U.S. patent application number 12/417453 was filed with the patent office on 2009-08-13 for alpha-amylase variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Carsten Andersen, Henrik Bisgaard-Frantzen, Soren Kjaerulff, Allan Svendsen.
Application Number | 20090203109 12/417453 |
Document ID | / |
Family ID | 34519637 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203109 |
Kind Code |
A1 |
Svendsen; Allan ; et
al. |
August 13, 2009 |
Alpha-Amylase Variants
Abstract
The invention relates to a variant of a parent Termamyl-like
.alpha.-amylase, comprising mutations in two, three, four, five or
six regions/positions. The variants have increased stability at
high temperatures (relative to the parent). The invention also
relates to a DNA construct comprising a DNA sequence encoding an
.alpha.-amylase variant of the invention, a recombinant expression
vector which carries a DNA construct of the invention, a cell which
is transformed with a DNA construct of the invention, the use of an
.alpha.-amylase variant of the invention for washing and/or
dishwashing, textile desizing, starch liquefaction, a detergent
additive comprising an .alpha.-amylase variant of the invention, a
manual or automatic dishwashing detergent composition comprising an
.alpha.-amylase variant of the invention, a method for generating a
variant of a parent Termamyl-like .alpha.-amylase, which variant
exhibits increased.
Inventors: |
Svendsen; Allan; (Birkerod,
DK) ; Kjaerulff; Soren; (Vanlose, DK) ;
Bisgaard-Frantzen; Henrik; (Bagsvaerd, DK) ;
Andersen; Carsten; (Vaerlose, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
34519637 |
Appl. No.: |
12/417453 |
Filed: |
April 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11039565 |
Jan 19, 2005 |
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12417453 |
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09441313 |
Nov 16, 1999 |
6887986 |
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11039565 |
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09193068 |
Nov 16, 1998 |
6197565 |
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09441313 |
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Current U.S.
Class: |
435/202 |
Current CPC
Class: |
C12N 9/2417 20130101;
C11D 3/38618 20130101; C12N 9/2414 20130101; C07H 21/04 20130101;
C11D 3/38609 20130101 |
Class at
Publication: |
435/202 |
International
Class: |
C12N 9/28 20060101
C12N009/28 |
Claims
1. A variant alpha-amylase, said variant has an amino acid sequence
which is at least 80% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7 or
8, has alpha-amylase activity and comprises a substitution of
lysine at position 376, using the numbering of SEQ ID NO:4.
2. The variant of claim 1, wherein the variant has an amino acid
sequence which is at least 85% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
3. The variant of claim 1, wherein the variant has an amino acid
sequence which is at least 90% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
4. The variant of claim 1, wherein the variant has an amino acid
sequence which is at least 95% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
5. The variant of claim 1, wherein the variant has an amino acid
sequence which is at least 97% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
6. The variant of claim 1, wherein the variant has an amino acid
sequence which is at least 99% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
7. A variant alpha-amylase, said variant has an amino acid sequence
which is at least 80% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7 or
8, has alpha-amylase activity and comprises a substitution of
alanine at position 356, using the numbering of SEQ ID NO:4.
8. The variant of claim 7, wherein the variant has an amino acid
sequence which is at least 85% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
9. The variant of claim 7, wherein the variant has an amino acid
sequence which is at least 90% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
10. The variant of claim 7, wherein the variant has an amino acid
sequence which is at least 95% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
11. The variant of claim 7, wherein the variant has an amino acid
sequence which is at least 97% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
12. The variant of claim 7, wherein the variant has an amino acid
sequence which is at least 99% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
13. A variant alpha-amylase, said variant has an amino acid
sequence which is at least 80% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8, has alpha-amylase activity and comprises a
substitution of phenylalanine at position 358, using the numbering
of SEQ ID NO:4.
14. The variant of claim 13, wherein the variant has an amino acid
sequence which is at least 85% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
15. The variant of claim 13, wherein the variant has an amino acid
sequence which is at least 90% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
16. The variant of claim 13, wherein the variant has an amino acid
sequence which is at least 95% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
17. The variant of claim 13, wherein the variant has an amino acid
sequence which is at least 97% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
18. The variant of claim 13, wherein the variant has an amino acid
sequence which is at least 99% identical to SEQ ID NO:1, 2, 3, 4,
5, 6, 7 or 8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/039,565 filed Jan. 19, 2005 which is a continuation of U.S.
application Ser. No. 09/441,313, filed Nov. 16, 1999, which is a
continuation-in-part of U.S. application Ser. No. 09/193,068 filed
on Nov. 16, 1998, the contents of which are fully incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel variants of parent
Termamyl-like .alpha.-amylases with altered properties relative of
the parent alpha-amylase. Said properties include increased
stability, e.g., at acidic pH, e.g., at low calcium concentrations
and/or high temperatures. Such variants are suitable for a number
of applications, in particular, industrial starch processing (e.g.,
starch liquefaction or saccharification).
BACKGROUND OF THE INVENTION
[0003] .alpha.-Amylases (.alpha.-1,4-glucan-4-glucanohydrolases, EC
3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of
starch and other linear and branched 1,4-glucosidic oligo- and
polysaccharides.
[0004] There is a very extensive body of patent and scientific
literature relating to this industrially very important class of
enzymes. A number of .alpha.-amylase such as Termamyl-like
.alpha.-amylases variants are known from, e.g., WO 90/11352, WO
95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
[0005] WO 96/23874 provides the three-dimensional, X-ray crystal
structural data for a Termamyl-like .alpha.-amylase which consists
of the 300 N-terminal amino acid residues of the B.
amyloliquefaciens.alpha.-amylase and amino acids 301-483 of the
C-terminal end of the B. licheniformis .alpha.-amylase comprising
the amino acid sequence (the latter being available commercially
under the tradename Termamyl.TM.), and which is thus closely
related to the industrially important Bacillus .alpha.-amylases
(which in the present context are embraced within the meaning of
the term "Termamyl-like .alpha.-amylases", and which include, inter
alia, the B. licheniformis, B. amyloliquefaciens and B.
stearothermophilus .alpha.-amylases). WO 96/23874 further describes
methodology for designing, on the basis of an analysis of the
structure of a parent Termamyl-like .alpha.-amylase, variants of
the parent Termamyl-like .alpha.-amylase which exhibit altered
properties relative to the parent.
BRIEF DISCLOSURE OF THE INVENTION
[0006] The present invention relates to novel .alpha.-amylolytic
variants (mutants) of a Termamyl-like .alpha.-amylase, in
particular variants exhibiting increased stability at acidic pH at
high temperatures (relative to the parent) which are advantageous
in connection with, e.g., the industrial processing of starch
(starch liquefaction, saccharification and the like) as described
in U.S. Pat. No. 3,912,590 and EP patent publications Nos. 252,730
and 63,909.
Starch Conversion
[0007] A "traditional" starch conversion process degrading starch
to lower molecular weight carbohydrate components such as sugars or
fat replacers includes a debranching step.
"Starch to Sugar" Conversion
[0008] In the case of converting starch into a sugar the starch is
depolymerized. A such depolymerization process consists of a
pretreatment step and two or three consecutive process steps, viz.
a liquefaction process, a saccharification process and dependent on
the desired end product optionally an isomerization process.
Pre-Treatment of Native Starch
[0009] Native starch consists of microscopic granules which are
insoluble in water at room temperature. When an aqueous starch
slurry is heated, the granules swell and eventually burst,
dispersing the starch molecules into the solution. During this
"gelatinization" process there is a dramatic increase in viscosity.
As the solids level is 30-40% in a typically industrial process,
the starch has to be thinned or "liquefied" so that it can be
handled. This reduction in viscosity is today mostly obtained by
enzymatic degradation.
Liquefaction
[0010] During the liquefaction step, the long chained starch is
degraded into branched and linear shorter units (maltodextrins) by
an .alpha.-amylase (e.g., Termamyl.TM. SEQ ID NO: 4 herein). The
liquefaction process is carried out at 105-110.degree. C. for 5 to
10 minutes followed by 1-2 hours at 95.degree. C. The pH lies
between 5.5 and 6.2. In order to ensure an optimal enzyme stability
under these conditions, 1 mM of calcium is added (40 ppm free
calcium ions). After this treatment the liquefied starch will have
a "dextrose equivalent" (DE) of 10-15.
Saccharification
[0011] After the liquefaction process the maltodextrins are
converted into dextrose by addition of a glucoamylase (e.g.,
AMG.TM.) and a debranching enzyme, such as an isoamylase (U.S. Pat.
No. 4,335,208) or a pullulanase (e.g., Promozyme.TM.) (U.S. Pat.
No. 4,560,651). Before this step the pH is reduced to a value below
4.5, maintaining the high temperature (above 95.degree. C.) to
inactivate the liquefying .alpha.-amylase to reduce the formation
of short oligosaccharide called "panose precursors" which cannot be
hydrolyzed properly by the debranching enzyme.
[0012] The temperature is lowered to 60.degree. C., and
glucoamylase and debranching enzyme are added. The saccharification
process proceeds for 24-72 hours.
