ile Gln Ala Phe Arg Leu Gln Gly Pro
Arg Phe Leu Thr Val Pro Ala 130 135 140 Gly Glu Glu Gly Pro Asp Leu
Asp Ala Leu Glu Glu Val Leu Lys Arg 145 150 155 160 Glu Arg Pro Arg
Phe Leu Tyr Leu Ile Pro Ser Phe Gln Asn Pro Thr 165 170 175 Gly Gly
Leu Thr Pro Leu Pro Ala Arg Lys Arg Leu Leu Gln Met Val 180 185 190
Met Glu Arg Gly Leu Val Val Val Glu Asp Asp Ala Tyr Arg Glu Leu 195
200 205 Tyr Phe Gly Glu Ala Arg Leu Pro Ser Leu Phe Glu Leu Ala Arg
Glu 210 215 220 Ala Gly Tyr Pro Gly Val Ile Tyr Leu Gly Ser Phe Ser
Lys Val Leu 225 230 235 240 Ser Pro Gly Leu Arg Val Ala Phe Ala Val
Ala His Pro Glu Ala Leu 245 250 255 Gln Lys Leu Val Gln Ala Lys Gln
Gly Ala Asp Leu His Thr Pro Met 260 265 270 Leu Asn Gln Met Leu Val
His Glu Leu Leu Lys Glu Gly Phe Ser Glu 275 280 285 Arg Leu Glu Arg
Val Arg Arg Val Tyr Arg Glu Lys Ala Gln Ala Met 290 295 300 Leu His
Ala Leu Asp Arg Glu Val Pro Lys Glu Val Arg Tyr Thr Arg 305 310 315
320 Pro Lys Gly Gly Met Phe Val Trp Met Glu Leu Pro Lys Gly Leu Ser
325 330 335 Ala Glu Gly Leu Phe Arg Arg Ala Leu Glu Glu Asn Val Ala
Phe Val 340 345 350 Pro Gly Gly Pro Phe Phe Ala Asn Gly Gly Gly Glu
Asn Thr Leu Arg 355 360 365 Leu Ser Tyr Ala Thr Leu Asp Arg Glu Gly
Ile Ala Glu Gly Val Arg 370 375 380 Arg Leu Gly Arg Ala Leu Lys Gly
Leu Leu Ala Leu Val Leu Glu 385 390 395 701248DNAThermotoga
maritimaCDS(1)..(1248) 70atg gtc gtc aat ctg gaa ggt aaa atc tct
aaa atc ggt caa aat atg 48Met Val Val Asn Leu Glu Gly Lys Ile Ser
Lys Ile Gly Gln Asn Met 1 5 10 15 aaa tcg agc att atc cgt gaa atc
ctg aaa ttc gct gcg gat aaa gac 96Lys Ser Ser Ile Ile Arg Glu Ile
Leu Lys Phe Ala Ala Asp Lys Asp 20 25 30 gcg att agc ttt ggc ggt
ggc gtg ccg gat ccg gaa acc ttc ccg cgt 144Ala Ile Ser Phe Gly Gly
Gly Val Pro Asp Pro Glu Thr Phe Pro Arg 35 40 45 aaa gaa ctg gca
gaa atc gct aaa gaa atc atc gaa aaa gaa tac cat 192Lys Glu Leu Ala
Glu Ile Ala Lys Glu Ile Ile Glu Lys Glu Tyr His 50 55 60 tac acc
ctg caa tac tct acc acg gaa ggc gat ccg gtt ctg aaa cag 240Tyr Thr
Leu Gln Tyr Ser Thr Thr Glu Gly Asp Pro Val Leu Lys Gln 65 70 75 80
caa att ctg aaa ctg ctg gaa cgt atg tac ggt att acc ggc ctg gat
288Gln Ile Leu Lys Leu Leu Glu Arg Met Tyr Gly Ile Thr Gly Leu Asp
85 90 95 gaa gac aac ctg atc ttt acg gtc ggc tca cag caa gcc ctg
gat ctg 336Glu Asp Asn Leu Ile Phe Thr Val Gly Ser Gln Gln Ala Leu
Asp Leu 100 105 110 att ggt aaa ctg ttc ctg gat gac gaa tcg tat tgc
gtt ctg gat gac 384Ile Gly Lys Leu Phe Leu Asp Asp Glu Ser Tyr Cys
Val Leu Asp Asp 115 120 125 ccg gca tac ctg ggt gca atc aac gca ttt
cgc cag tat ctg gcc aat 432Pro Ala Tyr Leu Gly Ala Ile Asn Ala Phe
Arg Gln Tyr Leu Ala Asn 130 135 140 ttc gtg gtt gtc ccg ctg gaa gat
gac ggc atg gat ctg aac gtg ctg 480Phe Val Val Val Pro Leu Glu Asp
Asp Gly Met Asp Leu Asn Val Leu 145 150 155 160 gaa cgt aaa ctg tca
gaa ttt gac aaa aac ggt aaa atc aaa caa gtt 528Glu Arg Lys Leu Ser
Glu Phe Asp Lys Asn Gly Lys Ile Lys Gln Val 165 170 175 aaa ttc atc
tac gtg gtt agc aac ttc cat aat ccg gca ggt gtg acc 576Lys Phe Ile
Tyr Val Val Ser Asn Phe His Asn Pro Ala Gly Val Thr 180 185 190 acg
tct ctg gaa aaa cgc aaa gcg ctg gtt gaa att gcc gaa aaa tac 624Thr
Ser Leu Glu Lys Arg Lys Ala Leu Val Glu Ile Ala Glu Lys Tyr 195 200
205 gac ctg ttt atc gtc gaa gat gac ccg tat ggc gct ctg cgc tac gaa
672Asp Leu Phe Ile Val Glu Asp Asp Pro Tyr Gly Ala Leu Arg Tyr Glu
210 215 220 ggt gaa acc gtg gac ccg att ttt aaa atc ggt ggc ccg gaa
cgt gtc 720Gly Glu Thr Val Asp Pro Ile Phe Lys Ile Gly Gly Pro Glu
Arg Val 225 230 235 240 gtg ctg ctg aac acg ttc agt aaa gtt ctg gca
ccg ggt ctg cgc att 768Val Leu Leu Asn Thr Phe Ser Lys Val Leu Ala
Pro Gly Leu Arg Ile 245 250 255 ggc atg gtc gct ggt tcc aaa gaa ttc
atc cgt aaa atc gtt cag gca 816Gly Met Val Ala Gly Ser Lys Glu Phe
Ile Arg Lys Ile Val Gln Ala 260 265 270 aaa caa agt gct gat ctg tgc
tcc ccg gca att acc cac cgt ctg gca 864Lys Gln Ser Ala Asp Leu Cys
Ser Pro Ala Ile Thr His Arg Leu Ala 275 280 285 gca cgc tat ctg gaa
cgt tac gac ctg ctg gaa cag ctg aaa ccg acc 912Ala Arg Tyr Leu Glu
Arg Tyr Asp Leu Leu Glu Gln Leu Lys Pro Thr 290 295 300 atc gaa ctg
tat cgt cgc aaa cgc acg gtg atg ctg aat gca ctg gaa 960Ile Glu Leu
Tyr Arg Arg Lys Arg Thr Val Met Leu Asn Ala Leu Glu 305 310 315 320
gaa tac ttt tca gat att ccg ggc gtt aaa tgg gtc aaa tcg gaa ggt
1008Glu Tyr Phe Ser Asp Ile Pro Gly Val Lys Trp Val Lys Ser Glu Gly
325 330 335 ggc ctg ttc atc tgg ctg acc ctg ccg gaa ggt ttt gat acg
tgg gaa 1056Gly Leu Phe Ile Trp Leu Thr Leu Pro Glu Gly Phe Asp Thr
Trp Glu 340 345 350 atg ttc gaa tat gcc aaa cgc aaa aaa gtg ttt tac
gtt ccg ggt cgt 1104Met Phe Glu Tyr Ala Lys Arg Lys Lys Val Phe Tyr
Val Pro Gly Arg 355 360 365 gtc ttc aaa gtg tat gat gaa ccg agc ccg
tct atg cgt ctg tcc ttt 1152Val Phe Lys Val Tyr Asp Glu Pro Ser Pro
Ser Met Arg Leu Ser Phe 370 375 380 tgt ctg ccg ccg gac gaa aaa atc
gtg gaa ggc atc aaa cgt ctg cgt 1200Cys Leu Pro Pro Asp Glu Lys Ile
Val Glu Gly Ile Lys Arg Leu Arg 385 390 395 400 gaa gtt gtg ctg gaa
tac ggt aaa gaa aaa cat ctg ctg ctc gag tga 1248Glu Val Val Leu Glu
Tyr Gly Lys Glu Lys His Leu Leu Leu Glu 405 410 415
71415PRTThermotoga maritima 71Met Val Val Asn Leu Glu Gly Lys Ile
Ser Lys Ile Gly Gln Asn Met 1 5 10 15 Lys Ser Ser Ile Ile Arg Glu
Ile Leu Lys Phe Ala Ala Asp Lys Asp 20 25 30 Ala Ile Ser Phe Gly
Gly Gly Val Pro Asp Pro Glu Thr Phe Pro Arg 35 40 45 Lys Glu Leu
Ala Glu Ile Ala Lys Glu Ile Ile Glu Lys Glu Tyr His 50 55 60 Tyr
Thr Leu Gln Tyr Ser Thr Thr Glu Gly Asp Pro Val Leu Lys Gln 65 70
75 80 Gln Ile Leu Lys Leu Leu Glu Arg Met Tyr Gly Ile Thr Gly Leu
Asp 85 90 95 Glu Asp Asn Leu Ile Phe Thr Val Gly Ser Gln Gln Ala
Leu Asp Leu 100 105 110 Ile Gly Lys Leu Phe Leu Asp Asp Glu Ser Tyr
Cys Val Leu Asp Asp 115 120 125 Pro Ala Tyr Leu Gly Ala Ile Asn Ala
Phe Arg Gln Tyr Leu Ala Asn 130 135 140 Phe Val Val Val Pro Leu Glu
Asp Asp Gly Met Asp Leu Asn Val Leu 145 150 155 160 Glu Arg Lys Leu
Ser Glu Phe Asp Lys Asn Gly Lys Ile Lys Gln Val 165 170 175 Lys Phe
Ile Tyr Val Val Ser Asn Phe His Asn Pro Ala Gly Val Thr 180 185 190
Thr Ser Leu Glu Lys Arg Lys Ala Leu Val Glu Ile Ala Glu Lys Tyr 195
200 205 Asp Leu Phe Ile Val Glu Asp Asp Pro Tyr Gly Ala Leu Arg Tyr
Glu 210 215 220 Gly Glu Thr Val Asp Pro Ile Phe Lys Ile Gly Gly Pro
Glu Arg Val 225 230 235 240 Val Leu Leu Asn Thr Phe Ser Lys Val Leu
Ala Pro Gly Leu Arg Ile 245 250 255 Gly Met Val Ala Gly Ser Lys Glu
Phe Ile Arg Lys Ile Val Gln Ala 260 265 270 Lys Gln Ser Ala Asp Leu
Cys Ser Pro Ala Ile Thr His Arg Leu Ala 275 280 285 Ala Arg Tyr Leu
Glu Arg Tyr Asp Leu Leu Glu Gln Leu Lys Pro Thr 290 295 300 Ile Glu
Leu Tyr Arg Arg Lys Arg Thr Val Met Leu Asn Ala Leu Glu 305 310 315
320 Glu Tyr Phe Ser Asp Ile Pro Gly Val Lys Trp Val Lys Ser Glu Gly
325 330
335 Gly Leu Phe Ile Trp Leu Thr Leu Pro Glu Gly Phe Asp Thr Trp Glu
340 345 350 Met Phe Glu Tyr Ala Lys Arg Lys Lys Val Phe Tyr Val Pro
Gly Arg 355 360 365 Val Phe Lys Val Tyr Asp Glu Pro Ser Pro Ser Met
Arg Leu Ser Phe 370 375 380 Cys Leu Pro Pro Asp Glu Lys Ile Val Glu
Gly Ile Lys Arg Leu Arg 385 390 395 400 Glu Val Val Leu Glu Tyr Gly
Lys Glu Lys His Leu Leu Leu Glu 405 410 415 721218DNAPyrococcus
horikoshiiCDS(1)..(1218) 72atg ctg ggc gat gtg gaa cgc ttc ttc tcg
aaa aaa gct ctg gaa atg 48Met Leu Gly Asp Val Glu Arg Phe Phe Ser
Lys Lys Ala Leu Glu Met 1 5 10 15 cgt gcg tct gaa gtc cgt gaa ctg
ctg aaa ctg gtg gaa acc agt gat 96Arg Ala Ser Glu Val Arg Glu Leu
Leu Lys Leu Val Glu Thr Ser Asp 20 25 30 att atc tcc ctg gcg ggc
ggt ctg ccg aac ccg aaa acg ttc ccg aaa 144Ile Ile Ser Leu Ala Gly
Gly Leu Pro Asn Pro Lys Thr Phe Pro Lys 35 40 45 gaa att atc cgt
gat atc ctg gtt gaa atc atg gaa aaa tat gca gac 192Glu Ile Ile Arg
Asp Ile Leu Val Glu Ile Met Glu Lys Tyr Ala Asp 50 55 60 aaa gct
ctg caa tac ggc acc acg aaa ggt ttt acc ccg ctg cgt gaa 240Lys Ala
Leu Gln Tyr Gly Thr Thr Lys Gly Phe Thr Pro Leu Arg Glu 65 70 75 80
acg ctg atg aaa tgg ctg ggc aaa cgc tat ggt att tcc cag gat aat
288Thr Leu Met Lys Trp Leu Gly Lys Arg Tyr Gly Ile Ser Gln Asp Asn
85 90 95 gac att atg atc acc agc ggt tct cag caa gcc ctg gat ctg
att ggc 336Asp Ile Met Ile Thr Ser Gly Ser Gln Gln Ala Leu Asp Leu
Ile Gly 100 105 110 cgc gtg ttc ctg aac ccg ggt gac atc gtg gtt gtc
gaa gca ccg acc 384Arg Val Phe Leu Asn Pro Gly Asp Ile Val Val Val
Glu Ala Pro Thr 115 120 125 tac ctg gcg gcc ctg caa gct ttt aat ttc
tat gaa ccg cag tac att 432Tyr Leu Ala Ala Leu Gln Ala Phe Asn Phe
Tyr Glu Pro Gln Tyr Ile 130 135 140 caa atc ccg ctg gat gac gaa ggc
atg aaa gtt gaa atc ctg gaa gaa 480Gln Ile Pro Leu Asp Asp Glu Gly
Met Lys Val Glu Ile Leu Glu Glu 145 150 155 160 aaa ctg aaa gaa ctg
aaa agc cag ggt aaa aaa gtc aaa gtg gtt tat 528Lys Leu Lys Glu Leu
Lys Ser Gln Gly Lys Lys Val Lys Val Val Tyr 165 170 175 acc gtg ccg
acg ttc caa aac ccg gcg ggt gtg acc atg aat gaa gat 576Thr Val Pro
Thr Phe Gln Asn Pro Ala Gly Val Thr Met Asn Glu Asp 180 185 190 cgt
cgc aaa tat ctg ctg gaa ctg gcc tca gaa tac gac ttt atc gtc 624Arg
Arg Lys Tyr Leu Leu Glu Leu Ala Ser Glu Tyr Asp Phe Ile Val 195 200
205 gtg gaa gat gac ccg tat ggc gaa ctg cgt tac tcg ggt aac ccg gaa
672Val Glu Asp Asp Pro Tyr Gly Glu Leu Arg Tyr Ser Gly Asn Pro Glu
210 215 220 aag aaa att aaa gcc ctg gat aat gaa ggc cgc gtt atc tac
ctg ggt 720Lys Lys Ile Lys Ala Leu Asp Asn Glu Gly Arg Val Ile Tyr
Leu Gly 225 230 235 240 acc ttt agc aaa att ctg gca ccg ggc ttc cgt
atc ggt tgg atg gtc 768Thr Phe Ser Lys Ile Leu Ala Pro Gly Phe Arg
Ile Gly Trp Met Val 245 250 255 ggc gat ccg ggt att atc cgc aaa atg
gaa att gca aaa cag tct acc 816Gly Asp Pro Gly Ile Ile Arg Lys Met
Glu Ile Ala Lys Gln Ser Thr 260 265 270 gac ctg tgc acg aac gtt ttt
ggc caa gtt gtc gct tgg cgt tat gtc 864Asp Leu Cys Thr Asn Val Phe
Gly Gln Val Val Ala Trp Arg Tyr Val 275 280 285 gat ggc ggt tac ctg
gaa aaa cat att ccg gaa atc cgc aaa ttt tat 912Asp Gly Gly Tyr Leu
Glu Lys His Ile Pro Glu Ile Arg Lys Phe Tyr 290 295 300 aaa ccg cgt
cgc gat gca atg ctg gaa gct ctg gaa gaa ttc atg ccg 960Lys Pro Arg
Arg Asp Ala Met Leu Glu Ala Leu Glu Glu Phe Met Pro 305 310 315 320
gaa ggt gtc aaa tgg acc aaa ccg gaa ggc ggt atg ttt att tgg gtg
1008Glu Gly Val Lys Trp Thr Lys Pro Glu Gly Gly Met Phe Ile Trp Val
325 330 335 acg ctg ccg gat ggc atc gac agc aag aaa atg ctg gaa cgt
gcg atc 1056Thr Leu Pro Asp Gly Ile Asp Ser Lys Lys Met Leu Glu Arg
Ala Ile 340 345 350 aaa aaa ggc gtg gcc tat gtt ccg ggt gaa gcg ttt
tac gcc cac cgt 1104Lys Lys Gly Val Ala Tyr Val Pro Gly Glu Ala Phe
Tyr Ala His Arg 355 360 365 gat gtg aaa aac acc atg cgc ctg aat ttc
acg tat gtt gac gaa gac 1152Asp Val Lys Asn Thr Met Arg Leu Asn Phe
Thr Tyr Val Asp Glu Asp 370 375 380 aaa atc atg gaa ggt atc aaa cgc
ctg gca gaa acg atc aaa gaa gaa 1200Lys Ile Met Glu Gly Ile Lys Arg
Leu Ala Glu Thr Ile Lys Glu Glu 385 390 395 400 ctg aaa gcg ctc gag
tga 1218Leu Lys Ala Leu Glu 405 73405PRTPyrococcus horikoshii 73Met
Leu Gly Asp Val Glu Arg Phe Phe Ser Lys Lys Ala Leu Glu Met 1 5 10
15 Arg Ala Ser Glu Val Arg Glu Leu Leu Lys Leu Val Glu Thr Ser Asp
20 25 30 Ile Ile Ser Leu Ala Gly Gly Leu Pro Asn Pro Lys Thr Phe
Pro Lys 35 40 45 Glu Ile Ile Arg Asp Ile Leu Val Glu Ile Met Glu
Lys Tyr Ala Asp 50 55 60 Lys Ala Leu Gln Tyr Gly Thr Thr Lys Gly
Phe Thr Pro Leu Arg Glu 65 70 75 80 Thr Leu Met Lys Trp Leu Gly Lys
Arg Tyr Gly Ile Ser Gln Asp Asn 85 90 95 Asp Ile Met Ile Thr Ser
Gly Ser Gln Gln Ala Leu Asp Leu Ile Gly 100 105 110 Arg Val Phe Leu
Asn Pro Gly Asp Ile Val Val Val Glu Ala Pro Thr 115 120 125 Tyr Leu
Ala Ala Leu Gln Ala Phe Asn Phe Tyr Glu Pro Gln Tyr Ile 130 135 140
Gln Ile Pro Leu Asp Asp Glu Gly Met Lys Val Glu Ile Leu Glu Glu 145
150 155 160 Lys Leu Lys Glu Leu Lys Ser Gln Gly Lys Lys Val Lys Val
Val Tyr 165 170 175 Thr Val Pro Thr Phe Gln Asn Pro Ala Gly Val Thr
Met Asn Glu Asp 180 185 190 Arg Arg Lys Tyr Leu Leu Glu Leu Ala Ser
Glu Tyr Asp Phe Ile Val 195 200 205 Val Glu Asp Asp Pro Tyr Gly Glu
Leu Arg Tyr Ser Gly Asn Pro Glu 210 215 220 Lys Lys Ile Lys Ala Leu
Asp Asn Glu Gly Arg Val Ile Tyr Leu Gly 225 230 235 240 Thr Phe Ser
Lys Ile Leu Ala Pro Gly Phe Arg Ile Gly Trp Met Val 245 250 255 Gly
Asp Pro Gly Ile Ile Arg Lys Met Glu Ile Ala Lys Gln Ser Thr 260 265
270 Asp Leu Cys Thr Asn Val Phe Gly Gln Val Val Ala Trp Arg Tyr Val
275 280 285 Asp Gly Gly Tyr Leu Glu Lys His Ile Pro Glu Ile Arg Lys
Phe Tyr 290 295 300 Lys Pro Arg Arg Asp Ala Met Leu Glu Ala Leu Glu
Glu Phe Met Pro 305 310 315 320 Glu Gly Val Lys Trp Thr Lys Pro Glu
Gly Gly Met Phe Ile Trp Val 325 330 335 Thr Leu Pro Asp Gly Ile Asp
Ser Lys Lys Met Leu Glu Arg Ala Ile 340 345 350 Lys Lys Gly Val Ala
Tyr Val Pro Gly Glu Ala Phe Tyr Ala His Arg 355 360 365 Asp Val Lys
Asn Thr Met Arg Leu Asn Phe Thr Tyr Val Asp Glu Asp 370 375 380 Lys
Ile Met Glu Gly Ile Lys Arg Leu Ala Glu Thr Ile Lys Glu Glu 385 390
395 400 Leu Lys Ala Leu Glu 405 741173DNAPhormidium
lapideumCDS(1)..(1173) 74atg aaa ctg gct gcc cgt gtt gaa agt gtg
tcc ccg agt atg acc ctg 48Met Lys Leu Ala Ala Arg Val Glu Ser Val
Ser Pro Ser Met Thr Leu 1 5 10 15 att att gat gcg aaa gca aaa gcg
atg aaa gcg gaa ggc att gat gtg 96Ile Ile Asp Ala Lys Ala Lys Ala
Met Lys Ala Glu Gly Ile Asp Val 20 25 30 tgc agt ttt tcc gcc ggt
gaa ccg gac ttc aac acg ccg aaa cat atc 144Cys Ser Phe Ser Ala Gly
Glu Pro Asp Phe Asn Thr Pro Lys His Ile 35 40 45 gtt gaa gcg gcc
aaa gca gct ctg gaa cag ggt aaa acc cgt tat ggt 192Val Glu Ala Ala
Lys Ala Ala Leu Glu Gln Gly Lys Thr Arg Tyr Gly 50 55 60 ccg gcg
gcc ggt gaa ccg cgt ctg cgt gaa gcg att gcc cag aaa ctg 240Pro Ala
Ala Gly Glu Pro Arg Leu Arg Glu Ala Ile Ala Gln Lys Leu 65 70 75 80
caa cgt gat aac ggc ctg tgt tac ggt gcg gac aac atc ctg gtt acc
288Gln Arg Asp Asn Gly Leu Cys Tyr Gly Ala Asp Asn Ile Leu Val Thr
85 90 95 aat ggc ggt aaa cag agt att ttt aat ctg atg ctg gcg atg
atc gaa 336Asn Gly Gly Lys Gln Ser Ile Phe Asn Leu Met Leu Ala Met
Ile Glu 100 105 110 ccg ggt gat gaa gtg att atc ccg gcc ccg ttc tgg
gtc tcc tat ccg 384Pro Gly Asp Glu Val Ile Ile Pro Ala Pro Phe Trp
Val Ser Tyr Pro 115 120 125 gaa atg gtg aaa ctg gcc gaa ggc acg ccg
gtt att ctg ccg acc acg 432Glu Met Val Lys Leu Ala Glu Gly Thr Pro
Val Ile Leu Pro Thr Thr 130 135 140 gtc gaa acc cag ttt aaa gtg agc
ccg gaa cag att cgc caa gcg atc 480Val Glu Thr Gln Phe Lys Val Ser
Pro Glu Gln Ile Arg Gln Ala Ile 145 150 155 160 acc ccg aaa acg aaa
ctg ctg gtt ttc aac acc ccg tct aat ccg acg 528Thr Pro Lys Thr Lys
Leu Leu Val Phe Asn Thr Pro Ser Asn Pro Thr 165 170 175 ggt atg gtt
tac acc ccg gat gaa gtc cgt gca att gct cag gtc gca 576Gly Met Val
Tyr Thr Pro Asp Glu Val Arg Ala Ile Ala Gln Val Ala 180 185 190 gtg
gaa gca ggt ctg tgg gtg ctg agt gat gaa atc tac gaa aaa atc 624Val
Glu Ala Gly Leu Trp Val Leu Ser Asp Glu Ile Tyr Glu Lys Ile 195 200
205 ctg tac gat gac gca caa cat ctg agt atc ggt gca gct tcc ccg gaa
672Leu Tyr Asp Asp Ala Gln His Leu Ser Ile Gly Ala Ala Ser Pro Glu
210 215 220 gcg tat gaa cgc agc gtg gtt tgc tct ggc ttt gcg aaa acc
tac gcc 720Ala Tyr Glu Arg Ser Val Val Cys Ser Gly Phe Ala Lys Thr
Tyr Ala 225 230 235 240 atg acg ggt tgg cgt gtt ggt ttc ctg gca ggt
ccg gtt ccg ctg gtc 768Met Thr Gly Trp Arg Val Gly Phe Leu Ala Gly
Pro Val Pro Leu Val 245 250 255 aaa gca gcc acc aaa att cag ggt cac
tca acg tcg aac gtc tgc acc 816Lys Ala Ala Thr Lys Ile Gln Gly His
Ser Thr Ser Asn Val Cys Thr 260 265 270 ttt gca caa tat ggc gct atc
