U.S. patent application number 14/173483 was filed with the patent office on 2014-07-24 for 2-deoxy-scyllo-inosose synthase.
This patent application is currently assigned to ASAHI KASEI CHEMICALS CORPORATION. The applicant listed for this patent is Asahi Kasei Chemicals Corporation. Invention is credited to Shinichi Imazu, Kazunobu Konishi.
Application Number | 20140206050 14/173483 |
Document ID | / |
Family ID | 42780613 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206050 |
Kind Code |
A1 |
Konishi; Kazunobu ; et
al. |
July 24, 2014 |
2-Deoxy-Scyllo-Inosose Synthase
Abstract
An object of the present invention is to provide a DOI synthase
having properties such as stability to heat and pH, which are
superior to those of conventional enzymes, and a method for
producing DOI using the above-mentioned enzyme. The present
invention provides a 2-deoxy-scyllo-inosose synthase having the
properties described in the following (1), (2), (4), (6) and (7),
and also having the properties described in the following (3)
and/or (5): (1) action: the enzyme has a function to convert
glucose-6-phosphate to 2-deoxy-scyllo-inosose; (2) optimum pH
range: pH 7.0 to 7.7; (3) stable pH range: pH 6.0 to 8.0; (4)
optimum temperature range: 55.degree. C. to 70.degree. C.; (5)
stable temperature range: 20.degree. C. to 46.degree. C.; (6)
coenzyme used: NAD.sup.+; and (7) molecular weight: 39,000 to
42,000.
Inventors: |
Konishi; Kazunobu; (Tokyo,
JP) ; Imazu; Shinichi; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Asahi Kasei Chemicals Corporation |
Tokyo |
|
JP |
|
|
Assignee: |
ASAHI KASEI CHEMICALS
CORPORATION
Tokyo
JP
|
Family ID: |
42780613 |
Appl. No.: |
14/173483 |
Filed: |
February 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13258932 |
Dec 8, 2011 |
8703466 |
|
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PCT/JP2010/002210 |
Mar 26, 2010 |
|
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14173483 |
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Current U.S.
Class: |
435/148 |
Current CPC
Class: |
C12P 7/26 20130101; C12N
9/93 20130101 |
Class at
Publication: |
435/148 |
International
Class: |
C12P 7/26 20060101
C12P007/26 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2009 |
JP |
2009-075802 |
Mar 26, 2009 |
JP |
2009-075803 |
Mar 26, 2009 |
JP |
2009-075804 |
Mar 26, 2009 |
JP |
2009-075805 |
Mar 26, 2009 |
JP |
2009-075806 |
Mar 27, 2009 |
JP |
2009-078713 |
Claims
1-15. (canceled)
16. A method of producing 2-deoxy-scyllo-inosose from
glucose-6-phosphate, wherein a microorganism capable of expressing
2-deoxy-scyllo-inosose synthase is cultured in a medium containing
a cobalt ion at a concentration of 0.1 to 50 mg/L in cobalt
chloride hexahydrate equivalent.
17. The method according to claim 16, wherein a raw material that
contains polysaccharide comprising glucose as a constituent, or
glucose, is used.
18. The method according to claim 16, wherein the
2-deoxy-scyllo-inosose. synthase is a 2-deoxy-scyllo-inosose
synthase having the properties described in the following (1), (2),
(4), (6) and (7), and also having the properties described in the
following (3) and/or (5): (1) action: the enzyme has a function to
convert glucose-6-phosphate to 2-deoxy-scyllo-inosose; (2) optimum
pH range: pH 7.0 to 7.7; (3) stable pH range: pH 6.0 to 8.0; (4)
optimum temperature range: 55.degree. C. to 70.degree. C.; (5)
stable temperature range: 20.degree. C. to 50.degree. C.; (6)
coenzyme used: NAD+; and (7) molecular weight: 39,000 to
42,000.
19. The method according to claim 18, wherein the
2-deoxy-scyllo-inosose synthase has the property described in the
following (8): (8) specific activity: 1.0 jlmol/min/mg or greater
(reaction temperature: 65.degree. C.).
20. The method according to claim 18, wherein the
2-deoxy-scyllo-inosose synthase has the property described in the
following (9): (9) cofactor: activity being improved by addition of
Co2+ ion.
21. The method according to claim 18, wherein the stable
temperature range is 20.degree. C. to 50.degree. C.
22. The method according to claim 18, wherein the
2-deoxy-scyllo-inosose synthase has any one of the following amino
acid sequences: (a) the amino acid sequence shown in any one of SEQ
ID NOS: 2, 4, 6, 8, 10 and 12; (b) an amino acid sequence having
sequence identity of 80% or more with the amino acid sequence shown
in any one of SEQ ID NOS: 2, 4, 6, 8, 10 and 12, and having high
temperature stability and/or wide range pH stability; and (c) an
amino acid sequence comprising a deletion, addition and/or
substitution of one or multiple amino acids with respect to the
amino acid sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10
and 12, and having high temperature stability and/or wide range pH
stability.
Description
TECHNICAL FIELD
[0001] The present invention relates to a heat-resistant
2-deoxy-scyllo-inosose synthase, a 2-deoxy-scyllo-inosose synthase
gene, a recombinant vector, a transformant, and a method for
producing 2-deoxy-scyllo-inosose.
BACKGROUND ART
[0002] At present, a majority of aromatic compounds, such as
catechol, are produced from petroleum as a raw material. From the
viewpoint of the problem of depleted oil resources or reduction in
the amount of carbon dioxide emissions, it is desired to develop a
novel environment-conscious production process in which a biomass
is used.
[0003] On the other hand, it has been found that
2-deoxy-scyllo-inosose (hereinafter referred to as DOI) can be
converted to an industrially useful aromatic compound (e.g.
catechol, etc.) (Non-Patent Document 1), and it has been reported
that this DOI can be synthesized from glucose that is one
constituent of biomass (Non-Patent Document 1). An enzyme that
plays an important role in this process of producing DOI is a
2-deoxy-scyllo-inosose synthase (hereinafter referred to as a DOI
synthase) for converting glucose-6-phosphate to DOI. Since the DOI
is not only converted to the aforementioned aromatic compound but
can also be an intermediate of various types of useful compounds,
the DOI synthase has received a great deal of attention.
[0004] A DOI synthase has been isolated and purified from
microorganisms belonging to Bacillus circulans that is a
butirosin-producing bacterium in 1997 (Non-Patent Document 2), and
the gene sequence thereof has been then published (Patent Document
1). Other than this DOI synthase, a DOI synthase derived from a
Streptoalloteichus hindustanus JCM3268 strain (Non-Patent Document
3) and the like have been discovered so far.
PRIOR ART REFERENCES
Patent Documents
[0005] Patent Document 1: JP Patent Publication (Kokai) No.
2000-236881
Non-Patent Documents
[0005] [0006] Non-Patent Document 1: Tetrahedron Letters 41,
1935-1938, 2000 [0007] Non-Patent Document 2: The Journal of
Antibiotics 50(5), 424-428, 1997 [0008] Non-Patent Document 3: The
Journal of Antibiotics 59(6), 358-361, 2006
SUMMARY OF INVENTION
Object to be Solved by the Invention
[0009] However, various types of properties of the above-described
DOI synthase, such as stability and specific activity, have not
been satisfactory. In addition, the analysis of the enzyme directed
towards the development of an industrial production method at a
scale of several thousands of tons and studies regarding the
improvement of functions have rarely been carried out. For example,
the DOI synthase (BtrC) derived from microorganisms belonging to
Bacillus circulans has a serious problem regarding stability and
the like. Moreover, the DOI synthase derived from the
Streptoalloteichus hindustanus JCM3268 strain has significantly low
specific activity, and thus, the industrial application of this
enzyme has been difficult. An object to be solved by the present
invention is to provide a DOI synthase having properties such as
stability to heat and pH, which are superior to those of
conventional enzymes, and a method for producing DOI using the
above-mentioned enzyme.
Means for Solving the Object
[0010] As a result of intensive studies directed towards achieving
the aforementioned object, the present inventors have discovered a
DOI synthase having heat stability and/or pH stability that are
superior to those of conventional enzymes, thereby completing the
present invention.
[0011] Specifically, the present invention relates to a DOI
synthase, a DOI synthase gene, a recombinant vector, a transformant
and a method for producing DOI, which will be described below.
[1] A 2-deoxy-scyllo-inosose synthase having the properties
described in the following (1), (2), (4), (6) and (7), and also
having the properties described in the following (3) and/or (5):
(1) action: the enzyme has a function to convert
glucose-6-phosphate to 2-deoxy-scyllo-inosose; (2) optimum pH
range: pH 7.0 to 7.7; (3) stable pH range: pH 6.0 to 8.0; (4)
optimum temperature range: 55.degree. C. to 70.degree. C.; (5)
stable temperature range: 20.degree. C. to 46.degree. C.; (6)
coenzyme used: NAD.sup.+; and (7) molecular weight: 39,000 to
42,000. [2] The 2-deoxy-scyllo-inosose synthase according to [1]
above, which has the property described in the following (8): (8)
specific activity: 1.0 pmol/min/mg or greater (reaction
temperature: 65.degree. C.). [3] The 2-deoxy-scyllo-inosose
synthase according to [1] or [2] above, which has the property
described in the following (9): (9) cofactor: activity being
improved by addition of Co.sup.2+ ion. [4] The
2-deoxy-scyllo-inosose synthase according to any one of [1] to [3]
above, wherein the stable temperature range is 20.degree. C. to
60.degree. C. [5] A 2-deoxy-scyllo-inosose synthase having any one
of the following amino acid sequences: (a) the amino acid sequence
shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10 and 12; (b) an amino
acid sequence having homology of 80% or more with the amino acid
sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10 and 12, and
having high temperature stability and/or wide range pH stability;
and (c) an amino acid sequence comprising a deletion, addition
and/or substitution of one or multiple amino acids with respect to
the amino acid sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8,
10 and 12, and having high temperature stability and/or wide range
pH stability. [6] A 2-deoxy-scyllo-inosose synthase gene having a
nucleotide sequence encoding any one of the following amino acid
sequences: (a) the amino acid sequence shown in any one of SEQ ID
NOS: 2, 4, 6, 8, 10 and 12; (b) an amino acid sequence having
homology of 80% or more with the amino acid sequence shown in any
one of SEQ ID NOS: 2, 4, 6, 8, 10 and 12, and having high
temperature stability and/or wide range pH stability; and (c) an
amino acid sequence comprising a deletion, addition and/or
substitution of one or multiple amino acids with respect to the
amino acid sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10
and 12, and having high temperature stability and/or wide range pH
stability. [7] A 2-deoxy-scyllo-inosose synthase gene having any
one of the following nucleotide sequences: (a) the nucleotide
sequence shown in any one of SEQ ID NOS: 1, 3, 5, 7, 9 and 11; (b)
a nucleotide sequence having homology of 80% or more with the
nucleotide sequence shown in any one of SEQ ID NOS: 1, 3, 5, 7, 9
and 11, and encoding a 2-deoxy-scyllo-inosose synthase having high
temperature stability and/or wide range pH stability; and (c) a
nucleotide sequence comprising a deletion, addition and/or
substitution of one or multiple nucleotides with respect to the
nucleotide sequence shown in any one of SEQ ID NOS: 1, 3, 5, 7, 9
and 11, and encoding a 2-deoxy-scyllo-inosose synthase having high
temperature stability and/or wide range pH stability. [8] A
recombinant vector which comprises the 2-deoxy-scyllo-inosose
synthase gene according to [6] or [7] above. [9] A transformant
which is obtained by introducing the 2-deoxy-scyllo-inosose
synthase gene according to [6] or [7] above or the recombinant
vector according to [8] above into a host. [10] A
2-deoxy-scyllo-inosose synthase which is obtained by culturing the
transformant according to [9] above. [11] A method for producing
2-deoxy-scyllo-inosose, wherein the 2-deoxy-scyllo-inosose synthase
according to any one of [1] to [5] and [10] above is used. [12] A
method for producing 2-deoxy-scyllo-inosose, using, as a raw
material, glucose, polysaccharide comprising glucose as a
constituent, and/or glucose-6-phosphate, wherein the
2-deoxy-scyllo-inosose synthase gene according to [6] or [7] above
or the transformant according to [9] above is utilized. [13] A
method for producing 2-deoxy-scyllo-inosose from
glucose-6-phosphate, wherein a microorganism capable of expressing
2-deoxy-scyllo-inosose synthase is cultured in a medium containing
a cobalt ion. [14] The method according to [13] above, wherein a
raw material that contains polysaccharide comprising glucose as a
constituent, or glucose, is used. [15] The method according to [13]
or [14] above, wherein the 2-deoxy-scyllo-inosose synthase is the
2-deoxy-scyllo-inosose synthase according to any one of [1] to [5]
above.
Advantageous Effects of Invention
[0012] The DOI synthase of the present invention can be used under
high temperature conditions in the production of DOI. Moreover,
strict temperature control is not required during the production of
DOI, depending on production conditions. Furthermore, the present
DOI synthase enables the production of DOI under acidic conditions
which is a pH range wherein DOI is stable. Further, strict pH
control is not required during the production of DOI, depending on
production conditions.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 shows the optimum pH range of a purified DOI synthase
(DOLS-1) (Example 3).
[0014] FIG. 2 shows the stable pH range of a purified DOI synthase
(DOIS-1) (Example 3).