[0013] Normally, when denaturing the .alpha.-amylase after the
liquefaction step about 0.2-0.5% of the saccharification product is
the branched trisaccharide 6.sup.2-.alpha.-glucosyl maltose
(panose) which cannot be degraded by a pullulanase. If active
amylase from the liquefaction step is present during
saccharification (i.e., no denaturing), this level can be as high
as 1-2%, which is highly undesirable as it lowers the
saccharification yield significantly.
Isomerization
[0014] When the desired final sugar product is e.g. high fructose
syrup the dextrose syrup may be converted into fructose. After the
saccharification process the pH is increased to a value in the
range of 6-8, preferably pH 7.5, and the calcium is removed by ion
exchange. The dextrose syrup is then converted into high fructose
syrup using, e.g., an immobilized glucoseisomerase (such as
Sweetzyme.TM.).
[0015] In the context of the invention the term "acidic pH" means a
pH below 7.0, especially below the pH range in which industrial
starch liquefaction processes are traditionally performed, as
described above, which is between pH 5.5 and 6.2.
[0016] In the context of the present invention the term "low
Calcium concentration" means concentrations below the normal level
used in traditional industrial starch liquefaction processes, such
as between 0-40 ppm, preferably between 10-30 ppm, such as between
15-25 ppm Calcium. Normal concentrations vary depending of the
concentration of free Ca.sup.2+ in the corn. Normally a dosage
corresponding to 1 mM (40 ppm) is added which together with the
level in corn gives between 40 and 60 ppm free Ca.sup.2+.
[0017] In the context of the invention the term "high temperature"
means temperatures between 95 and 160.degree. C., especially the
temperature range in which industrial starch liquefaction processes
are normally performed, which is between 95 and 105.degree. C.
[0018] The invention further relates to DNA constructs encoding
variants of the invention, to methods for preparing variants of the
invention, and to the use of variants of the invention, alone or in
combination with other .alpha.-amylolytic enzymes, in various
industrial processes, in particular starch liquefaction.
Nomenclature
[0019] In the present description and claims, the conventional
one-letter and three-letter codes for amino acid residues are used.
For ease of reference, .alpha.-amylase variants of the invention
are described by use of the following nomenclature:
Original amino acid(s):position(s):substituted amino acid(s)
[0020] According to this nomenclature, for instance the
substitution of alanine for asparagine in position 30 is shown
as:
[0021] Ala30Asn or A30N
a deletion of alanine in the same position is shown as:
[0022] Ala30* or A30*
and insertion of an additional amino acid residue, such as lysine,
is shown as:
[0023] Ala30AlaLys or A30AK
[0024] A deletion of a consecutive stretch of amino acid residues,
such as amino acid residues 30-33, is indicated as (30-33)* or
.DELTA.(A30-N33).
[0025] Where a specific .alpha.-amylase contains a "deletion" in
comparison with other .alpha.-amylases and an insertion is made in
such a position this is indicated as:
[0026] *36Asp or *36D
for insertion of an aspartic acid in position 36 Multiple mutations
are separated by plus signs, i.e.:
[0027] Ala30Asp+Glu34Ser or A30D+E34S
representing mutations in positions 30 and 34 substituting aspartic
acid and serine for alanine and glutamic acid, respectively.
Multiple mutation may also be separated as follows, i.e., meaning
the same as the plus sign:
[0028] Ala30Asp/Glu34Ser or A30D/E34S
[0029] When one or more alternative amino acid residues may be
inserted in a given position it is indicated as
[0030] A30N,E or
[0031] A30N or A30E
[0032] Furthermore, when a position suitable for modification is
identified herein without any specific modification being
suggested, it is to be understood that any amino acid residue may
be substituted for the amino acid residue present in the
position.
[0033] Thus, for instance, when a modification of an alanine in
position 30 is mentioned, but not specified, it is to be understood
that the alanine may be deleted or substituted for any other amino
acid, i.e., any one of:
R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.
BRIEF DESCRIPTION OF THE DRAWING
[0034] FIG. 1 is an alignment of the amino acid sequences of six
parent Termamyl-like .alpha.-amylases in the context of the
invention. The numbers on the Extreme left designate the respective
amino acid sequences as follows:
1: SEQ ID NO: 2,
[0035] 2: amylase
3: SEQ ID NO: 1,
4: SEQ ID NO: 5,
5: SEQ ID NO: 4,
6: SEQ ID NO: 3.
[0036] FIG. 2 shows the PCR strategy used in Example 1.
DETAILED DISCLOSURE OF THE INVENTION
The Termamyl-Like .alpha.-Amylase
[0037] It is well known that a number of .alpha.-amylases produced
by Bacillus spp. are highly homologous on the amino acid level. For
instance, the B. licheniformis .alpha.-amylase comprising the amino
acid sequence shown in SEQ ID NO: 4 (commercially available as
Termamyl.TM.) has been found to be about 89% homologous with the B.
amyloliquefaciens .alpha.-amylase comprising the amino acid
sequence shown in SEQ ID NO: 5 and about 79% homologous with the B.
stearothermophilus .alpha.-amylase comprising the amino acid
sequence shown in SEQ ID NO: 3. Further homologous .alpha.-amylases
include an .alpha.-amylase derived from a strain of the Bacillus
sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which
are described in detail in WO 95/26397, and the .alpha.-amylase
described by Tsukamoto et al., Biochemical and Biophysical Research
Communications, 151 (1988), pp. 25-31.
[0038] Still further homologous .alpha.-amylases include the
.alpha.-amylase produced by the B. licheniformis strain described
in EP 0252666 (ATCC 27811), and the .alpha.-amylases identified in
WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B.
licheniformis .alpha.-amylases are Optitherm.TM. and Takatherm.TM.
(available from Solvay), Maxamyl.TM. (available from
Gist-brocades/Genencor), Spezym AA.TM. and Spezyme Delta AA.TM.
(available from Genencor), and Keistase.TM. (available from
Daiwa).
[0039] Because of the substantial homology found between these
.alpha.-amylases, they are considered to belong to the same class
of .alpha.-amylases, namely the class of "Termamyl-like
.alpha.-amylases".
[0040] Accordingly, in the present context, the term "Termamyl-like
.alpha.-amylase" is intended to indicate an .alpha.-amylase which,
at the amino acid level, exhibits a substantial homology to
Termamyl.TM., i.e., the B. licheniformis .alpha.-amylase having the
amino acid sequence shown in SEQ ID NO: 4 herein. In other words, a
Termamyl-like .alpha.-amylase is an .alpha.-amylase which has the
amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8
herein, and the amino acid sequence shown in SEQ ID NO: 1 of WO
95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7
herein) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino
acid sequence shown as SEQ ID NO: 8 herein) or in Tsukamoto et al.,
1988, (which amino acid sequence is shown in SEQ ID NO: 6 herein)
or i) which displays at least 60% homology (identity), preferred at
least 70%, more preferred at least 75%, even more preferred at
least 80%, especially at least 85%, especially preferred at least
90%, especially at least 95%, even especially more preferred at
least 97%, especially at least 99% homology with at least one of
said amino acid sequences shown in SEQ ID NOS 1: or 2 or 3 or 4 or
5 or 6 or 7 or 8 and/or ii) displays immunological cross-reactivity
with an antibody raised against one or more of said
.alpha.-amylases, and/or iii) is encoded by a DNA sequence which
hybridizes, under the low to very high stringency conditions (said
conditions described below) to the DNA sequences encoding the
above-specified .alpha.-amylases which are apparent from SEQ ID
NOS: 9, 10, 11, 12, and 32, respectively, of the present
application (which encodes the amino acid sequences shown in SEQ ID
NOS: 1, 2, 3, 4, and 5 herein, respectively), from SEQ ID NO: 4 of
WO 95/26397 (which DNA sequence, together with the stop codon TAA,
is shown in SEQ ID NO: 13 herein and encodes the amino acid
sequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO
95/26397 (shown in SEQ ID NO: 14 herein), respectively.
[0041] In connection with property i), the "homology" (identity)
may be determined by use of any conventional algorithm, preferably
by use of the gap programme from the GCG package version 8 (August
1994) using default values for gap penalties, i.e., a gap creation
penalty of 3.0 and gap extension penalty of 0.1 (Genetic Computer
Group (1991) Programme Manual for the GCG Package, version 8, 575
Science Drive, Madison, Wis., USA 53711).
[0042] The parent Termamyl-like .alpha.-amylase backbone may in an
embodiment have an amino acid sequence which has a degree of
identity to SEQ ID NO: 4 of at least 65%, preferably at least 70%,
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, even more preferably at least about 90%,
even more preferably at least 95%, even more preferably at least
97%, and even more preferably at least 99% identity determined as
described above
[0043] A structural alignment between Termamyl.RTM. (SEQ ID NO: 4)
and a Termamyl-like .alpha.-amylase may be used to identify
equivalent/corresponding positions in other Termamyl-like
.alpha.-amylases. One method of obtaining said structural alignment
is to use the Pile Up programme from the GCG package using default
values of gap penalties, i.e., a gap creation penalty of 3.0 and
gap extension penalty of 0.1. Other structural alignment methods
include the hydrophobic cluster analysis (Gaboriaud et al., (1987),
FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T;
Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
[0044] For example, the corresponding positions, of target residues
found in the C-domain of the B. licheniformis .alpha.-amylase, in
the amino acid sequences of a number of Termamyl-like
.alpha.-amylases which have already been mentioned are as
follows:
TABLE-US-00001 Termamyl-like .alpha.-amylase B. lich. (SEQ ID NO:
4) S356 Y358 E376 S417 A420 B. amylo. (SEQ ID NO: 5) S356 Y358 E376
S417 A420 B. stearo. (SEQ ID NO: 3) ---- Y361 ---- ---- ---- Bac.WO
95/26397 ---- Y363 ---- S419 ---- (SEQ ID NO: 2) Bac.WO 95/26397
---- Y363 ---- ---- ---- (SEQ ID NO: 1)
[0045] As will be described further below mutations of these
conserved amino acid residues are very important in relation to
increasing the stability at acidic pH and/or at low calcium
concentration at high temperatures.