gca gct tac gaa aat tct cag gat tgt 864Phe Ala Gln Tyr Gly Ala Ile
Ala Ala Tyr Glu Asn Ser Gln Asp Cys 275 280 285 gtg caa gaa atg ctg
gcg gcc ttt gcg gaa cgt cgc cgt tat atg ctg 912Val Gln Glu Met Leu
Ala Ala Phe Ala Glu Arg Arg Arg Tyr Met Leu 290 295 300 gat gca ctg
aat gct atg ccg ggt ctg gaa tgt ccg aaa ccg gac ggc 960Asp Ala Leu
Asn Ala Met Pro Gly Leu Glu Cys Pro Lys Pro Asp Gly 305 310 315 320
gcg ttt tac atg ttc ccg tca att gcc aaa acc ggt cgc agc tct ctg
1008Ala Phe Tyr Met Phe Pro Ser Ile Ala Lys Thr Gly Arg Ser Ser Leu
325 330 335 gat ttt tgc tcg gaa ctg ctg gac cag cac caa gtg gca acg
gtt ccg 1056Asp Phe Cys Ser Glu Leu Leu Asp Gln His Gln Val Ala Thr
Val Pro 340 345 350 ggt gca gct ttc ggc gct gat gac tgt atc cgt ctg
agc tat gca acc 1104Gly Ala Ala Phe Gly Ala Asp Asp Cys Ile Arg Leu
Ser Tyr Ala Thr 355 360 365 gac ctg gac acg atc aaa cgc ggt atg gaa
cgc ctg gaa aaa ttt ctg 1152Asp Leu Asp Thr Ile Lys Arg Gly Met Glu
Arg Leu Glu Lys Phe Leu 370 375 380 cac ggc att ctg ctc gag tga
1173His Gly Ile Leu Leu Glu 385 390 75390PRTPhormidium lapideum
75Met Lys Leu Ala Ala Arg Val Glu Ser Val Ser Pro Ser Met Thr Leu 1
5 10 15 Ile Ile Asp Ala Lys Ala Lys Ala Met Lys Ala Glu Gly Ile Asp
Val 20 25 30 Cys Ser Phe Ser Ala Gly Glu Pro Asp Phe Asn Thr Pro
Lys His Ile 35 40 45 Val Glu Ala Ala Lys Ala Ala Leu Glu Gln Gly
Lys Thr Arg Tyr Gly 50 55 60 Pro Ala Ala Gly Glu Pro Arg Leu Arg
Glu Ala Ile Ala Gln Lys Leu 65 70 75 80 Gln Arg Asp Asn Gly Leu Cys
Tyr Gly Ala Asp Asn Ile Leu Val Thr 85 90 95 Asn Gly Gly Lys Gln
Ser Ile Phe Asn Leu Met Leu Ala Met Ile Glu 100 105 110 Pro Gly Asp
Glu Val Ile Ile Pro Ala Pro Phe Trp Val Ser Tyr Pro 115 120 125 Glu
Met Val Lys Leu Ala Glu Gly Thr Pro Val Ile Leu Pro Thr Thr 130 135
140 Val Glu Thr Gln Phe Lys Val Ser Pro Glu Gln Ile Arg Gln Ala Ile
145 150 155 160 Thr Pro Lys Thr Lys Leu Leu Val Phe Asn Thr Pro Ser
Asn Pro Thr 165 170 175 Gly Met Val Tyr Thr Pro Asp Glu Val Arg Ala
Ile Ala Gln Val Ala 180 185 190 Val Glu Ala Gly Leu Trp Val Leu Ser
Asp Glu Ile Tyr Glu Lys Ile 195 200 205 Leu Tyr Asp Asp Ala Gln His
Leu Ser Ile Gly Ala Ala Ser Pro Glu 210 215 220 Ala Tyr Glu Arg Ser
Val Val Cys Ser Gly Phe Ala Lys Thr Tyr Ala 225 230 235 240 Met Thr
Gly Trp Arg Val Gly Phe Leu Ala Gly Pro Val Pro Leu Val 245 250 255
Lys Ala Ala Thr Lys Ile Gln Gly His Ser Thr Ser Asn Val Cys Thr 260
265 270 Phe Ala Gln Tyr Gly Ala Ile Ala Ala Tyr Glu Asn Ser Gln Asp
Cys 275 280 285 Val Gln Glu Met Leu Ala Ala Phe Ala Glu Arg Arg Arg
Tyr Met Leu 290 295 300 Asp Ala Leu Asn Ala Met Pro Gly Leu Glu Cys
Pro Lys Pro Asp Gly 305 310 315 320 Ala Phe Tyr Met Phe Pro Ser Ile
Ala Lys Thr Gly Arg Ser Ser Leu 325 330 335 Asp Phe Cys Ser Glu Leu
Leu Asp Gln His Gln Val Ala Thr Val Pro 340 345 350 Gly Ala Ala Phe
Gly Ala Asp Asp Cys Ile Arg Leu Ser Tyr Ala Thr 355 360 365 Asp Leu
Asp Thr Ile Lys Arg Gly Met Glu Arg Leu Glu Lys Phe Leu 370 375 380
His Gly Ile Leu Leu Glu 385 390 761164DNAThermus
thermophilusCDS(1)..(1164) 76atg cgt ggt ctg tcg cgt cgt gtc caa
gca atg aaa ccg tca gca acc 48Met Arg Gly Leu Ser Arg Arg Val Gln
Ala Met Lys Pro Ser Ala Thr 1 5 10 15 gtc gcc gtt aat gcc aaa gcc
ctg gaa ctg cgt cgt cag ggt gtc gat 96Val Ala Val Asn Ala Lys Ala
Leu Glu Leu Arg Arg Gln Gly Val Asp 20 25 30 ctg gtg gca ctg acc
gct ggc gaa ccg gat ttt gac acg ccg gaa cat 144Leu Val Ala Leu Thr
Ala Gly Glu Pro Asp Phe Asp Thr Pro Glu His 35 40 45 gtt aaa gaa
gcg gca cgt cgc gca ctg gca caa ggt aaa acc aaa tat 192Val Lys Glu
Ala Ala Arg Arg Ala Leu Ala Gln Gly Lys Thr Lys Tyr 50 55 60 gca
ccg ccg gcg ggt att ccg gaa ctg cgt gaa gca ctg gct gaa aaa 240Ala
Pro Pro Ala Gly Ile Pro Glu Leu Arg Glu Ala Leu Ala Glu Lys 65 70
75 80 ttc cgt cgc gaa aac ggt ctg agc gtg acg ccg gaa gaa acc atc
gtt 288Phe Arg Arg Glu Asn Gly Leu Ser Val Thr Pro Glu Glu Thr Ile
Val 85 90 95 acg gtc ggc ggt aaa cag gcg ctg ttt aac ctg ttt caa
gcc att ctg 336Thr Val Gly Gly Lys Gln Ala Leu Phe Asn Leu Phe Gln
Ala Ile Leu 100 105 110 gat ccg ggc gac gaa gtg atc gtt ctg tca ccg
tat tgg gtg tcg tac 384Asp Pro Gly Asp Glu Val Ile Val Leu Ser Pro
Tyr Trp Val Ser Tyr 115 120 125 ccg gaa atg gtt cgt ttt gcg ggc ggt
gtg gtt gtc gaa gtg gaa acc 432Pro Glu Met Val Arg Phe Ala Gly Gly
Val Val Val Glu Val Glu Thr 130 135 140 ctg ccg gaa gaa ggt ttc gtc
ccg gat ccg gaa cgt gtg cgt cgc gca 480Leu Pro Glu Glu Gly Phe Val
Pro Asp Pro Glu Arg Val Arg Arg Ala 145 150 155 160 att acc ccg cgc
acg aaa gct ctg gtg gtt aac tct ccg aac aat ccg 528Ile Thr Pro Arg
Thr Lys Ala Leu Val Val Asn Ser Pro Asn Asn Pro 165 170 175 acc ggc
gca gtt tat ccg aaa gaa gtc ctg gaa gca ctg gca cgt ctg 576Thr Gly
Ala Val Tyr Pro Lys Glu Val Leu Glu Ala Leu Ala Arg Leu 180 185 190
gca gtt gaa cat gat ttt tac ctg gtc agc gac gaa atc tat gaa cat
624Ala Val Glu His Asp Phe Tyr Leu Val Ser Asp Glu Ile Tyr Glu His
195 200 205 ctg ctg tac gaa ggt gaa cac ttc tcc ccg ggt cgt gtc gca
ccg gaa 672Leu Leu Tyr Glu Gly Glu His Phe Ser Pro Gly Arg Val Ala
Pro Glu 210 215 220 cac acc ctg acg gtg aac ggt gca gct aaa gca ttt
gct atg acc ggc 720His Thr Leu Thr Val Asn Gly Ala Ala Lys Ala Phe
Ala Met Thr Gly 225 230 235 240 tgg cgc att ggt tat gca tgc ggc ccg
aaa gaa gtg atc aaa gcg atg 768Trp Arg Ile Gly Tyr Ala Cys Gly Pro
Lys Glu Val Ile Lys Ala Met 245 250 255 gcc tca gtt agc tct cag agt
acc acg tcc ccg gat acg att gca caa 816Ala Ser Val Ser Ser Gln Ser
Thr Thr Ser Pro Asp Thr Ile Ala Gln 260 265 270 tgg gct acc ctg gaa
gcg ctg acg aat cag gaa gcg tcg cgt gcc ttt 864Trp Ala Thr Leu Glu
Ala Leu Thr Asn Gln Glu Ala Ser Arg Ala Phe 275 280 285 gtt gaa atg
gca cgc gaa gct tat cgt cgc cgt cgc gac ctg ctg ctg 912Val Glu Met
Ala Arg Glu Ala Tyr Arg Arg Arg Arg Asp Leu Leu Leu 290 295 300 gaa
ggt ctg acc gca ctg ggc ctg aaa gct gtt cgt ccg agc ggt gcg 960Glu
Gly Leu Thr Ala Leu Gly Leu Lys Ala Val Arg Pro Ser Gly Ala 305 310
315 320 ttc tac gtc ctg atg gat acg tct ccg atc gca ccg gac gaa gtt
cgt 1008Phe Tyr Val Leu Met Asp Thr Ser Pro Ile Ala Pro Asp Glu Val
Arg 325 330 335 gcg gcc gaa cgt ctg ctg gaa gcc ggt gtg gca gtc gtg
ccg ggc acc 1056Ala Ala Glu Arg Leu Leu Glu Ala Gly Val Ala Val Val
Pro Gly Thr 340 345 350 gat ttt gca gct ttc ggc cac gtg cgc ctg tca
tac gcc acc tcc gaa 1104Asp Phe Ala Ala Phe Gly His Val Arg Leu Ser
Tyr Ala Thr Ser Glu 355 360 365 gaa aat ctg cgt aaa gcc ctg gaa cgt
ttt gct cgt gtc ctg ggt cgt 1152Glu Asn Leu Arg Lys Ala Leu Glu Arg
Phe Ala Arg Val Leu Gly Arg 370 375 380 gcg ctc gag tga 1164Ala Leu
Glu 385 77387PRTThermus thermophilus 77Met Arg Gly Leu Ser Arg Arg
Val Gln Ala Met Lys Pro Ser Ala Thr 1 5 10 15 Val Ala Val Asn Ala
Lys Ala Leu Glu Leu Arg Arg Gln Gly Val Asp 20 25 30 Leu Val Ala
Leu Thr Ala Gly Glu Pro Asp Phe Asp Thr Pro Glu His 35 40 45 Val
Lys Glu Ala Ala Arg Arg Ala Leu Ala Gln Gly Lys Thr Lys Tyr 50 55
60 Ala Pro Pro Ala Gly Ile Pro Glu Leu Arg Glu Ala Leu Ala Glu Lys
65 70 75 80 Phe Arg Arg Glu Asn Gly Leu Ser Val Thr Pro Glu Glu Thr
Ile Val 85 90 95 Thr Val Gly Gly Lys Gln Ala Leu Phe Asn Leu Phe
Gln Ala Ile Leu 100 105 110 Asp Pro Gly Asp Glu Val Ile Val Leu Ser
Pro Tyr Trp Val Ser Tyr 115 120 125 Pro Glu Met Val Arg Phe Ala Gly
Gly Val Val Val Glu Val Glu Thr 130 135 140 Leu Pro Glu Glu Gly Phe
Val Pro Asp Pro Glu Arg Val Arg Arg Ala 145 150 155 160 Ile Thr Pro
Arg Thr Lys Ala Leu Val Val Asn Ser Pro Asn Asn Pro 165 170 175 Thr
Gly Ala Val Tyr Pro Lys Glu Val Leu Glu Ala Leu Ala Arg Leu 180 185
190 Ala Val Glu His Asp Phe Tyr Leu Val Ser Asp Glu Ile Tyr Glu His
195 200 205 Leu Leu Tyr Glu Gly Glu His Phe Ser Pro Gly Arg Val Ala
Pro Glu 210 215 220 His Thr Leu Thr Val Asn Gly Ala Ala Lys Ala Phe
Ala Met Thr Gly 225 230 235 240 Trp Arg Ile Gly Tyr Ala Cys Gly Pro
Lys Glu Val Ile Lys Ala Met 245 250 255 Ala Ser Val Ser Ser Gln Ser
Thr Thr Ser Pro Asp Thr Ile Ala Gln 260 265 270 Trp Ala Thr Leu Glu
Ala Leu Thr Asn Gln Glu Ala Ser Arg Ala Phe 275 280 285 Val Glu Met
Ala Arg Glu Ala Tyr Arg Arg Arg Arg Asp Leu Leu Leu 290 295 300 Glu
Gly Leu Thr Ala Leu Gly Leu Lys Ala Val Arg Pro Ser Gly Ala 305 310
315 320 Phe Tyr Val Leu Met Asp Thr Ser Pro Ile Ala Pro Asp Glu Val
Arg 325 330 335 Ala Ala Glu Arg Leu Leu Glu Ala Gly Val Ala Val Val
Pro Gly Thr 340 345 350 Asp Phe Ala Ala Phe Gly His Val Arg Leu Ser
Tyr Ala Thr Ser Glu 355 360 365 Glu Asn Leu Arg Lys Ala Leu Glu Arg
Phe Ala Arg Val Leu Gly Arg 370 375 380 Ala Leu Glu 385
781176DNAPyrococcus horikoshiiCDS(1)..(1176) 78atg gcc ctg tcg gac
cgt ctg gaa ctg gtg tcg gcg agt gaa att cgt 48Met Ala Leu Ser Asp
Arg Leu Glu Leu Val Ser Ala Ser Glu Ile Arg 1 5 10 15 aaa ctg ttt
gat att gcc gct ggt atg aaa gat gtt att agt ctg ggc 96Lys Leu Phe
Asp Ile Ala Ala Gly Met Lys Asp Val Ile Ser Leu Gly 20 25 30 att
ggt gaa ccg gat ttc gac acc ccg cag cat atc aaa gaa tat gct 144Ile
Gly Glu Pro Asp Phe Asp Thr Pro Gln His Ile Lys Glu Tyr Ala 35 40
45 aaa gaa gcg ctg gat aaa ggt ctg acg cac tac ggc ccg aac att ggt
192Lys Glu Ala Leu Asp Lys Gly Leu Thr His Tyr Gly Pro Asn Ile Gly
50 55 60 ctg ctg gaa ctg cgc gaa gcc atc gca gaa aaa ctg aaa aaa
cag aac 240Leu Leu Glu Leu Arg Glu Ala Ile Ala Glu Lys Leu Lys Lys
Gln Asn 65 70 75 80 ggc att gaa gct gat ccg aaa acc gaa atc atg gtt
ctg ctg ggc gcc 288Gly Ile Glu Ala Asp Pro Lys Thr Glu Ile Met Val
Leu Leu Gly Ala 85 90 95 aat caa gca ttt ctg atg ggt ctg agt gcc
ttc ctg aaa gac ggc gaa 336Asn Gln Ala Phe Leu Met Gly Leu Ser Ala
Phe Leu Lys Asp Gly Glu 100 105 110 gaa gtg ctg att ccg acc ccg gct
ttc gtc tcc tat gct ccg gcg gtg 384Glu Val Leu Ile Pro Thr Pro Ala
Phe Val Ser Tyr Ala Pro Ala Val 115 120 125 atc ctg gcg ggc ggt aaa
ccg gtt gaa gtc ccg acg tat gaa gaa gat 432Ile Leu Ala Gly Gly Lys
Pro Val Glu Val Pro Thr Tyr Glu Glu Asp 130 135 140 gaa ttt cgc ctg
aat gtt gac gaa ctg aaa aaa tac gtc acc gat aaa 480Glu Phe Arg Leu
Asn Val Asp Glu Leu Lys Lys Tyr Val Thr Asp Lys 145 150 155 160 acg
cgt gcc ctg att atc aac tca ccg tgc aat ccg acc ggt gcc gtt 528Thr
Arg Ala Leu Ile Ile Asn Ser Pro Cys Asn Pro Thr Gly Ala Val 165 170
175 ctg acg aaa aaa gat ctg gaa gaa att gca gac ttt gtg gtt gaa cat
576Leu Thr Lys Lys Asp Leu Glu Glu Ile Ala Asp Phe Val Val Glu His
180 185 190 gat ctg att gtg atc tcg gac gaa gtt tat gaa cat ttc att
tac gat 624Asp Leu Ile Val Ile Ser Asp Glu Val Tyr Glu His Phe Ile
Tyr Asp 195 200 205 gac gct cgc cac tac agc atc gcg tct ctg gat ggc
atg ttt gaa cgt 672Asp Ala Arg His Tyr Ser Ile Ala Ser Leu Asp Gly
Met Phe Glu Arg 210 215 220 acc atc acg gtg aac ggc ttt agc aaa acc
ttc gca atg acg ggt tgg 720Thr Ile Thr Val Asn Gly Phe Ser Lys Thr
Phe Ala Met Thr Gly Trp 225 230 235 240 cgt ctg ggt ttt gtg gca gca
ccg tct tgg att atc gaa cgt atg gtt 768Arg Leu Gly Phe Val Ala Ala
Pro Ser Trp Ile Ile Glu Arg Met Val 245 250 255 aaa ttc cag atg tac
aac gcg acc tgt ccg gtc acg ttc att caa tac 816Lys Phe Gln Met Tyr
Asn Ala Thr Cys Pro Val Thr Phe Ile Gln Tyr 260 265 270 gca gct gcg
aaa gcc ctg aaa gat gaa cgc tct tgg aaa gca gtt gaa 864Ala Ala Ala
Lys Ala Leu Lys Asp Glu Arg Ser Trp Lys Ala Val Glu 275 280 285 gaa
atg cgt aaa gaa tat gac cgt cgc cgt aaa ctg gtg tgg aaa cgt 912Glu
Met Arg Lys Glu Tyr Asp Arg Arg Arg Lys Leu Val Trp Lys Arg 290 295
300 ctg aac gaa atg ggt ctg ccg acc gtt aaa ccg aaa ggc gcg ttt tac
960Leu Asn Glu Met Gly Leu Pro Thr Val Lys Pro Lys Gly Ala Phe Tyr
305 310 315 320 att ttc ccg cgc atc cgt gat acc ggc ctg acg agt aaa
aaa ttc tcc 1008Ile Phe Pro Arg Ile Arg Asp Thr Gly Leu Thr Ser Lys
Lys Phe Ser 325 330 335 gaa ctg atg ctg aaa gaa gct cgc gtc gca gtc
gtg ccg ggt tca gca 1056Glu Leu Met Leu Lys Glu Ala Arg Val Ala Val
Val Pro Gly Ser Ala 340 345 350 ttt ggt aaa gca ggc gaa ggt tat gtg
cgt att tcg tat gcc acc gca 1104Phe Gly Lys Ala Gly Glu Gly Tyr Val
Arg Ile Ser Tyr Ala Thr Ala 355 360 365 tac gaa aaa ctg gaa gaa gcg
atg gac cgt atg gaa cgt gtg ctg aaa 1152Tyr Glu Lys Leu Glu Glu Ala
Met Asp Arg Met Glu Arg Val Leu Lys 370 375 380 gaa cgc aaa ctg gtg
ctc gag tga 1176Glu Arg Lys Leu Val Leu Glu 385 390
79391PRTPyrococcus horikoshii 79Met Ala Leu Ser Asp Arg Leu Glu Leu
Val Ser Ala Ser Glu Ile Arg 1 5 10 15 Lys Leu Phe Asp Ile Ala Ala
Gly Met Lys Asp Val Ile Ser Leu Gly 20 25 30 Ile Gly Glu Pro Asp
Phe Asp Thr Pro Gln His Ile Lys Glu Tyr Ala 35 40 45 Lys Glu Ala
Leu Asp Lys Gly Leu Thr His Tyr Gly Pro Asn Ile Gly 50 55 60 Leu
Leu Glu Leu Arg Glu Ala Ile Ala Glu Lys Leu Lys Lys Gln Asn 65 70
75 80 Gly Ile Glu Ala Asp Pro Lys Thr Glu Ile Met Val Leu Leu Gly
Ala 85 90 95 Asn Gln Ala Phe Leu Met Gly Leu Ser Ala Phe Leu Lys
Asp Gly Glu 100 105 110 Glu Val Leu Ile Pro Thr Pro Ala Phe Val Ser
Tyr Ala Pro Ala Val 115 120 125 Ile Leu Ala Gly Gly Lys Pro Val Glu
Val Pro Thr Tyr Glu Glu Asp 130 135 140 Glu Phe Arg Leu Asn Val Asp
Glu Leu Lys Lys Tyr Val Thr Asp Lys 145 150 155 160 Thr Arg Ala Leu
Ile Ile Asn Ser Pro Cys Asn Pro Thr Gly Ala Val 165 170 175 Leu Thr
Lys Lys Asp Leu Glu Glu Ile Ala Asp Phe Val Val Glu His 180 185 190
Asp Leu Ile Val Ile Ser Asp Glu Val Tyr Glu His Phe Ile Tyr Asp 195
200 205 Asp Ala Arg His Tyr Ser Ile Ala Ser Leu Asp Gly Met Phe Glu
Arg 210 215 220 Thr Ile Thr Val Asn Gly Phe Ser Lys Thr Phe Ala Met
Thr Gly Trp 225 230 235 240 Arg Leu Gly Phe Val Ala Ala Pro Ser Trp
Ile Ile Glu Arg Met Val 245 250 255 Lys Phe Gln Met Tyr Asn Ala Thr
Cys Pro Val Thr Phe Ile Gln Tyr 260 265 270 Ala Ala Ala Lys Ala Leu
Lys Asp Glu Arg Ser Trp Lys Ala Val Glu 275 280 285 Glu Met Arg Lys
Glu Tyr Asp Arg Arg Arg Lys Leu Val Trp Lys Arg 290 295 300 Leu Asn
Glu Met Gly Leu Pro Thr Val Lys Pro Lys Gly Ala Phe Tyr 305 310 315
320 Ile Phe Pro Arg Ile Arg Asp Thr Gly Leu Thr Ser Lys Lys Phe Ser
325 330 335 Glu Leu Met Leu Lys Glu Ala Arg Val Ala Val Val Pro Gly
Ser Ala 340 345 350 Phe Gly Lys Ala Gly Glu Gly Tyr Val Arg Ile Ser
Tyr Ala Thr Ala 355 360 365 Tyr Glu Lys Leu Glu Glu Ala Met Asp Arg
Met Glu Arg Val Leu Lys 370 375 380 Glu Arg Lys Leu Val Leu Glu 385
390 801119DNAMethanococcus jannaschiiCDS(1)..(1119) 80atg ctg tct
aaa cgc ctg ctg aat ttt gaa tct ttt gaa gtt atg gac 48Met Leu Ser
Lys Arg Leu Leu Asn Phe Glu Ser Phe Glu Val Met Asp 1 5 10 15 atc
ctg gca ctg gca cag aaa ctg gaa tcg gaa ggt aaa aaa gtg att 96Ile
Leu Ala Leu Ala Gln Lys Leu Glu Ser Glu Gly Lys Lys Val Ile 20 25
30 cat ctg gaa atc ggt gaa ccg gat ttt aac acc ccg aaa ccg att gtt
144His Leu Glu Ile Gly Glu Pro Asp Phe Asn Thr Pro Lys Pro Ile Val
35 40 45 gac gaa ggt atc aaa agc ctg aaa gaa ggc aaa acc cac tat
acg gat 192Asp Glu Gly Ile Lys Ser Leu Lys Glu Gly Lys Thr His Tyr
Thr Asp 50 55 60 tct cgt ggc att ctg gaa ctg cgc gaa aaa atc agt
gaa ctg tac aaa 240Ser Arg Gly Ile Leu Glu
Leu Arg Glu Lys Ile Ser Glu Leu Tyr Lys 65 70 75 80 gac aaa tac aaa
gca gat atc atc ccg gac aat att atc att acg ggc 288Asp Lys Tyr Lys
Ala Asp Ile Ile Pro Asp Asn Ile Ile Ile Thr Gly 85 90 95 ggt agc
tct ctg ggt ctg ttt ttc gct ctg agt tcc atc att gat gac 336Gly Ser
Ser Leu Gly Leu Phe Phe Ala Leu Ser Ser Ile Ile Asp Asp 100 105 110
ggc gat gaa gtg ctg att cag aac ccg tgc tat ccg tgt tac aaa aat
384Gly Asp Glu Val Leu Ile Gln Asn Pro Cys Tyr Pro Cys Tyr Lys Asn
115 120 125 ttt atc cgt ttc ctg ggt gca aaa ccg gtc ttt tgc gat ttc
acc gtg 432Phe Ile Arg Phe Leu Gly Ala Lys Pro Val Phe Cys Asp Phe
Thr Val 130 135 140 gaa agt ctg gaa gaa gca ctg tcc gac aaa acg aaa
gct atc att atc 480Glu Ser Leu Glu Glu Ala Leu Ser Asp Lys Thr Lys
Ala Ile Ile Ile 145 150 155 160 aac tca ccg tcg aat ccg ctg ggc gaa