[0015] FIG. 3 shows the optimum temperature range of a purified DOI
synthase (DOIS-1) (Example 3).
[0016] FIG. 4 shows the stable temperature range of a purified DOI
synthase (DOLS-1) (Example 3).
[0017] FIG. 5 shows the stable temperature range of a purified DOI
synthase (DOLS-5) (Example 5).
[0018] FIG. 6 shows the stable temperature range of a known
purified DOI synthase (BtrC) (Comparative Example 1).
[0019] FIG. 7 shows the stable pH range of a known purified DOI
synthase (BtrC) (Comparative Example 1).
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0020] The present invention will be specifically described
below.
[DOI Synthase]
[0021] The DOI synthase in the embodiment for carrying out the
present invention (hereinafter referred to as "the present
embodiment") has the following properties:
(1) action: the present enzyme has a function to convert
glucose-6-phosphate to DOI; and (2) stability: the present enzyme
shows high temperature stability and/or wide range pH
stability.
[0022] Preferably, the DOI synthase according to the present
invention has the properties described in the following (1), (2),
(4), (6) and (7), and also having the properties described in the
following (3) and/or (5):
(1) action: the present enzyme has a function to convert
glucose-6-phosphate to 2-deoxy-scyllo-inosose; (2) optimum pH
range: pH 7.0 to 7.7; (3) stable pH range: pH 6.0 to 8.0; (4)
optimum temperature range: 55.degree. C. to 70.degree. C.; (5)
stable temperature range: 20.degree. C. to 46.degree. C.
(preferably 20.degree. C. to 60.degree. C.); (6) coenzyme used:
NAD.sup.+; and (7) molecular weight: 39,000 to 42,000.
[0023] More preferably, the DOI synthase according to the present
invention has the properties described in the following (8) and/or
(9), in addition to the above-described properties:
(8) specific activity: 1.0 pmol/min/mg or greater (reaction
temperature: 65.degree. C.); and (9) cofactor: activity being
improved by addition of Co.sup.2+ ion.
[0024] Specific examples of the amino acid sequences of the DOI
synthase of the present invention are shown in SEQ ID NOS: 2, 4, 6,
8, 10 and 12. The DOI synthase having the amino acid sequence shown
in SEQ ID NO: 2, 4, 6, 8, 10 or 12 preferably has properties of
high temperature stability and/or wide range pH stability. The DOI
synthase having the amino acid sequence shown in SEQ ID NO: 2, 4,
6, 8, 10 or 12 preferably has excellent properties of high
temperature stability and/or wide range pH stability in the present
embodiment.
[0025] In the present embodiment, the DOI synthase may be a protein
having homology of preferably 80% or more, more preferably 85% or
more, further preferably 90% or more, still further preferably 95%
or more, and still further preferably 99% or more, with at least
one amino acid sequence selected from the group consisting of the
amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and
12.
[0026] Moreover, in the present embodiment, the DOI synthase may
comprise a deletion, substitution and/or addition of one or
multiple amino acids with respect to at least one amino acid
sequence selected from the group consisting of the amino acid
sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and 12. The number of
amino acids which are deleted, substituted and/or added is
preferably 1 to 50, more preferably 1 to 30, further preferably 1
to 20, still further preferably 1 to 10, still further preferably 1
to 5, and still further preferably 1 to 3.
[0027] An enzyme, which has an amino acid sequence having homology
of 80% or more with at least one amino acid sequence selected from
the group consisting of the amino acid sequences shown in SEQ ID
NOS: 2, 4, 6, 8, 10 and 12, which has excellent properties of high
temperature stability and/or wide range pH stability, and which has
DOI synthase activity, is included in the present invention.
[0028] An enzyme, which has an amino acid sequence comprising a
deletion, substitution and/or addition of one or multiple amino
acids with respect to at least one amino acid sequence selected
from the group consisting of the amino acid sequences shown in SEQ
ID NOS: 2, 4, 6, 8, 10 and 12, which has excellent properties of
high temperature stability and/or wide range pH stability, and
which has DOI synthase activity, is included in the present
invention.
[0029] As a method of causing a mutation such as a deletion,
substitution or addition to amino acids in a DOI synthase, there
can be applied known methods such as a PCR method, an error-prone
PCR method, a DNA shuffling method or a means for producing a
chimeric enzyme.
[0030] The homology between the amino acid sequences of DOI
synthases can be calculated using sequence analysis tools, such as
a BESTFIT program provided by UWGCG Package (Devereux et al.,
(1984) Nucleic Acids Research 12, pp. 387-395), or PILEUP or BLAST
algorisms (Altschul S. F. (1993) J Mol Evol 36: 290-300; Altschul
S. F. (1990) J Mol Biol 215: 403-10).
[0031] Moreover, DOI synthases retaining high temperature stability
can be selected from the DOI synthases obtained by the
above-described methods by measuring the activities of the obtained
enzymes after completion of a heat treatment.
[DOI Synthase Gene]
[0032] The DOI synthase gene of the present embodiment may be
either a natural DOI synthase gene that is isolated and/or
extracted from metagenome or microorganisms, or a DOI synthase gene
synthesized based on its nucleotide sequence by a known method such
as a PCR method or an artificial synthetic method. Furthermore, in
order to produce a novel chimeric gene of DOI enzyme, a known DOI
synthase gene may be used in combination. Examples of such a known
DOI synthase gene include DOI synthase genes derived from a
Paenibacillus sp. NBRC13157 strain, a Streptoalloteichus
hindustanus JCM3268 strain, and a Streptomyces fradiae NBRC12773
strain, etc.
[0033] Specific examples of the nucleotide sequence of the DOI
synthase gene according to the present embodiment are shown in SEQ
ID NOS: 1, 3, 5, 7, 9 and 11.
[0034] A transformant is obtained from a recombinant vector
comprising a DOI synthase gene having at least one gene sequence
selected from the group consisting of the nucleotide sequences
shown in SEQ ID NOS: 1, 3, 5, 7, 9 and 11, and a host. Thereafter,
the obtained transformant is cultured, so as to obtain a DOI
synthase.
[0035] A DOI synthase obtained from the DOI synthase gene having
the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11
preferably has properties of high temperature stability and/or wide
range pH stability.
[0036] In the present embodiment, the DOI synthase gene may have a
nucleotide sequence having homology of preferably 80% or more, more
preferably 85% or more, further preferably 90% or more, still
further preferably 95% or more, and still further preferably 99% or
more, with the DOI synthase gene having the nucleotide sequence
shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11. A DOI synthase gene, which
has a nucleotide sequence having homology of 80% or more with at
least one nucleotide sequence selected from the group consisting of
the nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9 and 11,
which has excellent properties of high temperature stability and/or
wide range pH stability, and which encodes an enzyme having DOI
synthase activity, is included in the present invention.
[0037] In the present embodiment, the DOI synthase gene may
comprise a deletion, addition and/or substitution of one or
multiple nucleotides with respect to the nucleotide sequence shown
in any one of SEQ ID NOS: 1, 3, 5, 7, 9 and 11. A DOI synthase
gene, which has a nucleotide sequence comprising a deletion,
addition and/or substitution of one or multiple nucleotides with
respect to the nucleotide sequence shown in any one of SEQ ID NOS:
1, 3, 5, 7, 9 and 11, and encoding a DOI synthase having high
temperature stability and/or wide range pH stability, is included
in the present invention. The number of nucleotides which are
deleted, added and/or substituted is preferably 1 to 50, more
preferably 1 to 30, further preferably 1 to 20, still further
preferably 1 to 10, still further preferably 1 to 5, and still
further preferably 1 to 3.
[0038] In the present embodiment, there may also be used a DOI
synthase gene having a nucleotide sequence encoding the amino acid
sequence described in any one of the following (a), (b) and
(c):
(a) the amino acid sequence shown in any one of SEQ ID NOS: 2, 4,
6, 8, 10 and 12; (b) an amino acid sequence having homology of 80%
or more with the amino acid sequence shown in any one of SEQ ID
NOS: 2, 4, 6, 8, 10 and 12, and having high temperature stability
and/or wide range pH stability; and (c) an amino acid sequence
comprising a deletion, addition and/or substitution of one or
multiple amino acids with respect to the amino acid sequence shown
in any one of SEQ ID NOS: 2, 4, 6, 8, 10 and 12, and having high
temperature stability and/or wide range pH stability.
[Recombinant Vector and Transformant]
[0039] The recombinant vector of the present embodiment can be
obtained by ligating (inserting) the gene of the present embodiment
to (into) a known vector such as a plasmid. The type of such a
known vector is not particularly limited, as long as it is able to
replicate in a host. Examples of such a vector include plasmid DNA
and phage DNA.
[0040] Examples of the aforementioned plasmid DNA include an
Escherichia coli-derived plasmid (e.g. pBR322, pBR325, pUC18,
pUC119, pTrcHis, pBlueBacHis, etc.), a Bacillus subtilis-derived
plasmid (e.g. pUB110, pTPS, etc.), and a yeast-derived plasmid
(e.g. YEp13, YEp24, YCp50, pYE52, etc.). An example of the
aforementioned phage DNA is 2, phage.
[0041] In order to insert the gene of the present embodiment into
the aforementioned vector, there is applied a method comprising:
first cleaving purified DNA by suitable restriction enzymes, and
then inserting the thus cleaved DNA into the suitable restriction
site or multicloning site of vector DNA, so as to ligate it to the
vector.
[0042] In order to allow a foreign gene to express in a host, a
suitable promoter needs to be located before a structural gene. The
type of the promoter is not particularly limited, and any given
promoter known to function in a host can be used Such promoters
will be described in detail for each type of host in the subsequent
section regarding transformant. Moreover, if necessary, a
cis-element such as an enhancer, a splicing signal, a poly(A)
addition signal, a ribosome binding sequence (SD sequence), a
terminator sequence and the like may be disposed.
[0043] The transformant of the present embodiment can be obtained
by introducing the recombinant vector of the present embodiment
into a host such that a gene of interest can be expressed therein.
Herein, the type of the host is not particularly limited, as long
as it is capable of expressing the DNA of the present embodiment.
Examples of such a host include: bacteria belonging to genus
Escherichia such as Escherichia coli, genus Bacillus such as
Bacillus subtilis, genus Pseudomonas such as Pseudomonas putida,
and genus Rhizobium such as Rhizobium meliloti; yeast such as
Saccharomyces cerevisiae; animal cells such as COS cells and CHO
cells; and insect cells such as Sf19 and Sf21.
[0044] In addition, a host used as the transformant of the present
embodiment preferably has a function to synthesize
glucose-6-phosphate from a monosaccharide such as glucose,
fructose, galactose or xylose. Also, such a host preferably has a
function to decompose a polysaccharide formed by ligation of two or
more monosaccharides, as necessary.
[0045] From the viewpoint of efficient expression of a DOI
synthase, when a bacterium such as Escherichia coli is used as a
host, it is desired that the recombinant vector of the present
embodiment can autonomously replicate in each bacterium, and also
that the recombinant vector is constituted with a promoter, a
ribosome-binding sequence, the gene of the present embodiment, and
a transcription termination sequence. Moreover, a gene that
controls the promoter may also be comprised in the vector. Examples
of Escherichia coli include E. coli K12, DH1 and DH10B
(Invitrogen), BL21-CodonPlus(DE3)-RIL (Stratagene), and TOP10F.
Examples of Bacillus subtilis include B. subtilis MI114 and
207-21.
[0046] The type of a promoter is not particularly limited, as long
as it functions in a host such as Escherichia coli. Examples of
such a promoter used herein include: Escherichia coli-derived
promoters such as a gapA promoter, a gadA promoter, a tip promoter,
a lac promoter, a PL promoter or a PR promoter; and phage-derived
promoters such as a T7 promoter.
[0047] The type of a method of introducing a recombinant vector
into a bacterium is not particularly limited, as long as it enables
introduction of DNA in the bacterium. Examples of such an
introduction method include a method using a calcium ion (Cohen, S
N et al., Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)) and an
electroporation method.
[0048] When a yeast is used as a host, S. cerevisiae, Pichia
pastoris and the like are used, for example. In this case, as a
promoter, a promoter capable of being expressed in such a yeast can
be used Examples of such a promoter include a gal1 promoter, a
gal10 promoter, a heat shock protein promoter, an MF.alpha.1
promoter, a PHO5 promoter, and an AOX promoter.
[0049] Examples of a method of introducing a recombinant vector
into a yeast include an electroporation method (Becker, D. M. et
al.: Methods. Enzymol., 194: 180 (1990)), a spheroplast method
(Hinnen, A. et al.: Proc Natl. Acad. Sci. USA, 75: 1929 (1978)),
and a lithium acetate method (Itch, H.: J. Bacteriol., 153: 163
(1983)).
[Production of DOI Synthase]
[0050] The enzyme of the present embodiment can be obtained by
culturing the transformant of the present embodiment in a suitable
medium, and then collecting a protein having the activity of the
enzyme from the culture. A method of culturing the transformant of
the present embodiment may be determined depending on the type of a
host. For example, in the case of a transformant whose host is a
microorganism such as Escherichia coli or a yeast, either a natural
medium or a synthetic medium may be used, as long as it contains a
carbon source, a nitrogen source, inorganic salts and the like,
which can be assimilated by the microorganism, and is able to
efficiently culture the transformant.