[0046] Property ii) (see above) of the .alpha.-amylase, i.e., the
immunological cross reactivity, may be assayed using an antibody
raised against, or reactive with, at least one epitope of the
relevant Termamyl-like .alpha.-amylase. The antibody, which may
either be monoclonal or polyclonal, may be produced by methods
known in the art, e.g., as described by Hudson et al., Practical
Immunology, Third edition (1989), Blackwell Scientific
Publications. The immunological cross-reactivity may be determined
using assays known in the art, examples of which are Western
Blotting or radial immunodiffusion assay, e.g., as described by
Hudson et al., 1989. In this respect, immunological
cross-reactivity between the .alpha.-amylases having the amino acid
sequences SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8 respectively, have
been found.
[0047] The oligonucleotide probe used in the characterization of
the Termamyl-like .alpha.-amylase in accordance with property iii)
above may suitably be prepared on the basis of the full or partial
nucleotide or amino acid sequence of the .alpha.-amylase in
question.
[0048] Suitable conditions for testing hybridization involve
presoaking in 5.times.SSC and prehybridizing for 1 hour at
.about.40.degree. C. in a solution of 20% formamide,
5.times.Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50
mg of denatured sonicated calf thymus DNA, followed by
hybridization in the same solution supplemented with 100 mM ATP for
18 hours at .about.40.degree. C., followed by three times washing
of the filter in 2.times.SSC, 0.2% SDS at 40.degree. C. for 30
minutes (low stringency), preferred at 50.degree. C. (medium
stringency), more preferably at 65.degree. C. (high stringency),
even more preferably at .about.75.degree. C. (very high
stringency). More details about the hybridization method can be
found in Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor, 1989.
[0049] In the present context, "derived from" is intended not only
to indicate an .alpha.-amylase produced or producible by a strain
of the organism in question, but also an .alpha.-amylase encoded by
a DNA sequence isolated from such strain and produced in a host
organism transformed with said DNA sequence. Finally, the term is
intended to indicate an .alpha.-amylase which is encoded by a DNA
sequence of synthetic and/or cDNA origin and which has the
identifying characteristics of the .alpha.-amylase in question. The
term is also intended to indicate that the parent .alpha.-amylase
may be a variant of a naturally occurring .alpha.-amylase, i.e., a
variant which is the result of a modification (insertion,
substitution, deletion) of one or more amino acid residues of the
naturally occurring .alpha.-amylase.
Parent Hybrid .alpha.-Amylases
[0050] The parent .alpha.-amylase (backbone) may be a hybrid
.alpha.-amylase, i.e., an .alpha.-amylase which comprises a
combination of partial amino acid sequences derived from at least
two .alpha.-amylases.
[0051] The parent hybrid .alpha.-amylase may be one which on the
basis of amino acid homology and/or immunological cross-reactivity
and/or DNA hybridization (as defined above) can be determined to
belong to the Termamyl-like .alpha.-amylase family. In this case,
the hybrid .alpha.-amylase is typically composed of at least one
part of a Termamyl-like .alpha.-amylase and part(s) of one or more
other .alpha.-amylases selected from Termamyl-like .alpha.-amylases
or non-Termamyl-like .alpha.-amylases of microbial (bacterial or
fungal) and/or mammalian origin.
[0052] Thus, the parent hybrid .alpha.-amylase may comprise a
combination of partial amino acid sequences deriving from at least
two Termamyl-like .alpha.-amylases, or from at least one
Termamyl-like and at least one non-Termamyl-like bacterial
.alpha.-amylase, or from at least one Termamyl-like and at least
one fungal .alpha.-amylase. The Termamyl-like .alpha.-amylase from
which a partial amino acid sequence derives may, e.g., be any of
those specific Termamyl-like .alpha.a-mylase referred to
herein.
[0053] For instance, the parent .alpha.-amylase may comprise a
C-terminal part of an .alpha.-amylase derived from a strain of B.
licheniformis, and a N-terminal part of an .alpha.-amylase derived
from a strain of B. amyloliquefaciens or from a strain of B.
stearothermophilus. For instance, the parent .alpha.-amylase may
comprise at least 430 amino acid residues of the C-terminal part of
the B. licheniformis .alpha.-amylase. A such hybrid Termamyl-like
.alpha.-amylase may be identical to the Bacillus licheniformis
.alpha.-amylase shown in SEQ ID NO: 4, except that the N-terminal
35 amino acid residues (of the mature protein) is replaced with the
N-terminal 33 amino acid residues of the mature protein of the
Bacillus amyloliquefaciens .alpha.-amylase (BAN) shown in SEQ ID
NO: 5. A such hybrid may also consist of an amino acid segment
corresponding to the 68 N-terminal amino acid residues of the B.
stearothermophilus .alpha.-amylase having the amino acid sequence
shown in SEQ ID NO: 3 and an amino acid segment corresponding to
the 415 C-terminal amino acid residues of the B. licheniformis
.alpha.-amylase having the amino acid sequence shown in SEQ ID NO:
4.
[0054] The non-Termamyl-like .alpha.-amylase may, e.g., be a fungal
.alpha.-amylase, a mammalian or a plant .alpha.-amylase or a
bacterial .alpha.-amylase (different from a Termamyl-like
.alpha.-amylase). Specific examples of such .alpha.-amylases
include the Aspergillus oryzae TAKA .alpha.-amylase, the A. niger
acid .alpha.-amylase, the Bacillus subtilis .alpha.-amylase, the
porcine pancreatic .alpha.-amylase and a barley .alpha.-amylase.
All of these .alpha.-amylases have elucidated structures which are
markedly different from the structure of a typical Termamyl-like
.alpha.-amylase as referred to herein.
[0055] The fungal .alpha.-amylases mentioned above, i.e. derived
from A. niger and A. oryzae, are highly homologous on the amino
acid level and generally considered to belong to the same family of
.alpha.-amylases. The fungal .alpha.-amylase derived from
Aspergillus oryzae is commercially available under the tradename
Fungamyl.TM..
[0056] Furthermore, when a particular variant of a Termamyl-like
.alpha.-amylase (variant of the invention) is referred to--in a
conventional manner--by reference to modification (e.g., deletion
or substitution) of specific amino acid residues in the amino acid
sequence of a specific Termamyl-like .alpha.-amylase, it is to be
understood that variants of another Termamyl-like .alpha.-amylase
modified in the equivalent position(s) (as determined from the best
possible amino acid sequence alignment between the respective amino
acid sequences) are encompassed thereby.
[0057] A preferred embodiment of a variant of the invention is one
derived from a B. licheniformis .alpha.-amylase (as parent
Termamyl-like .alpha.-amylase), e.g., one of those referred to
above, such as the B. licheniformis .alpha.-amylase having the
amino acid sequence shown in SEQ ID NO: 4.
Altered Properties of Variants of the Invention
[0058] The following discusses the relationship between
alterations/mutations which may be present in variants of the
invention, and desirable alterations in properties (relative to
those a parent, Termamyl-like .alpha.-amylase) which may result
therefrom.
Increased Stability at Acidic pH and/or Low Calcium Concentration
at High Temperatures
[0059] The present invention relates to a variant of a parent
Termamyl-like .alpha.-amylase, which variant .alpha.-amylase has
been altered in comparison to the parent .alpha.-amylase in one or
more solvent exposed amino acid residues on the surface of the
.alpha.-amylase to increase the overall hydrophibicity of the
.alpha.-amylase and/or to increase the overall numbers of methyl
groups in the sidechains of said solvent exposed amino acid
residues on the surface.
[0060] In a preferred embodiment one or more solvent exposed amino
acid residues on a concav surface with inwards bend are altered to
more hydrophobic amino acid residues.
[0061] In another preferred embodiment one or more solvent exposed
amino acid residues on a convex surface are altered to increase the
number of methyl groups in the sidechain.
[0062] The present invention relates to an .alpha.-amylase variant
of a parent Termamyl-like .alpha.-amylase, comprising an alteration
at one or more positions selected from the group of:
E376, S417, A420, S356, Y358;
[0063] wherein (a) the alteration(s) are independently
[0064] (i) an insertion of an amino acid downstream of the amino
acid which occupies the position,
[0065] (ii) a deletion of the amino acid which occupies the
position, or
[0066] (iii) a substitution of the amino acid which occupies the
position with a different amino acid,
(b) the variant has .alpha.-amylase activity and (c) each position
corresponds to a position of the amino acid sequence of the parent
Termamyl-like .alpha.-amylase having the amino acid sequence of SEQ
ID NO: 4.