gtc att gat cgc gaa atc tat 528Asn Ser Pro Ser Asn Pro Leu Gly Glu
Val Ile Asp Arg Glu Ile Tyr 165 170 175 gaa ttt gcg tac gaa aac att
ccg tac att atc tcc gac gaa atc tat 576Glu Phe Ala Tyr Glu Asn Ile
Pro Tyr Ile Ile Ser Asp Glu Ile Tyr 180 185 190 aat ggt ctg gtg tac
gaa ggc aaa tgt tat tct gcc att gaa ttc gat 624Asn Gly Leu Val Tyr
Glu Gly Lys Cys Tyr Ser Ala Ile Glu Phe Asp 195 200 205 gaa aac ctg
gaa aaa acc att ctg atc aat ggt ttt agc aaa ctg tac 672Glu Asn Leu
Glu Lys Thr Ile Leu Ile Asn Gly Phe Ser Lys Leu Tyr 210 215 220 gcg
atg acg ggt tgg cgt att ggc tac gtt atc agt aac gat gaa atc 720Ala
Met Thr Gly Trp Arg Ile Gly Tyr Val Ile Ser Asn Asp Glu Ile 225 230
235 240 atc gaa gcc att ctg aaa ctg caa caa aac ctg ttt att agc gca
ccg 768Ile Glu Ala Ile Leu Lys Leu Gln Gln Asn Leu Phe Ile Ser Ala
Pro 245 250 255 acc atc tct caa tat gcg gcc ctg aaa gct ttc gaa aaa
gaa acg gaa 816Thr Ile Ser Gln Tyr Ala Ala Leu Lys Ala Phe Glu Lys
Glu Thr Glu 260 265 270 cgc gaa att aac agc atg atc aaa gaa ttc gat
cgt cgc cgt cgc ctg 864Arg Glu Ile Asn Ser Met Ile Lys Glu Phe Asp
Arg Arg Arg Arg Leu 275 280 285 gtg ctg aaa tac gtt aaa gac ttt ggt
tgg gaa gtt aac aat ccg att 912Val Leu Lys Tyr Val Lys Asp Phe Gly
Trp Glu Val Asn Asn Pro Ile 290 295 300 ggc gcc tat tac gtc ttc ccg
aac atc ggt gaa gat ggc cgt gaa ttc 960Gly Ala Tyr Tyr Val Phe Pro
Asn Ile Gly Glu Asp Gly Arg Glu Phe 305 310 315 320 gcg tac aaa ctg
ctg aaa gaa aaa ttc gtc gcc ctg acc ccg ggc att 1008Ala Tyr Lys Leu
Leu Lys Glu Lys Phe Val Ala Leu Thr Pro Gly Ile 325 330 335 ggt ttt
ggc tca aaa ggc aaa aat tac att cgc atc tcg tat gcc aac 1056Gly Phe
Gly Ser Lys Gly Lys Asn Tyr Ile Arg Ile Ser Tyr Ala Asn 340 345 350
agt tac gaa aac atc aaa gaa ggt ctg gaa cgc atc aaa gaa ttc ctg
1104Ser Tyr Glu Asn Ile Lys Glu Gly Leu Glu Arg Ile Lys Glu Phe Leu
355 360 365 aac aaa ctc gag tga 1119Asn Lys Leu Glu 370
81372PRTMethanococcus jannaschii 81Met Leu Ser Lys Arg Leu Leu Asn
Phe Glu Ser Phe Glu Val Met Asp 1 5 10 15 Ile Leu Ala Leu Ala Gln
Lys Leu Glu Ser Glu Gly Lys Lys Val Ile 20 25 30 His Leu Glu Ile
Gly Glu Pro Asp Phe Asn Thr Pro Lys Pro Ile Val 35 40 45 Asp Glu
Gly Ile Lys Ser Leu Lys Glu Gly Lys Thr His Tyr Thr Asp 50 55 60
Ser Arg Gly Ile Leu Glu Leu Arg Glu Lys Ile Ser Glu Leu Tyr Lys 65
70 75 80 Asp Lys Tyr Lys Ala Asp Ile Ile Pro Asp Asn Ile Ile Ile
Thr Gly 85 90 95 Gly Ser Ser Leu Gly Leu Phe Phe Ala Leu Ser Ser
Ile Ile Asp Asp 100 105 110 Gly Asp Glu Val Leu Ile Gln Asn Pro Cys
Tyr Pro Cys Tyr Lys Asn 115 120 125 Phe Ile Arg Phe Leu Gly Ala Lys
Pro Val Phe Cys Asp Phe Thr Val 130 135 140 Glu Ser Leu Glu Glu Ala
Leu Ser Asp Lys Thr Lys Ala Ile Ile Ile 145 150 155 160 Asn Ser Pro
Ser Asn Pro Leu Gly Glu Val Ile Asp Arg Glu Ile Tyr 165 170 175 Glu
Phe Ala Tyr Glu Asn Ile Pro Tyr Ile Ile Ser Asp Glu Ile Tyr 180 185
190 Asn Gly Leu Val Tyr Glu Gly Lys Cys Tyr Ser Ala Ile Glu Phe Asp
195 200 205 Glu Asn Leu Glu Lys Thr Ile Leu Ile Asn Gly Phe Ser Lys
Leu Tyr 210 215 220 Ala Met Thr Gly Trp Arg Ile Gly Tyr Val Ile Ser
Asn Asp Glu Ile 225 230 235 240 Ile Glu Ala Ile Leu Lys Leu Gln Gln
Asn Leu Phe Ile Ser Ala Pro 245 250 255 Thr Ile Ser Gln Tyr Ala Ala
Leu Lys Ala Phe Glu Lys Glu Thr Glu 260 265 270 Arg Glu Ile Asn Ser
Met Ile Lys Glu Phe Asp Arg Arg Arg Arg Leu 275 280 285 Val Leu Lys
Tyr Val Lys Asp Phe Gly Trp Glu Val Asn Asn Pro Ile 290 295 300 Gly
Ala Tyr Tyr Val Phe Pro Asn Ile Gly Glu Asp Gly Arg Glu Phe 305 310
315 320 Ala Tyr Lys Leu Leu Lys Glu Lys Phe Val Ala Leu Thr Pro Gly
Ile 325 330 335 Gly Phe Gly Ser Lys Gly Lys Asn Tyr Ile Arg Ile Ser
Tyr Ala Asn 340 345 350 Ser Tyr Glu Asn Ile Lys Glu Gly Leu Glu Arg
Ile Lys Glu Phe Leu 355 360 365 Asn Lys Leu Glu 370
821200DNAThermotoga maritimaCDS(1)..(1200) 82atg gat gtc ttt agc
gac cgt gtc ctg ctg acc gaa gaa tca ccg atc 48Met Asp Val Phe Ser
Asp Arg Val Leu Leu Thr Glu Glu Ser Pro Ile 1 5 10 15 cgc aaa ctg
gtt ccg ttt gct gaa atg gcg aaa aaa cgc ggc gtc cgt 96Arg Lys Leu
Val Pro Phe Ala Glu Met Ala Lys Lys Arg Gly Val Arg 20 25 30 att
cat cac ctg aac atc ggt cag ccg gat ctg aaa acc ccg gaa gtg 144Ile
His His Leu Asn Ile Gly Gln Pro Asp Leu Lys Thr Pro Glu Val 35 40
45 ttt ttc gaa cgc atc tat gaa aat aaa ccg gaa gtg gtt tat tac agc
192Phe Phe Glu Arg Ile Tyr Glu Asn Lys Pro Glu Val Val Tyr Tyr Ser
50 55 60 cat agc gcg ggc att tgg gaa ctg cgt gaa gcg ttt gcc agc
tat tac 240His Ser Ala Gly Ile Trp Glu Leu Arg Glu Ala Phe Ala Ser
Tyr Tyr 65 70 75 80 aaa cgt cgc caa cgc gtt gat gtc aaa ccg gaa aac
gtg ctg gtt acc 288Lys Arg Arg Gln Arg Val Asp Val Lys Pro Glu Asn
Val Leu Val Thr 85 90 95 aat ggc ggt tct gaa gcc att ctg ttt agt
ttc gca gtc atc gct aac 336Asn Gly Gly Ser Glu Ala Ile Leu Phe Ser
Phe Ala Val Ile Ala Asn 100 105 110 ccg ggt gac gaa att ctg gtg ctg
gaa ccg ttt tat gcg aac tac aat 384Pro Gly Asp Glu Ile Leu Val Leu
Glu Pro Phe Tyr Ala Asn Tyr Asn 115 120 125 gca ttc gct aaa att gcc
ggc gtg aaa ctg atc ccg gtt acg cgt cgc 432Ala Phe Ala Lys Ile Ala
Gly Val Lys Leu Ile Pro Val Thr Arg Arg 130 135 140 atg gaa gaa ggt
ttt gcg atc ccg cag aac ctg gaa tcg ttc atc aat 480Met Glu Glu Gly
Phe Ala Ile Pro Gln Asn Leu Glu Ser Phe Ile Asn 145 150 155 160 gaa
cgt acc aaa ggc att gtt ctg agc aac ccg tgc aat ccg acg ggc 528Glu
Arg Thr Lys Gly Ile Val Leu Ser Asn Pro Cys Asn Pro Thr Gly 165 170
175 gtc gtg tat ggt aaa gat gaa atg cgt tac ctg gtt gaa att gcc gaa
576Val Val Tyr Gly Lys Asp Glu Met Arg Tyr Leu Val Glu Ile Ala Glu
180 185 190 cgc cac ggc ctg ttt ctg atc gtc gac gaa gtg tac agt gaa
att gtg 624Arg His Gly Leu Phe Leu Ile Val Asp Glu Val Tyr Ser Glu
Ile Val 195 200 205 ttt cgc ggt gaa ttc gcg tca gcc ctg tcg atc gaa
agc gat aaa gtt 672Phe Arg Gly Glu Phe Ala Ser Ala Leu Ser Ile Glu
Ser Asp Lys Val 210 215 220 gtc gtg att gac agt gtt tcc aaa aaa ttc
tct gcg tgc ggc gcc cgt 720Val Val Ile Asp Ser Val Ser Lys Lys Phe
Ser Ala Cys Gly Ala Arg 225 230 235 240 gtc ggt tgt ctg atc acc cgc
aac gaa gaa ctg att agt cat gca atg 768Val Gly Cys Leu Ile Thr Arg
Asn Glu Glu Leu Ile Ser His Ala Met 245 250 255 aaa ctg gct cag ggt
cgt ctg gca ccg ccg ctg ctg gaa caa atc ggc 816Lys Leu Ala Gln Gly
Arg Leu Ala Pro Pro Leu Leu Glu Gln Ile Gly 260 265 270 tcc gtg ggt
ctg ctg aat ctg gat gac tca ttt ttc gat ttt gtt cgt 864Ser Val Gly
Leu Leu Asn Leu Asp Asp Ser Phe Phe Asp Phe Val Arg 275 280 285 gaa
acc tat cgt gaa cgc gtt gaa acg gtc ctg aaa aaa ctg gaa gaa 912Glu
Thr Tyr Arg Glu Arg Val Glu Thr Val Leu Lys Lys Leu Glu Glu 290 295
300 cac ggc ctg aaa cgc ttt acc aaa ccg tcc ggt gca ttc tac att acg
960His Gly Leu Lys Arg Phe Thr Lys Pro Ser Gly Ala Phe Tyr Ile Thr
305 310 315 320 gct gaa ctg ccg gtg gaa gac gcg gaa gaa ttt gcc cgc
tgg atg ctg 1008Ala Glu Leu Pro Val Glu Asp Ala Glu Glu Phe Ala Arg
Trp Met Leu 325 330 335 acc gat ttc aat atg gac ggc gaa acc acg atg
gtt gca ccg ctg cgt 1056Thr Asp Phe Asn Met Asp Gly Glu Thr Thr Met
Val Ala Pro Leu Arg 340 345 350 ggt ttt tat ctg acg ccg ggc ctg ggt
aaa aaa gaa att cgc atc gct 1104Gly Phe Tyr Leu Thr Pro Gly Leu Gly
Lys Lys Glu Ile Arg Ile Ala 355 360 365 tgt gtg ctg gaa aaa gat ctg
ctg tct cgt gcg att gat gtt ctg atg 1152Cys Val Leu Glu Lys Asp Leu
Leu Ser Arg Ala Ile Asp Val Leu Met 370 375 380 gaa ggt ctg aaa atg
ttc tgt agc agc cgt atc tcc tgt ctc gag tga 1200Glu Gly Leu Lys Met
Phe Cys Ser Ser Arg Ile Ser Cys Leu Glu 385 390 395
83399PRTThermotoga maritima 83Met Asp Val Phe Ser Asp Arg Val Leu
Leu Thr Glu Glu Ser Pro Ile 1 5 10 15 Arg Lys Leu Val Pro Phe Ala
Glu Met Ala Lys Lys Arg Gly Val Arg 20 25 30 Ile His His Leu Asn
Ile Gly Gln Pro Asp Leu Lys Thr Pro Glu Val 35 40 45 Phe Phe Glu
Arg Ile Tyr Glu Asn Lys Pro Glu Val Val Tyr Tyr Ser 50 55 60 His
Ser Ala Gly Ile Trp Glu Leu Arg Glu Ala Phe Ala Ser Tyr Tyr 65 70
75 80 Lys Arg Arg Gln Arg Val Asp Val Lys Pro Glu Asn Val Leu Val
Thr 85 90 95 Asn Gly Gly Ser Glu Ala Ile Leu Phe Ser Phe Ala Val
Ile Ala Asn 100 105 110 Pro Gly Asp Glu Ile Leu Val Leu Glu Pro Phe
Tyr Ala Asn Tyr Asn 115 120 125 Ala Phe Ala Lys Ile Ala Gly Val Lys
Leu Ile Pro Val Thr Arg Arg 130 135 140 Met Glu Glu Gly Phe Ala Ile
Pro Gln Asn Leu Glu Ser Phe Ile Asn 145 150 155 160 Glu Arg Thr Lys
Gly Ile Val Leu Ser Asn Pro Cys Asn Pro Thr Gly 165 170 175 Val Val
Tyr Gly Lys Asp Glu Met Arg Tyr Leu Val Glu Ile Ala Glu 180 185 190
Arg His Gly Leu Phe Leu Ile Val Asp Glu Val Tyr Ser Glu Ile Val 195
200 205 Phe Arg Gly Glu Phe Ala Ser Ala Leu Ser Ile Glu Ser Asp Lys
Val 210 215 220 Val Val Ile Asp Ser Val Ser Lys Lys Phe Ser Ala Cys
Gly Ala Arg 225 230 235 240 Val Gly Cys Leu Ile Thr Arg Asn Glu Glu
Leu Ile Ser His Ala Met 245 250 255 Lys Leu Ala Gln Gly Arg Leu Ala
Pro Pro Leu Leu Glu Gln Ile Gly 260 265 270 Ser Val Gly Leu Leu Asn
Leu Asp Asp Ser Phe Phe Asp Phe Val Arg 275 280 285 Glu Thr Tyr Arg
Glu Arg Val Glu Thr Val Leu Lys Lys Leu Glu Glu 290 295 300 His Gly
Leu Lys Arg Phe Thr Lys Pro Ser Gly Ala Phe Tyr Ile Thr 305 310 315
320 Ala Glu Leu Pro Val Glu Asp Ala Glu Glu Phe Ala Arg Trp Met Leu
325 330 335 Thr Asp Phe Asn Met Asp Gly Glu Thr Thr Met Val Ala Pro
Leu Arg 340 345 350 Gly Phe Tyr Leu Thr Pro Gly Leu Gly Lys Lys Glu
Ile Arg Ile Ala 355 360 365 Cys Val Leu Glu Lys Asp Leu Leu Ser Arg
Ala Ile Asp Val Leu Met 370 375 380 Glu Gly Leu Lys Met Phe Cys Ser
Ser Arg Ile Ser Cys Leu Glu 385 390 395 841263DNASaccharomyces
cerevisiaeCDS(1)..(1263) 84atg tca gcc acc ctg ttc aat aat atc gaa
ctg ctg ccg ccg gac gcc 48Met Ser Ala Thr Leu Phe Asn Asn Ile Glu
Leu Leu Pro Pro Asp Ala 1 5 10 15 ctg ttt ggt atc aaa caa cgc tat
ggt caa gat caa cgc gcc acc aaa 96Leu Phe Gly Ile Lys Gln Arg Tyr
Gly Gln Asp Gln Arg Ala Thr Lys 20 25 30 gtt gac ctg ggc att ggt
gca tat cgt gat gac aat ggc aaa ccg tgg 144Val Asp Leu Gly Ile Gly
Ala Tyr Arg Asp Asp Asn Gly Lys Pro Trp 35 40 45 gtc ctg ccg agt
gtg aaa gcg gcc gaa aaa ctg atc cat aac gat agc 192Val Leu Pro Ser
Val Lys Ala Ala Glu Lys Leu Ile His Asn Asp Ser 50 55 60 tct tac
aat cac gaa tac ctg ggc atc acc ggt ctg ccg agc ctg acg 240Ser Tyr
Asn His Glu Tyr Leu Gly Ile Thr Gly Leu Pro Ser Leu Thr 65 70 75 80
tct aac gca gct aaa att atc ttt ggt acc cag agc gac gcg ctg caa
288Ser Asn Ala Ala Lys Ile Ile Phe Gly Thr Gln Ser Asp Ala Leu Gln
85 90 95 gaa gat cgc gtc att tct gtg cag tca ctg tcg ggc acg ggt
gca ctg 336Glu Asp Arg Val Ile Ser Val Gln Ser Leu Ser Gly Thr Gly
Ala Leu 100 105 110 cat atc agt gct aaa ttt ttc tcc aaa ttt ttc ccg
gac aaa ctg gtt 384His Ile Ser Ala Lys Phe Phe Ser Lys Phe Phe Pro
Asp Lys Leu Val 115 120 125 tac ctg tca aaa ccg acc tgg gca aac cac
atg gct att ttc gaa aat 432Tyr Leu Ser Lys Pro Thr Trp Ala Asn His
Met Ala Ile Phe Glu Asn 130 135 140 cag ggc ctg aaa acc gct acg tat
ccg tac tgg gcc aat gaa acg aaa 480Gln Gly Leu Lys Thr Ala Thr Tyr
Pro Tyr Trp Ala Asn Glu Thr Lys 145 150 155 160 tcg ctg gat ctg aac
ggc ttt ctg aat gcg att caa aaa gcc ccg gaa 528Ser Leu
Asp Leu Asn Gly Phe Leu Asn Ala Ile Gln Lys Ala Pro Glu 165 170 175
ggt tca atc ttc gtg ctg cat tcg tgc gcg cac aat ccg acc ggt ctg
576Gly Ser Ile Phe Val Leu His Ser Cys Ala His Asn Pro Thr Gly Leu
180 185 190 gac ccg acg agt gaa cag tgg gtt caa att gtc gat gcg atc
gcc tcc 624Asp Pro Thr Ser Glu Gln Trp Val Gln Ile Val Asp Ala Ile
Ala Ser 195 200 205 aaa aac cat atc gcg ctg ttt gat acc gcc tat cag
ggc ttc gca acg 672Lys Asn His Ile Ala Leu Phe Asp Thr Ala Tyr Gln
Gly Phe Ala Thr 210 215 220 ggt gat ctg gac aaa gat gca tac gct gtt
cgt ctg ggc gtc gaa aaa 720Gly Asp Leu Asp Lys Asp Ala Tyr Ala Val
Arg Leu Gly Val Glu Lys 225 230 235 240 ctg agt acc gtt tcc ccg gtg
ttt gtt tgc caa tca ttc gcg aaa aac 768Leu Ser Thr Val Ser Pro Val
Phe Val Cys Gln Ser Phe Ala Lys Asn 245 250 255 gcc ggc atg tat ggt
gaa cgc gtc ggt tgt ttt cat ctg gct ctg acc 816Ala Gly Met Tyr Gly
Glu Arg Val Gly Cys Phe His Leu Ala Leu Thr 260 265 270 aaa cag gcg
caa aat aaa acc att aaa ccg gca gtg acg tct cag ctg 864Lys Gln Ala
Gln Asn Lys Thr Ile Lys Pro Ala Val Thr Ser Gln Leu 275 280 285 gcg
aaa att atc cgt tca gaa gtg tcg aac ccg ccg gca tac ggc gct 912Ala
Lys Ile Ile Arg Ser Glu Val Ser Asn Pro Pro Ala Tyr Gly Ala 290 295
300 aaa atc gtt gcc aaa ctg ctg gaa acc ccg gaa ctg acg gaa cag tgg
960Lys Ile Val Ala Lys Leu Leu Glu Thr Pro Glu Leu Thr Glu Gln Trp
305 310 315 320 cac aaa gat atg gtg acc atg agt tcc cgc att acg aaa
atg cgt cat 1008His Lys Asp Met Val Thr Met Ser Ser Arg Ile Thr Lys
Met Arg His 325 330 335 gcg ctg cgc gac cac ctg gtc aaa ctg ggc acc
ccg ggt aac tgg gat 1056Ala Leu Arg Asp His Leu Val Lys Leu Gly Thr
Pro Gly Asn Trp Asp 340 345 350 cat atc gtg aat cag tgt ggc atg ttt
agc ttc acc ggt ctg acg ccg 1104His Ile Val Asn Gln Cys Gly Met Phe
Ser Phe Thr Gly Leu Thr Pro 355 360 365 caa atg gtt aaa cgt ctg gaa
gaa acc cac gcc gtg tat ctg gtt gca 1152Gln Met Val Lys Arg Leu Glu
Glu Thr His Ala Val Tyr Leu Val Ala 370 375 380 agc ggt cgc gcg tct
att gcc ggc ctg aac cag ggt aat gtc gaa tat 1200Ser Gly Arg Ala Ser
Ile Ala Gly Leu Asn Gln Gly Asn Val Glu Tyr 385 390 395 400 gtc gca
aaa gcc att gac gaa gtg gtc cgt ttc tac gca acc gaa gca 1248Val Ala
Lys Ala Ile Asp Glu Val Val Arg Phe Tyr Ala Thr Glu Ala 405 410 415
aaa ctg ctc gag tga 1263Lys Leu Leu Glu 420 85420PRTSaccharomyces
cerevisiae 85Met Ser Ala Thr Leu Phe Asn Asn Ile Glu Leu Leu Pro
Pro Asp Ala 1 5 10 15 Leu Phe Gly Ile Lys Gln Arg Tyr Gly Gln Asp
Gln Arg Ala Thr Lys 20 25 30 Val Asp Leu Gly Ile Gly Ala Tyr Arg
Asp Asp Asn Gly Lys Pro Trp 35 40 45 Val Leu Pro Ser Val Lys Ala
Ala Glu Lys Leu Ile His Asn Asp Ser 50 55 60 Ser Tyr Asn His Glu
Tyr Leu Gly Ile Thr Gly Leu Pro Ser Leu Thr 65 70 75 80 Ser Asn Ala
Ala Lys Ile Ile Phe Gly Thr Gln Ser Asp Ala Leu Gln 85 90 95 Glu
Asp Arg Val Ile Ser Val Gln Ser Leu Ser Gly Thr Gly Ala Leu 100 105
110 His Ile Ser Ala Lys Phe Phe Ser Lys Phe Phe Pro Asp Lys Leu Val
115 120 125 Tyr Leu Ser Lys Pro Thr Trp Ala Asn His Met Ala Ile Phe
Glu Asn 130 135 140 Gln Gly Leu Lys Thr Ala Thr Tyr Pro Tyr Trp Ala
Asn Glu Thr Lys 145 150 155 160 Ser Leu Asp Leu Asn Gly Phe Leu Asn
Ala Ile Gln Lys Ala Pro Glu 165 170 175 Gly Ser Ile Phe Val Leu His
Ser Cys Ala His Asn Pro Thr Gly Leu 180 185 190 Asp Pro Thr Ser Glu
Gln Trp Val Gln Ile Val Asp Ala Ile Ala Ser 195 200 205 Lys Asn His
Ile Ala Leu Phe Asp Thr Ala Tyr Gln Gly Phe Ala Thr 210 215 220 Gly
Asp Leu Asp Lys Asp Ala Tyr Ala Val Arg Leu Gly Val Glu Lys 225 230
235 240 Leu Ser Thr Val Ser Pro Val Phe Val Cys Gln Ser Phe Ala Lys
Asn 245 250 255 Ala Gly Met Tyr Gly Glu Arg Val Gly Cys Phe His Leu
Ala Leu Thr 260 265 270 Lys Gln Ala Gln Asn Lys Thr Ile Lys Pro Ala
Val Thr Ser Gln Leu 275 280 285 Ala Lys Ile Ile Arg Ser Glu Val Ser
Asn Pro Pro Ala Tyr Gly Ala 290 295 300 Lys Ile Val Ala Lys Leu Leu
Glu Thr Pro Glu Leu Thr Glu Gln Trp 305 310 315 320 His Lys Asp Met
Val Thr Met Ser Ser Arg Ile Thr Lys Met Arg His 325 330 335 Ala Leu
Arg Asp His Leu Val Lys Leu Gly Thr Pro Gly Asn Trp Asp 340 345 350
His Ile Val Asn Gln Cys Gly Met Phe Ser Phe Thr Gly Leu Thr Pro 355
360 365 Gln Met Val Lys Arg Leu Glu Glu Thr His Ala Val Tyr Leu Val
Ala 370 375 380 Ser Gly Arg Ala Ser Ile Ala Gly Leu Asn Gln Gly Asn
Val Glu Tyr 385 390 395 400 Val Ala Lys Ala Ile Asp Glu Val Val Arg
Phe Tyr Ala Thr Glu Ala 405 410 415 Lys Leu Leu Glu 420
861200DNAEubacterium rectaleCDS(1)..(1200) 86atg gtc gtc aat gaa
tca atg tat caa ctg ggc tcg gtc cgc tcg gca 48Met Val Val Asn Glu