[0051] During the culture, an antibiotic such as ampicillin or
tetracycline may be added to the medium, as necessary. In the case
of culturing a microorganism that has been transformed with an
expression vector comprising an inducible promoter, an inducer may
be added to the medium, as necessary. For example, in the case of
culturing a microorganism that has been transformed with an
expression vector comprising a lac promoter,
isopropyl-.beta.-thiogalactopyranoside (IPTG) or the like may be
added to the medium, and in the case of culturing a microorganism
that has been transformed with an expression vector comprising a
tip promoter, indole acrylic acid (IAA) or the like may be added to
the medium.
[0052] After completion of the culture, if the enzyme protein of
the present embodiment is produced inside the cell mass or cells,
the cells are disintegrated. On the other hand, the protein of the
present embodiment is secreted outside the cell mass or cells, the
culture solution is used as is, or the protein is recovered by
centrifugation or the like.
[0053] For isolation and/or purification of the protein, for
example, ammonium sulfate precipitation, gel filtration, ion
exchange chromatography, affinity chromatography and the like may
be used singly or in combination, as appropriate.
[0054] The term "activity" is used herein to mean the activity of
converting glucose-6-phosphate to DOI. A method of measuring an
activity value is as described below.
[0055] The activity of the DOI synthase of the present embodiment
can be confirmed by a method comprising adding the enzyme to a
reaction solution containing suitable glucose-6-phosphate serving
as a substrate and then detecting the generated DOI. The generated
DOI can be confirmed by applying the method described in a
Non-Patent Document (Journal of Biotechnology 129, 502-509
(2007)).
[0056] As a method of measuring the DOI synthase activity, there is
applied a method comprising: mixing 100 .mu.L of a DOI synthase
solution having an appropriate concentration into 900 .mu.L of a
150 mM Bis-Tris buffer (pH 7.0) containing 20 .mu.L of 1000 mM
glucose-6-phosphate solution, 50 .mu.L of 100 mM NAD.sup.+ solution
and 50 .mu.L of 100 mM cobalt chloride hexahydrate solution;
reacting the obtained mixture for approximately 5 to 60 minutes;
deactivating the enzyme; and then quantifying DOI.
[0057] Confirmation of the purification degree of the purified DOI
synthase and the measurement of a molecular weight can be carried
out by electrophoresis, gel filtration chromatography, etc. In
addition, the optimum temperature range or optimum pH range of the
enzyme may be obtained by measuring the enzyme activity, while
changing the reaction temperature or the reaction pH. Moreover, the
stable pH range or stable temperature range of the enzyme may also
be obtained by exposing the DOI synthase to various pH conditions
or temperature conditions for a certain period of time, and then
measuring the enzyme activity. Bis-Tris buffer (pH 5.5 to 8.0) and
Tris buffer (pH 7.4 to 8.0) may be used to evaluate the DOI
synthase under various pH conditions.
[0058] The term "optimum pH range" is used herein to mean a pH
range in which the activity value is 70 or greater, when the
highest activity value obtained by measuring the activity value of
the enzyme while changing pH is set at 100.
[0059] The term "stable pH range" is used herein to mean a pH range
in which the activity value is 70 or greater, when the highest
activity value obtained by measuring the activity value of the
enzyme while changing pH is set at 100.
[0060] The term "optimum temperature range" is used herein to mean
a temperature range in which the activity value is 50 or greater,
when the highest activity value obtained by measuring the activity
value of the enzyme while changing pH is set at 100.
[0061] The term "stable temperature range" is used herein to mean a
temperature range in which the activity value is 50 or greater,
when the highest activity value obtained by measuring the activity
value of the enzyme while changing pH is set at 100.
[0062] The term "high temperature stability" in the high
temperature stability of the DOI synthase of the present embodiment
is used herein to mean that the upper limit of the stable
temperature range is 46.degree. C., more preferably 50.degree. C.,
further preferably 60.degree. C., still further preferably
70.degree. C., still further preferably 80.degree. C., still
further preferably 90.degree. C., and particularly preferably
95.degree. C. The lower limit of the stable temperature range is
not particularly limited. Taking into consideration ordinary
operations, the lower limit of the stable temperature range may be,
for example, 20.degree. C., preferably 10.degree. C., more
preferably 5.degree. C., and further preferably 1.degree. C.
[0063] As the 2-deoxy-scyllo-inosose synthase of the present
embodiment having high temperature stability, an enzyme having a
residual enzyme activity of 50 or greater after incubation at
50.degree. C. for 1 hour can be selected, for example. The term
"residual enzyme activity" is used herein to indicate relative
activity obtained when the highest activity value is set at
100.
[0064] In general, in an industrial enzyme reaction, the
temperature needs to be controlled within a temperature range in
which the enzyme has high activity, and thus, the control of the
temperature becomes an extremely important factor.
[0065] From the viewpoint of the activity of bacteria, in the
synthesis of DOI, a strict temperature control, in which the
temperature is controlled at 37.degree. C. or lower, has
conventionally been required. If a DOI synthase having high
temperature stability is used, it becomes possible for the enzyme
to maintain high activity even in a high temperature range. As a
result, it becomes possible to produce DOI without such strict
temperature control.
[0066] The DOI synthase according to the present embodiment having
wide range pH stability has characteristics, in which the stable pH
range is preferably pH 6.0 to 7.0, more preferably pH 6.0 to 7.4,
further preferably pH 6.0 to 7.7, still further preferably pH 6.0
to 8.0, and particularly preferably pH 4.0 to 9.0.
[0067] From the viewpoint of maintaining the high activity of
bacteria, in the conventional DOI synthesis, a strict pH control,
in which the pH value is controlled at pH 6.0 to 7.0, has
conventionally been required. If a DOI synthase having wide range
pH stability is used, it becomes possible for the enzyme to
maintain high activity even in a wide pH range. As a result, it
becomes possible to produce DOI without such strict pH control.
[0068] The enzyme of the present embodiment is not required to have
high purity, unless its action is inhibited. Hence, purification is
not essential, and the enzyme of the present embodiment may
comprise other enzymes and the like.
[Production of DOI]
[0069] The DOI of the present embodiment can be obtained by a
fermentative production method comprising culturing the
transformant of the present embodiment and then collecting DOI from
the culture.
[0070] As a nutrient source for the above-described culture, there
can be used a medium that contains a carbon source, a nitrogen
source, inorganic salts and other organic nutrient sources. The
culture temperature is not particularly limited, as long as it is a
temperature at which the bacteria can grow. The culture time is not
particularly limited, and it may be approximately 1 to 7 days.
Thereafter, DOI may be recovered from the obtained culture cell
mass or culture supernatant.
[0071] A polysaccharide comprising glucose as a constituent may be
directly used as a carbon source in the culture, as long as it can
be assimilated by the transformant. More preferred examples of such
a carbon source include a monosaccharide, and a monosaccharide
derived from a raw material containing a polysaccharide such as
starch, rice bran or blackstrap molasses. A specific example is
D-glucose. When the expression of a promoter is an inducible type,
an inducer may be added on a timely basis.
[0072] Examples of a carbon source that can be used herein include:
sugars such as D-glucose, galactose, maltose, saccharose or
treharose; oils and fats; fatty acids; and n-paraffin Examples of
such oils and fats include rapeseed oil, coconut oil, palm oil and
palm kernel oil. Examples of such fatty acids include: saturated or
unsaturated fatty acids such as hexanoic acid, octanoic acid,
decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic
acid, linolenic acid or myristic acid; and fatty acid derivatives
such as fatty acid esters or salts.
[0073] Examples of an organic nutrient source include amino acids
such as adenine, histidine, leucine, uracil or tryptophan.
[0074] Examples of a nitrogen source include: ammonia; ammonium
salts such as ammonium chloride, ammonium sulfate or ammonium
phosphate; peptone; meat extract; and yeast extract.
[0075] Examples of inorganic salts include sodium hydrogen
phosphate, potassium dihydrogen phosphate, magnesium chloride,
magnesium sulfate and sodium chloride.
[0076] In the present invention, DOI can be produced from
glucose-6-phosphate by culturing microorganisms capable of
expressing a DOI synthase in a medium containing a cobalt ion.
[0077] In the method for producing DOI according to the present
embodiment, the cobalt ion is preferably used in the form of the
salt of a divalent cobalt ion. Specific examples include cobalt
chloride, cobalt sulfate and cobalt acetate.
[0078] When a cobalt ion is added to a culture solution, the
productivity of DOI is improved, when compared with a culture in
which no cobalt ions are added. If such cobalt ions are excessively
added to the culture solution, it may cause growth inhibition of
microorganisms. Accordingly, the cobalt concentration during the
culture may be set within a range that is sufficient for the enzyme
to possess activity and does not inhibit the growth of
microorganisms. The appropriate cobalt concentration depends on the
type of a host. When Escherichia coli is used as a host, for
example, it is preferable that the cobalt concentration immediately
after initiation of the culture be maintained at a concentration of
preferably 0.1 to 200 mg/L, more preferably 0.1 to 100 mg/L, and
further preferably 0.1 to 50 mg/L, relative to cobalt chloride
hexahydrate. The expression "immediately after initiation of the
culture" is used herein to mean a range from initiation of the main
culture to the time point at which the cell solution OD reaches 5.
The "cell solution OD" means the optical density of a culture
solution, and in the present embodiment, it indicates the
absorption intensity of a culture solution with respect to light at
600 nm.
[0079] When a high-density culture is applied, with an increase in
the concentration of a cell mass in the culture solution, carbon
sources such as glucose, which are raw materials of constituents in
a microorganism cell mass, such as proteins, nucleic acids, lipids
or vitamins, and which serve as energy sources necessary for the
growth of the microorganisms, become insufficient. In such a case,
carbon sources are continuously or intermittently supplied to the
culture solution. In the present embodiment, cobalt ions are
further added to the culture solution, so that cobalt ions
necessary for the expression of the activity of the enzyme can
preferably be supplied in appropriate amounts, continuously or
intermittently.
[0080] In the method for producing DOI of the present embodiment,
the added cobalt value is controlled to be preferably 0.0003 to 70,
more preferably 0.001 to 15, and further preferably 0.01 to 5. The
added cobalt value was calculated by the following formula:
Added cobalt value=[the total amount of cobalt chloride hexahydrate
(mg) added to the culture solution]/the amount of the culture
solution (L)/cell solution OD
[0081] When cobalt salts other than cobalt chloride hexahydrate is
used, the added cobalt value may be calculated relative to cobalt
chloride hexahydrate, and cobalt salts may be then added, so that
the amount of cobalt ions becomes an appropriate value.
[0082] As a method of recovering DOI from a culture solution, the
following method is applied.
[0083] After completion of the culture, a cell mass is removed from
the culture using a centrifuge or a filter, so as to obtain a
culture supernatant. This culture supernatant is further subjected
to a filtration treatment, so as to remove solids such as a cell
mass. Then, an ion exchange resin is added to the filtrate, and
elution is then carried out with distilled water. The resultant is
measured in terms of ICP emission analysis, pH and the like, and at
the same time, a fraction containing no impurities is separated.
Solvents are removed from the aqueous solution thereof, so as to
recover DOI.
[0084] The obtained DOI is analyzed, for example, by high
performance liquid chromatography, nuclear magnetic resonance,
etc.
[0085] Other than the above-described fermentative production
method, there is applied a method using glucose-6-phosphate
converted from glucose by the action of glucokinase or the like.
Using the DOI synthase of the present embodiment or a transformant,
DOI can be obtained from glucose-6-phosphate.
[0086] In view of the reaction rate or the stability of the enzyme,
the reaction temperature applied during the production of DOI using
the DOI synthase of the present embodiment and/or a transformant is
preferably 10.degree. C. to 95.degree. C., more preferably
20.degree. C. to 70.degree. C., and further preferably 30.degree.
C. to 60.degree. C.
[0087] The reaction pH can be adjusted in a broad range. In view of
the stability of the enzyme, the reaction pH is preferably pH 2.0
to 10.0, more preferably pH 4.0 to 8.0, and further preferably 6.0
to 8.0.
[0088] The reaction time depends on the amount of the enzyme used.
Taking into consideration industrial use thereof, the reaction time
is generally 20 minutes to 200 hours, and more preferably 6 to 80
hours. When a transformant is used as a DOI synthetic catalyst, it
may previously be subjected to a freezing treatment or various
types of crushing treatments.
[0089] However, the present embodiment is not limited to the
above-described reaction conditions or reaction embodiments, and it
may be selected, as appropriate.
[0090] Hereinafter, the present invention will be more specifically
described in the following examples. These examples are not
intended to limit the scope of the present invention.
EXAMPLES
Example 1
(1) Preparation of Chromosomal DNA
[0091] The chromosomal DNA of a Paenibacillus sp. NBRC13157 strain
was prepared according to an ordinary method.
[0092] The Paenibacillus sp. NBRC13157 strain was cultured at
30.degree. C. for 1 day on an NR agar plate (1% Bacto Tryptone,
0.2% Yeast Extract, 1% Ehrlich's bonito extract, and 1.5% Bacto
Agar; pH7.0), so as to form a colony. Thereafter, a platinum loop
of the colony was inoculated into 30 mL of an NR medium (1% Bacto
Tryptone, 0.2% Yeast Extract, and 1% Ehrlich's bonito extract;
pH7.0) that had been dispensed into a 150-mL Erlenmeyer flask. The
obtained mixture was cultured at 30.degree. C. at 180 rpm for 1
day. Thereafter, the obtained culture solution was centrifuged at
4.degree. C. at 12,000 g for 1 minute, the supernatant was then
removed, and the cell mass was then recovered.