[0067] In an embodiment the alteration is one of the following
substitutions:
E376A, R, D, C, Q, G, H, I, K, L, M, N, F, P, S, T, W, Y, V.
[0068] In a preferred embodiment the substitution is: E376K.
[0069] In an embodiment the alteration is one of the following
substitutions: S417A, R, D, C, E, Q, G, H, I, K, L, M, N, F, P, T,
W, Y, V;
In a preferred embodiment the substitution is S417T.
[0070] In an embodiment the alteration is one of the following
substitutions A420R, D, C, E, Q, G, H, I, K, L, M, N, F, P, S, T,
W, Y, V;
In a preferred embodiment the substitution is: A420Q, R.
[0071] In an embodiment the alteration is one of the following
substitutions: S356A, R, D, C, E, Q, G, H, I, K, L, M, N, F, P, T,
W, Y, V.
[0072] In an embodiment the alteration is one of the following
substitutions Y358A, R, D, C, E, Q, G, H, I, K, L, M, N, F, P, S,
T, W, V.
In a preferred embodiment the substitution is Y358F.
[0073] In an embodiment of the invention a variant comprises one or
more of the following substitutions: E376K, S417T, A420Q, R, S356A,
Y358F.
[0074] The increase in stability at acidic pH and/or low calcium
concentration at high temperatures may be determined using the
method described below in Example 2 illustrating the invention.
[0075] The parent Termamyl-like .alpha.-amylase used as the
backbone for preparing variants of the invention may be any
Termamyl-like .alpha.-amylases as defined above.
[0076] Specifically contemplated are parent Termamyl-like
.alpha.-amylases selected from the group derived from B.
licheniformis, such as B. licheniformis strain ATCC 27811, B.
amyloliquefaciens, B. stearothermophilus, Bacillus sp. NCIB 12289,
NCIB 12512, NCIB 12513 or DSM 9375, and the parent Termamyl-like
.alpha.-amylases depicted in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 and
8.
[0077] In an embodiment of the invention the parent Termamyl-like
.alpha.-amylase is a hybrid .alpha.-amylase being identical to the
Bacillus licheniformis .alpha.-amylase shown in SEQ ID NO: 4
(Termamyl), except that the N-terminal 35 amino acid residues (of
the mature protein) is replaced with the N-terminal 33 amino acid
residues of the mature protein of the Bacillus amyloliquefaciens
.alpha.-amylase (BAN) shown in SEQ ID NO: 5. The parent
Termamyl-like hybrid .alpha.-amylase may be the above mentioned
hybrid Termamyl-like .alpha.-amylase which further has the
following mutations: H156Y+181T+190F+209V+264S (using the numbering
in SEQ ID NO: 4). Said backbone is referred to below as
"LE174".
[0078] The parent .alpha.-amylase may advantageously further have a
mutation in one or more of the following positions: K176, I201 and
H205 (using the numbering in SEQ ID NO: 4), especially one or more
the following substitutions: K176R, I201F, and H205N (using the
numbering in SEQ ID NO: 4), such as specifically the following
substitutions: K176R+I201F+H205N (using the numbering in SEQ ID NO:
4).
[0079] The inventors have found that the above mentioned variants
have increased stability at pHs below 7.0 (i.e., acidic pH) and/or
at calcium concentration below 1 mM (40 ppm) (i.e., low calcium
concentrations) at temperatures in the range from 95 to 160.degree.
C. (i.e., high temperatures) relative to the parent Termamyl-like
.alpha.-amylase.
[0080] Alterations (e.g., by substitution) of one or more solvent
exposed amino acid residues which 1) increase the overall
hydrophobicity of the enzyme, or 2) increase the number of methyl
groups in the sidechains of the solvent exposed amino acid residues
improve the temperature stability. It is preferred to alter (e.g.,
by substitution) to more hydrophobic residues on a concav surface
with inwards bend. On a convex surface alterations (e.g., by
substitution) to amino acid residues with an increased number of
methyl groups in the sidechain are preferred.
[0081] Using the program CAST found on the internet at
http://sunrise.cbs.umn.edu/cast/ version 1.0 (release February
1998), (reference: Jie Liang, Herbert Edelsbrunner, and Clare
Woodward. 1998. Anatomy of protein Pockets and Cavities:
Measurements of binding site geometry and implications for ligand
design. Protein Science, 7, pp. 1884-1897), a concave area which
access to the surface can be identified. Access to the surface is
in the program defined as a probe with a diameter of 1.4 .ANG. can
pass in and out. Using default parameters in the CAST program
cancave cavities can be found using the Calcium depleted
alpha-amylase structure from B. licheniformis as found in the
Brookhaven database (1BPL):
[0082] Three types of interaction can be rationalised:
A. Interaction between the sidechain of the residue and the
protein, B. Interaction between the sidechain of the residue and
the surrounding water, C. Interaction between the water and the
protein.
[0083] Using the parent Termamyl-like .alpha.-amylase shown in SEQ
ID NO: 4 as the backbone the following positions are considered to
be solvent exposed and may suitably be altered:
E376, S417, A420, S356, Y358.
[0084] Corresponding and other solvent exposed positions on the
surface of other Termamyl-like .alpha.-amylase may be identified
using the dssp program by W. Kabsch and C. Sander, Biopolymers 22
(1983) pp. 2577-2637. The convex surfaces can be identified using
the AACAVI program part from the WHATIF package (G. Vriend, Whatif
and drug design program. J. Mol. Graph. 8, pp. 52-56. (1990)
version 19980317).
[0085] In an embodiment of the invention a variant comprises one or
more of the following substitutions: E376K, S417T, A420Q, R, S356A,
Y358F.
[0086] The inventors have found that the stability at acidic pH
and/or low calcium concentration at high temperatures may be
increased even more by combining mutations in the above mentioned
positions, i.e., E376, S417, A420, S356, Y358, (using the SEQ ID
NO: 4 numbering) with mutations in one or more of positions K176,
I201, and H205.
[0087] The following additional substitutions are preferred:
K176A, R, D, C, E, Q, G, H, I, L, M, N, F, P, S, T, W, Y, V;
I201A, R, D, C, E, Q, G, H, L, K, M, N, F, P, S, T, W, Y, V;
H205A, R, D, C, E, Q, G, I, L, K, M, N, F, P, S, T, W, Y, V;
[0088] As also shown in Example 2 illustrating the invention
combining the following mutations give increased stability:
K176+I201F+H205N+E376K+A420R or
K176+I201F+H205N+S417T+A420Q or
[0089] K176+I201F+H205N+S356A+Y358F using the hybrid
.alpha.-amylase referred to as LE174 as the parent Termamyl-like
.alpha.-amylase.
General Mutations in Variants of the Invention
[0090] It may be preferred that a variant of the invention
comprises one or more modifications in addition to those outlined
above. Thus, it may be advantageous that one or more proline
residues present in the part of the .alpha.-amylase variant which
is modified is/are replaced with a non-proline residue which may be
any of the possible, naturally occurring non-proline residues, and
which preferably is an alanine, glycine, serine, threonine, valine
or leucine.
[0091] Analogously, it may be preferred that one or more cysteine
residues present among the amino acid residues with which the
parent .alpha.-amylase is modified is/are replaced with a
non-cysteine residue such as serine, alanine, threonine, glycine,
valine or leucine.
[0092] Furthermore, a variant of the invention may--either as the
only modification or in combination with any of the above outlined
modifications--be modified so that one or more Asp and/or Glu
present in an amino acid fragment corresponding to the amino acid
fragment 185-209 of SEQ ID NO: 4 is replaced by an Asn and/or Gln,
respectively. Also of interest is the replacement, in the
Termamyl-like .alpha.-amylase, of one or more of the Lys residues
present in an amino acid fragment corresponding to the amino acid
fragment 185-209 of SEQ ID NO: 4 by an Arg.
[0093] It will be understood that the present invention encompasses
variants incorporating two or more of the above outlined
modifications.
[0094] Furthermore, it may be advantageous to introduce
point-mutations in any of the variants described herein.
Cloning a DNA Sequence Encoding an .alpha.-Amylase of the Invention
Cloning a DNA Sequence Encoding an a-Amylase Cloning a DNA Sequence
Encoding an a-Amylase Cloning a DNA Sequence Encoding an
a-Amylase
[0095] The DNA sequence encoding a parent .alpha.-amylase may be
isolated from any cell or microorganism producing the
.alpha.-amylase in question, using various methods well known in
the art. First, a genomic DNA and/or cDNA library should be
constructed using chromosomal DNA or messenger RNA from the
organism that produces the .alpha.-amylase to be studied. Then, if
the amino acid sequence of the .alpha.-amylase is known,
homologous, labelled oligonucleotide probes may be synthesized and
used to identify .alpha.-amylase-encoding clones from a genomic
library prepared from the organism in question. Alternatively, a
labelled oligonucleotide probe containing sequences homologous to a
known .alpha.-amylase gene could be used as a probe to identify
.alpha.-amylase-encoding clones, using hybridization and washing
conditions of lower stringency.