Ser Met Tyr Gln Leu Gly Ser Val Arg Ser Ala 1 5 10 15 atc cgt gaa
ctg ttc gaa tat ggc aaa aaa cgt gct gcg att gtt ggc 96Ile Arg Glu
Leu Phe Glu Tyr Gly Lys Lys Arg Ala Ala Ile Val Gly 20 25 30 aaa
gaa aac gtc tat gat ttt agc att ggt aat ccg tct atc ccg gcc 144Lys
Glu Asn Val Tyr Asp Phe Ser Ile Gly Asn Pro Ser Ile Pro Ala 35 40
45 ccg cag att gtt aac gac acc atc aaa gaa ctg gtg acg gat tat gac
192Pro Gln Ile Val Asn Asp Thr Ile Lys Glu Leu Val Thr Asp Tyr Asp
50 55 60 tct gtt gct ctg cat ggc tac acc agt gcg caa ggt gat gtg
gaa acg 240Ser Val Ala Leu His Gly Tyr Thr Ser Ala Gln Gly Asp Val
Glu Thr 65 70 75 80 cgt gcg gcc att gct gaa ttt ctg aac aat acc cat
ggc acg cac ttc 288Arg Ala Ala Ile Ala Glu Phe Leu Asn Asn Thr His
Gly Thr His Phe 85 90 95 aac gcc gac aat ctg tac atg acc atg ggt
gca gct gcg agc ctg tct 336Asn Ala Asp Asn Leu Tyr Met Thr Met Gly
Ala Ala Ala Ser Leu Ser 100 105 110 atc tgc ttt cgt gcc ctg acc agc
gat gcg tat gat gaa ttc att acg 384Ile Cys Phe Arg Ala Leu Thr Ser
Asp Ala Tyr Asp Glu Phe Ile Thr 115 120 125 atc gcg ccg tat ttt ccg
gaa tac aaa gtg ttc gtt aat gcc gca ggc 432Ile Ala Pro Tyr Phe Pro
Glu Tyr Lys Val Phe Val Asn Ala Ala Gly 130 135 140 gca cgc ctg gtc
gaa gtg ccg gca gat acc gaa cat ttt cag att gat 480Ala Arg Leu Val
Glu Val Pro Ala Asp Thr Glu His Phe Gln Ile Asp 145 150 155 160 ttc
gac gct ctg gaa gaa cgt atc aac gcg cac acg cgc ggc gtt att 528Phe
Asp Ala Leu Glu Glu Arg Ile Asn Ala His Thr Arg Gly Val Ile 165 170
175 atc aat agt ccg aac aat ccg tcc ggt acc gtc tat tca gaa gaa acg
576Ile Asn Ser Pro Asn Asn Pro Ser Gly Thr Val Tyr Ser Glu Glu Thr
180 185 190 atc aaa aaa ctg tcg gat ctg ctg gaa aag aaa agc aaa gaa
att ggc 624Ile Lys Lys Leu Ser Asp Leu Leu Glu Lys Lys Ser Lys Glu
Ile Gly 195 200 205 cgt ccg atc ttt att atc gcg gat gaa ccg tat cgc
gaa att gtt tac 672Arg Pro Ile Phe Ile Ile Ala Asp Glu Pro Tyr Arg
Glu Ile Val Tyr 210 215 220 gac ggt atc aaa gtg ccg ttc gtt acc aaa
tat tac gat aac acg ctg 720Asp Gly Ile Lys Val Pro Phe Val Thr Lys
Tyr Tyr Asp Asn Thr Leu 225 230 235 240 gtg tgc tat agt tac tcc aaa
tca ctg tcg ctg ccg ggc gaa cgt atc 768Val Cys Tyr Ser Tyr Ser Lys
Ser Leu Ser Leu Pro Gly Glu Arg Ile 245 250 255 ggt tac gtt ctg gtc
ccg gat gaa gtt tat gac aaa gca gaa ctg tac 816Gly Tyr Val Leu Val
Pro Asp Glu Val Tyr Asp Lys Ala Glu Leu Tyr 260 265 270 gct gcg gtc
tgc ggt gct ggt cgt gca ctg ggt tat gtg tgt gcg ccg 864Ala Ala Val
Cys Gly Ala Gly Arg Ala Leu Gly Tyr Val Cys Ala Pro 275 280 285 agt
ctg ttc cag aaa atg atc gtt aaa tgt caa ggc gcc acc ggt gat 912Ser
Leu Phe Gln Lys Met Ile Val Lys Cys Gln Gly Ala Thr Gly Asp 290 295
300 atc aac gca tat aaa gaa aat cgt gac ctg ctg tac gaa ggc ctg acc
960Ile Asn Ala Tyr Lys Glu Asn Arg Asp Leu Leu Tyr Glu Gly Leu Thr
305 310 315 320 cgc att ggt tat cac tgc ttc aaa ccg gat ggc gcc ttt
tac atg ttc 1008Arg Ile Gly Tyr His Cys Phe Lys Pro Asp Gly Ala Phe
Tyr Met Phe 325 330 335 gtg aaa gca ctg gaa gat gac tcc aat gct ttt
tgt gaa aaa gcg aaa 1056Val Lys Ala Leu Glu Asp Asp Ser Asn Ala Phe
Cys Glu Lys Ala Lys 340 345 350 gaa gaa gat gtc ctg att gtg gcc gca
gac ggt ttc ggt tgc ccg ggt 1104Glu Glu Asp Val Leu Ile Val Ala Ala
Asp Gly Phe Gly Cys Pro Gly 355 360 365 tgg gtc cgt atc tct tat tgt
gtg gat cgt gaa atg att aaa cac agc 1152Trp Val Arg Ile Ser Tyr Cys
Val Asp Arg Glu Met Ile Lys His Ser 370 375 380 atg ccg gcc ttt gaa
aaa atc tat aaa aaa tac aat aaa ctc gag tga 1200Met Pro Ala Phe Glu
Lys Ile Tyr Lys Lys Tyr Asn Lys Leu Glu 385 390 395
87399PRTEubacterium rectale 87Met Val Val Asn Glu Ser Met Tyr Gln
Leu Gly Ser Val Arg Ser Ala 1 5 10 15 Ile Arg Glu Leu Phe Glu Tyr
Gly Lys Lys Arg Ala Ala Ile Val Gly 20 25 30 Lys Glu Asn Val Tyr
Asp Phe Ser Ile Gly Asn Pro Ser Ile Pro Ala 35 40 45 Pro Gln Ile
Val Asn Asp Thr Ile Lys Glu Leu Val Thr Asp Tyr Asp 50 55 60 Ser
Val Ala Leu His Gly Tyr Thr Ser Ala Gln Gly Asp Val Glu Thr 65 70
75 80 Arg Ala Ala Ile Ala Glu Phe Leu Asn Asn Thr His Gly Thr His
Phe 85 90 95 Asn Ala Asp Asn Leu Tyr Met Thr Met Gly Ala Ala Ala
Ser Leu Ser 100 105 110 Ile Cys Phe Arg Ala Leu Thr Ser Asp Ala Tyr
Asp Glu Phe Ile Thr 115 120 125 Ile Ala Pro Tyr Phe Pro Glu Tyr Lys
Val Phe Val Asn Ala Ala Gly 130 135 140 Ala Arg Leu Val Glu Val Pro
Ala Asp Thr Glu His Phe Gln Ile Asp 145 150 155 160 Phe Asp Ala Leu
Glu Glu Arg Ile Asn Ala His Thr Arg Gly Val Ile 165 170 175 Ile Asn
Ser Pro Asn Asn Pro Ser Gly Thr Val Tyr Ser Glu Glu Thr 180 185 190
Ile Lys Lys Leu Ser Asp Leu Leu Glu Lys Lys Ser Lys Glu Ile Gly 195
200 205 Arg Pro Ile Phe Ile Ile Ala Asp Glu Pro Tyr Arg Glu Ile Val
Tyr 210 215 220 Asp Gly Ile Lys Val Pro Phe Val Thr Lys Tyr Tyr Asp
Asn Thr Leu 225 230 235 240 Val Cys Tyr Ser Tyr Ser Lys Ser Leu Ser
Leu Pro Gly Glu Arg Ile 245 250 255 Gly Tyr Val Leu Val Pro Asp Glu
Val Tyr Asp Lys Ala Glu Leu Tyr 260 265 270 Ala Ala Val Cys Gly Ala
Gly Arg Ala Leu Gly Tyr Val Cys Ala Pro 275 280 285 Ser Leu Phe Gln
Lys Met Ile Val Lys Cys Gln Gly Ala Thr Gly Asp 290 295 300 Ile Asn
Ala Tyr Lys Glu Asn Arg Asp Leu Leu Tyr Glu Gly Leu Thr 305 310 315
320 Arg Ile Gly Tyr His Cys Phe Lys Pro Asp Gly Ala Phe Tyr Met Phe
325 330 335 Val Lys Ala Leu Glu Asp Asp Ser Asn Ala Phe Cys Glu Lys
Ala Lys 340 345 350 Glu Glu Asp Val Leu Ile Val Ala Ala Asp Gly Phe
Gly Cys Pro Gly 355 360 365 Trp Val Arg Ile Ser Tyr Cys Val Asp Arg
Glu Met Ile Lys His Ser 370 375 380 Met Pro Ala Phe Glu Lys Ile Tyr
Lys Lys Tyr Asn Lys Leu Glu 385 390 395 881314DNABacillus
pumilusCDS(1)..(1314) 88atg tcg ggc ttt acg gca ctg tcg gaa gcg gaa
ctg aat gac ctg tat 48Met Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu
Leu Asn Asp Leu Tyr 1 5 10 15 gcg gca ctg caa gat gaa tat gaa acc
tac aaa tcc aaa aac ctg cat 96Ala Ala Leu Gln Asp Glu Tyr Glu Thr
Tyr Lys Ser Lys Asn Leu His 20 25 30 ctg gac atg agc cgt ggc aaa
ccg tct ccg aaa cag ctg gat ctg agt 144Leu Asp Met Ser Arg Gly Lys
Pro Ser Pro Lys Gln Leu Asp Leu Ser 35 40 45 atg ggt atg ctg gat
gtg gtt acc tcc aaa gac gca atg acc gct gaa 192Met Gly Met Leu Asp
Val Val Thr Ser Lys Asp Ala Met Thr Ala Glu 50 55 60 gat ggc acg
gac gtg cgc aac tat ggc ggt ctg acg ggt ctg agc gaa 240Asp Gly Thr
Asp Val Arg Asn Tyr Gly Gly Leu Thr Gly Leu Ser Glu 65 70 75 80 acc
aaa acg ttt ttc gcg gat gtt ctg aat ctg aaa ccg gaa caa att 288Thr
Lys Thr Phe Phe Ala Asp Val Leu Asn Leu Lys Pro Glu Gln Ile 85 90
95 atc att ggc ggt aac agc tct ctg aat atg atg cat gac acc atc gca
336Ile Ile Gly Gly Asn Ser Ser Leu Asn Met Met His Asp Thr Ile Ala
100 105 110 cgt gct atg acg cac ggc gtt tat gat tct aaa acc ccg tgg
ggt aaa 384Arg Ala Met Thr His Gly Val Tyr Asp Ser Lys Thr Pro Trp
Gly Lys 115 120 125 ctg ccg aaa gtc aaa ttt ctg gca ccg tca ccg ggt
tac gat cgt cac 432Leu Pro Lys Val Lys Phe Leu Ala Pro Ser Pro Gly
Tyr Asp Arg His 130 135 140 ttt tcg att tgc gaa ctg ttc aac atc gaa
atg att acc gtc gat atg 480Phe Ser Ile Cys Glu Leu Phe Asn Ile Glu
Met Ile Thr Val Asp Met 145 150 155 160 aaa gcc gac ggt ccg gat atg
gac cag gtg gaa aaa ctg gtt gcg gaa 528Lys Ala Asp Gly Pro Asp Met
Asp Gln Val Glu Lys Leu Val Ala Glu 165 170 175 gac gaa gcc atc aaa
ggc att tgg tgt gtt ccg aaa tat agc aat ccg 576Asp Glu Ala Ile Lys
Gly Ile Trp Cys Val Pro Lys
Tyr Ser Asn Pro 180 185 190 gat ggt att acc tac tct gat gaa gtc gtg
gac cgt ctg gcg agc atg 624Asp Gly Ile Thr Tyr Ser Asp Glu Val Val
Asp Arg Leu Ala Ser Met 195 200 205 aaa acg aaa gcc gat gac ttt cgc
atc ttc tgg gat gac gcg tat gcc 672Lys Thr Lys Ala Asp Asp Phe Arg
Ile Phe Trp Asp Asp Ala Tyr Ala 210 215 220 gtg cat cac ctg acc gat
acg ccg gac acc ctg aaa gat att ttt cag 720Val His His Leu Thr Asp
Thr Pro Asp Thr Leu Lys Asp Ile Phe Gln 225 230 235 240 gca gtc gaa
gaa gct ggc cat ccg cac cgt gtg ttt atg ttc gca tca 768Ala Val Glu
Glu Ala Gly His Pro His Arg Val Phe Met Phe Ala Ser 245 250 255 acc
tcg aaa atc acg ttt ccg ggc agc ggt att gcg ctg atg gcc agt 816Thr
Ser Lys Ile Thr Phe Pro Gly Ser Gly Ile Ala Leu Met Ala Ser 260 265
270 tcc ctg gat aac gtt agt ttc acc cag aaa caa ctg tcc atc cag acg
864Ser Leu Asp Asn Val Ser Phe Thr Gln Lys Gln Leu Ser Ile Gln Thr
275 280 285 att ggc ccg gat aaa atc aac caa ctg cgt cat ctg cgc ttt
ttc aaa 912Ile Gly Pro Asp Lys Ile Asn Gln Leu Arg His Leu Arg Phe
Phe Lys 290 295 300 aat ccg gaa ggt ctg aaa gaa cac atg cgc aaa cac
gcg gcc atc att 960Asn Pro Glu Gly Leu Lys Glu His Met Arg Lys His
Ala Ala Ile Ile 305 310 315 320 aaa ccg aaa ttt gat ctg gtg ctg tca
atc ctg gac gaa acc ctg ggc 1008Lys Pro Lys Phe Asp Leu Val Leu Ser
Ile Leu Asp Glu Thr Leu Gly 325 330 335 ggt aaa gat att gcc gaa tgg
cat aaa ccg aac ggc ggt tac ttc att 1056Gly Lys Asp Ile Ala Glu Trp
His Lys Pro Asn Gly Gly Tyr Phe Ile 340 345 350 tcg ctg aat acc ctg
gat cac tgc gca aaa gct gtt gtc cag aaa gca 1104Ser Leu Asn Thr Leu
Asp His Cys Ala Lys Ala Val Val Gln Lys Ala 355 360 365 aaa gaa gct
ggt gtt acc atg acg ggt gca ggt gca acc tat ccg tac 1152Lys Glu Ala
Gly Val Thr Met Thr Gly Ala Gly Ala Thr Tyr Pro Tyr 370 375 380 ggt
aaa gat ccg ctg gac cgt aat atc cgc att gca ccg acc ttt ccg 1200Gly
Lys Asp Pro Leu Asp Arg Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390
395 400 agt ctg gaa gaa ctg gaa caa gct atc gat att ttc acg ctg tgt
gtt 1248Ser Leu Glu Glu Leu Glu Gln Ala Ile Asp Ile Phe Thr Leu Cys
Val 405 410 415 caa ctg gtg tct atc gaa aaa ctg ctg tcc aaa aaa tct
caa tct gct 1296Gln Leu Val Ser Ile Glu Lys Leu Leu Ser Lys Lys Ser
Gln Ser Ala 420 425 430 ccg acc gtg ctc gag tga 1314Pro Thr Val Leu
Glu 435 89437PRTBacillus pumilus 89Met Ser Gly Phe Thr Ala Leu Ser
Glu Ala Glu Leu Asn Asp Leu Tyr 1 5 10 15 Ala Ala Leu Gln Asp Glu
Tyr Glu Thr Tyr Lys Ser Lys Asn Leu His 20 25 30 Leu Asp Met Ser
Arg Gly Lys Pro Ser Pro Lys Gln Leu Asp Leu Ser 35 40 45 Met Gly
Met Leu Asp Val Val Thr Ser Lys Asp Ala Met Thr Ala Glu 50 55 60
Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Thr Gly Leu Ser Glu 65
70 75 80 Thr Lys Thr Phe Phe Ala Asp Val Leu Asn Leu Lys Pro Glu
Gln Ile 85 90 95 Ile Ile Gly Gly Asn Ser Ser Leu Asn Met Met His
Asp Thr Ile Ala 100 105 110 Arg Ala Met Thr His Gly Val Tyr Asp Ser
Lys Thr Pro Trp Gly Lys 115 120 125 Leu Pro Lys Val Lys Phe Leu Ala
Pro Ser Pro Gly Tyr Asp Arg His 130 135 140 Phe Ser Ile Cys Glu Leu
Phe Asn Ile Glu Met Ile Thr Val Asp Met 145 150 155 160 Lys Ala Asp
Gly Pro Asp Met Asp Gln Val Glu Lys Leu Val Ala Glu 165 170 175 Asp
Glu Ala Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro 180 185
190 Asp Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Leu Ala Ser Met
195 200 205 Lys Thr Lys Ala Asp Asp Phe Arg Ile Phe Trp Asp Asp Ala
Tyr Ala 210 215 220 Val His His Leu Thr Asp Thr Pro Asp Thr Leu Lys
Asp Ile Phe Gln 225 230 235 240 Ala Val Glu Glu Ala Gly His Pro His
Arg Val Phe Met Phe Ala Ser 245 250 255 Thr Ser Lys Ile Thr Phe Pro
Gly Ser Gly Ile Ala Leu Met Ala Ser 260 265 270 Ser Leu Asp Asn Val
Ser Phe Thr Gln Lys Gln Leu Ser Ile Gln Thr 275 280 285 Ile Gly Pro
Asp Lys Ile Asn Gln Leu Arg His Leu Arg Phe Phe Lys 290 295 300 Asn
Pro Glu Gly Leu Lys Glu His Met Arg Lys His Ala Ala Ile Ile 305 310
315 320 Lys Pro Lys Phe Asp Leu Val Leu Ser Ile Leu Asp Glu Thr Leu
Gly 325 330 335 Gly Lys Asp Ile Ala Glu Trp His Lys Pro Asn Gly Gly
Tyr Phe Ile 340 345 350 Ser Leu Asn Thr Leu Asp His Cys Ala Lys Ala
Val Val Gln Lys Ala 355 360 365 Lys Glu Ala Gly Val Thr Met Thr Gly
Ala Gly Ala Thr Tyr Pro Tyr 370 375 380 Gly Lys Asp Pro Leu Asp Arg
Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390 395 400 Ser Leu Glu Glu
Leu Glu Gln Ala Ile Asp Ile Phe Thr Leu Cys Val 405 410 415 Gln Leu
Val Ser Ile Glu Lys Leu Leu Ser Lys Lys Ser Gln Ser Ala 420 425 430
Pro Thr Val Leu Glu 435 90 1296DNABacillus
cellulosilyticusCDS(1)..(1296) 90atg agc gat tac acc gtg ctg tca
acg caa gaa ctg caa caa gtc cac 48Met Ser Asp Tyr Thr Val Leu Ser
Thr Gln Glu Leu Gln Gln Val His 1 5 10 15 atg gac ctg ctg gaa aaa
ttc aac aaa ctg aaa gac gaa aat ctg gcg 96Met Asp Leu Leu Glu Lys
Phe Asn Lys Leu Lys Asp Glu Asn Leu Ala 20 25 30 ctg gat atg agt
cgt ggt aaa ccg tcc ccg gat caa ctg gac ctg tca 144Leu Asp Met Ser
Arg Gly Lys Pro Ser Pro Asp Gln Leu Asp Leu Ser 35 40 45 aac ggc
atg ctg gat att atc tcg gcg gac acc ccg ctg aaa gcc gaa 192Asn Gly
Met Leu Asp Ile Ile Ser Ala Asp Thr Pro Leu Lys Ala Glu 50 55 60
gat ggt acg gac gtg cgc aat tat ggc ggt ctg gat ggc ctg ccg gaa
240Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Asp Gly Leu Pro Glu
65 70 75 80 gcg aaa gcc ttt ttc agc aac att ctg aat gtt agc tct aac
gaa att 288Ala Lys Ala Phe Phe Ser Asn Ile Leu Asn Val Ser Ser Asn
Glu Ile 85 90 95 atc att ggc ggt aac agt tcc ctg aat ctg atg cat
gat acc gtt gca 336Ile Ile Gly Gly Asn Ser Ser Leu Asn Leu Met His
Asp Thr Val Ala 100 105 110 cgt gct atg ctg ttt ggt gtc aat gac ggc
gaa acg gcg tgg gcc aaa 384Arg Ala Met Leu Phe Gly Val Asn Asp Gly
Glu Thr Ala Trp Ala Lys 115 120 125 ctg ccg aaa gtc aaa ttc ctg tgc
ccg agc ccg ggt tac gat cgt cac 432Leu Pro Lys Val Lys Phe Leu Cys
Pro Ser Pro Gly Tyr Asp Arg His 130 135 140 ttc tct att tgt gaa ctg
ttc aac atc gaa atg att cgc gtt gat atg 480Phe Ser Ile Cys Glu Leu
Phe Asn Ile Glu Met Ile Arg Val Asp Met 145 150 155 160 ctg gaa gac
ggc ccg aac atg gat cag atc gaa aaa ctg gtg caa gaa 528Leu Glu Asp
Gly Pro Asn Met Asp Gln Ile Glu Lys Leu Val Gln Glu 165 170 175 gac
gaa agt atc aaa ggt att tgg tgc gtt ccg aaa tat agt aac ccg 576Asp
Glu Ser Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro 180 185
190 gat ggc att acc tac tcc gat gaa gtg gtt gac cgt ttt gca agc atg
624Asp Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Phe Ala Ser Met
195 200 205 aaa acg aaa gct aaa gac ttt cgc att ttc tgg gat gac gcg
tat acc 672Lys Thr Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp Asp Ala
Tyr Thr 210 215 220 gtg cat cac ctg acg gat aaa ccg gac gaa ctg aaa
aat atc ctg acc 720Val His His Leu Thr Asp Lys Pro Asp Glu Leu Lys
Asn Ile Leu Thr 225 230 235 240 gca tgt aaa cag gct ggc aac gaa gat
cgc gtt ctg atc ttt tca tcg 768Ala Cys Lys Gln Ala Gly Asn Glu Asp
Arg Val Leu Ile Phe Ser Ser 245 250 255 acc agc aaa att acg ttc gcg
ggc ggt ggc atc ggt gtc ctg gcc agc 816Thr Ser Lys Ile Thr Phe Ala
Gly Gly Gly Ile Gly Val Leu Ala Ser 260 265 270 tct gaa aac aac atc
cag tac ttc aaa aaa ctg ctg gca atg caa acc 864Ser Glu Asn Asn Ile
Gln Tyr Phe Lys Lys Leu Leu Ala Met Gln Thr 275 280 285 atc ggc ccg
gat aaa ctg aat cag atc cgt cat att cgc ttt ttc aaa 912Ile Gly Pro
Asp Lys Leu Asn Gln Ile Arg His Ile Arg Phe Phe Lys 290 295 300 aac
gtg gaa aat ctg tca acc cac atg aaa aaa cac gcc tcg atc att 960Asn
Val Glu Asn Leu Ser Thr His Met Lys Lys His Ala Ser Ile Ile 305 310
315 320 aaa ccg aaa ttt gat atg gtc ctg aac aaa ctg gaa agc gaa atc
ggt 1008Lys Pro Lys Phe Asp Met Val Leu Asn Lys Leu Glu Ser Glu Ile
Gly 325 330 335 ggc aaa aac att ggt tct tgg gtg gaa ccg aat ggt ggc
tat ttt att 1056Gly Lys Asn Ile Gly Ser Trp Val Glu Pro Asn Gly Gly
Tyr Phe Ile 340 345 350 agt ttc aac acc ctg gat ggt tgc gcc aaa acg
gtc gtg tcc atg gcg 1104Ser Phe Asn Thr Leu Asp Gly Cys Ala Lys Thr
Val Val Ser Met Ala 355 360 365 aaa gaa gcc ggc gtt aaa ctg acc ggt
gca ggc gct acg ttt ccg tac 1152Lys Glu Ala Gly Val Lys Leu Thr Gly
Ala Gly Ala Thr Phe Pro Tyr 370 375 380 ggt cac gat ccg cgt gac cgc
aat atc cgt att gca ccg acc ttc ccg 1200Gly His Asp Pro Arg Asp Arg
Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390 395 400 tct ctg atc gaa