[0093] The obtained cell mass was suspended in a lysis buffer (50
mM Tris-HCl (pH 8.0), 20 mM EDTA, and 50 mM glucose), and was fully
washed. The cell mass was recovered by centrifugation, and it was
then re-suspended in a lysis buffer. To the suspension, lysozyme
was added, and the obtained mixture was then incubated at
37.degree. C. for 45 minutes. Subsequently, SDS and RNase were
added to the reaction solution, and the obtained mixture was then
incubated at 37.degree. C. for 45 minutes. Thereafter, Proteinase K
was added to the reaction solution, and the obtained mixture was
then gently shaken at 50.degree. C. for 60 minutes. The obtained
solution was treated with phenol-chloroform and chloroform, and was
then subjected to ethanol precipitation. The precipitated nucleic
acid was recovered by winding it around a glass pipette. This
nucleic acid was washed with 70% ethanol, was dried, and was then
re-suspended in TE. By this operation, approximately 100 .mu.g of
chromosomal DNA was prepared.
(2) Isolation of DOI Synthase Gene
[0094] PCR primers for amplification of a DOI synthase gene from
the chromosomal DNA prepared in (1) above, were synthesized. That
is, oligo DNA having the sequence shown in SEQ ID NO: 15 was
synthesized as a sense primer, and oligo DNA having the sequence
shown in SEQ ID NO: 16 was synthesized as an antisense primer.
[0095] Using the thus obtained PCR primers, and also using the
chromosomal DNA prepared in (1) above as a template, a DOI synthase
gene was amplified according to a PCR method, and as a result, a
PCR product consisting of 1107 base pairs was obtained.
[0096] The gene sequence of the obtained PCR product was confirmed
by analyzing it with a DNA sequencer, so as to obtain a known DOI
synthase (BtrC) gene having the nucleotide sequence shown in SEQ ID
NO: 13. The amino acid sequence corresponding to this gene was
shown in SEQ ID NO: 14 (BtrC).
(3) Construction of Expression Plasmid Vector of DOI Synthase Gene
and Transformation
[0097] A blunt end treatment and phosphorylation were performed on
the PCR product obtained in (2) above, and the resultant product
was then ligated to a plasmid formed by linking an Escherichia
coli-derived gapA promoter, an SD sequence and a terminator to
pUC19. Into this plasmid vector, a gapA promoter capable of
efficiently transcribing a gene ligated as a foreign gene in
Escherichia coli had been introduced. Thus, even in a case in which
recombinant microorganisms are cultured in a medium containing
glucose, the DOI synthase can be efficiently expressed and
produced.
[0098] The competent cells of Escherichia coli JM109 strain
prepared by a calcium chloride method were transformed with the
thus obtained plasmid vector according to a heat shock method, so
as to prepare recombinant microorganisms.
(4) Obtainment of Novel DOI Synthase Gene
[0099] Mutations were introduced into the plasmid vector obtained
in (3) above according to an ordinary method, so as to obtain
heat-resistant DOI synthase genes having the nucleotide sequences
shown in SEQ ID NO: 1 (DOLS-1), SEQ ID NO: 3 (DOIS-2), SEQ ID NO: 5
(DOLS-3), SEQ ID NO: 7 (DOIS-4), SEQ ID NO: 9 (DOIS-5) and SEQ ID
NO: 11 (DOLS-6). Also using the thus obtained DOI synthase genes,
transformants were obtained in the same manner as that of (3)
above. Moreover, amino acid sequences corresponding to the
nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 are shown in SEQ ID
NO: 2 (DOIS-1), SEQ ID NO: 4 (DOLS-2), SEQ ID NO: 6 (DOLS-3), SEQ
ID NO: 8 (DOIS-4), SEQ ID NO: 10 (DOIS-5) and SEQ ID NO: 12
(DOLS-6).
[0100] The novel DOI synthase can be selected by subjecting various
DOI synthases produced by ordinary methods to a heat treatment or
various pH treatments, and then measuring the activity thereof.
Example 2
Obtainment of Purified Enzyme
[0101] The transformant of DOI synthase (DOLS-1) produced in
Example 1 was cultured at 37.degree. C. for 1 day on an LB plate
containing 100 mg/L ampicillin, so as to form a colony.
[0102] Subsequently, 30 mL of an LB medium containing 100 mg/L
ampicillin was placed in a 150-mL Erlenmeyer flask, and the colony
formed in the above-described plate was inoculated into the medium
using a platinum loop. The obtained mixture was subjected to a
rotary shaking culture at 37.degree. C. at 180 rpm for 3 to 8
hours, until OD (600 nm) became approximately 0.5. The obtained
reaction solution was used as a pre-culture solution in the main
culture.
[0103] 100 ml of an LB medium containing 2 g/L glucose and 100 mg/L
ampicillin was placed into each of thirty-six 500-mL Erlenmeyer
flask, and 0.5 mL of the pre-culture solution was then added to
each of the Erlenmeyer flasks. The obtained mixture was subjected
to a rotary shaking culture at 37.degree. C. at 180 rpm for 16
hours.
[0104] Subsequently, the culture solution was centrifuged at
4.degree. C. at 10,000 g.times.30 minutes to remove the
supernatant, and the cell mass was then recovered while washing the
residue with a 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg/L
cobalt chloride hexahydrate several times. The recovered cell mass
was cryopreserved at -80.degree. C.
[0105] This cryopreserved cell mass was suspended in a 50 mM
Tris-HCl buffer (pH 7.7) containing 0.2 mg/L cobalt chloride
hexahydrate, and 180 mg of lysozyme (derived from albumen;
manufactured by Sigma) and 60 .mu.L of deoxyribonucleoase I
(bovine-derived recombinant solution, manufactured by WaKo) were
then added to the suspension. The obtained mixture was stirred at
37.degree. C. at 120 rpm for 5 hours to disintegrate the cell
mass.
[0106] After completion of the cell mass disintegration, the
resultant solution was centrifuged at 4.degree. C. at 10,000
g.times.30 minutes to remove the cell mass residue, so as to
recover a supernatant.
[0107] This supernatant was brought to 30% saturation by addition
of ammonium sulfate, and it was then stirred at 4.degree. C. for a
while. Thereafter, the generated precipitate was removed by
centrifugation at 4.degree. C. at 10,000 g.times.30 minutes, so as
to recover a supernatant.
[0108] This supernatant was brought to 40.0% saturation by further
addition of ammonium sulfate, and it was then stirred at 4.degree.
C. for a while. Thereafter, the generated precipitate was
centrifuged at 4.degree. C. at 10,000 g.times.30 minutes to remove
a supernatant, so as to recover a precipitate. Subsequently, this
precipitate was dissolved in 50 mM Tris-HCl buffer (pH 7.7)
containing 0.2 mg/L cobalt chloride hexahydrate.
[0109] The dissolved solution was concentrated at 4.degree. C.
using an ultrafiltration membrane having a mean fractional
molecular weight of 10,000, and 50 mM Tris-HCl buffer (pH 7.7)
containing 0.2 mg/L cobalt chloride hexahydrate was then added to
the concentrate, followed by concentration again. This desalting
operation was repeatedly carried out two or three times.
[0110] The thus obtained enzyme solution was adsorbed on "DEAE
Sepharose FF" (GE Healthcare Biosciences) that had been
equilibrated with 50 mM Tris-HCl buffer (pH 7.7), and the enzyme
was then eluted by a concentration gradient method using 50 mM
Tris-HCl buffer (pH 7.7) containing 0 to 0.4 M sodium chloride.
[0111] The active fractions of DOI synthases as eluted above were
collected, and the collected fraction was then concentrated using
an ultrafiltration membrane having a mean fractional molecular
weight of 10,000. The concentrate was suspended in 50 mM Tris-HCl
buffer (pH 7.7) containing 10% by weight of ammonium sulfate. The
thus obtained solution was adsorbed on "HiTrap Phenyl FF (high
sub)" (GE Healthcare Biosciences) that had been equilibrated with
50 mM Tris-HCl buffer (pH 7.7) containing 10% by weight of ammonium
sulfate, and the enzyme was then eluted by a concentration gradient
method using 50 mM Tris-HCl buffer (pH 7.7) containing 10% to 0% by
weight of ammonium sulfate.
[0112] The thus eluted active fractions were collected, and the
collected fraction was then concentrated using an ultrafiltration
membrane having a mean fractional molecular weight of 10,000. The
concentrate was adsorbed on "Mono Q 5/50 GL" (GE Healthcare
Biosciences) that had been equilibrated with 50 mM Tris-HCl buffer
(pH 7.7), and the enzyme was then eluted by a concentration
gradient method using 50 mM Tris-HCl buffer (pH 7.7) containing 0
to 0.2 M sodium chloride.
[0113] The thus eluted active fractions were collected, and the
collected fraction was then concentrated using an ultrafiltration
membrane having a mean fractional molecular weight of 10,000. The
concentrate was filled into "HiLoad 16/60 Superdex 200" (GE
Healthcare Biosciences) that had been equilibrated with a 50 mM
Tris-HCl buffer (pH 7.7) containing 0.2 mg/L cobalt chloride
hexahydrate and 0.1 M NaCl, and the enzyme was then eluted using
the same buffer as described above.
[0114] The thus eluted active fractions were collected, and the
collected fraction was then concentrated using an ultrafiltration
membrane having a mean fractional molecular weight of 10,000, so as
to obtain a purified DOI synthase (DOLS-1).
[0115] A purified DOI synthase (DOIS-2), a purified DOI synthase
(DOIS-3), a purified DOI synthase (DOIS-4), a purified DOI synthase
(DOLS-5), a purified DOI synthase (DOLS-6) and a known DOI synthase
(BtrC) were obtained in the same manner as that in the
above-described series of experiments for obtaining a purified
enzyme.
Example 3
Evaluation of Novel DOI Synthases
[0116] Using the purified DOI synthases obtained in Example 2, an
experiment was carried out to examine their actions.
(1) Optimum pH Range
[0117] In the measurement of the enzyme activity at various pH
values, an appropriate amount of the purified DOI synthase (DOIS-1)
was added to the reaction solution, and the reaction solution was
then adjusted so that it comprised 20 mM glucose-6-phosphate
disodium salt (Oriental Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast
Co., Ltd.), 5 mM cobalt chloride hexahydrate and 100 mM of various
types of buffers. As buffers, Bis-Tris buffer (pH 6.0 to 7.7) and
Tris buffer (pH 7.4 to 8.0) were used. The reaction temperature was
set at 30.degree. C., and the activity was measured by quantifying
the generated DOI. Relative activity was obtained while setting the
highest activity value at 100. The results are shown in FIG. 1. The
optimum pH range for the enzyme of the present invention was found
to be pH 7.0 to 7.7.
(2) Stable pH Range
[0118] Using 100 mM buffers having its pH value in the range from
pH 5.5 to 8.0, the purified DOI synthase (DOLS-1) was incubated at
each pH value at 30.degree. C. for 60 minutes. Thereafter, the
residual enzyme activity was measured. Bis-Tris buffer (pH 5.5 to
8.0) and Tris buffer (pH 7.4 to 8.0) were used in the
incubation.
[0119] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. Relative activity was obtained while
setting the highest activity value at 100. The results are shown in
FIG. 2. The stable pH range for the present enzyme was found to be
pH 6.0 to 8.0. The purified DOI synthase (DOLS-2) and the purified
DOI synthase (DOLS-3) had the same results as those for DOIS-1.
Such wide range pH stability was a novel property, which had not
been possessed by the existing enzymes.
(3) Optimum Temperature Range
[0120] The enzyme activity of the purified DOI synthase (DOLS-1)
was measured under various reaction temperature conditions (a
reaction temperature range from 10.degree. C. to 70.degree. C.) in
100 mM Bis-Tris buffer (pH 7.0) which comprised 20 mM
glucose-6-phosphate disodium salt (Oriental Yeast Co., Ltd.), 5 mM
NAD+ (Oriental Yeast Co., Ltd.) and 5 mM cobalt chloride
hexahydrate. Relative activity was obtained while setting the
highest activity value at 100. The results are shown in FIG. 3. The
optimum temperature range for the present enzyme was found to be
55.degree. C. to 70.degree. C. The specific activity at a reaction
temperature of 65.degree. C. was extremely high (1.8 .mu.mol
(DOI)/min/mg (enzyme)).
(4) Stable Temperature Range
[0121] 100 mM Bis-Tris buffer (pH 7.0), to which the purified DOI
synthase (DOIS-1) had been added and which had been adjusted to
comprise 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM cobalt
chloride hexahydrate, was incubated at various temperatures in a
temperature range from 25.degree. C. to 60.degree. C. for 1 hour.
Thereafter, the residual enzyme activity in each case was
measured.
[0122] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. Relative activity was obtained while
setting the highest activity value at 100. The results are shown in
FIG. 4. The stable temperature range for the purified DOI synthase
(DOLS-1) was found to be 50.degree. C. or lower. The purified DOI
synthase (DOLS-2) and the purified DOI synthase (DOIS-3) had the
same results as those for DOIS-1. Such high temperature stability
was a novel property, which had not been possessed by the existing
enzymes.
[0123] Moreover, an experiment regarding stable temperature range
was carried out by incubating the enzyme in the absence of NAD+ and
cobalt ions known to be act as stabilizers. As a result, the
relative activity of the purified DOI synthase (DOIS-1) was 38 at
an incubation temperature of 46.degree. C. The relative activity
thereof was 89 at an incubation temperature of 42.degree. C. and
was 100 at an incubation temperature of 37.degree. C.