[0096] Yet another method for identifying .alpha.-amylase-encoding
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming
.alpha.-amylase-negative bacteria with the resulting genomic DNA
library, and then plating the transformed bacteria onto agar
containing a substrate for .alpha.-amylase, thereby allowing clones
expressing the .alpha.-amylase to be identified.
[0097] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g. the
phosphoroamidite method described by S. L. Beaucage and M. H.
Caruthers (1981) or the method described by Matthes et al. (1984).
In the phosphoroamidite method, oligonucleotides are synthesized,
e.g., in an automatic DNA synthesizer, purified, annealed, ligated
and cloned in appropriate vectors.
[0098] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et
al. (1988).
Site-Directed Mutagenesis
[0099] Once an .alpha.-amylase-encoding DNA sequence has been
isolated, and desirable sites for mutation identified, mutations
may be introduced using synthetic oligonucleotides. These
oligonucleotides contain nucleotide sequences flanking the desired
mutation sites; mutant nucleotides are inserted during
oligonucleotide synthesis. In a specific method, a single-stranded
gap of DNA, bridging the .alpha.-amylase-encoding sequence, is
created in a vector carrying the .alpha.-amylase gene. Then the
synthetic nucleotide, bearing the desired mutation, is annealed to
a homologous portion of the single-stranded DNA. The remaining gap
is then filled in with DNA polymerase I (Klenow fragment) and the
construct is ligated using T4 ligase. A specific example of this
method is described in Morinaga et al. (1984). U.S. Pat. No.
4,760,025 discloses the introduction of oligonucleotides encoding
multiple mutations by performing minor alterations of the cassette.
However, an even greater variety of mutations can be introduced at
any one time by the Morinaga method, because a multitude of
oligonucleotides, of various lengths, can be introduced.
[0100] Another method for introducing mutations into
.alpha.-amylase-encoding DNA sequences is described in Nelson and
Long (1989). It involves the 3-step generation of a PCR fragment
containing the desired mutation introduced by using a chemically
synthesized DNA strand as one of the primers in the PCR reactions.
From the PCR-generated fragment, a DNA fragment carrying the
mutation may be isolated by cleavage with restriction endonucleases
and reinserted into an expression plasmid.
Random Mutagenesis
[0101] Random mutagenesis is suitably performed either as localised
or region-specific random mutagenesis in at least three parts of
the gene translating to the amino acid sequence shown in question,
or within the whole gene.
[0102] The random mutagenesis of a DNA sequence encoding a parent
.alpha.-amylase may be conveniently performed by use of any method
known in the art.
[0103] In relation to the above, a further aspect of the present
invention relates to a method for generating a variant of a parent
.alpha.-amylase, e.g., wherein the variant exhibits altered or
increased thermal stability relative to the parent, the method
comprising:
[0104] (a) subjecting a DNA sequence encoding the parent
.alpha.-amylase to random mutagenesis,
[0105] (b) expressing the mutated DNA sequence obtained in step (a)
in a host cell, and
[0106] (c) screening for host cells expressing an .alpha.-amylase
variant which has an altered property (i.e. thermal stability)
relative to the parent .alpha.-amylase.
[0107] Step (a) of the above method of the invention is preferably
performed using doped primers.
[0108] For instance, the random mutagenesis may be performed by use
of a suitable physical or chemical mutagenizing agent, by use of a
suitable oligonucleotide, or by subjecting the DNA sequence to PCR
generated mutagenesis. Furthermore, the random mutagenesis may be
performed by use of any combination of these mutagenizing agents.
The mutagenizing agent may, e.g., be one which induces transitions,
transversions, inversions, scrambling, deletions, and/or
insertions.
[0109] Examples of a physical or chemical mutagenizing agent
suitable for the present purpose include ultraviolet (UV)
ir-radiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide
analogues. When such agents are used, the mutagenesis is typically
performed by incubating the DNA sequence encoding the parent enzyme
to be mutagenized in the presence of the mutagenizing agent of
choice under suitable conditions for the mutagenesis to take place,
and selecting for mutated DNA having the desired properties.
[0110] When the mutagenesis is performed by the use of an
oligonucleotide, the oligonucleotide may be doped or spiked with
the three non-parent nucleotides during the synthesis of the
oligonucleotide at the positions which are to be changed. The
doping or spiking may be done so that codons for unwanted amino
acids are avoided. The doped or spiked oligonucleotide can be
incorporated into the DNA encoding the alpha-amylase enzyme by any
published technique, using e.g. PCR, LCR or any DNA polymerase and
ligase as deemed appropriate.
[0111] Preferably, the doping is carried out using "constant random
doping", in which the percentage of wild-type and mutation in each
position is predefined. Furthermore, the doping may be directed
toward a preference for the introduction of certain nucleotides,
and thereby a preference for the introduction of one or more
specific amino acid residues. The doping may be made, e.g., so as
to allow for the introduction of 90% wild type and 10% mutations in
each position. An additional consideration in the choice of a
doping scheme is based on genetic as well as protein-structural
constraints. The doping scheme may be made by using the DOPE
program which, inter alia, ensures that introduction of stop codons
is avoided.
[0112] When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent .alpha.-amylase is
subjected to PCR under conditions that increase the
misincorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol. 1, 1989, pp. 11-15).
[0113] A mutator strain of E. coli (Fowler et al., Molec. Gen.
Genet., 133, 1974, pp. 179-191), S. cereviseae or any other
microbial organism may be used for the random mutagenesis of the
DNA encoding the .alpha.-amylase by, e.g., transforming a plasmid
containing the parent glycosylase into the mutator strain, growing
the mutator strain with the plasmid and isolating the mutated
plasmid from the mutator strain. The mutated plasmid may be
subsequently transformed into the expression organism.
[0114] The DNA sequence to be mutagenized may be conveniently
present in a genomic or cDNA library prepared from an organism
expressing the parent alpha-amylase. Alternatively, the DNA
sequence may be present on a suitable vector such as a plasmid or a
bacteriophage, which as such may be incubated with or other-wise
exposed to the mutagenising agent. The DNA to be mutagenized may
also be present in a host cell either by being integrated in the
genome of said cell or by being present on a vector harboured in
the cell. Finally, the DNA to be mutagenized may be in isolated
form. It will be understood that the DNA sequence to be subjected
to random mutagenesis is preferably a cDNA or a genomic DNA
sequence.
[0115] In some cases it may be convenient to amplify the mutated
DNA sequence prior to performing the expression step b) or the
screening step c). Such amplification may be performed in
accordance with methods known in the art, the presently preferred
method being PCR-generated amplification using oligonucleotide
primers prepared on the basis of the DNA or amino acid sequence of
the parent enzyme.
[0116] Subsequent to the incubation with or exposure to the
mutagenising agent, the mutated DNA is expressed by culturing a
suitable host cell carrying the DNA sequence under conditions
allowing expression to take place. The host cell used for this
purpose may be one which has been transformed with the mutated DNA
sequence, optionally present on a vector, or one which was carried
the DNA sequence encoding the parent enzyme during the mutagenesis
treatment. Examples of suitable host cells are the following: gram
positive bacteria such as Bacillus subtilis, Bacillus
licheniformis, Bacillus lentus, Bacillus brevis, Bacillus
stearothermophilus, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus, Bacillus megaterium, Bacillus thuringiensis, Streptormyces
lividans or Streptomyces murinus; and gram-negative bacteria such
as E. coli.
[0117] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
Localized Random Mutagenesis
[0118] The random mutagenesis may be advantageously localized to a
part of the parent .alpha.-amylase in question. This may, e.g., be
advantageous when certain regions of the enzyme have been
identified to be of particular importance for a given property of
the enzyme, and when modified are expected to result in a variant
having improved properties. Such regions may normally be identified
when the tertiary structure of the parent enzyme has been
elucidated and related to the function of the enzyme.
[0119] The localized, or region-specific, random mutagenesis is
conveniently performed by use of PCR generated mutagenesis
techniques as described above or any other suitable technique known
in the art. Alternatively, the DNA sequence encoding the part of
the DNA sequence to be modified may be isolated, e.g., by insertion
into a suitable vector, and said part may be subsequently subjected
to mutagenesis by use of any of the mutagenesis methods discussed
above.
Alternative Methods of Providing .alpha.-Amylase Variants
[0120] Alternative methods for providing variants of the invention
include gene shuffling method known in the art including the
methods, e.g., described in WO 95/22625 (from Affymax Technologies
N.V.) and WO 96/00343 (from Novo Nordisk A/S)..sub.--
Expression of .alpha.-Amylase Variants of the Invention
[0121] According to the invention, a DNA sequence encoding the
variant produced by methods described above, or by any alternative
methods known in the art, can be expressed, in enzyme form, using
an expression vector which typically includes control sequences
encoding a promoter, operator, ribosome binding site, translation
initiation signal, and, optionally, a repressor gene or various
activator genes.