ctg gaa cgc gct atg gat gtc ttc tgc ctg tgt gtg 1248Ser Leu Ile Glu
Leu Glu Arg Ala Met Asp Val Phe Cys Leu Cys Val 405 410 415 caa ctg
gcg tcg gtg gaa aaa ctg ctg aaa gaa caa ctg ctc gag tga 1296Gln Leu
Ala Ser Val Glu Lys Leu Leu Lys Glu Gln Leu Leu Glu 420 425 430
91431PRTBacillus cellulosilyticus 91Met Ser Asp Tyr Thr Val Leu Ser
Thr Gln Glu Leu Gln Gln Val His 1 5 10 15 Met Asp Leu Leu Glu Lys
Phe Asn Lys Leu Lys Asp Glu Asn Leu Ala 20 25 30 Leu Asp Met Ser
Arg Gly Lys Pro Ser Pro Asp Gln Leu Asp Leu Ser 35 40 45 Asn Gly
Met Leu Asp Ile Ile Ser Ala Asp Thr Pro Leu Lys Ala Glu 50 55 60
Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Asp Gly Leu Pro Glu 65
70 75 80 Ala Lys Ala Phe Phe Ser Asn Ile Leu Asn Val Ser Ser Asn
Glu Ile 85 90 95 Ile Ile Gly Gly Asn Ser Ser Leu Asn Leu Met His
Asp Thr Val Ala 100 105 110 Arg Ala Met Leu Phe Gly Val Asn Asp Gly
Glu Thr Ala Trp Ala Lys 115 120 125 Leu Pro Lys Val Lys Phe Leu Cys
Pro Ser Pro Gly Tyr Asp Arg His 130 135 140 Phe Ser Ile Cys Glu Leu
Phe Asn Ile Glu Met Ile Arg Val Asp Met 145 150 155 160 Leu Glu Asp
Gly Pro Asn Met Asp Gln Ile Glu Lys Leu Val Gln Glu 165 170 175 Asp
Glu Ser Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro 180 185
190 Asp Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Phe Ala Ser Met
195 200 205 Lys Thr Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp Asp Ala
Tyr Thr 210 215 220 Val His His Leu Thr Asp Lys Pro Asp Glu Leu Lys
Asn Ile Leu Thr 225 230 235 240 Ala Cys Lys Gln Ala Gly Asn Glu Asp
Arg Val Leu Ile Phe Ser Ser 245 250 255 Thr Ser Lys Ile Thr Phe Ala
Gly Gly Gly Ile Gly Val Leu Ala Ser 260 265 270 Ser Glu Asn Asn Ile
Gln Tyr Phe Lys Lys Leu Leu Ala Met Gln Thr 275 280 285 Ile Gly Pro
Asp Lys Leu Asn Gln Ile Arg His Ile Arg Phe Phe Lys 290 295 300 Asn
Val Glu Asn Leu Ser Thr His Met Lys Lys His Ala Ser Ile Ile 305 310
315 320 Lys Pro Lys Phe Asp Met Val Leu Asn Lys Leu Glu Ser Glu Ile
Gly 325 330 335 Gly Lys Asn Ile Gly Ser Trp Val Glu Pro Asn Gly Gly
Tyr Phe Ile 340 345 350 Ser Phe Asn Thr Leu Asp Gly Cys Ala Lys Thr
Val Val Ser Met Ala 355 360 365 Lys Glu Ala Gly Val Lys Leu Thr Gly
Ala Gly Ala Thr Phe Pro Tyr 370 375 380 Gly His Asp Pro Arg Asp Arg
Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390 395 400 Ser Leu Ile Glu
Leu Glu Arg Ala Met Asp Val Phe Cys Leu Cys Val 405 410 415 Gln Leu
Ala Ser Val Glu Lys Leu Leu Lys Glu Gln Leu Leu Glu 420 425 430
921185DNABacillus sp.CDS(1)..(1185) 92atg aaa gaa ctg ctg gca aac
cgt gtg aaa acc ctg acc ccg tct acg 48Met Lys Glu Leu Leu Ala Asn
Arg Val Lys Thr Leu Thr Pro Ser Thr 1 5 10 15 acc ctg gcg att acc
gca aaa gcg aaa gaa atg aaa gcg cag ggt att 96Thr Leu Ala Ile Thr
Ala Lys Ala Lys Glu Met Lys Ala Gln Gly Ile 20 25 30 gat gtg atc
ggt ctg ggt gca ggt gaa ccg gac ttt aac acc ccg cag 144Asp Val Ile
Gly Leu Gly Ala Gly Glu Pro Asp Phe Asn Thr Pro Gln 35 40 45 aat
att atg gat gcg gcc atc gac tcg atg cag caa ggc tat acc aaa 192Asn
Ile Met Asp Ala Ala Ile Asp Ser Met Gln Gln Gly Tyr Thr Lys 50 55
60 tac acg ccg agc ggc ggt ctg ccg gca ctg aaa cag gct atc atc gaa
240Tyr Thr Pro Ser Gly Gly Leu Pro Ala Leu Lys Gln Ala Ile Ile Glu
65 70 75 80 aaa ttc aaa cgt gat aac caa ctg gaa tat aaa ccg aat gaa
att atc 288Lys Phe Lys Arg Asp Asn Gln Leu Glu Tyr Lys Pro Asn Glu
Ile Ile 85 90 95 gtc ggt gtg ggc gcg aaa cat gtg ctg tac acc ctg
ttc cag gtt att 336Val Gly Val Gly Ala Lys His Val Leu Tyr Thr Leu
Phe Gln Val Ile 100 105 110 ctg aac gaa ggt gac gaa gtt att atc ccg
atc ccg tat tgg gtt tct 384Leu Asn Glu Gly Asp Glu Val Ile Ile Pro
Ile Pro Tyr Trp Val Ser 115 120 125
tac ccg gaa cag gtc aaa ctg gcc ggc ggt gtt ccg gtc tat att gaa
432Tyr Pro Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val Tyr Ile Glu
130 135 140 gca acc agt gaa cag aac tac aaa att acg gct gaa caa ctg
aaa aat 480Ala Thr Ser Glu Gln Asn Tyr Lys Ile Thr Ala Glu Gln Leu
Lys Asn 145 150 155 160 gcg atc acc gat aaa acg aaa gcc gtc att atc
aac agc ccg tct aat 528Ala Ile Thr Asp Lys Thr Lys Ala Val Ile Ile
Asn Ser Pro Ser Asn 165 170 175 ccg acc ggc atg gtg tat acg cgt gaa
gaa ctg gaa gat att gca aaa 576Pro Thr Gly Met Val Tyr Thr Arg Glu
Glu Leu Glu Asp Ile Ala Lys 180 185 190 atc gct ctg gaa aac aat att
ctg atc gtg tcc gac gaa att tat gaa 624Ile Ala Leu Glu Asn Asn Ile
Leu Ile Val Ser Asp Glu Ile Tyr Glu 195 200 205 aaa ctg ctg tac aac
ggt gcc gaa cat ttc agt att gca cag atc tcc 672Lys Leu Leu Tyr Asn
Gly Ala Glu His Phe Ser Ile Ala Gln Ile Ser 210 215 220 gaa gaa gtt
aaa gca caa acc att gtc atc aat ggc gtg agt aaa tcc 720Glu Glu Val
Lys Ala Gln Thr Ile Val Ile Asn Gly Val Ser Lys Ser 225 230 235 240
cac tca atg acg ggc tgg cgc att ggt tat gca gct ggc aac gcg gat
768His Ser Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Asn Ala Asp
245 250 255 att atc aat gcc atg acc gac ctg gca tcg cac agc acg tct
aac ccg 816Ile Ile Asn Ala Met Thr Asp Leu Ala Ser His Ser Thr Ser
Asn Pro 260 265 270 acc acg gct agc cag tat gcg gcc atc gaa gcg tac
aat ggt ccg caa 864Thr Thr Ala Ser Gln Tyr Ala Ala Ile Glu Ala Tyr
Asn Gly Pro Gln 275 280 285 gat agt gtg gaa gaa atg cgt aaa gcg ttt
gaa tcc cgc ctg gaa acc 912Asp Ser Val Glu Glu Met Arg Lys Ala Phe
Glu Ser Arg Leu Glu Thr 290 295 300 att tat ccg aaa ctg tca gct atc
ccg ggt ttt aaa gtg gtt aaa ccg 960Ile Tyr Pro Lys Leu Ser Ala Ile
Pro Gly Phe Lys Val Val Lys Pro 305 310 315 320 cag ggc gcc ttc tac
ctg ctg ccg gat gtt tct gaa gca gct caa aaa 1008Gln Gly Ala Phe Tyr
Leu Leu Pro Asp Val Ser Glu Ala Ala Gln Lys 325 330 335 acc ggc ttt
gca agc gtc gac gaa ttc gcg tct gcc ctg ctg acg gaa 1056Thr Gly Phe
Ala Ser Val Asp Glu Phe Ala Ser Ala Leu Leu Thr Glu 340 345 350 gcg
aat gtg gcc gtt att ccg ggt agc ggt ttc ggt gca ccg tca acc 1104Ala
Asn Val Ala Val Ile Pro Gly Ser Gly Phe Gly Ala Pro Ser Thr 355 360
365 att cgc atc tcg tac gca acc tct ctg aac ctg att gaa gaa gcg att
1152Ile Arg Ile Ser Tyr Ala Thr Ser Leu Asn Leu Ile Glu Glu Ala Ile
370 375 380 gaa cgc att gac cgt ttt gtg aaa ctc gag tga 1185Glu Arg
Ile Asp Arg Phe Val Lys Leu Glu 385 390 93394PRTBacillus sp. 93Met
Lys Glu Leu Leu Ala Asn Arg Val Lys Thr Leu Thr Pro Ser Thr 1 5 10
15 Thr Leu Ala Ile Thr Ala Lys Ala Lys Glu Met Lys Ala Gln Gly Ile
20 25 30 Asp Val Ile Gly Leu Gly Ala Gly Glu Pro Asp Phe Asn Thr
Pro Gln 35 40 45 Asn Ile Met Asp Ala Ala Ile Asp Ser Met Gln Gln
Gly Tyr Thr Lys 50 55 60 Tyr Thr Pro Ser Gly Gly Leu Pro Ala Leu
Lys Gln Ala Ile Ile Glu 65 70 75 80 Lys Phe Lys Arg Asp Asn Gln Leu
Glu Tyr Lys Pro Asn Glu Ile Ile 85 90 95 Val Gly Val Gly Ala Lys
His Val Leu Tyr Thr Leu Phe Gln Val Ile 100 105 110 Leu Asn Glu Gly
Asp Glu Val Ile Ile Pro Ile Pro Tyr Trp Val Ser 115 120 125 Tyr Pro
Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val Tyr Ile Glu 130 135 140
Ala Thr Ser Glu Gln Asn Tyr Lys Ile Thr Ala Glu Gln Leu Lys Asn 145
150 155 160 Ala Ile Thr Asp Lys Thr Lys Ala Val Ile Ile Asn Ser Pro
Ser Asn 165 170 175 Pro Thr Gly Met Val Tyr Thr Arg Glu Glu Leu Glu
Asp Ile Ala Lys 180 185 190 Ile Ala Leu Glu Asn Asn Ile Leu Ile Val
Ser Asp Glu Ile Tyr Glu 195 200 205 Lys Leu Leu Tyr Asn Gly Ala Glu
His Phe Ser Ile Ala Gln Ile Ser 210 215 220 Glu Glu Val Lys Ala Gln
Thr Ile Val Ile Asn Gly Val Ser Lys Ser 225 230 235 240 His Ser Met
Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Asn Ala Asp 245 250 255 Ile
Ile Asn Ala Met Thr Asp Leu Ala Ser His Ser Thr Ser Asn Pro 260 265
270 Thr Thr Ala Ser Gln Tyr Ala Ala Ile Glu Ala Tyr Asn Gly Pro Gln
275 280 285 Asp Ser Val Glu Glu Met Arg Lys Ala Phe Glu Ser Arg Leu
Glu Thr 290 295 300 Ile Tyr Pro Lys Leu Ser Ala Ile Pro Gly Phe Lys
Val Val Lys Pro 305 310 315 320 Gln Gly Ala Phe Tyr Leu Leu Pro Asp
Val Ser Glu Ala Ala Gln Lys 325 330 335 Thr Gly Phe Ala Ser Val Asp
Glu Phe Ala Ser Ala Leu Leu Thr Glu 340 345 350 Ala Asn Val Ala Val
Ile Pro Gly Ser Gly Phe Gly Ala Pro Ser Thr 355 360 365 Ile Arg Ile
Ser Tyr Ala Thr Ser Leu Asn Leu Ile Glu Glu Ala Ile 370 375 380 Glu
Arg Ile Asp Arg Phe Val Lys Leu Glu 385 390 941239DNASinorhizobium
melilotiCDS(1)..(1239) 94atg acc atc aat gcc acg gtt aaa gaa gcg
ggt ttc cag ccg gcg tcg 48Met Thr Ile Asn Ala Thr Val Lys Glu Ala
Gly Phe Gln Pro Ala Ser 1 5 10 15 cgt att agt agt atc ggt gtg tct
gaa atc ctg aaa atc ggt gcc cgc 96Arg Ile Ser Ser Ile Gly Val Ser
Glu Ile Leu Lys Ile Gly Ala Arg 20 25 30 gcg gcc gca atg aaa cgt
gaa ggc aaa ccg gtt att atc ctg ggc gca 144Ala Ala Ala Met Lys Arg
Glu Gly Lys Pro Val Ile Ile Leu Gly Ala 35 40 45 ggt gaa ccg gat
ttt gac acc ccg gaa cat gtc aaa caa gct gcg agc 192Gly Glu Pro Asp
Phe Asp Thr Pro Glu His Val Lys Gln Ala Ala Ser 50 55 60 gat gcc
att cac cgc ggt gaa acc aaa tat acg gca ctg gac ggc acg 240Asp Ala
Ile His Arg Gly Glu Thr Lys Tyr Thr Ala Leu Asp Gly Thr 65 70 75 80
ccg gaa ctg aaa aaa gct atc cgc gaa aaa ttt caa cgt gaa aac ggt
288Pro Glu Leu Lys Lys Ala Ile Arg Glu Lys Phe Gln Arg Glu Asn Gly
85 90 95 ctg gcg tac gaa ctg gat gaa att acc gtg gct acg ggc gcg
aaa cag 336Leu Ala Tyr Glu Leu Asp Glu Ile Thr Val Ala Thr Gly Ala
Lys Gln 100 105 110 atc ctg ttc aat gcc atg atg gca tct ctg gat ccg
ggt gac gaa gtt 384Ile Leu Phe Asn Ala Met Met Ala Ser Leu Asp Pro
Gly Asp Glu Val 115 120 125 att atc ccg acc ccg tat tgg acg tca tac
tcg gat att gtc cat atc 432Ile Ile Pro Thr Pro Tyr Trp Thr Ser Tyr
Ser Asp Ile Val His Ile 130 135 140 tgc gaa ggt aaa ccg gtg ctg att
gct tgt gac gcg agc tct ggc ttt 480Cys Glu Gly Lys Pro Val Leu Ile
Ala Cys Asp Ala Ser Ser Gly Phe 145 150 155 160 cgt ctg acc gcc gaa
aaa ctg gaa gca gca atc acc ccg cgt acg cgt 528Arg Leu Thr Ala Glu
Lys Leu Glu Ala Ala Ile Thr Pro Arg Thr Arg 165 170 175 tgg gtc ctg
ctg aac agc ccg tct aat ccg agc ggt gct gcg tat tct 576Trp Val Leu
Leu Asn Ser Pro Ser Asn Pro Ser Gly Ala Ala Tyr Ser 180 185 190 gcc
gca gat tac cgc ccg ctg ctg gaa gtt ctg ctg cgt cat ccg cac 624Ala
Ala Asp Tyr Arg Pro Leu Leu Glu Val Leu Leu Arg His Pro His 195 200
205 gtc tgg ctg ctg gtt gat gac atg tat gaa cac atc gtg tac gat ggc
672Val Trp Leu Leu Val Asp Asp Met Tyr Glu His Ile Val Tyr Asp Gly
210 215 220 ttt cgc ttc gtt acc ccg gcg cag ctg gaa ccg ggt ctg aaa
aac cgt 720Phe Arg Phe Val Thr Pro Ala Gln Leu Glu Pro Gly Leu Lys
Asn Arg 225 230 235 240 acc ctg acg gtg aat ggc gtt agc aaa gct tat
gcg atg acg ggt tgg 768Thr Leu Thr Val Asn Gly Val Ser Lys Ala Tyr
Ala Met Thr Gly Trp 245 250 255 cgt att ggt tac gcg ggc ggt ccg cgt
gaa ctg atc aaa gcc atg gca 816Arg Ile Gly Tyr Ala Gly Gly Pro Arg
Glu Leu Ile Lys Ala Met Ala 260 265 270 gtg gtt cag agt caa gcc acc
tcc tgc ccg agt tcc att tca cag gct 864Val Val Gln Ser Gln Ala Thr
Ser Cys Pro Ser Ser Ile Ser Gln Ala 275 280 285 gcg tcg gtg gca gca
ctg aac ggt ccg caa gat ttt ctg aaa gaa cgc 912Ala Ser Val Ala Ala
Leu Asn Gly Pro Gln Asp Phe Leu Lys Glu Arg 290 295 300 acc gaa agc
ttc cag cgt cgc cgt gac ctg gtc gtg aac ggt ctg aat 960Thr Glu Ser
Phe Gln Arg Arg Arg Asp Leu Val Val Asn Gly Leu Asn 305 310 315 320
gcg att gat ggc ctg gac tgc cgt gtt ccg gaa ggt gct ttt tat acc
1008Ala Ile Asp Gly Leu Asp Cys Arg Val Pro Glu Gly Ala Phe Tyr Thr
325 330 335 ttc tca ggc tgt gcg ggt gtt ctg ggc aaa gtc acg ccg tcg
ggc aaa 1056Phe Ser Gly Cys Ala Gly Val Leu Gly Lys Val Thr Pro Ser
Gly Lys 340 345 350 cgc atc aaa acc gat acg gac ttt tgt gcc tat ctg
ctg gaa gat gcc 1104Arg Ile Lys Thr Asp Thr Asp Phe Cys Ala Tyr Leu
Leu Glu Asp Ala 355 360 365 cat gtg gca gtt gtc ccg ggt agt gca ttc
ggc ctg tcc ccg ttt ttc 1152His Val Ala Val Val Pro Gly Ser Ala Phe
Gly Leu Ser Pro Phe Phe 370 375 380 cgt att agt tac gcg acc tcc gaa
gcc gaa ctg aaa gaa gcc ctg gaa 1200Arg Ile Ser Tyr Ala Thr Ser Glu
Ala Glu Leu Lys Glu Ala Leu Glu 385 390 395 400 cgc att gct gcc gcc
tgt gac cgt ctg tcg ctc gag tga 1239Arg Ile Ala Ala Ala Cys Asp Arg
Leu Ser Leu Glu 405 410 95412PRTSinorhizobium meliloti 95Met Thr
Ile Asn Ala Thr Val Lys Glu Ala Gly Phe Gln Pro Ala Ser 1 5 10 15
Arg Ile Ser Ser Ile Gly Val Ser Glu Ile Leu Lys Ile Gly Ala Arg 20
25 30 Ala Ala Ala Met Lys Arg Glu Gly Lys Pro Val Ile Ile Leu Gly
Ala 35 40 45 Gly Glu Pro Asp Phe Asp Thr Pro Glu His Val Lys Gln
Ala Ala Ser 50 55 60 Asp Ala Ile His Arg Gly Glu Thr Lys Tyr Thr
Ala Leu Asp Gly Thr 65 70 75 80 Pro Glu Leu Lys Lys Ala Ile Arg Glu
Lys Phe Gln Arg Glu Asn Gly 85 90 95 Leu Ala Tyr Glu Leu Asp Glu
Ile Thr Val Ala Thr Gly Ala Lys Gln 100 105 110 Ile Leu Phe Asn Ala
Met Met Ala Ser Leu Asp Pro Gly Asp Glu Val 115 120 125 Ile Ile Pro
Thr Pro Tyr Trp Thr Ser Tyr Ser Asp Ile Val His Ile 130 135 140 Cys
Glu Gly Lys Pro Val Leu Ile Ala Cys Asp Ala Ser Ser Gly Phe 145 150
155 160 Arg Leu Thr Ala Glu Lys Leu Glu Ala Ala Ile Thr Pro Arg Thr
Arg 165 170 175 Trp Val Leu Leu Asn Ser Pro Ser Asn Pro Ser Gly Ala
Ala Tyr Ser 180 185 190 Ala Ala Asp Tyr Arg Pro Leu Leu Glu Val Leu
Leu Arg His Pro His 195 200 205 Val Trp Leu Leu Val Asp Asp Met Tyr
Glu His Ile Val Tyr Asp Gly 210 215 220 Phe Arg Phe Val Thr Pro Ala
Gln Leu Glu Pro Gly Leu Lys Asn Arg 225 230 235 240 Thr Leu Thr Val
Asn Gly Val Ser Lys Ala Tyr Ala Met Thr Gly Trp 245 250 255 Arg Ile
Gly Tyr Ala Gly Gly Pro Arg Glu Leu Ile Lys Ala Met Ala 260 265 270
Val Val Gln Ser Gln Ala Thr Ser Cys Pro Ser Ser Ile Ser Gln Ala 275
280 285 Ala Ser Val Ala Ala Leu Asn Gly Pro Gln Asp Phe Leu Lys Glu
Arg 290 295 300 Thr Glu Ser Phe Gln Arg Arg Arg Asp Leu Val Val Asn
Gly Leu Asn 305 310 315 320 Ala Ile Asp Gly Leu Asp Cys Arg Val Pro
Glu Gly Ala Phe Tyr Thr 325 330 335 Phe Ser Gly Cys Ala Gly Val Leu
Gly Lys Val Thr Pro Ser Gly Lys 340 345 350 Arg Ile Lys Thr Asp Thr
Asp Phe Cys Ala Tyr Leu Leu Glu Asp Ala 355 360 365 His Val Ala Val
Val Pro Gly Ser Ala Phe Gly Leu Ser Pro Phe Phe 370 375 380 Arg Ile
Ser Tyr Ala Thr Ser Glu Ala Glu Leu Lys Glu Ala Leu Glu 385 390 395
400 Arg Ile Ala Ala Ala Cys Asp Arg Leu Ser Leu Glu 405 410
96999DNAMethanothermobacter thermautotrophicusCDS(1)..(999) 96atg
cgt ctg tgg cgt gct ctg tat cgc ccg ccg acc att acc tac ccg 48Met
Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile Thr Tyr Pro 1 5 10
15 tca aaa agc ccg gaa gtc att att atg tct tgt gaa gcg tcc ggc aaa
96Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys
20 25 30 att tgg ctg aac ggt gaa atg gtt gaa tgg gaa gaa gca acc
gtt cat 144Ile Trp Leu Asn Gly Glu Met Val Glu Trp Glu Glu Ala Thr
Val His 35 40 45 gtc ctg tca cat gtg gtt cac tat ggc agc tct gtg
ttt gaa ggt att 192Val Leu Ser His Val Val His Tyr Gly Ser Ser Val
Phe Glu Gly Ile 50 55 60 cgt tgc tac cgc aat tcg aaa ggt agc gcg
atc ttt cgt ctg cgc gaa 240Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala
Ile Phe Arg Leu Arg Glu 65 70 75 80 cac gtt aaa cgt ctg ttc gat tcc
gcc aaa att tat cgc atg gac atc 288His Val Lys Arg Leu Phe Asp Ser
Ala Lys Ile Tyr Arg Met Asp Ile 85 90 95 ccg tac acc cag gaa caa
att tgc gat gcc atc gtt gaa acg gtc cgt 336Pro Tyr Thr Gln Glu Gln
Ile Cys Asp Ala Ile Val Glu Thr Val Arg 100 105 110 gaa aac ggt ctg
gaa gaa tgt tat atc cgt ccg gtc gtg ttc cgc ggc 384Glu Asn Gly Leu
Glu Glu Cys Tyr Ile Arg Pro Val Val Phe Arg Gly 115 120 125 tac ggt
gaa atg ggc gtg cat ccg gtt aat tgt ccg gtg gac gtt gca 432Tyr Gly
Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp Val Ala 130 135 140
gtc gca gca tgg gaa tgg ggt gca tat ctg ggt gca gaa gca ctg gaa
480Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu
145 150 155 160 gtg ggc gtt gat gca ggt gtt tct acc tgg cgt cgc atg
gct ccg aac 528Val Gly Val Asp Ala Gly Val Ser Thr Trp