(5) Molecular Weight
[0124] The molecular weight of the enzyme was measured by
SDS-polyacrylamide gel electrophoresis using "Ready-Gel J" (Bio-Rad
Laboratories, Inc.) having a separated gel concentration of 10%. As
a result, the molecular weight of the purified DOI synthase
(DOLS-1) was found to be approximately 40,000. This molecular
weight was almost the same as the putative molecular weight
(40,656) assumed from its amino acid sequence.
Example 4
Evaluation of Novel DOI Synthases
[0125] Using the purified DOI synthase (DOIS-4) obtained in Example
2, an experiment was carried out to examine its action.
(Evaluation of Temperature Stability)
[0126] 100 mM Bis-Tris buffer (pH 7.0), to which the purified DOI
synthase (DOLS-4) had been added and which had been adjusted to
comprise 5 mM cobalt chloride hexahydrate, was incubated at each
temperature of 25.degree. C. and 42.degree. C. for 1 hour.
Thereafter, the residual enzyme activity in each case was
measured.
[0127] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. When the residual activity obtained
after incubation at 25.degree. C. was set at 100, the activity
value obtained after incubation at 42.degree. C. became extremely
high (74).
(Measurement of Specific Activity)
[0128] The specific activity at a reaction temperature of
60.degree. C. became an extremely high value (1.2 .mu.mol
(DOI)/min/mg (enzyme)).
Example 5
Evaluation of Novel DOI Synthases
[0129] Using the purified DOI synthase (DOLS-5) obtained in Example
2, an experiment was carried out to examine its action.
(Evaluation of pH Stability)
[0130] Using 100 mM buffers each having a pH value in a pH range
from pH 5.5 to 8.0, the purified DOI synthase (DOLS-5) was
incubated at each pH value at 30.degree. C. for 60 minutes, and
thereafter, the residual enzyme activity was measured. Bis-Tris
buffer (pH 5.5 to 8.0) and Tris buffer (pH 7.4 to 8.0) were used in
the incubation.
[0131] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. Relative activity was obtained while
setting the highest activity value at 100. The results are shown in
FIG. 5. As shown in FIG. 5, the present enzyme showed high
stability in an extremely wide pH range. This high stability was a
novel property, which had not been possessed by the existing
enzymes.
Example 6
Evaluation of Novel DOI Synthases
[0132] Using the purified DOI synthase (DOLS-6) obtained in Example
2, an experiment was carried out to examine its action.
(Evaluation of pH Stability)
[0133] Using 100 mM buffers each having a pH value in a pH range
from pH 5.5 to 8.0, the purified DOI synthase (DOLS-6) was
incubated at each pH value at 30.degree. C. for 60 minutes, and
thereafter, the residual enzyme activity was measured. Bis-Tris
buffer (pH 5.5 to 8.0) was used in the incubation.
[0134] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. When the residual activity at pH 8.0
at which the highest activity had been obtained was set at 100, the
activity value was 70 or greater in a pH range from pH 6.5 to 8.0,
and the residual activity at pH 6.0 was an extremely high value
(53). This high pH stability was a novel property, which had not
been possessed by the existing enzymes.
Example 7
Method for Producing DOI
[0135] The transformant of DOI synthase (DOLS-1) produced in
Example 1 was cultured at 37.degree. C. for 1 day on an LB plate
containing 100 mg/L ampicillin, so as to form a colony.
[0136] Subsequently, 30 mL of an LB medium containing 100 mg/L
ampicillin was placed in a 150-mL Erlenmeyer flask, and the colony
formed in the above-described plate was inoculated into the medium
using a platinum loop. The obtained mixture was subjected to a
rotary shaking culture at 37.degree. C. at 180 rpm for 3 to 8
hours, until OD (600 nm) became approximately 0.5. The obtained
reaction solution was used as a pre-culture solution in the main
culture.
[0137] 100 ml of an LB medium containing 2 g/L glucose and 100 mg/L
ampicillin was placed into each of thirty-six 500-mL Erlenmeyer
flask, and 0.5 mL of the pre-culture solution was then added to
each of the Erlenmeyer flasks. The obtained mixture was subjected
to a rotary shaking culture at 37.degree. C. at 180 rpm for 16
hours.
[0138] Subsequently, the culture solution was centrifuged at
4.degree. C. at 10,000 g.times.30 minutes to remove the
supernatant, and the cell mass was then recovered while washing the
residue with 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg/L
cobalt chloride hexahydrate several times. The recovered cell mass
was cryopreserved at -80.degree. C.
[0139] In the synthesis of DOI, a solution prepared by suspending
the above-described cryopreserved cell mass in 50 mM Tris-HCl
buffer (pH 7.7) containing 0.2 mg/L cobalt chloride hexahydrate was
used as a DOI synthetic catalyst. The composition of a reaction
solution used in the DOI synthetic reaction was 85 mM
glucose-6-phosphate disodium salt (Oriental Yeast Co., Ltd.), 0.1
mM NAD+ (Oriental Yeast Co., Ltd.) and 1 mM cobalt chloride
hexahydrate, wherein cell mass OD was 30. Using this reaction
solution, the DOI synthetic reaction was initiated. The pH at the
initiation of the reaction was adjusted to be pH 7.7, and the
synthetic reaction was carried out at a reaction temperature of
46.degree. C. for 2.5 hours. As a result, 1% by weight of DOI was
generated. After completion of the reaction, hydrochloric acid was
added to the reaction solution, so that the pH was decreased to pH
6 or less. Subsequently, this reaction solution was centrifuged at
4.degree. C. at 10,000 g.times.30 minutes to recover a supernatant.
It was then filtrated with a filter to remove a cell mass
residue.
[0140] The synthesized DOI solution was subjected to a purification
treatment using a cation exchange resin, in which Amberlite
IR120BNa (manufactured by Organo Corp.) was reproduced to H.sup.+
type, and an anion exchange resin, in which Amberlite IRA96SB
(manufactured by Organo Corp.) was reproduced to OFF type. In this
purification treatment, a column treatment was used in combination
with a batch treatment, and a fraction, in which P, Co and Na had
not been detected and comprised a DOI solution, was recovered by an
ICP emission analysis method.
[0141] Subsequently, to the obtained DOI solution, activated carbon
Carborafin (manufactured by Japan EnviroChemicals, Ltd.) was added
in an amount of 0.5 g with respect to 1 g of DOI contained in the
DOI solution. The obtained mixture was stirred at room temperature
for 1 hour, so that a trace amount of impurity was adsorbed on the
activated carbon. One hour later, the activated carbon was removed
by a filtration operation, and the DOI solution was then recovered.
DOI was concentrated by subjecting the recovered DOI solution to
vacuum concentration, and the concentrate was then freeze-dried to
obtain DOI powders.
Example 8
[0142] The transformant of DOI synthase (DOLS-1) produced in
Example 1 was cultured at 37.degree. C. for 1 day on an LB plate
containing 100 mg/L ampicillin, so as to form a colony.
[0143] Subsequently, 30 mL of an LB medium containing 100 mg/L
ampicillin was placed in a 150-mL Erlenmeyer flask, and the colony
formed in the above-described plate was inoculated into the medium
using a platinum loop. The obtained mixture was subjected to a
rotary shaking culture at 37.degree. C. at 180 rpm for 3 to 8
hours, until OD became approximately 0.5. The reaction solution was
used as a pre-culture solution in the main culture.
[0144] 100 mL of medium A containing the cobalt chloride
hexahydrate each having a different concentration shown in Table 1
below was placed in a 500-mL Erlenmeyer flask, and 0.5 mL of the
pre-culture solution was then added thereto. The obtained mixture
was subjected to a rotary shaking culture at 37.degree. C. at 180
rpm for 18 hours. Eighteen hours after initiation of the culture,
the cell solution OD under various medium conditions, added cobalt
value, and relative value (DOI productivity) obtained when the DOI
concentration in the supernatant of a cobalt ion-non-added medium
was set at 100, are shown in Table 2 below
TABLE-US-00001 TABLE 1 Medium A Potassium dihydrogen phosphate 2
g/L Diammonium hydrogen phosphate 3 g/L Magnesium sulfate
heptahydrate 0.2 mg/L Thiamine hydrochloride 20 mg/L Yeast extract
3 g/L Glucose 10 g/L Cobalt chloride hexahydrate 0 to 20 mg/L (*)
The above substances were dissolved in distilled water, and the pH
value was then adjusted to pH 7.0.
TABLE-US-00002 TABLE 2 Added concentration Cell Added Relative
value of cobalt chloride solution cobalt (DOI hexahydrate (mg/L) OD
value productivity) 0 4.2 0.00 100 0.1 4.3 0.02 114 0.5 4.4 0.11
118 1 4.3 0.23 124 3 4.2 0.71 131 5 4.3 1.17 125 7 4.0 1.77 114 10
4.3 2.30 122 20 4.6 4.37 122
Comparative Example 1
Evaluation of Known DOI Synthase
[0145] The known purified DOI synthase (BrtC) having the amino acid
sequence shown in SEQ ID NO: 14 was subjected to the following
experiments in the same manner as that of Example 3-(4) "Stable
temperature range."
[0146] 100 mM Bis-Tris buffer (pH 7.0), to which the purified DOI
synthase (BtrC) had been added and which had been adjusted to
comprise 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM cobalt
chloride hexahydrate, was incubated at various temperatures in a
temperature range from 25.degree. C. to 60.degree. C. for 1 hour.
Thereafter, the residual enzyme activity in each case was
measured.
[0147] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. Relative activity was obtained while
setting the highest activity value at 100. The results are shown in
FIG. 6. The stable temperature range of the purified known DOI
synthase (BtrC) was found to be 42.degree. C. or lower. The
relative activity at 46.degree. C. was 23, and the relative
activity at 50.degree. C. was 0. Thus, the purified known DOI
synthase (BtrC) showed significantly low heat stability.
[0148] Moreover, an experiment regarding stable temperature range
was carried out by incubating the enzyme in the absence of NAD+ and
cobalt ions known to be act as stabilizers. As a result, the
relative activity of the purified known DOI synthase (BtrC) was 0
at incubation temperatures of 42.degree. C. and 46.degree. C., and
the relative activity thereof at 37.degree. C. was 14. Also from
this experiment, it was found that the existing enzyme has
significantly low heat stability.
[0149] The purified known DOI synthase (BrtC) having the amino acid
sequence shown in SEQ ID NO: 14 was subjected to the following
experiments in the same manner as that of Example 3-(2) "Stable pH
range."
[0150] The purified known DOI synthase (BrtC) was incubated at each
pH value at 30.degree. C. for 60 minutes, and the residual enzyme
activity was then measured. Bis-Tris buffer (pH 5.5 to 8.0) and
Tris buffer (pH 7.4 to 8.0) were used in the incubation.
[0151] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. Relative activity was obtained while
setting the highest activity value at 100. The results are shown in
FIG. 7. As shown in FIG. 7, the relative activity at pH 6 was
extremely low (27), and the present enzyme was almost completely
deactivated at pH 5.5.
Comparative Example 2
Evaluation of Known DOI Synthase
[0152] The purified known DOI synthase (BrtC) having the amino acid
sequence shown in SEQ ID NO: 14 was subjected to the following
experiments in the same manner as that of Example 3.
[0153] 100 mM Bis-Tris buffer (pH 7.0), to which the purified known
DOI synthase (BtrC) had been added and which had been adjusted to
comprise 5 mM cobalt chloride hexahydrate, was incubated at
temperatures of 25.degree. C. and 42.degree. C. for 1 hour.
Thereafter, the residual enzyme activity in each case was
measured.
[0154] The residual enzyme activity was measured at a reaction
temperature of 30.degree. C. in 100 mM Bis-Tris buffer (pH 7.0)
which comprised 20 mM glucose-6-phosphate disodium salt (Oriental
Yeast Co., Ltd.), 5 mM NAD+ (Oriental Yeast Co., Ltd.) and 5 mM
cobalt chloride hexahydrate. When the residual activity obtained
after completion of the incubation at 25.degree. C. was set at 100,
the activity value after completion of the incubation of 42.degree.
C. became 0. Thus, the purified known DOI synthase (BtrC) showed
significantly low heat stability.
INDUSTRIAL APPLICABILITY
[0155] According to the present invention, it becomes possible to
produce DOI that is extremely useful as a precursor of industrially
useful aromatic compounds (catechol, etc.) by an efficient
production process.