[0122] The recombinant expression vector carrying the DNA sequence
encoding an .alpha.-amylase variant of the invention may be any
vector which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e., a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid, a bacteriophage or an
extrachromosomal element, minichromosome or an artificial
chromosome. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0123] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA sequence
encoding an .alpha.-amylase variant of the invention, especially in
a bacterial host, are the promoter of the lac operon of E. coli,
the Streptomyces coelicolor agarase gene dagA promoters, the
promoters of the Bacillus licheniformis .alpha.-amylase gene
(amyL), the promoters of the Bacillus stearothermophilus maltogenic
amylase gene (amyM), the promoters of the Bacillus
amyloliquefaciens .alpha.-amylase (amyQ), the promoters of the
Bacillus subtilis xylA and xylB genes etc. For transcription in a
fungal host, examples of useful promoters are those derived from
the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, A. niger neutral .alpha.-amylase, A. niger
acid stable .alpha.-amylase, A. niger glucoamylase, Rhizomucor
miehei lipase, A. oryzae alkaline protease, A. oryzae triose
phosphate isomerase or A. nidulans acetamidase.
[0124] The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes,
poly-adenylation sequences operably connected to the DNA sequence
encoding the .alpha.-amylase variant of the invention. Termination
and polyadenylation sequences may suitably be derived from the same
sources as the promoter.
[0125] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. Examples of such
sequences are the origins of replication of plasmids pUC19,
pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0126] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
such as the dal genes from B. subtilis or B. licheniformis, or one
which confers antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracyclin resistance. Furthermore, the vector
may comprise Aspergillus selection markers such as amdS, argB, niaD
and sC, a marker giving rise to hygromycin resistance, or the
selection may be accomplished by co-transformation, e.g. as
described in WO 91/17243.
[0127] While intracellular expression may be advantageous in some
respects, e.g. when using certain bacteria as host cells, it is
generally preferred that the expression is extracellular. In
general, the Bacillus .alpha.-amylases mentioned herein comprise a
preregion permitting secretion of the expressed protease into the
culture medium. If desirable, this preregion may be replaced by a
different preregion or signal sequence, conveniently accomplished
by substitution of the DNA sequences encoding the respective
preregions.
[0128] The procedures used to ligate the DNA construct of the
invention encoding an .alpha.-amylase variant, the promoter,
terminator and other elements, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor, 1989).
[0129] The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant production of
an .alpha.-amylase variant of the invention. The cell may be
transformed with the DNA construct of the invention encoding the
variant, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is generally
considered to be an advantage as the DNA sequence is more likely to
be stably maintained in the cell. Integration of the DNA constructs
into the host chromosome may be performed according to conventional
methods, e.g. by homologous or heterologous recombination.
Alternatively, the cell may be transformed with an expression
vector as described above in connection with the different types of
host cells.
[0130] The cell of the invention may be a cell of a higher organism
such as a mammal or an insect, but is preferably a microbial cell,
e.g., a bacterial or a fungal (including yeast) cell.
[0131] Examples of suitable bacteria are grampositive bacteria such
as Bacillus subztilis, Bacillus licheniformis, Bacillus lentus,
Bacillus brevis, Bacillus stearothermophilus, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus
thuringiensis, or Streptomyces lividans or Streptomyces murinus, or
gramnegative bacteria such as E. coli. The transformation of the
bacteria may, for instance, be effected by protoplast
transformation or by using competent cells in a manner known per
se.
[0132] The yeast organism may favourably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces
cerevisiae. The filamentous fungus may advantageously belong to a
species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus
niger. Fungal cells may be transformed by a process involving
protoplast formation and transformation of the protoplasts followed
by regeneration of the cell wall in a manner known per se. A
suitable procedure for transformation of Aspergillus host cells is
described in EP 238 023.
[0133] In a yet further aspect, the present invention relates to a
method of producing an .alpha.-amylase variant of the invention,
which method comprises cultivating a host cell as described above
under conditions conducive to the production of the variant and
recovering the variant from the cells and/or culture medium.
[0134] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of the .alpha.-amylase variant of the
invention. Suitable media are available from commercial suppliers
or may be prepared according to published recipes (e.g., as
described in catalogues of the American Type Culture
Collection).
[0135] The .alpha.-amylase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such as
ion exchange chromatography, affinity chromatography, or the
like.
INDUSTRIAL APPLICATIONS
[0136] The .alpha.-amylase variants of this invention possesses
valuable properties allowing for a variety of industrial
applications. An enzyme variant of the invention are applicable as
a component in washing, dishwashing and hard-surface cleaning
detergent compositions. Numerous variants are particularly useful
in the production of sweeteners and ethanol from starch, and/or for
textile desizing. Conditions for conventional starch-conversion
processes, including starch liquefaction and/or saccharification
processes, are described in, e.g., U.S. Pat. No. 3,912,590 and in
EP patent publications Nos. 252,730 and 63,909.
Production of Sweeteners from Starch:
[0137] A "traditional" process for conversion of starch to fructose
syrups normally consists of three consecutive enzymatic processes,
viz. a liquefaction process followed by a saccharification process
and an isomerization process. During the liquefaction process,
starch is degraded to dextrins by an .alpha.-amylase (e.g.
Termamyl.TM.) at pH values between 5.5 and 6.2 and at temperatures
of 95-160.degree. C. for a period of approx. 2 hours. In order to
ensure an optimal enzyme stability under these conditions, 1 mM of
calcium is added (40 ppm free calcium ions).
[0138] After the liquefaction process the dextrins are converted
into dextrose by addition of a glucoamylase (e.g. AMG.TM.) and a
debranching enzyme, such as an isoamylase or a pullulanase (e.g.
Promozyme.TM.). Before this step the pH is reduced to a value below
4.5, maintaining the high temperature (above 95.degree. C.), and
the liquefying .alpha.-amylase activity is denatured. The
temperature is lowered to 60.degree. C., and glucoamylase and
debranching enzyme are added. The saccharification process proceeds
for 24-72 hours.
[0139] After the saccharification process the pH is increased to a
value in the range of 6-8, preferably pH 7.5, and the calcium is
removed by ion exchange. The dextrose syrup is then converted into
high fructose syrup using, e.g., an immobilized glucoseisomerase
(such as Sweetzyme.TM.).
[0140] At least 1 enzymatic improvements of this process could be
envisaged. Reduction of the calcium dependency of the liquefying
.alpha.-amylase. Addition of free calcium is required to ensure
adequately high stability of the .alpha.-amylase, but free calcium
strongly inhibits the activity of the glucoseisomerase and needs to
be removed, by means of an expensive unit operation, to an extent
which reduces the level of free calcium to below 3-5 ppm. Cost
savings could be obtained if such an operation could be avoided and
the liquefaction process could be performed without addition of
free calcium ions.
[0141] To achieve that, a less calcium-dependent Termamyl-like
.alpha.-amylase which is stable and highly active at low
concentrations of free calcium (<40 ppm) is required. Such a
Termamyl-like .alpha.-amylase should have a pH optimum at a pH in
the range of 4.5-6.5, preferably in the range of 4.5-5.5.
Detergent Compositions
[0142] As mentioned above, variants of the invention may suitably
be incorporated in detergent compositions. Reference is made, for
example, to WO 96/23874 and WO 97/07202 for further details
concerning relevant ingredients of detergent compositions (such as
laundry or dishwashing detergents), appropriate methods of
formulating the variants in such detergent compositions, and for
examples of relevant types of detergent compositions.
[0143] Detergent compositions comprising a variant of the invention
may additionally comprise one or more other enzymes, such as a
lipase, cutinase, protease, cellulase, peroxidase or laccase,
and/or another .alpha.-amylase.
[0144] .alpha.-amylase variants of the invention may be
incorporated in detergents at conventionally employed
concentrations. It is at present contemplated that a variant of the
invention may be incorporated in an amount corresponding to
0.00001-1 mg (calculated as pure, active enzyme protein) of
.alpha.-amylase per liter of wash/dishwash liquor using
conventional dosing levels of detergent.
Materials and Methods
Enzymes:
[0145] LE174 hybrid alpha-amylase variant: LE174 is a hybrid
Termamyl-like alpha-amylase being identical to the Termamyl
sequence, i.e., the Bacillus licheniformis .alpha.-amylase shown in
SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of
the mature protein) has been replaced by the N-terminal 33 residues
of BAN (mature protein), i.e., the Bacillus amyloliquefaciens
alpha-amylase shown in SEQ ID NO: 5, which further have following
mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in
SEQ ID NO: 4).
Construction of pSNK101
[0146] This E. coli/Bacillus shuttle vector can be used to
introduce mutations without expression of .alpha.-amylase in E.
coli and then be modified in such way that the .alpha.-amylase is
active in Bacillus. The vector was constructed as follows: The
.alpha.-amylase gene in the pX vector (pDN1528 with the following
alterations within amyL: BAN (1-33), H156Y, A181T, N190F, A209V,
Q264S, the plasmid pDN1528 is further described in Example 1) was
inactivated by interruption in the PstI site in the 5' coding
region of the alpha-amylase gene by a 1.2 kb fragment containing an
E. coli origin fragment. This fragment was amplified from the pUC19
(GenBank Accession #:X02514) using the forward primer 1:
5'-gacctgcagtcaggcaacta-3' (SEQ ID NO: 28) and the reverse primer
1: 5'-tagagtcgacctgcaggcat-3' (SEQ ID NO: 29). The PCR amplicon and
the pX plasmid containing the .alpha.-amylase gene were digested
with PstI at 37.degree. C. for 2 hours. The pX vector fragment and
the E. coli origin amplicon were ligated at room temperature. for 1
hour and transformed in E. coli by electrotransformation. The
resulting vector is designated pSnK101.