Arg Arg Met Ala Pro Asn 165 170 175 acg atg ccg aat atg gca aaa gct
ggc ggt aac tat ctg aat tca cag 576Thr Met Pro Asn Met Ala Lys Ala
Gly Gly Asn Tyr Leu Asn Ser Gln 180 185 190 ctg gca aaa atg gaa gct
gtg cgc cat ggc tac gat gaa gcg att atg 624Leu Ala Lys Met Glu Ala
Val Arg His Gly Tyr Asp Glu Ala Ile Met 195 200 205 ctg gac tat cac
ggt tac atc tct gaa ggc agt ggt gaa aac att ttt 672Leu Asp Tyr His
Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe 210 215 220 ctg gtc
tcg gaa ggc gaa atc tat acc ccg cct gtg agt tcc tca ctg 720Leu Val
Ser Glu Gly Glu Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu 225 230 235
240 ctg cgt ggt att acg cgc gat agc gtg att aaa atc gca cgt acc gaa
768Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala Arg Thr Glu
245 250 255 ggc gtc acg gtg cac gaa gaa ccg att acc cgc gaa atg ctg
tac atc 816Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu
Tyr Ile 260 265 270 gcg gat gaa gcc ttt ttc acc ggc acg gca gct gaa
att acc ccg atc 864Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu
Ile Thr Pro Ile 275 280 285 cgt agc gtt gac ggc att gaa atc ggt gct
ggt cgt cgc ggt ccg gtc 912Arg Ser Val Asp Gly Ile Glu Ile Gly Ala
Gly Arg Arg Gly Pro Val 290 295 300 acg aaa ctg ctg caa gat gaa ttt
ttc cgc atc atc cgt gcc gaa acc 960Thr Lys Leu Leu Gln Asp Glu Phe
Phe Arg Ile Ile Arg Ala Glu Thr 305 310 315 320 gaa gat agc ttt ggc
tgg ctg acc tac att ctc gag tga 999Glu Asp Ser Phe Gly Trp Leu Thr
Tyr Ile Leu Glu 325 330 97332PRTMethanothermobacter
thermautotrophicus 97Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro
Thr Ile Thr Tyr Pro 1 5 10 15 Ser Lys Ser Pro Glu Val Ile Ile Met
Ser Cys Glu Ala Ser Gly Lys 20 25 30 Ile Trp Leu Asn Gly Glu Met
Val Glu Trp Glu Glu Ala Thr Val His 35 40 45 Val Leu Ser His Val
Val His Tyr Gly Ser Ser Val Phe Glu Gly Ile 50 55 60 Arg Cys Tyr
Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg Leu Arg Glu 65 70 75 80 His
Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile 85 90
95 Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg
100 105 110 Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg Pro Val Val Phe
Arg Gly 115 120 125 Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro
Val Asp Val Ala 130 135 140 Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu
Gly Ala Glu Ala Leu Glu 145 150 155 160 Val Gly Val Asp Ala Gly Val
Ser Thr Trp Arg Arg Met Ala Pro Asn 165 170 175 Thr Met Pro Asn Met
Ala Lys Ala Gly Gly Asn Tyr Leu Asn Ser Gln 180 185 190 Leu Ala Lys
Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile Met 195 200 205 Leu
Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe 210 215
220 Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu
225 230 235 240 Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala
Arg Thr Glu 245 250 255 Gly Val Thr Val His Glu Glu Pro Ile Thr Arg
Glu Met Leu Tyr Ile 260 265 270 Ala Asp Glu Ala Phe Phe Thr Gly Thr
Ala Ala Glu Ile Thr Pro Ile 275 280 285 Arg Ser Val Asp Gly Ile Glu
Ile Gly Ala Gly Arg Arg Gly Pro Val 290 295 300 Thr Lys Leu Leu Gln
Asp Glu Phe Phe Arg Ile Ile Arg Ala Glu Thr 305 310 315 320 Glu Asp
Ser Phe Gly Trp Leu Thr Tyr Ile Leu Glu 325 330
981605DNALactobacillus acidophilusCDS(1)..(1605) 98atg gac aac tcc
gaa gaa aaa aaa ctg gaa gcc ctg ggt gcc ttt gaa 48Met Asp Asn Ser
Glu Glu Lys Lys Leu Glu Ala Leu Gly Ala Phe Glu 1 5 10 15 atc tca
cgt aaa atg ctg gcg ctg gcg cag aaa aat gaa aaa agc aac 96Ile Ser
Arg Lys Met Leu Ala Leu Ala Gln Lys Asn Glu Lys Ser Asn 20 25 30
att ttt ctg aat gcg ggc cgt ggt aac ccg aat tgg atc cag acc ctg
144Ile Phe Leu Asn Ala Gly Arg Gly Asn Pro Asn Trp Ile Gln Thr Leu
35 40 45 gca cgt ctg gca ttt gtg cgt ctg gtt caa ttc ggt gtt acg
gaa tct 192Ala Arg Leu Ala Phe Val Arg Leu Val Gln Phe Gly Val Thr
Glu Ser 50 55 60 aaa ctg acc att aac aat ggt atc atg gcc ggc tat
att aac acg gat 240Lys Leu Thr Ile Asn Asn Gly Ile Met Ala Gly Tyr
Ile Asn Thr Asp 65 70 75 80 ggc atc cgt gaa cgc ctg ttt gca ttc ctg
gat ccg gac aaa aac gat 288Gly Ile Arg Glu Arg Leu Phe Ala Phe Leu
Asp Pro Asp Lys Asn Asp 85 90 95 gaa gac aaa ttc ctg atc gat gcc
gtg aac tac tgc cat acc gaa ctg 336Glu Asp Lys Phe Leu Ile Asp Ala
Val Asn Tyr Cys His Thr Glu Leu 100 105 110 ggt ctg aat cgt gac aaa
gtg gtt gca gaa tgg gtt aac ggc gca gtc 384Gly Leu Asn Arg Asp Lys
Val Val Ala Glu Trp Val Asn Gly Ala Val 115 120 125 gct aac aat tat
ccg gtc ccg gat cgc tgt ctg gtg aac acg gaa aaa 432Ala Asn Asn Tyr
Pro Val Pro Asp Arg Cys Leu Val Asn Thr Glu Lys 130 135 140 atc atc
aac tat ttt ctg caa gaa ctg tca tac aaa gat gca aat ctg 480Ile Ile
Asn Tyr Phe Leu Gln Glu Leu Ser Tyr Lys Asp Ala Asn Leu 145 150 155
160 gct gaa caa acc gac ctg ttt ccg acg gaa ggc ggt acc gcg gcc att
528Ala Glu Gln Thr Asp Leu Phe Pro Thr Glu Gly Gly Thr Ala Ala Ile
165 170 175 gtt tac gcg ttc cat tcg ctg gcc gaa aac cac ctg ctg aaa
aaa ggt 576Val Tyr Ala Phe His Ser Leu Ala Glu Asn His Leu Leu Lys
Lys Gly 180 185 190 gat aaa atc gcc atc aac gaa ccg atc ttc acc ccg
tac ctg cgt atc 624Asp Lys Ile Ala Ile Asn Glu Pro Ile Phe Thr Pro
Tyr Leu Arg Ile 195 200 205 ccg gaa ctg aaa gat tat gaa ctg gtt gaa
gtc gac ctg cac agc tat 672Pro Glu Leu Lys Asp Tyr Glu Leu Val Glu
Val Asp Leu His Ser Tyr 210 215 220 gag aaa aac gat tgg gaa att gaa
ccg aat gaa atc gaa aaa ctg aaa 720Glu Lys Asn Asp Trp Glu Ile Glu
Pro Asn Glu Ile Glu Lys Leu Lys 225 230 235 240 gac ccg agc att aaa
gcg ctg atc gtc gtg aac ccg acg aat ccg acc 768Asp Pro Ser Ile Lys
Ala Leu Ile Val Val Asn Pro Thr Asn Pro Thr 245 250 255 tct aaa gaa
ttt gat acc aac gcg ctg aat gcc att aaa cag gct gtc 816Ser Lys Glu
Phe Asp Thr Asn Ala Leu Asn Ala Ile Lys Gln Ala Val 260 265 270 gag
aaa aac ccg aaa ctg atg att atc agc gac gaa gtg tat ggt gcc 864Glu
Lys Asn Pro Lys Leu Met Ile Ile Ser Asp Glu Val Tyr Gly Ala 275 280
285 ttt gtt ccg aac ttc aaa agc atc tat tct gtt gtc ccg tac aat acg
912Phe Val Pro Asn Phe Lys Ser Ile Tyr Ser Val Val Pro Tyr Asn Thr
290 295 300 atg ctg gtt tat agt tac tcc aaa ctg ttt ggt tgc acc ggc
tgg cgc 960Met Leu Val Tyr Ser Tyr Ser Lys Leu Phe Gly Cys Thr Gly
Trp Arg 305 310 315 320 ctg ggc gtt att gct ctg aac gag aaa aac gtc
ttc gat gac aat atc 1008Leu Gly Val Ile Ala Leu Asn Glu Lys Asn Val
Phe Asp Asp Asn Ile 325 330 335 gcg cat ctg gat aaa gtg gaa ctg cgt
cag ctg cac aaa cgc tac agc 1056Ala His Leu Asp Lys Val Glu Leu Arg
Gln Leu His Lys Arg Tyr Ser 340 345 350 tct gtg gtt ctg gat ccg gac
aaa atg aaa ttt att gat cgt ctg tgt 1104Ser Val Val Leu Asp Pro Asp
Lys Met Lys Phe Ile Asp Arg Leu Cys 355 360 365 gcg gac tca cgc tcg
atc ggt ctg tat cat acg gcc ggc ctg tca acc 1152Ala Asp Ser Arg Ser
Ile Gly Leu Tyr His Thr Ala Gly Leu Ser Thr 370 375 380 ccg cag caa
att atg gaa gca ctg ttc tcg atg acc cac ctg ctg acc 1200Pro Gln Gln
Ile Met Glu Ala Leu Phe Ser Met Thr His Leu Leu Thr 385 390 395 400
agt acg aac ggc ggt tcc gat gac ccg tac att gat atc gca cgt aaa
1248Ser Thr Asn Gly Gly Ser Asp Asp Pro Tyr Ile Asp Ile Ala Arg Lys
405 410 415 ctg gtg tct gaa cgc tat gat cag ctg cat gac gca atg caa
gct ccg 1296Leu Val Ser Glu Arg Tyr Asp Gln Leu His Asp Ala Met Gln
Ala Pro 420 425 430 aaa gat gaa acc gac acg aat acc cac tat tac tcc
ctg att gat atc 1344Lys Asp Glu Thr Asp Thr Asn Thr His Tyr Tyr Ser
Leu Ile Asp Ile 435 440 445 tat cgt ctg gcg gaa aaa atc tac ggc aaa
gaa ttt cgc gat tat ctg 1392Tyr Arg Leu Ala Glu Lys Ile Tyr Gly Lys
Glu Phe Arg Asp Tyr Leu 450 455 460 acg aac aat ttt gaa cag gtg gac
ttc ctg ctg aaa ctg gct gag aaa 1440Thr Asn Asn Phe Glu Gln Val Asp
Phe Leu Leu Lys Leu Ala Glu Lys 465 470 475 480 aac ggt gtc gtg ctg
gtc gat ggc gtg ggt ttc ggc gcg aaa ccg ggc 1488Asn Gly Val Val Leu
Val Asp Gly Val Gly Phe Gly Ala Lys Pro Gly 485 490 495 gaa ctg cgc
gtt agt caa gca aat ctg ccg acc gaa gat tat gct ctg 1536Glu Leu Arg
Val Ser Gln Ala Asn Leu Pro Thr Glu Asp Tyr Ala Leu 500 505 510 att
ggc aaa caa gtc ctg gaa ctg ctg aaa gaa tac tat gaa gaa ttt 1584Ile
Gly Lys Gln Val Leu Glu Leu Leu Lys Glu Tyr Tyr Glu Glu Phe 515 520
525 aaa cag aat aat ctc gag taa 1605Lys Gln Asn Asn Leu Glu 530
99534PRTLactobacillus acidophilus 99Met Asp Asn Ser Glu Glu Lys Lys
Leu Glu Ala Leu Gly Ala Phe Glu 1 5 10 15 Ile Ser Arg Lys Met Leu
Ala Leu Ala Gln Lys Asn Glu Lys Ser Asn 20 25 30 Ile Phe Leu Asn
Ala Gly Arg Gly Asn Pro Asn Trp Ile Gln Thr Leu 35 40 45 Ala Arg
Leu Ala Phe Val Arg Leu Val Gln Phe Gly Val Thr Glu Ser 50 55 60
Lys Leu Thr Ile Asn Asn Gly Ile Met Ala Gly Tyr Ile Asn Thr Asp 65
70 75 80 Gly Ile Arg Glu Arg Leu Phe Ala Phe Leu Asp Pro Asp Lys
Asn Asp 85 90 95 Glu Asp Lys Phe Leu Ile Asp Ala Val Asn Tyr Cys
His Thr Glu Leu 100 105 110 Gly Leu Asn Arg Asp Lys Val Val Ala Glu
Trp Val Asn Gly Ala Val 115 120 125 Ala Asn Asn Tyr Pro Val Pro Asp
Arg Cys Leu Val Asn Thr Glu Lys 130 135 140 Ile Ile Asn Tyr Phe Leu
Gln Glu Leu Ser Tyr Lys Asp Ala Asn Leu 145 150 155 160 Ala Glu Gln
Thr Asp Leu Phe Pro Thr Glu Gly Gly Thr Ala Ala Ile 165 170 175 Val
Tyr Ala Phe His Ser Leu Ala Glu Asn His Leu Leu Lys Lys Gly 180 185
190 Asp Lys Ile Ala Ile Asn Glu Pro Ile Phe Thr Pro Tyr Leu Arg Ile
195 200 205 Pro Glu Leu Lys Asp Tyr Glu Leu Val Glu Val Asp Leu His
Ser Tyr 210 215 220 Glu Lys Asn Asp Trp Glu Ile Glu Pro Asn Glu Ile
Glu Lys Leu Lys 225 230 235 240 Asp Pro Ser Ile Lys Ala Leu Ile Val
Val Asn Pro Thr Asn Pro Thr 245 250 255 Ser Lys Glu Phe Asp Thr Asn
Ala Leu Asn Ala Ile Lys Gln Ala Val 260 265 270 Glu Lys Asn Pro Lys
Leu Met Ile Ile Ser Asp Glu Val Tyr Gly Ala 275 280 285 Phe Val Pro
Asn Phe Lys Ser Ile Tyr Ser Val Val Pro Tyr Asn Thr 290 295 300 Met
Leu Val Tyr Ser Tyr Ser Lys Leu Phe Gly Cys Thr Gly Trp Arg 305 310
315 320 Leu Gly Val Ile Ala Leu Asn Glu Lys Asn Val Phe Asp Asp Asn
Ile 325 330 335 Ala His Leu Asp Lys Val Glu Leu Arg Gln Leu His Lys
Arg Tyr Ser 340 345 350 Ser Val Val Leu Asp Pro Asp Lys Met Lys Phe
Ile Asp Arg Leu Cys 355 360 365 Ala Asp Ser Arg Ser Ile Gly Leu Tyr
His Thr Ala Gly Leu Ser Thr 370 375 380 Pro Gln Gln Ile Met Glu Ala
Leu Phe Ser Met Thr His Leu Leu Thr 385 390 395 400 Ser Thr Asn Gly
Gly Ser Asp Asp Pro Tyr Ile Asp Ile Ala Arg Lys 405 410 415 Leu Val
Ser Glu Arg Tyr Asp Gln Leu His Asp Ala Met Gln Ala Pro 420 425 430
Lys Asp Glu Thr Asp Thr Asn Thr His Tyr Tyr Ser Leu Ile Asp Ile 435
440 445 Tyr Arg Leu Ala Glu Lys Ile Tyr Gly Lys Glu Phe Arg Asp Tyr
Leu 450 455 460 Thr Asn Asn Phe Glu Gln Val Asp Phe Leu Leu Lys Leu
Ala Glu Lys 465 470 475 480 Asn Gly Val Val Leu Val Asp Gly Val Gly
Phe Gly Ala Lys Pro Gly 485 490 495 Glu Leu Arg Val Ser Gln Ala Asn
Leu Pro Thr Glu Asp Tyr Ala Leu 500 505 510 Ile Gly Lys Gln Val Leu
Glu Leu Leu Lys Glu Tyr Tyr Glu Glu Phe 515 520 525 Lys Gln Asn Asn
Leu Glu 530 1001209DNASinorhizobium melilotiCDS(1)..(1209) 100atg
gcc ttc ctg gcg gat gca ctg agt cgt gtt aaa ccg tcg gca acc 48Met
Ala Phe Leu Ala Asp Ala Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5 10
15 atc gct gtg tcg cag aaa gcc cgt gaa ctg aaa gca aaa ggc cgt gat
96Ile Ala Val Ser Gln Lys Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp
20 25 30 gtg att ggc ctg ggt gcg ggc gaa ccg gat ttt gac acc ccg
gac aac 144Val Ile Gly Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro
Asp Asn 35 40 45 atc aaa aaa gcg gcc att gat gcc atc gac cgc ggc
gaa acc aaa tat 192Ile Lys Lys Ala Ala Ile Asp Ala Ile Asp Arg Gly
Glu Thr Lys Tyr 50 55 60 acg cct gtg agc ggt att ccg gaa ctg cgt
gaa gcg atc gcc aaa aaa 240Thr Pro Val Ser Gly Ile Pro Glu Leu Arg
Glu Ala Ile Ala Lys Lys 65 70 75 80 ttc aaa cgc gaa aac aac ctg gat
tac acc gca gct cag acg att gtt 288Phe Lys Arg Glu Asn Asn Leu Asp
Tyr Thr Ala Ala Gln Thr Ile Val 85 90 95 ggc acc ggc ggt aaa caa
atc ctg ttt aac gcg ttc atg gcc acc ctg 336Gly Thr Gly Gly Lys Gln
Ile Leu Phe Asn Ala Phe Met Ala Thr Leu 100
105 110 aat ccg ggt gat gaa gtg gtt att ccg gca ccg tat tgg gtg tct
tac 384Asn Pro Gly Asp Glu Val Val Ile Pro Ala Pro Tyr Trp Val Ser
Tyr 115 120 125 ccg gaa atg gtt gct ctg tgc ggc ggt acg ccg gtg ttt
gtt ccg acc 432Pro Glu Met Val Ala Leu Cys Gly Gly Thr Pro Val Phe
Val Pro Thr 130 135 140 cgt cag gaa aac aat ttc aaa ctg aaa gca gaa
gat ctg gac cgc gct 480Arg Gln Glu Asn Asn Phe Lys Leu Lys Ala Glu
Asp Leu Asp Arg Ala 145 150 155 160 atc acc ccg aaa acg aaa tgg ttt
gtt ttc aac agc ccg tct aat ccg 528Ile Thr Pro Lys Thr Lys Trp Phe
Val Phe Asn Ser Pro Ser Asn Pro 165 170 175 tca ggc gcg gcc tat tcg
cat gaa gaa ctg aaa gca ctg acc gat gtc 576Ser Gly Ala Ala Tyr Ser
His Glu Glu Leu Lys Ala Leu Thr Asp Val 180 185 190 ctg atg aaa cat
ccg cac gtc tgg gtg ctg acg gat gac atg tat gaa 624Leu Met Lys His
Pro His Val Trp Val Leu Thr Asp Asp Met Tyr Glu 195 200 205 cac ctg
acc tac ggt gac ttt cgt ttc gcc acg ccg gtt gaa gtc gaa 672His Leu
Thr Tyr Gly Asp Phe Arg Phe Ala Thr Pro Val Glu Val Glu 210 215 220
ccg ggc ctg tac gaa cgc acc ctg acg atg aat ggt gtg tca aaa gcg
720Pro Gly Leu Tyr Glu Arg Thr Leu Thr Met Asn Gly Val Ser Lys Ala
225 230 235 240 tat gcg atg acc ggt tgg cgt att ggc tac gca gct ggt
ccg ctg cat 768Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly
Pro Leu His 245 250 255 ctg att aaa gcg atg gat atg atc caa ggc cag
caa acg agt ggt gcg 816Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln
Gln Thr Ser Gly Ala 260 265 270 gcc tcc atc gca cag tgg gca gct gtt
gaa gct ctg aac ggc ccg caa 864Ala Ser Ile Ala Gln Trp Ala Ala Val
Glu Ala Leu Asn Gly Pro Gln 275 280 285 gat ttc atc ggt cgc aac aaa
gaa atc ttc cag ggc cgt cgc gac ctg 912Asp Phe Ile Gly Arg Asn Lys
Glu Ile Phe Gln Gly Arg Arg Asp Leu 290 295 300 gtc gtg agc atg ctg
aac cag gcc aaa ggc att tct tgc ccg acc ccg 960Val Val Ser Met Leu
Asn Gln Ala Lys Gly Ile Ser Cys Pro Thr Pro 305 310 315 320 gaa ggt
gca ttt tat gtc tac ccg agt tgt gcg ggt ctg att ggc aaa 1008Glu Gly
Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys 325 330 335
acc gcc ccg tcc ggt aaa gtc atc gaa acg gat gaa gac ttc gtg tcc
1056Thr Ala Pro Ser Gly Lys Val Ile Glu Thr Asp Glu Asp Phe Val Ser
340 345 350 gaa ctg ctg gaa acc gaa ggc gtt gcg gtt gtc cac ggt tca
gcc ttt 1104Glu Leu Leu Glu Thr Glu Gly Val Ala Val Val His Gly Ser
Ala Phe 355 360 365 ggt ctg ggc ccg aat ttc cgt att tcg tat gcg acg
tcc gaa gct ctg 1152Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr
Ser Glu Ala Leu 370 375 380 ctg gaa gaa gcc tgc cgt cgc att cag cgt
ttt tgt gcc gcc tgt cgt 1200Leu Glu Glu Ala Cys Arg Arg Ile Gln Arg
Phe Cys Ala Ala Cys Arg 385 390 395 400 ctc gag taa 1209Leu Glu
101402PRTSinorhizobium meliloti 101Met Ala Phe Leu Ala Asp Ala Leu
Ser Arg Val Lys Pro Ser Ala Thr 1 5 10 15 Ile Ala Val Ser Gln Lys
Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp 20 25 30 Val Ile Gly Leu
Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35 40 45 Ile Lys
Lys Ala Ala Ile Asp Ala Ile Asp Arg Gly Glu Thr Lys Tyr 50 55 60
Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Glu Ala Ile Ala Lys Lys 65
70 75 80 Phe Lys Arg Glu Asn Asn Leu Asp Tyr Thr Ala Ala Gln Thr
Ile Val 85 90 95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe
Met Ala Thr Leu 100 105 110 Asn Pro Gly Asp Glu Val Val Ile Pro Ala
Pro Tyr Trp Val Ser Tyr 115 120 125 Pro Glu Met Val Ala Leu Cys Gly
Gly Thr Pro Val Phe Val Pro Thr 130 135 140 Arg Gln Glu Asn Asn Phe
Lys Leu Lys Ala Glu Asp Leu Asp Arg Ala 145 150 155 160 Ile Thr Pro
Lys Thr Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165 170 175 Ser
Gly Ala Ala Tyr Ser His Glu Glu Leu Lys Ala Leu Thr Asp Val 180 185