Sequence CWU 1
1
1611107DNAPaenibacillus sp.CDS(1)..(1107) 1atg acg act aaa caa att
tgt ttt gcg gac cgg tgt ttt aac ttt gca 48Met Thr Thr Lys Gln Ile
Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 ttc ggc gaa cag
att ttg gaa tcg att gcc gcc tat att cac cgg gat 96Phe Gly Glu Gln
Ile Leu Glu Ser Ile Ala Ala Tyr Ile His Arg Asp 20 25 30 gaa ttc
gat caa tat atc gtg att tcg gac tcg ggg gta ccg gac tcg 144Glu Phe
Asp Gln Tyr Ile Val Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45
att gtt cat cat gcg gcc gaa tac ttc ggc aga ctc gcc cct gta cat
192Ile Val His His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val His
50 55 60 att ctt cgc ttt cag ggc gga gaa gaa tac aaa aca ctt gca
acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala
Thr Val 65 70 75 80 aca aat ttg caa gag cag gca att gct ctg gga gcc
aac cga aga acc 288Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala
Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt ggc gga ggg tta acc
gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val Gly Gly Gly Leu Thr
Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc atg atg ttt cgc ggg
att gcg ctt att cac gtt ccg acc 384Ala Ala Gly Met Met Phe Arg Gly
Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg ttt ttg gcg gcc tcc
gat tcg gtt ctt tcg att aag cag gct gtt 432Thr Phe Leu Ala Ala Ser
Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135 140 aat tta acg agc
gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc 480Asn Leu Thr Ser
Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 ttc
gtg ttc gcc gat acc cga atc ttg gcg gag tcg ccg ccc cgt cag 528Phe
Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165 170
175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat atg ctg att ctg gaa
576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu
180 185 190 aac gag cac atg gaa ttt aca gag gat gat tta aat gca gcc
aat gtg 624Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ala Ala
Asn Val 195 200 205 tat act ccg agg cag ctg gag acg ttt atc aac ttc
tgc ata tcg gcc 672Tyr Thr Pro Arg Gln Leu Glu Thr Phe Ile Asn Phe
Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta agc gaa gat att tac
gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu Ser Glu Asp Ile Tyr
Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt gag tac ggc cat acg
atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe Glu Tyr Gly His Thr
Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255 cag gga ggg atc acg
cac gga gaa gcc att gca gtg ggc atg att tac 816Gln Gly Gly Ile Thr
His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 gcc gct aaa
ata gcg aac cgg atg aac ctg ctg tcc gaa cag gac gtg 864Ala Ala Lys
Ile Ala Asn Arg Met Asn Leu Leu Ser Glu Gln Asp Val 275 280 285 tcc
acc cat tac tgg ctt tta aat aaa atc ggg gcc ttg cag gag ctt 912Ser
Thr His Tyr Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Glu Leu 290 295
300 ccg ctc cga gcg gac gcg gat tcg gtc ttc cat tat tta atc cac gat
960Pro Leu Arg Ala Asp Ala Asp Ser Val Phe His Tyr Leu Ile His Asp
305 310 315 320 aac aag agg ggc tac att aag ctg gat gag gat aat ttg
ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu
Gly Met Ile 325 330 335 tta ctt gag gga atc ggt cga ccg gcg gtt cat
aac caa tcg ctg ctt 1056Leu Leu Glu Gly Ile Gly Arg Pro Ala Val His
Asn Gln Ser Leu Leu 340 345 350 aca ccg gtc aag aaa tcg ctc ata aaa
gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val Lys Lys Ser Leu Ile Lys
Glu Val Ile Arg Glu Gly Leu 355 360 365 taa
11072368PRTPaenibacillus sp. 2Met Thr Thr Lys Gln Ile Cys Phe Ala
Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly Glu Gln Ile Leu Glu
Ser Ile Ala Ala Tyr Ile His Arg Asp 20 25 30 Glu Phe Asp Gln Tyr
Ile Val Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 Ile Val His
His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val His 50 55 60 Ile
Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala Thr Val 65 70
75 80 Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala Asn Arg Arg
Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val
Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu
Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala Ser Asp Ser Val
Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr Ser Gly Lys Asn
Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 Phe Val Phe Ala
Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165 170 175 Val Lys
Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185 190
Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ala Ala Asn Val 195
200 205 Tyr Thr Pro Arg Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile Ser
Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys Lys
Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His Thr Ile Gly His
Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile Thr His Gly Glu
Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala Lys Ile Ala Asn
Arg Met Asn Leu Leu Ser Glu Gln Asp Val 275 280 285 Ser Thr His Tyr
Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Glu Leu 290 295 300 Pro Leu
Arg Ala Asp Ala Asp Ser Val Phe His Tyr Leu Ile His Asp 305 310 315
320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met Ile
325 330 335 Leu Leu Glu Gly Ile Gly Arg Pro Ala Val His Asn Gln Ser
Leu Leu 340 345 350 Thr Pro Val Lys Lys Ser Leu Ile Lys Glu Val Ile
Arg Glu Gly Leu 355 360 365 31113DNAPaenibacillus sp.CDS(1)..(1113)
3atg acg act aaa caa att tgt ttt gcg gac cgg tgt ttt aac ttt gca
48Met Thr Thr Lys Gln Ile Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1
5 10 15 ttc ggc gaa cag att ttg gaa tcg att gcc gcc tat att cac cgg
gat 96Phe Gly Glu Gln Ile Leu Glu Ser Ile Ala Ala Tyr Ile His Arg
Asp 20 25 30 gaa ttc gat caa tat atc gtg att tcg gac tcg ggg gta
ccg gac tcg 144Glu Phe Asp Gln Tyr Ile Val Ile Ser Asp Ser Gly Val
Pro Asp Ser 35 40 45 att gtt cat cat gcg gcc gaa tac ttc ggc aga
ctc gcc cct gta cat 192Ile Val His His Ala Ala Glu Tyr Phe Gly Arg
Leu Ala Pro Val His 50 55 60 att ctt cgc ttt cag ggc gga gaa gaa
tac aaa aca ctt gca acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu
Tyr Lys Thr Leu Ala Thr Val 65 70 75 80 aca aat ttg caa gag cag gca
att gct ctg gga gcc aac cga aga acc 288Thr Asn Leu Gln Glu Gln Ala
Ile Ala Leu Gly Ala Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt
ggc gga ggg tta acc gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val
Gly Gly Gly Leu Thr Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc
atg atg ttt cgc ggg att gcg ctt att cac gtt ccg acc 384Ala Ala Gly
Met Met Phe Arg Gly Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg
ttt ttg gcg gcc tcc gat tcg gtt ctt tcg att aag cag gct gtt 432Thr
Phe Leu Ala Ala Ser Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135
140 aat tta acg agc gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc
480Asn Leu Thr Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg
145 150 155 160 ttc gtg ttc gcc gat acc cga atc ttg gcg gag tcg ccg
ccc cgt cag 528Phe Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro
Pro Arg Gln 165 170 175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat
atg ctg att ctg gaa 576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn
Met Leu Ile Leu Glu 180 185 190 aac gag cac atg gaa ttt aca gag gat
gat tta aat gca gcc aat gtg 624Asn Glu His Met Glu Phe Thr Glu Asp
Asp Leu Asn Ala Ala Asn Val 195 200 205 tat act ccg agg cag ctg gag
acg ttt atc aac ttc tgc ata tcg gcc 672Tyr Thr Pro Arg Gln Leu Glu
Thr Phe Ile Asn Phe Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta
agc gaa gat att tac gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu
Ser Glu Asp Ile Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt
gag tac ggc cat acg atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe
Glu Tyr Gly His Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255
cag gga ggg atc acg cac gga gaa gcc att gca gtg ggc atg att tac
816Gln Gly Gly Ile Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr
260 265 270 gcc gct aaa ata gcg aac cgg atg aac ctg ctg tcc gaa cag
gac gtg 864Ala Ala Lys Ile Ala Asn Arg Met Asn Leu Leu Ser Glu Gln
Asp Val 275 280 285 tcc acc cat tac tgg ctt tta aat aaa atc ggg gcc
ttg cag gag ctt 912Ser Thr His Tyr Trp Leu Leu Asn Lys Ile Gly Ala
Leu Gln Glu Leu 290 295 300 ccg ctc cga gcg gac gcg gat tcg gtc ttc
cat tat tta atc cac gat 960Pro Leu Arg Ala Asp Ala Asp Ser Val Phe
His Tyr Leu Ile His Asp 305 310 315 320 aac aag agg ggc tac att aag
ctg gat gag gat aat ttg ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys
Leu Asp Glu Asp Asn Leu Gly Met Ile 325 330 335 tta ctt gag gga atc
ggt cga ccg gcg gtt cat aac caa tcg ctg ctt 1056Leu Leu Glu Gly Ile
Gly Arg Pro Ala Val His Asn Gln Ser Leu Leu 340 345 350 aca ccg gtc
aag aaa tcg ctc ata aaa gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val
Lys Lys Ser Leu Ile Lys Glu Val Ile Arg Glu Gly Leu 355 360 365 tgg
gga tga 1113Trp Gly 370 4370PRTPaenibacillus sp. 4Met Thr Thr Lys
Gln Ile Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly
Glu Gln Ile Leu Glu Ser Ile Ala Ala Tyr Ile His Arg Asp 20 25 30
Glu Phe Asp Gln Tyr Ile Val Ile Ser Asp Ser Gly Val Pro Asp Ser 35
40 45 Ile Val His His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val
His 50 55 60 Ile Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu
Ala Thr Val 65 70 75 80 Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly
Ala Asn Arg Arg Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu
Thr Gly Asn Val Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg
Gly Ile Ala Leu Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala
Ser Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr
Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160
Phe Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165
170 175 Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu
Glu 180 185 190 Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ala
Ala Asn Val 195 200 205 Tyr Thr Pro Arg Gln Leu Glu Thr Phe Ile Asn
Phe Cys Ile Ser Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile
Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His
Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile
Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala
Lys Ile Ala Asn Arg Met Asn Leu Leu Ser Glu Gln Asp Val 275 280 285
Ser Thr His Tyr Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Glu Leu 290
295 300 Pro Leu Arg Ala Asp Ala Asp Ser Val Phe His Tyr Leu Ile His
Asp 305 310 315 320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn
Leu Gly Met Ile 325 330 335 Leu Leu Glu Gly Ile Gly Arg Pro Ala Val
His Asn Gln Ser Leu Leu 340 345 350 Thr Pro Val Lys Lys Ser Leu Ile
Lys Glu Val Ile Arg Glu Gly Leu 355 360 365 Trp Gly 370
51107DNAPaenibacillus sp.CDS(1)..(1107) 5atg acg act aaa caa att
tgt ttt gcg gac cgg tgt ttt aac ttt gca 48Met Thr Thr Lys Gln Ile
Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 ttc ggc gaa cag
att ttg gaa tcg att gcc gcc tat att cac cgg gat 96Phe Gly Glu Gln
Ile Leu Glu Ser Ile Ala Ala Tyr Ile His Arg Asp 20 25 30 gaa ttc
gat caa tat atc atg att tcg gac tcg ggg gta ccg gac tcg 144Glu Phe
Asp Gln Tyr Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45
att gtt cat cat gcg gcc gaa tac ttc ggc aga ctc gcc cct gta cat
192Ile Val His His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val His
50 55 60 att ctt cgc ttt cag ggc gga gaa gaa tac aaa aca ctt gca
acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala
Thr Val 65 70 75 80 aca aat ttg caa gag cag gca att gct ctg gga gcc
aac cga aga acc 288Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala
Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt ggc gga ggg tta acc
gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val Gly Gly Gly Leu Thr
Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc atg atg ttt cgc ggg
att gcg ctt att cac gtt ccg acc 384Ala Ala Gly Met Met Phe Arg Gly
Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg ttt ttg gcg gcc tcc
gat tcg gtt ctt tcg att aag cag gct gtt 432Thr Phe Leu Ala Ala Ser
Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135 140 aat tta acg agc
gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc
480Asn Leu Thr Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg
145 150 155 160 ttc gtg ttc gcc gat acc cga atc ttg gcg gag tcg ccg
ccc cgt cag 528Phe Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro
Pro Arg Gln 165 170 175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat
atg ctg att ctg gaa 576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn
Met Leu Ile Leu Glu 180 185 190 aac gag cac atg gaa ttt aca gag gat
gat tta aat tca gcc aat gtg 624Asn Glu His Met Glu Phe Thr Glu Asp
Asp Leu Asn Ser Ala Asn Val 195 200 205 tat tct ccg aag cag ctg gag
acg ttt atc aac ttc tgc ata tcg gcc 672Tyr Ser Pro Lys Gln Leu Glu
Thr Phe Ile Asn Phe Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta
agc gaa gat att tac gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu
Ser Glu Asp Ile Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt
gag tac ggc cat acg atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe
Glu Tyr Gly His Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255
cag gga ggg atc acg cac gga gaa gcc att gca gtg ggc atg att tac
816Gln Gly Gly Ile Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr
260 265 270 gcc gct aaa ata gcg aac cgg atg aac ctg ctg tcc gaa cag
gac gtg 864Ala Ala Lys Ile Ala Asn Arg Met Asn Leu Leu Ser Glu Gln
Asp Val 275 280 285 tcc acc cat tac tgg ctt tta aat aaa atc ggg gcc
ttg cag gag ctt 912Ser Thr His Tyr Trp Leu Leu Asn Lys Ile Gly Ala
Leu Gln Glu Leu 290 295 300 ccg ctc cga gcg gac gcg gat tcg gtc ttc
cat tat tta atc cac gat 960Pro Leu Arg Ala Asp Ala Asp Ser Val Phe
His Tyr Leu Ile His Asp 305 310 315 320 aac aag agg ggc tac att aag
ctg gat gag gat aat ttg ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys
Leu Asp Glu Asp Asn Leu Gly Met Ile 325 330 335 tta ctt gag gga atc
ggt cga ccg gcg gtt cat aac caa tcg ctg ctt 1056Leu Leu Glu Gly Ile
Gly Arg Pro Ala Val His Asn Gln Ser Leu Leu 340 345 350 aca ccg gtc
aag aaa tcg ctc ata aaa gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val
Lys Lys Ser Leu Ile Lys Glu Val Ile Arg Glu Gly Leu 355 360 365 taa
11076368PRTPaenibacillus sp. 6Met Thr Thr Lys Gln Ile Cys Phe Ala
Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly Glu Gln Ile Leu Glu
Ser Ile Ala Ala Tyr Ile His Arg Asp 20 25 30 Glu Phe Asp Gln Tyr
Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 Ile Val His
His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val His 50 55 60 Ile
Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala Thr Val 65 70
75 80 Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala Asn Arg Arg
Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val
Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu
Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala Ser Asp Ser Val
Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr Ser Gly Lys Asn
Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 Phe Val Phe Ala
Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165 170 175 Val Lys
Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185 190
Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala Asn Val 195
200 205 Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile Ser
Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys Lys
Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His Thr Ile Gly His
Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile Thr His Gly Glu
Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala Lys Ile Ala Asn
Arg Met Asn Leu Leu Ser Glu Gln Asp Val 275 280 285 Ser Thr His Tyr
Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Glu Leu 290 295 300 Pro Leu
Arg Ala Asp Ala Asp Ser Val Phe His Tyr Leu Ile His Asp 305 310 315
320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met Ile
325 330 335 Leu Leu Glu Gly Ile Gly Arg Pro Ala Val His Asn Gln Ser
Leu Leu 340 345 350 Thr Pro Val Lys Lys Ser Leu Ile Lys Glu Val Ile
Arg Glu Gly Leu 355 360 365 71107DNAPaenibacillus sp.CDS(1)..(1107)
7atg acg act aaa caa att tgt ttt gcg gac cgg tgt ttt aac ttt gca
48Met Thr Thr Lys Gln Ile Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1
5 10 15 ttc ggc gaa cat gtt ttg gaa tcg gtt gaa tcc tat att ccc cgg
gat 96Phe Gly Glu His Val Leu Glu Ser Val Glu Ser Tyr Ile Pro Arg
Asp 20 25 30 gaa ttc gat caa tat atc atg att tcg gac tcg ggg gta
ccg gac tcg 144Glu Phe Asp Gln Tyr Ile Met Ile Ser Asp Ser Gly Val
Pro Asp Ser 35 40 45 att gtt cat cat gcg gcc gaa tac ttc ggc aga
ctc gcc cct gta cat 192Ile Val His His Ala Ala Glu Tyr Phe Gly Arg
Leu Ala Pro Val His 50 55 60 att ctt cgc ttt cag ggc gga gaa gaa
tac aaa aca ctt gca acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu
Tyr Lys Thr Leu Ala Thr Val 65 70 75 80 aca aat ttg caa gag cag gca
att gct ctg gga gcc aac cga aga acc 288Thr Asn Leu Gln Glu Gln Ala
Ile Ala Leu Gly Ala Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt
ggc gga ggg tta acc gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val
Gly Gly Gly Leu Thr Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc
atg atg ttt cgc ggg att gcg ctt att cac gtt ccg acc 384Ala Ala Gly
Met Met Phe Arg Gly Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg
ttt ttg gcg gcc tcc gat tcg gtt ctt tcg att aag cag gct gtt 432Thr
Phe Leu Ala Ala Ser Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135
140 aat tta acg agc gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc
480Asn Leu Thr Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg
145 150 155 160 ttc gtg ttc gcc gat acc cga atc ttg gcg gag tcg ccg
ccc cgt cag 528Phe Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro
Pro Arg Gln 165 170 175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat
atg ctg att ctg gaa 576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn
Met Leu Ile Leu Glu 180 185 190 aac gag cac atg gaa ttt aca gag gat
gat tta aat tca gcc aat gtg 624Asn Glu His Met Glu Phe Thr Glu Asp
Asp Leu Asn Ser Ala Asn Val 195 200 205 tat tct ccg aag cag ctg gag
acg ttt atc aac ttc tgc ata tcg gcc 672Tyr Ser Pro Lys Gln Leu Glu
Thr Phe Ile Asn Phe Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta
agc gaa gat att tac gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu
Ser Glu Asp Ile Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt
gag tac ggc cat acg atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe
Glu Tyr Gly His Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255
cag gga ggg atc acg cac gga gaa gcc att gca gtg ggc atg att tac
816Gln Gly Gly Ile Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr
260 265 270 gcc gct aaa ata gcg aac cgg atg aac ctg atg ccc gaa cat
gac gtg 864Ala Ala Lys Ile Ala Asn Arg Met Asn Leu Met Pro Glu His
Asp Val 275 280 285 tcc gcc cat tac tgg ctt tta aat aaa atc ggg gcc
ttg cag gag ctt 912Ser Ala His Tyr Trp Leu Leu Asn Lys Ile Gly Ala
Leu Gln Glu Leu 290 295 300 ccg ctc cga gcg gac gcg gat tcg gtc ttc
cat tat tta atc cac gat 960Pro Leu Arg Ala Asp Ala Asp Ser Val Phe
His Tyr Leu Ile His Asp 305 310 315 320 aac aag agg ggc tac att aag
ctg gat gag gat aat ttg ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys
Leu Asp Glu Asp Asn Leu Gly Met Ile 325 330 335 tta ctt gag gga atc
ggt cga ccg gcg gtt cat aac caa tcg ctg ctt 1056Leu Leu Glu Gly Ile
Gly Arg Pro Ala Val His Asn Gln Ser Leu Leu 340 345 350 aca ccg gtc
aag aaa tcg ctc ata aaa gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val
Lys Lys Ser Leu Ile Lys Glu Val Ile Arg Glu Gly Leu 355 360 365 taa
11078368PRTPaenibacillus sp. 8Met Thr Thr Lys Gln Ile Cys Phe Ala
Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly Glu His Val Leu Glu
Ser Val Glu Ser Tyr Ile Pro Arg Asp 20 25 30 Glu Phe Asp Gln Tyr
Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 Ile Val His
His Ala Ala Glu Tyr Phe Gly Arg Leu Ala Pro Val His 50 55 60 Ile
Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala Thr Val 65 70
75 80 Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala Asn Arg Arg
Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val
Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu
Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala Ser Asp Ser Val
Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr Ser Gly Lys Asn
Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 Phe Val Phe Ala
Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165 170 175 Val Lys
Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185 190
Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala Asn Val 195
200 205 Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile Ser
Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys Lys
Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His Thr Ile Gly His
Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile Thr His Gly Glu
Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala Lys Ile Ala Asn
Arg Met Asn Leu Met Pro Glu His Asp Val 275 280 285 Ser Ala His Tyr
Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Glu Leu 290 295 300 Pro Leu
Arg Ala Asp Ala Asp Ser Val Phe His Tyr Leu Ile His Asp 305 310 315
320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met Ile
325 330 335 Leu Leu Glu Gly Ile Gly Arg Pro Ala Val His Asn Gln Ser
Leu Leu 340 345 350 Thr Pro Val Lys Lys Ser Leu Ile Lys Glu Val Ile
Arg Glu Gly Leu 355 360 365 91107DNAPaenibacillus sp.CDS(1)..(1107)
9atg acg act aaa caa att tgt ttt gcg gac cgg tgt ttt aac ttt gca
48Met Thr Thr Lys Gln Ile Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1
5 10 15 ttc ggc gaa cat gtt ttg gaa tcg gtt gaa tcc tat att ccc cgg
gat 96Phe Gly Glu His Val Leu Glu Ser Val Glu Ser Tyr Ile Pro Arg
Asp 20 25 30 gaa ttc gat caa tat atc atg att tcg gac tcg ggg gta
ccg gac tcg 144Glu Phe Asp Gln Tyr Ile Met Ile Ser Asp Ser Gly Val
Pro Asp Ser 35 40 45 att gtt cat tat gcg gcc gaa tac ttc ggc aaa
ctc gcc cct gta cat 192Ile Val His Tyr Ala Ala Glu Tyr Phe Gly Lys
Leu Ala Pro Val His 50 55 60 att ctt cgc ttt cag ggc gga gaa gaa
tac aaa aca ctt gca acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu
Tyr Lys Thr Leu Ala Thr Val 65 70 75 80 aca aat ttg caa gag cag gca
att gct ctg gga gcc aac cga aga acc 288Thr Asn Leu Gln Glu Gln Ala
Ile Ala Leu Gly Ala Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt
ggc gga ggg tta acc gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val
Gly Gly Gly Leu Thr Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc
atg atg ttt cgc ggg att gcg ctt att cac gtt ccg acc 384Ala Ala Gly
Met Met Phe Arg Gly Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg
ttt ttg gcg gcc tcc gat tcg gtt ctt tcg att aag cag gct gtt 432Thr
Phe Leu Ala Ala Ser Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135
140 aat tta acg agc gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc
480Asn Leu Thr Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg
145 150 155 160 ttc gtg ttc gcc gat acc cga atc ttg gcg gag tcg ccg
ccc cgt cag 528Phe Val Phe Ala Asp Thr Arg Ile Leu Ala Glu Ser Pro
Pro Arg Gln 165 170 175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat
atg ctg att ctg gaa 576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn
Met Leu Ile Leu Glu 180 185 190 aac gag cac atg gaa ttt aca gag gat
gat tta aat tca gcc aat gtg 624Asn Glu His Met Glu Phe Thr Glu Asp
Asp Leu Asn Ser Ala Asn Val 195 200 205 tat tct ccg aag cag ctg gag
acg ttt atc aac ttc tgc ata tcg gcc 672Tyr Ser Pro Lys Gln Leu Glu
Thr Phe Ile Asn Phe Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta
agc gaa gat att tac gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu
Ser Glu Asp Ile Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt
gag tac ggc cat acg atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe
Glu Tyr Gly His Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255
cag gga ggg atc acg cac gga gaa gcc att gca gtg ggc atg att tac
816Gln Gly Gly Ile Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr
260 265 270 gcc gct aaa ata gcg aac cgg atg aac ctg atg ccc gaa cat
gac gtg 864Ala Ala Lys Ile Ala Asn Arg Met Asn Leu Met Pro Glu His
Asp Val 275 280 285 tcc gcc cat tac tgg ctt tta aat aaa atc ggg gcc
ttg cag gat att 912Ser Ala His Tyr Trp Leu Leu Asn Lys Ile Gly Ala
Leu Gln Asp Ile 290 295 300 ccg ctc aaa tcg gac ccg gat tcg atc ttc
cat tat tta atc cac gat 960Pro Leu Lys Ser Asp Pro Asp Ser Ile Phe
His Tyr Leu Ile His
Asp 305 310 315 320 aac aag agg ggc tac att aag ctg gat gag gat aat
ttg ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn
Leu Gly Met Ile 325 330 335 tta ctt gag gga atc ggt cga ccg gcg gtt
cat aac caa tcg ctg ctt 1056Leu Leu Glu Gly Ile Gly Arg Pro Ala Val
His Asn Gln Ser Leu Leu 340 345 350 aca ccg gtc aag aaa tcg ctc ata
aaa gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val Lys Lys Ser Leu Ile
Lys Glu Val Ile Arg Glu Gly Leu 355 360 365 taa
110710368PRTPaenibacillus sp. 10Met Thr Thr Lys Gln Ile Cys Phe Ala
Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly Glu His Val Leu Glu
Ser Val Glu Ser Tyr Ile Pro Arg Asp 20 25 30 Glu Phe Asp Gln Tyr
Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 Ile Val His
Tyr Ala Ala Glu Tyr Phe Gly Lys Leu Ala Pro Val His 50 55 60 Ile
Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ala Thr Val 65 70
75 80 Thr Asn Leu Gln Glu Gln Ala Ile Ala Leu Gly Ala Asn Arg Arg
Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val
Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu
Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala Ser Asp Ser Val
Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr Ser Gly Lys Asn
Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 Phe Val Phe Ala
Asp Thr Arg Ile Leu Ala Glu Ser Pro Pro Arg Gln 165 170 175 Val Lys
Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185 190
Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala Asn Val 195
200 205 Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile Ser
Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys Lys
Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His Thr Ile Gly His
Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile Thr His Gly Glu
Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala Lys Ile Ala Asn
Arg Met Asn Leu Met Pro Glu His Asp Val 275 280 285 Ser Ala His Tyr
Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Asp Ile 290 295 300 Pro Leu
Lys Ser Asp Pro Asp Ser Ile Phe His Tyr Leu Ile His Asp 305 310 315
320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met Ile
325 330 335 Leu Leu Glu Gly Ile Gly Arg Pro Ala Val His Asn Gln Ser
Leu Leu 340 345 350 Thr Pro Val Lys Lys Ser Leu Ile Lys Glu Val Ile
Arg Glu Gly Leu 355 360 365 111107DNAPaenibacillus
sp.CDS(1)..(1107) 11atg acg act aaa caa att tgt ttt gcg gac cgg tgt
ttt aac ttt gca 48Met Thr Thr Lys Gln Ile Cys Phe Ala Asp Arg Cys
Phe Asn Phe Ala 1 5 10 15 ttc ggc gaa cat gtt ttg gaa tcg gtt gaa
tcc tat att ccc cgg gat 96Phe Gly Glu His Val Leu Glu Ser Val Glu
Ser Tyr Ile Pro Arg Asp 20 25 30 gaa ttc gat caa tat atc atg att
tcg gac tcg ggg gta ccg gac tcg 144Glu Phe Asp Gln Tyr Ile Met Ile
Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 att gtt cat tat gcg gcc
gaa tac ttc ggc aaa ctc gcc cct gta cat 192Ile Val His Tyr Ala Ala
Glu Tyr Phe Gly Lys Leu Ala Pro Val His 50 55 60 att ctt cgc ttt
cag ggc gga gaa gaa tac aaa aca ctt tca acc gtg 240Ile Leu Arg Phe
Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ser Thr Val 65 70 75 80 aca aat
ttg caa gag cgg gca att gct ctg gga gcc aac cga aga acc 288Thr Asn
Leu Gln Glu Arg Ala Ile Ala Leu Gly Ala Asn Arg Arg Thr 85 90 95
gct atc gta gcg gtt ggc gga ggg tta acc gga aac gtt gcc gga gtg
336Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val Ala Gly Val
100 105 110 gcg gcc ggc atg atg ttt cgc ggg att gcg ctt att cac gtt
ccg acc 384Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu Ile His Val
Pro Thr 115 120 125 acg ttt ttg gcg gcc tcc gat tcg gtt ctt tcg att
aag cag gct gtt 432Thr Phe Leu Ala Ala Ser Asp Ser Val Leu Ser Ile
Lys Gln Ala Val 130 135 140 aat tta acg agc gga aag aac ctg gtc ggc
ttt tat tat ccg cca cgc 480Asn Leu Thr Ser Gly Lys Asn Leu Val Gly
Phe Tyr Tyr Pro Pro Arg 145 150 155 160 ttc gtg ttc gcc gat acc cga
atc ttg tcg gag tcg ccg ccc cgt cag 528Phe Val Phe Ala Asp Thr Arg
Ile Leu Ser Glu Ser Pro Pro Arg Gln 165 170 175 gtg aaa gcg gga atg
tgc gag ctg gta aaa aat atg ctg att ctg gaa 576Val Lys Ala Gly Met
Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185 190 aac gag cac
atg gaa ttt aca gag gat gat tta aat tca gcc aat gtg 624Asn Glu His
Met Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala Asn Val 195 200 205 tat
tct ccg aag cag ctg gag acg ttt atc aac ttc tgc ata tcg gcc 672Tyr
Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile Ser Ala 210 215
220 aaa atg tcg gta tta agc gaa gat att tac gag aaa aag aag ggc ctg
720Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys Lys Lys Gly Leu
225 230 235 240 atc ttt gag tac ggc cat acg atc ggt cat gcg atc gag
ctt gcc gag 768Ile Phe Glu Tyr Gly His Thr Ile Gly His Ala Ile Glu
Leu Ala Glu 245 250 255 cag gga ggg atc acg cac gga gaa gcc att gca
gtg ggc atg att tac 816Gln Gly Gly Ile Thr His Gly Glu Ala Ile Ala
Val Gly Met Ile Tyr 260 265 270 gcc gct aaa ata gcg aac cgg atg aac
ctg atg ccc gaa cat gac gtg 864Ala Ala Lys Ile Ala Asn Arg Met Asn
Leu Met Pro Glu His Asp Val 275 280 285 tcc gcc cat tac tgg ctt tta
aat aaa atc ggg gcc ttg cag gat att 912Ser Ala His Tyr Trp Leu Leu
Asn Lys Ile Gly Ala Leu Gln Asp Ile 290 295 300 ccg ctc aaa tcg gac
ccg gat tcg atc ttc cat tat tta atc cac gat 960Pro Leu Lys Ser Asp
Pro Asp Ser Ile Phe His Tyr Leu Ile His Asp 305 310 315 320 aac aag
agg ggc tac att aag ctg gat gag gat aat ttg ggt atg att 1008Asn Lys
Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met Ile 325 330 335
tta ctt agc gga gtc ggt aaa ccg gcg atg tat aac caa acg ctg ctt
1056Leu Leu Ser Gly Val Gly Lys Pro Ala Met Tyr Asn Gln Thr Leu Leu
340 345 350 aca ccg gtc aga aaa acg ctc ata aaa gaa gtg atc cgg gaa
ggg ctg 1104Thr Pro Val Arg Lys Thr Leu Ile Lys Glu Val Ile Arg Glu
Gly Leu 355 360 365 taa 110712368PRTPaenibacillus sp. 12Met Thr Thr
Lys Gln Ile Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe
Gly Glu His Val Leu Glu Ser Val Glu Ser Tyr Ile Pro Arg Asp 20 25
30 Glu Phe Asp Gln Tyr Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser
35 40 45 Ile Val His Tyr Ala Ala Glu Tyr Phe Gly Lys Leu Ala Pro
Val His 50 55 60 Ile Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr
Leu Ser Thr Val 65 70 75 80 Thr Asn Leu Gln Glu Arg Ala Ile Ala Leu
Gly Ala Asn Arg Arg Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly
Leu Thr Gly Asn Val Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe
Arg Gly Ile Ala Leu Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala
Ala Ser Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu
Thr Ser Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155
160 Phe Val Phe Ala Asp Thr Arg Ile Leu Ser Glu Ser Pro Pro Arg Gln
165 170 175 Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile
Leu Glu 180 185 190 Asn Glu His Met Glu Phe Thr Glu Asp Asp Leu Asn
Ser Ala Asn Val 195 200 205 Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile
Asn Phe Cys Ile Ser Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp
Ile Tyr Glu Lys Lys Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly
His Thr Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly
Ile Thr His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala
Ala Lys Ile Ala Asn Arg Met Asn Leu Met Pro Glu His Asp Val 275 280
285 Ser Ala His Tyr Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Asp Ile
290 295 300 Pro Leu Lys Ser Asp Pro Asp Ser Ile Phe His Tyr Leu Ile
His Asp 305 310 315 320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp
Asn Leu Gly Met Ile 325 330 335 Leu Leu Ser Gly Val Gly Lys Pro Ala
Met Tyr Asn Gln Thr Leu Leu 340 345 350 Thr Pro Val Arg Lys Thr Leu
Ile Lys Glu Val Ile Arg Glu Gly Leu 355 360 365
131107DNAPaenibacillus sp.CDS(1)..(1107) 13atg acg act aaa caa att
tgt ttt gcg gac cgg tgt ttt aac ttt gca 48Met Thr Thr Lys Gln Ile
Cys Phe Ala Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 ttc ggc gaa cat
gtt ttg gaa tcg gtt gaa tcc tat att ccc cgg gat 96Phe Gly Glu His
Val Leu Glu Ser Val Glu Ser Tyr Ile Pro Arg Asp 20 25 30 gaa ttc
gat caa tat atc atg att tcg gac tcg ggg gta ccg gac tcg 144Glu Phe
Asp Gln Tyr Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45
att gtt cat tat gcg gcc gaa tac ttc ggc aaa ctc gcc cct gta cat
192Ile Val His Tyr Ala Ala Glu Tyr Phe Gly Lys Leu Ala Pro Val His
50 55 60 att ctt cgc ttt cag ggc gga gaa gaa tac aaa aca ctt tca
acc gtg 240Ile Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ser
Thr Val 65 70 75 80 aca aat ttg caa gag cgg gca att gct ctg gga gcc
aac cga aga acc 288Thr Asn Leu Gln Glu Arg Ala Ile Ala Leu Gly Ala
Asn Arg Arg Thr 85 90 95 gct atc gta gcg gtt ggc gga ggg tta acc
gga aac gtt gcc gga gtg 336Ala Ile Val Ala Val Gly Gly Gly Leu Thr
Gly Asn Val Ala Gly Val 100 105 110 gcg gcc ggc atg atg ttt cgc ggg
att gcg ctt att cac gtt ccg acc 384Ala Ala Gly Met Met Phe Arg Gly
Ile Ala Leu Ile His Val Pro Thr 115 120 125 acg ttt ttg gcg gcc tcc
gat tcg gtt ctt tcg att aag cag gct gtt 432Thr Phe Leu Ala Ala Ser
Asp Ser Val Leu Ser Ile Lys Gln Ala Val 130 135 140 aat tta acg agc
gga aag aac ctg gtc ggc ttt tat tat ccg cca cgc 480Asn Leu Thr Ser
Gly Lys Asn Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 ttc
gtg ttc gcc gat acc cga atc ttg tcg gag tcg ccg ccc cgt cag 528Phe
Val Phe Ala Asp Thr Arg Ile Leu Ser Glu Ser Pro Pro Arg Gln 165 170
175 gtg aaa gcg gga atg tgc gag ctg gta aaa aat atg ctg att ctg gaa
576Val Lys Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu
180 185 190 aac gac aac aag gaa ttt aca gag gat gat tta aat tca gcc
aat gtg 624Asn Asp Asn Lys Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala
Asn Val 195 200 205 tat tct ccg aag cag ctg gag acg ttt atc aac ttc
tgc ata tcg gcc 672Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe
Cys Ile Ser Ala 210 215 220 aaa atg tcg gta tta agc gaa gat att tac
gag aaa aag aag ggc ctg 720Lys Met Ser Val Leu Ser Glu Asp Ile Tyr
Glu Lys Lys Lys Gly Leu 225 230 235 240 atc ttt gag tac ggc cat acg
atc ggt cat gcg atc gag ctt gcc gag 768Ile Phe Glu Tyr Gly His Thr
Ile Gly His Ala Ile Glu Leu Ala Glu 245 250 255 cag gga ggg atc acg
cac gga gaa gcc att gca gtg ggc atg att tac 816Gln Gly Gly Ile Thr
His Gly Glu Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 gcc gct aaa
ata gcg aac cgg atg aac ctg atg ccc gaa cat gac gtg 864Ala Ala Lys
Ile Ala Asn Arg Met Asn Leu Met Pro Glu His Asp Val 275 280 285 tcc
gcc cat tac tgg ctt tta aat aaa atc ggg gcc ttg cag gat att 912Ser
Ala His Tyr Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Asp Ile 290 295
300 ccg ctc aaa tcg gac ccg gat tcg atc ttc cat tat tta atc cac gat
960Pro Leu Lys Ser Asp Pro Asp Ser Ile Phe His Tyr Leu Ile His Asp
305 310 315 320 aac aag agg ggc tac att aag ctg gat gag gat aat ttg
ggt atg att 1008Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu
Gly Met Ile 325 330 335 tta ctt agc gga gtc ggt aaa ccg gcg atg tat
aac caa acg ctg ctt 1056Leu Leu Ser Gly Val Gly Lys Pro Ala Met Tyr
Asn Gln Thr Leu Leu 340 345 350 aca ccg gtc aga aaa acg ctc ata aaa
gaa gtg atc cgg gaa ggg ctg 1104Thr Pro Val Arg Lys Thr Leu Ile Lys
Glu Val Ile Arg Glu Gly Leu 355 360 365 taa
110714368PRTPaenibacillus sp. 14Met Thr Thr Lys Gln Ile Cys Phe Ala
Asp Arg Cys Phe Asn Phe Ala 1 5 10 15 Phe Gly Glu His Val Leu Glu
Ser Val Glu Ser Tyr Ile Pro Arg Asp 20 25 30 Glu Phe Asp Gln Tyr
Ile Met Ile Ser Asp Ser Gly Val Pro Asp Ser 35 40 45 Ile Val His
Tyr Ala Ala Glu Tyr Phe Gly Lys Leu Ala Pro Val His 50 55 60 Ile
Leu Arg Phe Gln Gly Gly Glu Glu Tyr Lys Thr Leu Ser Thr Val 65 70
75 80 Thr Asn Leu Gln Glu Arg Ala Ile Ala Leu Gly Ala Asn Arg Arg
Thr 85 90 95 Ala Ile Val Ala Val Gly Gly Gly Leu Thr Gly Asn Val
Ala Gly Val 100 105 110 Ala Ala Gly Met Met Phe Arg Gly Ile Ala Leu
Ile His Val Pro Thr 115 120 125 Thr Phe Leu Ala Ala Ser Asp Ser Val
Leu Ser Ile Lys Gln Ala Val 130 135 140 Asn Leu Thr Ser Gly Lys Asn
Leu Val Gly Phe Tyr Tyr Pro Pro Arg 145 150 155 160 Phe Val Phe
Ala Asp Thr Arg Ile Leu Ser Glu Ser Pro Pro Arg Gln 165 170 175 Val
Lys Ala Gly Met Cys Glu Leu Val Lys Asn Met Leu Ile Leu Glu 180 185
190 Asn Asp Asn Lys Glu Phe Thr Glu Asp Asp Leu Asn Ser Ala Asn Val
195 200 205 Tyr Ser Pro Lys Gln Leu Glu Thr Phe Ile Asn Phe Cys Ile
Ser Ala 210 215 220 Lys Met Ser Val Leu Ser Glu Asp Ile Tyr Glu Lys
Lys Lys Gly Leu 225 230 235 240 Ile Phe Glu Tyr Gly His Thr Ile Gly
His Ala Ile Glu Leu Ala Glu 245 250 255 Gln Gly Gly Ile Thr His Gly
Glu Ala Ile Ala Val Gly Met Ile Tyr 260 265 270 Ala Ala Lys Ile Ala
Asn Arg Met Asn Leu Met Pro Glu His Asp Val 275 280 285 Ser Ala His
Tyr Trp Leu Leu Asn Lys Ile Gly Ala Leu Gln Asp Ile 290 295 300 Pro
Leu Lys Ser Asp Pro Asp Ser Ile Phe His Tyr Leu Ile His Asp 305 310
315 320 Asn Lys Arg Gly Tyr Ile Lys Leu Asp Glu Asp Asn Leu Gly Met
Ile 325 330 335 Leu Leu Ser Gly Val Gly Lys Pro Ala Met Tyr Asn Gln
Thr Leu Leu 340 345 350 Thr Pro Val Arg Lys Thr Leu Ile Lys Glu Val
Ile Arg Glu Gly Leu 355 360 365 1526DNAArtificial SequenceSynthetic
primer 15atgacgacta aacaaatttg ttttgc 261619DNAArtificial
SequenceSynthetic primer 16ttacagccct tcccggatc 19
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