[0147] This E. coli/Bacillus shuttle vector can be used to
introduce mutations without expression of .alpha.-amylase in E.
coli and then be modified in such way that the .alpha.-amylase is
active in Bacillus. The vector was constructed as follows: The
.alpha.-amylase gene in the pX vector (pDN1528 with the following
alterations within amyL: BAN (1-33), H156Y+A181T+N190F+A209V+Q264S,
the plasmid pDN1528 is further described in Example 1) was
inactivated by interruption in the PstI site in the 5'coding region
of the alpha-amylase gene by a 1.2 kb fragment containing an E.
coli origin fragment. This fragment was amplified from the pUC19
(GenBank Accession #:X02514) using the forward primer 2:
5'-gacctgcagtcaggcaacta-3' (SEQ ID NO: 30) and the reverse primer
2: 5'-tagagtcgacctgcaggcat-3' (SEQ ID NO: 31). The PCR amplicon and
the pX plasmid containing the .alpha.-amylase gene were digested
with PstI at 37.degree. C. for 2 hours. The pX vector fragment and
the E. coli origin amplicon were ligated at room temperature. for 1
hour and transformed in E. coli by electrotransformation. The
resulting vector is designated pSnK101.
Low pH Filter Assay
[0148] Bacillus libraries are plated on a sandwich of cellulose
acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and
nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell,
Dassel, Germany) on TY agar plates with 10 .mu.g/ml chloramphenicol
at 37.degree. C. for at least 21 hrs. The cellulose acetate layer
is located on the TY agar plate.
[0149] Each filter sandwich is specifically marked with a needle
after plating, but before incubation in order to be able to
localize positive variants on the filter and the nitrocellulose
filter with bound variants is transferred to a container with
citrate buffer, pH 4.5 and incubated at 90.degree. C. for 15 min.
The cellulose acetate filters with colonies are stored on the
TY-plates at room temperature until use. After incubation, residual
activity is detected on assay plates containing 1% agarose, 0.2%
starch in citrate buffer, pH 6.0. The assay plates with
nitrocellulose filters are marked the same way as the filter
sandwich and incubated for 2 hours. at 50.degree. C. After removal
of the filters the assay plates are stained with 10% Lugol
solution. Starch degrading variants are detected as white spots on
dark blue background and then identified on the storage plates.
Positive variants are rescreened twice under the same conditions as
the first screen.
Secondary Screening
[0150] Positive transformants after rescreening are picked from the
storage plate and tested in a secondary plate assay. Positive
transformants are grown for 22 hours at 37.degree. C. in 5 ml
LB+chloramphenicol. The Bacillus culture of each positive
transformant and a control LE174 variant were incubated in citrate
buffer, pH 4.5 at 90.degree. C. and samples were taken at 0, 10,
20, 30, 40, 60 and 80 minutes. A 3 microliter sample was spotted on
a assay plate. The assay plate was stained with 10% Lugol solution.
Improved variants were seen as variants with higher residual
activity detected as halos on the assay plate than the backbone.
The improved variants are determined by nucleotide sequencing.
Fermentation and Purification of .alpha.-Amylase Variants
[0151] A B. subtilis strain harbouring the relevant expression
plasmid is streaked on a LB-agar plate with 15 .mu.g/ml
chloramphenicol from -80.degree. C. stock, and grown overnight at
37.degree. C. The colonies are transferred to 100 ml BPX media
supplemented with 15 .mu.g/ml chloramphenicol in a 500 ml shaking
flask.
Composition of BPX Medium:
TABLE-US-00002 [0152] Potato starch 100 g/l Barley flour 50 g/l BAN
5000 SKB 0.1 g/l Sodium caseinate 10 g/l Soy Bean Meal 20 g/l
Na.sub.2HPO.sub.4, 12 H.sub.2O 9 g/l Pluronic .TM. 0.1 g/l
[0153] The culture is shaken at 37.degree. C. at 270 rpm for 5
days.
[0154] Cells and cell debris are removed from the fermentation
broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards
the supernatant is filtered to obtain a completely clear solution.
The filtrate is concentrated and washed on a UF-filter (10000 cut
off membrane) and the buffer is changed to 20 mM Acetate pH 5.5.
The UF-filtrate is applied on a S-sepharose F.F. and elution is
carried out by step elution with 0.2M NaCl in the same buffer. The
eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a
Q-sepharose F.F. and eluted with a linear gradient from 0-0.3M NaCl
over 6 column volumes. The fractions which contain the activity
(measured by the Phadebas assay) are pooled, pH was adjusted to pH
7.5 and remaining color was removed by a treatment with 0.5% W/vol.
active coal in 5 minutes.
Stability Determination
[0155] All the stability trials are made using the same set up. The
method is:
[0156] The enzyme is incubated under the relevant conditions (1-4).
Samples are taken at 0, 5, 10, 15 and 30 minutes and diluted 25
times (same dilution for all taken samples) in assay buffer (0.1M
50 mM Britton buffer pH 7.3) and the activity is measured using the
Phadebas assay (Pharmacia) under standard conditions pH 7.3,
37.degree. C.
[0157] The activity measured before incubation (0 minutes) is used
as reference (100%). The decline in percent is calculated as a
function of the incubation time. The table shows the residual
activity after 30 minutes of incubation.
Activity Determination-(KNU)
[0158] One Kilo alpha-amylase Unit (1 KNU) is the amount of enzyme
which breaks down 5.26 g starch (Merck, Amylum Solubile, Erg. B 6,
Batch 9947275) per hour in Novo Nordisk's standard method for
determination of alpha-amylase based upon the following
condition:
TABLE-US-00003 Substrate soluble starch Calcium content in solvent
0.0043 M Reaction time 7-20 minutes Temperature 37.degree. C. pH
5.6
Detailed description of Novo Nordisk's analytical method (AF 9) is
available on request.
Specific Activity Determination
Assay for .alpha.-Amylase Activity
[0159] .alpha.-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-coloured starch polymer which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0160] For every single measurement one tablet is suspended in a
tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic
acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2,
pH adjusted to the value of interest with NaOH). The test is
performed in a water bath at the temperature of interest. The
.alpha.-amylase to be tested is diluted in x ml of 50 mM
Britton-Robinson buffer. 1 ml of this .alpha.-amylase solution is
added to the 5 ml 50 mM Britton-Robinson buffer. The starch is
hydrolysed by the .alpha.-amylase giving soluble blue fragments.
The absorbance of the resulting blue solution, measured
spectrophotometrically at 620 nm, is a function of the
.alpha.-amylase activity.
[0161] It is important that the measured 620 nm absorbance after 10
or 15 minutes of incubation (testing time) is in the range of 0.2
to 2.0 absorbance units at 620 nm. In this absorbance range there
is linearity between activity and absorbance (Lambert-Beer law).
The dilution of the enzyme must therefore be adjusted to fit this
criterion. Under a specified set of conditions (temp., pH, reaction
time, buffer conditions) 1 mg of a given .alpha.-amylase will
hydrolyse a certain amount of substrate and a blue colour will be
produced. The colour intensity is measured at 620 nm. The measured
absorbance is directly proportional to the specific activity
(activity/mg of pure .alpha.-amylase protein) of the
.alpha.-amylase in question under the given set of conditions.
EXAMPLES
Example 1
Construction, by Random Mutagenesis, of Termamyl-Like LE174
.alpha.-Amylase Variants Having an Improved Stability at Low pH and
a Reduced Dependency on Calcium Ions for Stability Compared to the
Parent Enzyme.
Random Mutagenesis
[0162] To improve the stability at low pH and low calcium
concentration of the parent LE174 .alpha.-amylase variant random
mutagenesis in preselected regions was performed.
[0163] The regions were:
TABLE-US-00004 Region: Residue: SERI A425-Y438 SERII W411-L424
SERIII G397-G410 SERV T369-H382 SERVII G310-F323 SERIX
L346-P359
[0164] For each six region, random oligonucleotides are synthesized
using the same mutation rate (97% backbone and 1% of each of the
three remaining nucleotides giving 3% mutations) in each nucleotide
position in the above regions, e.g., 1. position in condon for
A425: 97% C, 1% A, 1% T, 1% G. The six random oligonucleotides and
if used complementary SOE helping primers are shown in tables 1-6:
with the four distribution of nucleotides below.