190 Leu Met Lys His Pro His Val Trp Val Leu Thr Asp Asp Met Tyr Glu
195 200 205 His Leu Thr Tyr Gly Asp Phe Arg Phe Ala Thr Pro Val Glu
Val Glu 210 215 220 Pro Gly Leu Tyr Glu Arg Thr Leu Thr Met Asn Gly
Val Ser Lys Ala 225 230 235 240 Tyr Ala Met Thr Gly Trp Arg Ile Gly
Tyr Ala Ala Gly Pro Leu His 245 250 255 Leu Ile Lys Ala Met Asp Met
Ile Gln Gly Gln Gln Thr Ser Gly Ala 260 265 270 Ala Ser Ile Ala Gln
Trp Ala Ala Val Glu Ala Leu Asn Gly Pro Gln 275 280 285 Asp Phe Ile
Gly Arg Asn Lys Glu Ile Phe Gln Gly Arg Arg Asp Leu 290 295 300 Val
Val Ser Met Leu Asn Gln Ala Lys Gly Ile Ser Cys Pro Thr Pro 305 310
315 320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly
Lys 325 330 335 Thr Ala Pro Ser Gly Lys Val Ile Glu Thr Asp Glu Asp
Phe Val Ser 340 345 350 Glu Leu Leu Glu Thr Glu Gly Val Ala Val Val
His Gly Ser Ala Phe 355 360 365 Gly Leu Gly Pro Asn Phe Arg Ile Ser
Tyr Ala Thr Ser Glu Ala Leu 370 375 380 Leu Glu Glu Ala Cys Arg Arg
Ile Gln Arg Phe Cys Ala Ala Cys Arg 385 390 395 400 Leu Glu
1021167DNAPyrococcus horikoshiiCDS(1)..(1167) 102atg gaa atc atg
gaa ttc gaa gaa gcg ttc aaa gaa gtc tac gaa atg 48Met Glu Ile Met
Glu Phe Glu Glu Ala Phe Lys Glu Val Tyr Glu Met 1 5 10 15 gtc aaa
ccg aaa tac aaa ctg ttc acg gca ggt ccg gtg gct tgc ttt 96Val Lys
Pro Lys Tyr Lys Leu Phe Thr Ala Gly Pro Val Ala Cys Phe 20 25 30
ccg gaa gtc ctg gaa att atg aaa gtg cag atg ttc tcg cat cgt agc
144Pro Glu Val Leu Glu Ile Met Lys Val Gln Met Phe Ser His Arg Ser
35 40 45 aaa gaa tat cgc aaa gtt cac atg gat acc gtc gaa cgt ctg
cgc gaa 192Lys Glu Tyr Arg Lys Val His Met Asp Thr Val Glu Arg Leu
Arg Glu 50 55 60 ttt ctg gaa gtc gaa aaa ggt gaa gtt ctg ctg gtc
ccg agc tct ggc 240Phe Leu Glu Val Glu Lys Gly Glu Val Leu Leu Val
Pro Ser Ser Gly 65 70 75 80 acc ggt att atg gaa gca tcc atc cgt aac
ggc gtt tca aaa ggc ggt 288Thr Gly Ile Met Glu Ala Ser Ile Arg Asn
Gly Val Ser Lys Gly Gly 85 90 95 aaa gtg ctg gtt acg att atc ggc
gcc ttc ggt aaa cgt tat aaa gaa 336Lys Val Leu Val Thr Ile Ile Gly
Ala Phe Gly Lys Arg Tyr Lys Glu 100 105 110 gtg gtt gaa tcc aat ggt
cgc aaa gca gtc gtg ctg gaa tac gaa ccg 384Val Val Glu Ser Asn Gly
Arg Lys Ala Val Val Leu Glu Tyr Glu Pro 115 120 125 ggc aaa gca gtg
aaa ccg gaa gat ctg gat gac gct ctg cgc aaa aac 432Gly Lys Ala Val
Lys Pro Glu Asp Leu Asp Asp Ala Leu Arg Lys Asn 130 135 140 ccg gac
gtg gaa gcg gtt acc att acg tac aac gaa acc tcg acg ggt 480Pro Asp
Val Glu Ala Val Thr Ile Thr Tyr Asn Glu Thr Ser Thr Gly 145 150 155
160 gtt ctg aat ccg ctg ccg gaa ctg gcg aaa gtc gcc aaa gaa cat gat
528Val Leu Asn Pro Leu Pro Glu Leu Ala Lys Val Ala Lys Glu His Asp
165 170 175 aaa ctg gtc ttt gtg gac gca gtg agc gct atg ggc ggt gct
gat atc 576Lys Leu Val Phe Val Asp Ala Val Ser Ala Met Gly Gly Ala
Asp Ile 180 185 190 aaa ttc gac aaa tgg ggc ctg gat gtt gtc ttt agt
tcc tca cag aaa 624Lys Phe Asp Lys Trp Gly Leu Asp Val Val Phe Ser
Ser Ser Gln Lys 195 200 205 gcg ttc ggt gtt ccg ccg ggt ctg gca att
ggt gcc ttt agc gaa cgt 672Ala Phe Gly Val Pro Pro Gly Leu Ala Ile
Gly Ala Phe Ser Glu Arg 210 215 220 ttc ctg gaa atc gcc gaa aaa atg
ccg gaa cgc ggt tgg tat ttt gac 720Phe Leu Glu Ile Ala Glu Lys Met
Pro Glu Arg Gly Trp Tyr Phe Asp 225 230 235 240 att ccg ctg tac gtg
aaa tac ctg aaa gaa aaa gaa tct acc ccg agt 768Ile Pro Leu Tyr Val
Lys Tyr Leu Lys Glu Lys Glu Ser Thr Pro Ser 245 250 255 acg ccg ccg
atg ccg caa gtg ttc ggc atc aat gtt gcc ctg cgt att 816Thr Pro Pro
Met Pro Gln Val Phe Gly Ile Asn Val Ala Leu Arg Ile 260 265 270 atc
gaa aaa atg ggc ggt aaa gaa aaa tgg ctg gaa atg tac gaa aaa 864Ile
Glu Lys Met Gly Gly Lys Glu Lys Trp Leu Glu Met Tyr Glu Lys 275 280
285 cgc gca aaa atg gtc cgt gaa ggt gtg cgc gaa att ggc ctg gat atc
912Arg Ala Lys Met Val Arg Glu Gly Val Arg Glu Ile Gly Leu Asp Ile
290 295 300 ctg gct gaa ccg ggt cat gaa tct ccg acc att acg gcg gtg
ctg acc 960Leu Ala Glu Pro Gly His Glu Ser Pro Thr Ile Thr Ala Val
Leu Thr 305 310 315 320 ccg ccg ggt atc aaa ggt gac gaa gtt tat gaa
gcc atg cgt aaa cgc 1008Pro Pro Gly Ile Lys Gly Asp Glu Val Tyr Glu
Ala Met Arg Lys Arg 325 330 335 ggc ttt gaa ctg gca aaa ggc tac ggt
tca gtt aaa gaa aaa acc ttt 1056Gly Phe Glu Leu Ala Lys Gly Tyr Gly
Ser Val Lys Glu Lys Thr Phe 340 345 350 cgt att ggc cac atg ggt tat
atg aaa ttc gaa gat atc caa gaa atg 1104Arg Ile Gly His Met Gly Tyr
Met Lys Phe Glu Asp Ile Gln Glu Met 355 360 365 ctg gac aat ctg cgt
gaa gtc atc aac gaa ctg aaa aaa caa aaa ggt 1152Leu Asp Asn Leu Arg
Glu Val Ile Asn Glu Leu Lys Lys Gln Lys Gly 370 375 380 atc aac ctc
gag taa 1167Ile Asn Leu Glu 385 103388PRTPyrococcus horikoshii
103Met Glu Ile Met Glu Phe Glu Glu Ala Phe Lys Glu Val Tyr Glu Met
1 5 10 15 Val Lys Pro Lys Tyr Lys Leu Phe Thr Ala Gly Pro Val Ala
Cys Phe 20 25 30 Pro Glu Val Leu Glu Ile Met Lys Val Gln Met Phe
Ser His Arg Ser 35 40 45 Lys Glu Tyr Arg Lys Val His Met Asp Thr
Val Glu Arg Leu Arg Glu 50 55 60 Phe Leu Glu Val Glu Lys Gly Glu
Val Leu Leu Val Pro Ser Ser Gly 65 70 75 80 Thr Gly Ile Met Glu Ala
Ser Ile Arg Asn Gly Val Ser Lys Gly Gly 85 90 95 Lys Val Leu Val
Thr Ile Ile Gly Ala Phe Gly Lys Arg Tyr Lys Glu 100 105 110 Val Val
Glu Ser Asn Gly Arg Lys Ala Val Val Leu Glu Tyr Glu Pro 115 120 125
Gly Lys Ala Val Lys Pro Glu Asp Leu Asp Asp Ala Leu Arg Lys Asn 130
135 140 Pro Asp Val Glu Ala Val Thr Ile Thr Tyr Asn Glu Thr Ser Thr
Gly 145 150 155 160 Val Leu Asn Pro Leu Pro Glu Leu Ala Lys Val Ala
Lys Glu His Asp 165 170 175 Lys Leu Val Phe Val Asp Ala Val Ser Ala
Met Gly Gly Ala Asp Ile 180 185 190 Lys Phe Asp Lys Trp Gly Leu Asp
Val Val Phe Ser Ser Ser Gln Lys 195 200 205 Ala Phe Gly Val Pro Pro
Gly Leu Ala Ile Gly Ala Phe Ser Glu Arg 210 215 220 Phe Leu Glu Ile
Ala Glu Lys Met Pro Glu Arg Gly Trp Tyr Phe Asp 225 230 235 240 Ile
Pro Leu Tyr Val Lys Tyr Leu Lys Glu Lys Glu Ser Thr Pro Ser 245 250
255 Thr Pro Pro Met Pro Gln Val Phe Gly Ile Asn Val Ala Leu Arg Ile
260 265 270 Ile Glu Lys Met Gly Gly Lys Glu Lys Trp Leu Glu Met Tyr
Glu Lys 275 280 285 Arg Ala Lys Met Val Arg Glu Gly Val Arg Glu Ile
Gly Leu Asp Ile 290 295 300 Leu Ala Glu Pro Gly His Glu Ser Pro Thr
Ile Thr Ala Val Leu Thr 305 310 315 320 Pro Pro Gly Ile Lys Gly Asp
Glu Val Tyr Glu Ala Met Arg Lys Arg 325 330 335 Gly Phe Glu Leu Ala
Lys Gly Tyr Gly Ser Val Lys Glu Lys Thr Phe 340 345 350 Arg Ile Gly
His Met Gly Tyr Met Lys Phe Glu Asp Ile Gln Glu Met 355 360 365 Leu
Asp Asn Leu Arg Glu Val Ile Asn Glu Leu Lys Lys Gln Lys Gly 370 375
380 Ile Asn Leu Glu 385 1041194DNAThermoanaerobacter
tengcongensisCDS(1)..(1194) 104atg aac ctg agc caa aac gca ctg caa
atc acg ccg agt atg acc ctg 48Met Asn Leu Ser Gln Asn Ala Leu Gln
Ile Thr Pro Ser Met Thr Leu 1 5 10 15 gaa atc acc gcc aaa gcc cgc
caa ctg aaa gcc gaa ggc gtc gat gtg 96Glu Ile Thr Ala Lys Ala Arg
Gln Leu Lys Ala Glu Gly Val Asp Val 20 25 30 att gac ttt ggc gtg
ggt gaa ccg gat ttc gac acc ccg gat tat atc 144Ile Asp Phe Gly Val
Gly Glu Pro Asp Phe Asp Thr Pro Asp Tyr Ile 35 40 45 aaa gaa gcg
gcc att gaa gcc atc aaa aaa ggt tat acc aaa tac acg 192Lys Glu Ala
Ala Ile Glu Ala Ile Lys Lys Gly Tyr Thr Lys Tyr Thr 50 55 60 ccg
gca tct ggc att ctg gaa ctg aaa aaa gct atc tgc gaa aaa ctg 240Pro
Ala Ser Gly Ile Leu Glu Leu Lys Lys Ala Ile Cys Glu Lys Leu 65 70
75 80 aaa cgt gaa aac ggt ctg ttt tat gaa ccg gaa cag att gtg gtt
tct 288Lys Arg Glu Asn Gly Leu Phe Tyr Glu Pro Glu Gln Ile Val Val
Ser 85 90 95 aat ggc gca aaa cat agt att tac aac gca ctg tcc gct
atc ctg aat 336Asn Gly Ala Lys His Ser Ile Tyr Asn Ala Leu Ser Ala
Ile Leu Asn 100 105 110 ccg ggt gat gaa gtt att atc ccg gtc ccg tat
tgg ctg agc tac ccg 384Pro Gly Asp Glu Val Ile Ile Pro Val Pro Tyr
Trp Leu Ser Tyr Pro 115 120 125 gaa atg gtg cgc ctg gcg tat ggc aaa
ccg gtt ttt gtc cag acc aaa 432Glu Met Val Arg Leu Ala Tyr Gly Lys
Pro Val Phe Val Gln Thr Lys 130 135 140 gaa gaa aac aac ttc aaa atc
acc gca gaa gaa ctg acg gca gct att 480Glu Glu Asn Asn Phe Lys Ile
Thr Ala Glu Glu Leu Thr Ala Ala Ile 145 150 155 160 aac ccg aaa acg
aaa gct ctg atc ctg aat
tca ccg aac aat ccg acc 528Asn Pro Lys Thr Lys Ala Leu Ile Leu Asn
Ser Pro Asn Asn Pro Thr 165 170 175 ggt gcg gtg tat acg cgt aaa gaa
ctg caa gat atc gcc gaa gtc gtg 576Gly Ala Val Tyr Thr Arg Lys Glu
Leu Gln Asp Ile Ala Glu Val Val 180 185 190 gaa gaa acc ggc att ttt
gtc atc tcg gac gaa gtg tat gaa aaa ctg 624Glu Glu Thr Gly Ile Phe
Val Ile Ser Asp Glu Val Tyr Glu Lys Leu 195 200 205 att tac gaa ggt
gaa cat gtt agt atc gct tcc ctg ggc gaa aaa att 672Ile Tyr Glu Gly
Glu His Val Ser Ile Ala Ser Leu Gly Glu Lys Ile 210 215 220 aaa gaa
ctg acc atc gtt gtc aac ggt atg agt aaa gcg tat gcc atg 720Lys Glu
Leu Thr Ile Val Val Asn Gly Met Ser Lys Ala Tyr Ala Met 225 230 235
240 acc ggc tgg cgt att ggt tac acg gcc agc tct ctg gat gtc gcg aaa
768Thr Gly Trp Arg Ile Gly Tyr Thr Ala Ser Ser Leu Asp Val Ala Lys
245 250 255 gtg atg gcc aat att cag tca cac acc acg tcg aac ccg aat
agc atc 816Val Met Ala Asn Ile Gln Ser His Thr Thr Ser Asn Pro Asn
Ser Ile 260 265 270 gcg caa tat gcc agc gtg acc gca ctg acg ggt gac
ggt gtt gcc att 864Ala Gln Tyr Ala Ser Val Thr Ala Leu Thr Gly Asp
Gly Val Ala Ile 275 280 285 aaa cgc atg gtc gaa gaa ttc aac aaa cgt
cgc ctg tac gcg gtg gaa 912Lys Arg Met Val Glu Glu Phe Asn Lys Arg
Arg Leu Tyr Ala Val Glu 290 295 300 cgt atc tct aaa atg aaa ggt ctg
aaa gca gtt cgc ccg caa ggc gct 960Arg Ile Ser Lys Met Lys Gly Leu
Lys Ala Val Arg Pro Gln Gly Ala 305 310 315 320 ttc tac gtg ttc gtt
aac atc gaa gaa tac gtg ggc aaa aaa gtt aac 1008Phe Tyr Val Phe Val
Asn Ile Glu Glu Tyr Val Gly Lys Lys Val Asn 325 330 335 ggt cgt aaa
atc aaa ggc agt ctg gat ttt gcg acc ctg ctg atc gaa 1056Gly Arg Lys
Ile Lys Gly Ser Leu Asp Phe Ala Thr Leu Leu Ile Glu 340 345 350 gaa
gca aac gtt gct gtg gtt ccg gcc ctg ccg ttc ggc atg gac aat 1104Glu
Ala Asn Val Ala Val Val Pro Ala Leu Pro Phe Gly Met Asp Asn 355 360
365 tat att cgc atc tcc tac gca acg agt atg gaa aac att gaa aaa ggt
1152Tyr Ile Arg Ile Ser Tyr Ala Thr Ser Met Glu Asn Ile Glu Lys Gly
370 375 380 ctg gat cgc att gaa aac ttc ctg aat aaa atc ctc gag taa
1194Leu Asp Arg Ile Glu Asn Phe Leu Asn Lys Ile Leu Glu 385 390 395
105397PRTThermoanaerobacter tengcongensis 105Met Asn Leu Ser Gln
Asn Ala Leu Gln Ile Thr Pro Ser Met Thr Leu 1 5 10 15 Glu Ile Thr
Ala Lys Ala Arg Gln Leu Lys Ala Glu Gly Val Asp Val 20 25 30 Ile
Asp Phe Gly Val Gly Glu Pro Asp Phe Asp Thr Pro Asp Tyr Ile 35 40
45 Lys Glu Ala Ala Ile Glu Ala Ile Lys Lys Gly Tyr Thr Lys Tyr Thr
50 55 60 Pro Ala Ser Gly Ile Leu Glu Leu Lys Lys Ala Ile Cys Glu
Lys Leu 65 70 75 80 Lys Arg Glu Asn Gly Leu Phe Tyr Glu Pro Glu Gln
Ile Val Val Ser 85 90 95 Asn Gly Ala Lys His Ser Ile Tyr Asn Ala
Leu Ser Ala Ile Leu Asn 100 105 110 Pro Gly Asp Glu Val Ile Ile Pro
Val Pro Tyr Trp Leu Ser Tyr Pro 115 120 125 Glu Met Val Arg Leu Ala
Tyr Gly Lys Pro Val Phe Val Gln Thr Lys 130 135 140 Glu Glu Asn Asn
Phe Lys Ile Thr Ala Glu Glu Leu Thr Ala Ala Ile 145 150 155 160 Asn
Pro Lys Thr Lys Ala Leu Ile Leu Asn Ser Pro Asn Asn Pro Thr 165 170
175 Gly Ala Val Tyr Thr Arg Lys Glu Leu Gln Asp Ile Ala Glu Val Val
180 185 190 Glu Glu Thr Gly Ile Phe Val Ile Ser Asp Glu Val Tyr Glu
Lys Leu 195 200 205 Ile Tyr Glu Gly Glu His Val Ser Ile Ala Ser Leu
Gly Glu Lys Ile 210 215 220 Lys Glu Leu Thr Ile Val Val Asn Gly Met
Ser Lys Ala Tyr Ala Met 225 230 235 240 Thr Gly Trp Arg Ile Gly Tyr
Thr Ala Ser Ser Leu Asp Val Ala Lys 245 250 255 Val Met Ala Asn Ile
Gln Ser His Thr Thr Ser Asn Pro Asn Ser Ile 260 265 270 Ala Gln Tyr
Ala Ser Val Thr Ala Leu Thr Gly Asp Gly Val Ala Ile 275 280 285 Lys
Arg Met Val Glu Glu Phe Asn Lys Arg Arg Leu Tyr Ala Val Glu 290 295
300 Arg Ile Ser Lys Met Lys Gly Leu Lys Ala Val Arg Pro Gln Gly Ala
305 310 315 320 Phe Tyr Val Phe Val Asn Ile Glu Glu Tyr Val Gly Lys
Lys Val Asn 325 330 335 Gly Arg Lys Ile Lys Gly Ser Leu Asp Phe Ala
Thr Leu Leu Ile Glu 340 345 350 Glu Ala Asn Val Ala Val Val Pro Ala
Leu Pro Phe Gly Met Asp Asn 355 360 365 Tyr Ile Arg Ile Ser Tyr Ala
Thr Ser Met Glu Asn Ile Glu Lys Gly 370 375 380 Leu Asp Arg Ile Glu
Asn Phe Leu Asn Lys Ile Leu Glu 385 390 395 1061284DNAClostridium
cellulolyticumCDS(1)..(1284) 106atg aaa agc tac aaa gac ctg agc aaa
gaa gaa ctg aaa tcc gaa atc 48Met Lys Ser Tyr Lys Asp Leu Ser Lys
Glu Glu Leu Lys Ser Glu Ile 1 5 10 15 gaa atc ctg gaa aaa cgc tac
aac gaa ttc aaa gca caa aac ctg aaa 96Glu Ile Leu Glu Lys Arg Tyr
Asn Glu Phe Lys Ala Gln Asn Leu Lys 20 25 30 ctg gat atg acc cgt
ggt aaa ccg tgc gct gaa cag ctg gac ctg tct 144Leu Asp Met Thr Arg
Gly Lys Pro Cys Ala Glu Gln Leu Asp Leu Ser 35 40 45 atg gat atg
ctg gac att ccg gcg gtg gaa ctg cgc aaa gcg gcc gat 192Met Asp Met
Leu Asp Ile Pro Ala Val Glu Leu Arg Lys Ala Ala Asp 50 55 60 ggc
acc gac tgt ttt aat tat ggc gtt ctg gat ggt att ccg gaa gca 240Gly
Thr Asp Cys Phe Asn Tyr Gly Val Leu Asp Gly Ile Pro Glu Ala 65 70
75 80 aaa gct ctg ttc gcc caa atg ctg gaa gtg agc acg gat gaa atc
atg 288Lys Ala Leu Phe Ala Gln Met Leu Glu Val Ser Thr Asp Glu Ile
Met 85 90 95 gtt ggc ggt aac agc tct ctg aat ctg atg tat gac acc
att gcg cgt 336Val Gly Gly Asn Ser Ser Leu Asn Leu Met Tyr Asp Thr
Ile Ala Arg 100 105 110 gcc atg tcg ctg ggc atc ctg ggt agc acg ccg
tgg tct aaa ctg aac 384Ala Met Ser Leu Gly Ile Leu Gly Ser Thr Pro
Trp Ser Lys Leu Asn 115 120 125 agt gtg aaa ttt ctg tgc ccg agc ccg
ggc tac gat cgc cat ttt gca 432Ser Val Lys Phe Leu Cys Pro Ser Pro
Gly Tyr Asp Arg His Phe Ala 130 135 140 att tgt gaa ctg ttc ggt atc
gaa atg att acc atc gat atg aaa cag 480Ile Cys Glu Leu Phe Gly Ile
Glu Met Ile Thr Ile Asp Met Lys Gln 145 150 155 160 gac ggc ccg gat
atg gac acg gtt gaa aaa ctg gtc tcc gaa gat gac 528Asp Gly Pro Asp
Met Asp Thr Val Glu Lys Leu Val Ser Glu Asp Asp 165 170 175 tca att
aaa ggt atc tgg tgc gtg ccg aaa tat tcc aat ccg gat ggc 576Ser Ile
Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro Asp Gly 180 185 190
att acc tac acg gat gaa gtg gtt gac cgt ttc tca aac ctg aaa ccg
624Ile Thr Tyr Thr Asp Glu Val Val Asp Arg Phe Ser Asn Leu Lys Pro
195 200 205 aaa gcc aaa gat ttt cgc atc ttc tgg gac aat gca tat tgc
gtt cat 672Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp Asn Ala Tyr Cys
Val His 210 215 220 cac ctg acc gaa aac ccg gat aaa ctg aaa aac atc
ctg aaa gct tgt 720His Leu Thr Glu Asn Pro Asp Lys Leu Lys Asn Ile
Leu Lys Ala Cys 225 230 235 240 aaa gat gcg ggt aac gac aat atg gtc
tac atc ttt agt tcc acg tca 768Lys Asp Ala Gly Asn Asp Asn Met Val
Tyr Ile Phe Ser Ser Thr Ser 245 250 255 aaa gtg tcg ttc ccg ggt gca
ggt gtc gca gtg atg gca acc tcg acg 816Lys Val Ser Phe Pro Gly Ala
Gly Val Ala Val Met Ala Thr Ser Thr 260 265 270 gaa aac atc aaa ggc
att aaa aaa tct ctg acc atc cag acg atc ggt 864Glu Asn Ile Lys Gly
Ile Lys Lys Ser Leu Thr Ile Gln Thr Ile Gly 275 280 285 cat gat aaa
att aat caa ctg cgt cac gcc aaa tac ttc aaa aac ctg 912His Asp Lys
Ile Asn Gln Leu Arg His Ala Lys Tyr Phe Lys Asn Leu 290 295 300 gat
ggt atc aac agc cac atg aaa aaa cac gca gac atc ctg aaa ccg 960Asp
Gly Ile Asn Ser His Met Lys Lys His Ala Asp Ile Leu Lys Pro 305 310
315 320 aaa ttt aac acc gtc ctg gaa att ttc gaa ggc gaa ctg ggc ggt
aaa 1008Lys Phe Asn Thr Val Leu Glu Ile Phe Glu Gly Glu Leu Gly Gly
Lys 325 330 335 gat atc gct tct tgg aac aaa ccg aat ggc ggt tat ttt
gtt agt ctg 1056Asp Ile Ala Ser Trp Asn Lys Pro Asn Gly Gly Tyr Phe
Val Ser Leu 340 345 350 aac acc atg gat aat tgt gcg aaa gaa gtt gct
aaa ctg gcg agt gaa 1104Asn Thr Met Asp Asn Cys Ala Lys Glu Val Ala
Lys Leu Ala Ser Glu 355 360 365 gcc ggc gtc gca ctg acc aaa gca ggc
gct acg ttc ccg tac ggt aac 1152Ala Gly Val Ala Leu Thr Lys Ala Gly
Ala Thr Phe Pro Tyr Gly Asn 370 375 380 gat ccg cgt gac cgc aat ctg
cgc att gcc ccg acc atg ccg ccg atc 1200Asp Pro Arg Asp Arg Asn Leu
Arg Ile Ala Pro Thr Met Pro Pro Ile 385 390 395 400 gaa gaa ctg aaa
aaa gcc att gaa gtc ctg gtc att tgt gtc caa ctg 1248Glu Glu Leu Lys
Lys Ala Ile Glu Val Leu Val Ile Cys Val Gln Leu 405 410 415 gtc tcc
gct aat aaa ctg ctg aat caa ctc gag taa 1284Val Ser Ala Asn Lys Leu
Leu Asn Gln Leu Glu 420 425 107427PRTClostridium cellulolyticum
107Met Lys Ser Tyr Lys Asp Leu Ser Lys Glu Glu Leu Lys Ser Glu Ile
1 5 10 15 Glu Ile Leu Glu Lys Arg Tyr Asn Glu Phe Lys Ala Gln Asn
Leu Lys 20 25 30 Leu Asp Met Thr Arg Gly Lys Pro Cys Ala Glu Gln
Leu Asp Leu Ser 35 40 45 Met Asp Met Leu Asp Ile Pro Ala Val Glu
Leu Arg Lys Ala Ala Asp 50 55 60 Gly Thr Asp Cys Phe Asn Tyr Gly
Val Leu Asp Gly Ile Pro Glu Ala 65 70 75 80 Lys Ala Leu Phe Ala Gln
Met Leu Glu Val Ser Thr Asp Glu Ile Met 85 90 95 Val Gly Gly Asn
Ser Ser Leu Asn Leu Met Tyr Asp Thr Ile Ala Arg 100 105 110 Ala Met
Ser Leu Gly Ile Leu Gly Ser Thr Pro Trp Ser Lys Leu Asn 115 120 125
Ser Val Lys Phe Leu Cys Pro Ser Pro Gly Tyr Asp Arg His Phe Ala 130
135 140 Ile Cys Glu Leu Phe Gly Ile Glu Met Ile Thr Ile Asp Met Lys
Gln 145 150 155 160 Asp Gly Pro Asp Met Asp Thr Val Glu Lys Leu Val
Ser Glu Asp Asp 165 170 175 Ser Ile Lys Gly Ile Trp Cys Val Pro Lys
Tyr Ser Asn Pro Asp Gly 180 185 190 Ile Thr Tyr Thr Asp Glu Val Val
Asp Arg Phe Ser Asn Leu Lys Pro 195 200 205 Lys Ala Lys Asp Phe Arg
Ile Phe Trp Asp Asn Ala Tyr Cys Val His 210 215 220 His Leu Thr Glu
Asn Pro Asp Lys Leu Lys Asn Ile Leu Lys Ala Cys 225 230 235 240 Lys
Asp Ala Gly Asn Asp Asn Met Val Tyr Ile Phe Ser Ser Thr Ser 245 250
255 Lys Val Ser Phe Pro Gly Ala Gly Val Ala Val Met Ala Thr Ser Thr
260 265 270 Glu Asn Ile Lys Gly Ile Lys Lys Ser Leu Thr Ile Gln Thr
Ile Gly 275 280 285 His Asp Lys Ile Asn Gln Leu Arg His Ala Lys Tyr
Phe Lys Asn Leu 290 295 300 Asp Gly Ile Asn Ser His Met Lys Lys His
Ala Asp Ile Leu Lys Pro 305 310 315 320 Lys Phe Asn Thr Val Leu Glu
Ile Phe Glu Gly Glu Leu Gly Gly Lys 325 330 335 Asp Ile Ala Ser Trp
Asn Lys Pro Asn Gly Gly Tyr Phe Val Ser Leu 340 345 350 Asn Thr Met
Asp Asn Cys Ala Lys Glu Val Ala Lys Leu Ala Ser Glu 355 360 365 Ala
Gly Val Ala Leu Thr Lys Ala Gly Ala Thr Phe Pro Tyr Gly Asn 370 375
380 Asp Pro Arg Asp Arg Asn Leu Arg Ile Ala Pro Thr Met Pro Pro Ile
385 390 395 400 Glu Glu Leu Lys Lys Ala Ile Glu Val Leu Val Ile Cys
Val Gln Leu 405 410 415 Val Ser Ala Asn Lys Leu Leu Asn Gln Leu Glu
420 425 1081299DNARhodococcus erythropolisCDS(1)..(1299) 108atg ccg
att caa acc cag att ggt ctg atg agc cac gaa gaa ctg acg 48Met Pro
Ile Gln Thr Gln Ile Gly Leu Met Ser His Glu Glu Leu Thr 1 5 10 15
agc gaa cac gaa agc caa agc gca aaa tac acg caa ctg caa gct caa
96Ser Glu His Glu Ser Gln Ser Ala Lys Tyr Thr Gln Leu Gln Ala Gln
20 25 30 aaa ctg gcg ctg gat ctg acc cgt ggt aaa ccg agc ccg gaa
cag ctg 144Lys Leu Ala Leu Asp Leu Thr Arg Gly Lys Pro Ser Pro Glu
Gln Leu 35 40 45 gac ctg tct gcc gaa ctg ctg acg ctg ccg ggc gat
ggt gac ttt cgt 192Asp Leu Ser Ala Glu Leu Leu Thr Leu Pro Gly Asp
Gly Asp Phe Arg 50 55 60 gat ggc agc ggt acc gac tgc cgc aat tat
ggt ggt ctg acg ggt ctg 240Asp Gly Ser Gly Thr Asp Cys Arg Asn Tyr
Gly Gly Leu Thr Gly Leu 65 70 75 80 ccg gaa ctg cgt gcg att ttc ggc
gaa ctg ctg ggt atc ccg gtg gcc 288Pro Glu Leu Arg Ala Ile Phe Gly
Glu Leu Leu Gly Ile Pro Val Ala 85 90 95 aac ctg ctg gcc ggt aac
aat gct tcc ctg gaa atc atg cat gat aac 336Asn Leu Leu Ala Gly Asn
Asn Ala Ser Leu Glu Ile Met His Asp Asn 100 105 110 gtg gtt ttt agt
ctg ctg cac ggt acc ccg gac tcc gca cgt ccg tgg 384Val Val Phe Ser
Leu Leu His Gly Thr Pro Asp Ser Ala Arg Pro Trp 115 120 125 gcc cag
gaa gaa aaa att aaa ttt ctg tgt ccg gct ccg ggc tac gat 432Ala Gln
Glu Glu Lys Ile Lys Phe Leu Cys Pro Ala Pro Gly Tyr Asp 130 135 140
cgc cat ttc gcg atc acg gaa agt ctg ggt att gaa atg atc gcc gtg
480Arg His Phe Ala Ile Thr Glu Ser Leu Gly Ile Glu Met Ile Ala Val
145 150 155 160 ccg atg aac cac gat ggc ccg gac gtc gtg aaa att gca
gaa ctg gtt 528Pro Met Asn His Asp Gly Pro Asp Val Val Lys Ile Ala
Glu Leu Val 165 170
175 gct tct gat ccg caa atc aaa ggc atg tgg gcg gtg ccg gtt tat gcc
576Ala Ser Asp Pro Gln Ile Lys Gly Met Trp Ala Val Pro Val Tyr Ala
180 185 190 aat ccg acc ggc gca gtt tac tca gaa gaa att gtc cgt acc
ctg gca 624Asn Pro Thr Gly Ala Val Tyr Ser Glu Glu Ile Val Arg Thr
Leu Ala 195 200 205 tcg atg ccg acg gcg gcc ccg gat ttt cgt ctg tat
tgg gac aac gca 672Ser Met Pro Thr Ala Ala Pro Asp Phe Arg Leu Tyr
Trp Asp Asn Ala 210 215 220 tac gct gtc cat ccg ctg gtt ggc gaa acc
gcg ccg agt tat gat att 720Tyr Ala Val His Pro Leu Val Gly Glu Thr
Ala Pro Ser Tyr Asp Ile 225 230 235 240 ctg tcc atg gca gct gaa gca
ggt cac ccg aac cgt ccg ctg gtc ttt 768Leu Ser Met Ala Ala Glu Ala
Gly His Pro Asn Arg Pro Leu Val Phe 245 250 255 gca agt acc tcc aaa
atc acg ttc gcg ggc gcc ggt gtg agc ttt ttc 816Ala Ser Thr Ser Lys
Ile Thr Phe Ala Gly Ala Gly Val Ser Phe Phe 260 265 270 ggt agc tct
gcg gaa aat ctg gcc tgg tac cag aaa ttc ctg ggc aaa 864Gly Ser Ser
Ala Glu Asn Leu Ala Trp Tyr Gln Lys Phe Leu Gly Lys 275 280 285 aaa
tct atc ggt ccg gat aaa gtt aac caa ctg cgt cat ctg cgc ttt 912Lys
Ser Ile Gly Pro Asp Lys Val Asn Gln Leu Arg His Leu Arg Phe 290 295
300 ttc ggc aat gct gac ggt gtc cgt gcg cac atg gaa aaa cac cgc gca
960Phe Gly Asn Ala Asp Gly Val Arg Ala His Met Glu Lys His Arg Ala
305 310 315 320 ttt ctg gct ccg aaa ttc gaa ctg gtg ctg cgt att ctg
gaa gat cgc 1008Phe Leu Ala Pro Lys Phe Glu Leu Val Leu Arg Ile Leu
Glu Asp Arg 325 330 335 ctg ggt gca agc aaa gtt gct tct tgg acc gaa
ccg aaa ggc ggt tat 1056Leu Gly Ala Ser Lys Val Ala Ser Trp Thr Glu
Pro Lys Gly Gly Tyr 340 345 350 ttt atc agc ctg gat gtt gtc gac ggc
acg gcg aaa cgc gtg att gaa 1104Phe Ile Ser Leu Asp Val Val Asp Gly
Thr Ala Lys Arg Val Ile Glu 355 360 365 ctg gcg aaa aac gca ggt atc
gca ctg acc gcg gcc ggt tca gcg ttt 1152Leu Ala Lys Asn Ala Gly Ile
Ala Leu Thr Ala Ala Gly Ser Ala Phe 370 375 380 ccg tac tcg acg gat
ccg gat gac cgt aat att cgt ctg gca ccg tca 1200Pro Tyr Ser Thr Asp
Pro Asp Asp Arg Asn Ile Arg Leu Ala Pro Ser 385 390 395 400 ttc ccg
tcg acc gcc gaa ctg gaa gtt gca atg gat ggt gtc gca acc 1248Phe Pro
Ser Thr Ala Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr 405 410 415
tgt gtc ctg ctg gcc gca acg gaa tca cgc ctg tca gaa atc ctc gag
1296Cys Val Leu Leu Ala Ala Thr Glu Ser Arg Leu Ser Glu Ile Leu Glu
420 425 430 taa 1299109432PRTRhodococcus erythropolis 109Met Pro
Ile Gln Thr Gln Ile Gly Leu Met Ser His Glu Glu Leu Thr 1 5 10 15
Ser Glu His Glu Ser Gln Ser Ala Lys Tyr Thr Gln Leu Gln Ala Gln 20
25 30 Lys Leu Ala Leu Asp Leu Thr Arg Gly Lys Pro Ser Pro Glu Gln
Leu 35 40 45 Asp Leu Ser Ala Glu Leu Leu Thr Leu Pro Gly Asp Gly
Asp Phe Arg 50 55 60 Asp Gly Ser Gly Thr Asp Cys Arg Asn Tyr Gly
Gly Leu Thr Gly Leu 65 70 75 80 Pro Glu Leu Arg Ala Ile Phe Gly Glu
Leu Leu Gly Ile Pro Val Ala 85 90 95 Asn Leu Leu Ala Gly Asn Asn
Ala Ser Leu Glu Ile Met His Asp Asn 100 105 110 Val Val Phe Ser Leu
Leu His Gly Thr Pro Asp Ser Ala Arg Pro Trp 115 120 125 Ala Gln Glu
Glu Lys Ile Lys Phe Leu Cys Pro Ala Pro Gly Tyr Asp 130 135 140 Arg
His Phe Ala Ile Thr Glu Ser Leu Gly Ile Glu Met Ile Ala Val 145 150
155 160 Pro Met Asn His Asp Gly Pro Asp Val Val Lys Ile Ala Glu Leu
Val 165 170 175 Ala Ser Asp Pro Gln Ile Lys Gly Met Trp Ala Val Pro
Val Tyr Ala 180 185 190 Asn Pro Thr Gly Ala Val Tyr Ser Glu Glu Ile
Val Arg Thr Leu Ala 195 200 205 Ser Met Pro Thr Ala Ala Pro Asp Phe
Arg Leu Tyr Trp Asp Asn Ala 210 215 220 Tyr Ala Val His Pro Leu Val
Gly Glu Thr Ala Pro Ser Tyr Asp Ile 225 230 235 240 Leu Ser Met Ala
Ala Glu Ala Gly His Pro Asn Arg Pro Leu Val Phe 245 250 255 Ala Ser
Thr Ser Lys Ile Thr Phe Ala Gly Ala Gly Val Ser Phe Phe 260 265 270
Gly Ser Ser Ala Glu Asn Leu Ala Trp Tyr Gln Lys Phe Leu Gly Lys 275
280 285 Lys Ser Ile Gly Pro Asp Lys Val Asn Gln Leu Arg His Leu Arg
Phe 290 295 300 Phe Gly Asn Ala Asp Gly Val Arg Ala His Met Glu Lys
His Arg Ala 305 310 315 320 Phe Leu Ala Pro Lys Phe Glu Leu Val Leu
Arg Ile Leu Glu Asp Arg 325 330 335 Leu Gly Ala Ser Lys Val Ala Ser
Trp Thr Glu Pro Lys Gly Gly Tyr 340 345 350 Phe Ile Ser Leu Asp Val
Val Asp Gly Thr Ala Lys Arg Val Ile Glu 355 360 365 Leu Ala Lys Asn
Ala Gly Ile Ala Leu Thr Ala Ala Gly Ser Ala Phe 370 375 380 Pro Tyr
Ser Thr Asp Pro Asp Asp Arg Asn Ile Arg Leu Ala Pro Ser 385 390 395
400 Phe Pro Ser Thr Ala Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr
405 410 415 Cys Val Leu Leu Ala Ala Thr Glu Ser Arg Leu Ser Glu Ile
Leu Glu 420 425 430 1101356DNASaccharophagus
degradansCDS(1)..(1356) 110atg caa gct ccg tac acc aat ctg aaa acg
cac cac aaa tgg gtc gaa 48Met Gln Ala Pro Tyr Thr Asn Leu Lys Thr
His His Lys Trp Val Glu 1 5 10 15 ttc tcc gcc gaa gac acc acg ctg
aac ctg agt aac gcc tca gtc gaa 96Phe Ser Ala Glu Asp Thr Thr Leu
Asn Leu Ser Asn Ala Ser Val Glu 20 25 30 cag ctg caa gaa tgg aaa
cag caa ctg tcg gcg gaa tat gat aac gtt 144Gln Leu Gln Glu Trp Lys
Gln Gln Leu Ser Ala Glu Tyr Asp Asn Val 35 40 45 ctg gcc cgt aaa
ctg aat ctg gac ctg acc cgc ggc aaa ccg agt gcg 192Leu Ala Arg Lys
Leu Asn Leu Asp Leu Thr Arg Gly Lys Pro Ser Ala 50 55 60 gaa cag
ctg agt ctg tcc gat gct atg gac ggc att ctg gcg ggt gat 240Glu Gln
Leu Ser Leu Ser Asp Ala Met Asp Gly Ile Leu Ala Gly Asp 65 70 75 80
tat att acg gcc agt ggc atc gac gtg cgt aac tac ggc ggt ctg gaa
288Tyr Ile Thr Ala Ser Gly Ile Asp Val Arg Asn Tyr Gly Gly Leu Glu
85 90 95 ggt atc ccg gaa gcg cgt gcg att ggc tcc gat atc ctg ggt
gtt ccg 336Gly Ile Pro Glu Ala Arg Ala Ile Gly Ser Asp Ile Leu Gly
Val Pro 100 105 110 gtc gaa aac gtt ctg gcc ggc ggt aat agc tct ctg
acc ctg atg tac 384Val Glu Asn Val Leu Ala Gly Gly Asn Ser Ser Leu
Thr Leu Met Tyr 115 120 125 cag acg atg gca att gct cat caa ttc ggt
ctg gct ggc gaa ggt agc 432Gln Thr Met Ala Ile Ala His Gln Phe Gly
Leu Ala Gly Glu Gly Ser 130 135 140 gcg tgg tct cag gaa ggc acc gtg
aaa ttt ctg tgc ccg gtt ccg ggt 480Ala Trp Ser Gln Glu Gly Thr Val
Lys Phe Leu Cys Pro Val Pro Gly 145 150 155 160 tat gat cgt cat tac
agc gtt tgt gaa cac ctg ggc atc gaa atg ctg 528Tyr Asp Arg His Tyr
Ser Val Cys Glu His Leu Gly Ile Glu Met Leu 165 170 175 acc gtc gcg
atg acc tct acg ggt ccg gat atg gac caa gtg gaa aaa 576Thr Val Ala
Met Thr Ser Thr Gly Pro Asp Met Asp Gln Val Glu Lys 180 185 190 atg
att gcg gcc gat ccg agc atc aaa ggc atg tgg tgc gtt ccg aaa 624Met
Ile Ala Ala Asp Pro Ser Ile Lys Gly Met Trp Cys Val Pro Lys 195 200
205 tat agt aat ccg acc ggt gtg gtt tac tcc gac gaa acg gtc gaa cgt
672Tyr Ser Asn Pro Thr Gly Val Val Tyr Ser Asp Glu Thr Val Glu Arg
210 215 220 att gca aac ctg ggc aat atc gct ggt aaa aac ttt cgc gtg
ttc tgg 720Ile Ala Asn Leu Gly Asn Ile Ala Gly Lys Asn Phe Arg Val
Phe Trp 225 230 235 240 gat aat gcg tat gcc att cat gat ctg tca gac
aac ccg gtt gca ctg 768Asp Asn Ala Tyr Ala Ile His Asp Leu Ser Asp
Asn Pro Val Ala Leu 245 250 255 gct aat atc ttt gaa gcc tgt aaa gca
gct ggc acc gaa gat tcg gtg 816Ala Asn Ile Phe Glu Ala Cys Lys Ala
Ala Gly Thr Glu Asp Ser Val 260 265 270 att cag ttc gca tca acc tcg
aaa gtc acg cac gcc ggc agc ggt gtg 864Ile Gln Phe Ala Ser Thr Ser
Lys Val Thr His Ala Gly Ser Gly Val 275 280 285 gca ttt atc gcg gcc
tcg gat acc aac ctg aaa ttt ttc aaa ctg gca 912Ala Phe Ile Ala Ala
Ser Asp Thr Asn Leu Lys Phe Phe Lys Leu Ala 290 295 300 ctg ggc ttc
atg acg att ggt ccg gat aaa gtg aat cag ctg cgt cat 960Leu Gly Phe
Met Thr Ile Gly Pro Asp Lys Val Asn Gln Leu Arg His 305 310 315 320
gcc aaa ttt ttc gca gct gac ggt gca ctg tca gct cac atg gcg aaa
1008Ala Lys Phe Phe Ala Ala Asp Gly Ala Leu Ser Ala His Met Ala Lys
325 330 335 cac gcg gcc att atc aaa ccg cgc ttt gcg agt gtt ctg aaa
cac ctg 1056His Ala Ala Ile Ile Lys Pro Arg Phe Ala Ser Val Leu Lys
His Leu 340 345 350 gaa gca gct ttc tcc gat aac gac ctg ggc gaa tgg
gaa agc gcg gat 1104Glu Ala Ala Phe Ser Asp Asn Asp Leu Gly Glu Trp
Glu Ser Ala Asp 355 360 365 ggc ggt tat ttt att tct ttc gac acc cgt
ccg ggt ctg gcc cag aaa 1152Gly Gly Tyr Phe Ile Ser Phe Asp Thr Arg
Pro Gly Leu Ala Gln Lys 370 375 380 gtc gtg aaa ctg gcc ggc gat gca
ggt gtg aaa ctg acc ccg gcg ggt 1200Val Val Lys Leu Ala Gly Asp Ala
Gly Val Lys Leu Thr Pro Ala Gly 385 390 395 400 gca acg ttt ccg tac
ggt aaa gat ccg cag gac tct aat att cgc atc 1248Ala Thr Phe Pro Tyr
Gly Lys Asp Pro Gln Asp Ser Asn Ile Arg Ile 405 410 415 gca ccg acc
gtg ccg acg gtt gat caa gtc gaa gaa gct atg caa gtc 1296Ala Pro Thr
Val Pro Thr Val Asp Gln Val Glu Glu Ala Met Gln Val 420 425 430 ttc
gtc ctg tgt gtg aaa ctg gcg tcg gtg gaa caa gca ctg gca aat 1344Phe
Val Leu Cys Val Lys Leu Ala Ser Val Glu Gln Ala Leu Ala Asn 435 440
445 tcg ctc gag taa 1356Ser Leu Glu 450 111451PRTSaccharophagus
degradans 111Met Gln Ala Pro Tyr Thr Asn Leu Lys Thr His His Lys
Trp Val Glu 1 5 10 15 Phe Ser Ala Glu Asp Thr Thr Leu Asn Leu Ser
Asn Ala Ser Val Glu 20 25 30 Gln Leu Gln Glu Trp Lys Gln Gln Leu
Ser Ala Glu Tyr Asp Asn Val 35 40 45 Leu Ala Arg Lys Leu Asn Leu
Asp Leu Thr Arg Gly Lys Pro Ser Ala 50 55 60 Glu Gln Leu Ser Leu
Ser Asp Ala Met Asp Gly Ile Leu Ala Gly Asp 65 70 75 80 Tyr Ile Thr
Ala Ser Gly Ile Asp Val Arg Asn Tyr Gly Gly Leu Glu 85 90 95 Gly
Ile Pro Glu Ala Arg Ala Ile Gly Ser Asp Ile Leu Gly Val Pro 100 105
110 Val Glu Asn Val Leu Ala Gly Gly Asn Ser Ser Leu Thr Leu Met Tyr
115 120 125 Gln Thr Met Ala Ile Ala His Gln Phe Gly Leu Ala Gly Glu
Gly Ser 130 135 140 Ala Trp Ser Gln Glu Gly Thr Val Lys Phe Leu Cys
Pro Val Pro Gly 145 150 155 160 Tyr Asp Arg His Tyr Ser Val Cys Glu
His Leu Gly Ile Glu Met Leu 165 170 175 Thr Val Ala Met Thr Ser Thr
Gly Pro Asp Met Asp Gln Val Glu Lys 180 185 190 Met Ile Ala Ala Asp
Pro Ser Ile Lys Gly Met Trp Cys Val Pro Lys 195 200 205 Tyr Ser Asn
Pro Thr Gly Val Val Tyr Ser Asp Glu Thr Val Glu Arg 210 215 220 Ile
Ala Asn Leu Gly Asn Ile Ala Gly Lys Asn Phe Arg Val Phe Trp 225 230
235 240 Asp Asn Ala Tyr Ala Ile His Asp Leu Ser Asp Asn Pro Val Ala
Leu 245 250 255 Ala Asn Ile Phe Glu Ala Cys Lys Ala Ala Gly Thr Glu
Asp Ser Val 260 265 270 Ile Gln Phe Ala Ser Thr Ser Lys Val Thr His
Ala Gly Ser Gly Val 275 280 285 Ala Phe Ile Ala Ala Ser Asp Thr Asn
Leu Lys Phe Phe Lys Leu Ala 290 295 300 Leu Gly Phe Met Thr Ile Gly
Pro Asp Lys Val Asn Gln Leu Arg His 305 310 315 320 Ala Lys Phe Phe
Ala Ala Asp Gly Ala Leu Ser Ala His Met Ala Lys 325 330 335 His Ala
Ala Ile Ile Lys Pro Arg Phe Ala Ser Val Leu Lys His Leu 340 345 350
Glu Ala Ala Phe Ser Asp Asn Asp Leu Gly Glu Trp Glu Ser Ala Asp 355
360 365 Gly Gly Tyr Phe Ile Ser Phe Asp Thr Arg Pro Gly Leu Ala Gln
Lys 370 375 380 Val Val Lys Leu Ala Gly Asp Ala Gly Val Lys Leu Thr
Pro Ala Gly 385 390 395 400 Ala Thr Phe Pro Tyr Gly Lys Asp Pro Gln
Asp Ser Asn Ile Arg Ile 405 410 415 Ala Pro Thr Val Pro Thr Val Asp
Gln Val Glu Glu Ala Met Gln Val 420 425 430 Phe Val Leu Cys Val Lys
Leu Ala Ser Val Glu Gln Ala Leu Ala Asn 435 440 445 Ser Leu Glu 450
11211PRTCorynebacterium ammoniagenesmisc_feature(3)..(3)Xaa can be
any naturally occurring amino acid 112Met Ser Xaa Ile Ala Gln Xaa
Ile Leu Asp Gln 1 5 10
* * * * *
References