TABLE-US-00005 TABLE 1 RSERI: 5'-GC GTT TTG CCG GCC GAC ATA 312 234
322 243 333 133 444 233 423 242 212 211 243 343 CAA ACC TGA
ATT-3'(SEQ ID NO: 15)
TABLE-US-00006 TABLE 2 RSERII: 5'-GC GTT TTG CCG GCC GAC ATA CAT
TCG CTT TGC CCC ACC GGG TCC GTC TGT TAT TAA TGC CGC 311 133 241 122
243 113 341 432 423 433 223 332 242 331 GCC GAC AAT GTC ATG
GTG-3'(SEQ ID NO: 16)
TABLE-US-00007 TABLE 3 RSERIII: 5'-GTC GCC TTC CCT TGT CCA 433 413
112 423 124 424 423 411 121 123 124 324 243 233 GTA CGC ATA CTG TTT
TCT-3'(SEQ ID NO: 17) Helping primer FSERIII: 5'-TGG ACA AGG GAA
GGC GAC AG-3'(SEQ ID NO: 18)
TABLE-US-00008 TABLE 4 RSERV: 5-TAA GAT CGG TTC AAT TTT 424 222 311
443 144 112 223 434 324 441 423 233 222 342 CCC GTA CAT ATC CCC GTA
GAA-3 (SEQ ID NO: 19) Helping primer FSERV: 5-AAA ATT GAA CCG ATC
TTA-3 (SEQ ID NO: 20)
TABLE-US-00009 TABLE 5 FSERVII: 5'-TT CCA TGC TGC ATC GAC ACA GGG
AGG CGG CTA TGA TAT GAG GAA ATT GCT GAA 344 213 442 342 223 311 431
233 422 411 123 442 213 122 TGT CGA TAA CCA-3'(SEQ ID NO: 21)
Helping primer RSERVII: 5'- TGT CGA TGC AGC ATG GAA - 3'(SEQ ID NO:
22)
TABLE-US-00010 TABLE 6 FSERIX: 5'-GT CCA AAC ATG GTT TAA GCC 432
243 221 343 222 212 232 313 114 441 123 244 121 333 TCA GGT TTT CTA
CGG GGA-3'(SEQ ID NO: 23) Helping primer RSERIX: 5'-GGC TTA AAC CAT
GTT TGG AC-3'(SEQ ID NO: 24)
[0165] Distribution of nucleotides in each mutated nucleotide
position
1:97% A, 1% T, 1% C, 1% G
2:97% T, 1% A, 1% C, 1% G
3:97% C, 1% A, 1% T, 1% G
4:97% G, 1% A, 1% T, 1% C
Construction of Plasmid Libraries
[0166] Two approximately 1.4 kb fragments were PCR amplified using
the primer 1B: 5'-CGA TTG CTG ACG CTG TTA TTT GCG-3' and the random
oligonucleotide apparent from table 1, respectively the random
oligonucleotide apparent from table 2. The vector pSnK101 and the
PCR fragments were digested with EcoRV and EagI for 2 hours. The
approximately 3.6 kb vector fragment and the approximately 1.3 kb
PCR fragments was purified and ligated overnight and transformed in
to E. coli and then further transformed into a Bacillus host starin
as described below. The random oligonucleotides apparent from
Tables 3-6 (which by a common term is designated aSER and bSER in
FIG. 2) for each region and specific B. licheniformis primers 1B
(SEQ ID NO: 26) and #63: 5'-CTA TCT TTG AAC ATA AAT TGA AAC C-3'
(SEQ ID NO: 27) covering the EcoRV and the EagI sites in the LE174
sequence are used to generate PCR-library-fragments by the overlap
extension method (Horton et al., Gene, 77 (1989), pp. 61-68) FIG. 2
shows the PCR strategy. The PCR fragments are cloned in the E.
coli/Bacillus shuttle vector pSNK101 (see Materials and Methods)
enabling mutagenesis in E. coli and immediate expression in
Bacillus subtilis preventing lethal accumulation of amylases in E.
coli. After establishing the cloned PCR fragments in E. coli, a
modified pUC19 fragment is digested out of the plasmid and the
promoter and the mutated Termamyl gene is physically connected and
expression can take place in the Bacillus host
Screening
[0167] The six libraries were screened in the low pH filter assays
described in the "Material and Methods" section above.
[0168] All variants listed in the table in Example 2 below was
prepared as described in Example 1.
Example 2
Measurement of Stability
[0169] Normally, industrial liquefaction processes is run at pH
6.0-6.2 with addition of about 40 ppm free calcium in order to
improve the stability at 95.degree. C.-105.degree. C. Variants of
the invention have been made in order to improve the stability
at
1. lower pH than pH 6.2 and/or 2. at free calcium levels lower than
40 ppm free calcium.
[0170] An assay which measures the stability at acidic pH, pH 5.0,
in the presence of 5 ppm free calcium was used to measure the
increase in stability.
[0171] 10 .mu.g of the variant was incubated under the following
conditions: A 0.1 M acetate solution, pH adjusted to pH 5.0,
containing 5 ppm calcium and 5% w/w common corn starch (free of
calcium). Incubation was made in a water bath at 95.degree. C. for
30 minutes.
Results:
[0172] Increased stability at pH 5.0, 5 ppm calcium incubated at
95.degree. C.
TABLE-US-00011 LE174 WITH LE174 WITH LE174 WITH LE174 WITH K176R +
K176R + I201F + K176R + I201F + K176R + I201F + MINUTES OF I201F +
H205N + E376K + H205N + S417T + H205N + S356A + INCUBATION H205N
A420R A420Q Y358F 0 100 100 100 100 5 65 61 66 66 10 58 53 60 59 15
51 48 55 56 30 36 39 45 49
Specific Activity Determination.
[0173] The specific activity was determined using the Phadebas
assay (Pharmacia) (described above) as activity/mg enzyme. The
activity was determined using the .alpha.-amylase assay described
in the Materials and Methods section herein.
LE174 with the following substitutions:
K176R+I201F+H205N
[0174] Specific activity determined: 13400NU/mg LE174 with the
following substitutions:
K176R+I201F+H205N+E376K+ A420R:
[0175] Specific activity determined: 14770NU/mg LE174 with the
following substitutions:
K176R+I201F+H205N+S417T+A420Q:
[0176] Specific activity determined:16670NU/mg LE174 with the
following substitutions:
K176R+I201F+H205N+S356A+Y358F:
[0177] Specific activity determined:15300NU/mg
REFERENCES CITED
[0178] Klein, C., et al., Biochemistry 1992, 31, 8740-8746, [0179]
Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283, [0180]
Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238, [0181] Larson,
S. B., J. Mol. Biol. (1994) 235, 1560-1584, [0182] Lawson, C. L.,
J. Mol. Biol. (1994) 236, 590-600, [0183] Qian, M., et al., J. Mol.
Biol. (1993) 231, 785-799, [0184] Brady, R. L., et al., Acta
Crystallogr. sect. B, 47, 527-535, [0185] Swift, H. J., et al.,
Acta Crystallogr. sect. B, 47, 535-544 [0186] A. Kadziola, Ph.D.
Thesis: "An alpha-amylase from Barley and its Complex with a
Substrate Analogue Inhibitor Studied by X-ray Crystallography",
Department of Chemistry University of Copenhagen 1993 [0187]
MacGregor, E. A., Food Hydrocolloids, 1987, Vol. 1, No. 5-6, p.
[0188] B. Diderichsen and L. Christiansen, Cloning of a maltogenic
.alpha.-amylase from Bacillus stearothermophilus, FEMS Microbiol.
letters: 56: pp. 53-60 (1988) [0189] Hudson et al., Practical
Immunology, Third edition (1989), Blackwell Scientific
Publications, [0190] Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989 [0191] S. L.
Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp.
1859-1869 [0192] Matthes et al., The EMBO J. 3, 1984, pp. 801-805.
[0193] R. K. Saiki et al., Science 239, 1988, pp. 487-491. [0194]
Morinaga et al., (1984, Biotechnology 2:646-639) [0195] Nelson and
Long, Analytical Biochemistry 180, 1989, pp. 147-151 [0196]
Hunkapiller et al., 1984, Nature 310:105-111 [0197] R. Higuchi, B.
Krummel, and R. K. Saiki (1988). A general method of in vitro
preparation and specific mutagenesis of DNA fragments: study of
protein and DNA interactions. Nucl. Acids Res. 16:7351-7367. [0198]
Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221. [0199] Gryczan
et al., 1978, J. Bacteriol. 134, pp. 318-329. [0200] S. D. Erlich,
1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682. [0201] Boel et al.,
1990, Biochemistry 29, pp. 6244-6249.
Sequence CWU 1
1
311485PRTBacillus species 1His His Asn Gly Thr Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn
Arg Leu Arg Asp Asp Ala Ala 20 25 30Asn Leu Lys Ser Lys Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn Asp
Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn Gln
Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95Ile Gln Val
Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Gly
Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120
125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg
Gln Leu Gln Asn Lys 165 170 175Ile Tyr Lys Phe Arg Gly Thr Gly Lys
Ala Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val
Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr Asn Thr
Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235
240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp
Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Asn Ser Gly 290 295 300Gly Tyr Tyr Asp Met Arg Asn Ile
Leu Asn Gly Ser Val Val Gln Lys305 310 315 320His Pro Thr His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu Ala
Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350Tyr
Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr
Gly Thr385 390 395 400Gln His Asp Tyr Phe Asp His His Asp Ile Ile
Gly Trp Thr Arg Glu 405 410 415Gly Asn Ser Ser His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Asn Lys Trp
Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445Gln Val Trp Arg Asp
Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460Asn Ala Asp
Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475
480Val Trp Val Lys Gln 4852485PRTBacillus species 2His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys
Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn
Gly 85 90 95Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly
Ala Asp 100 105 110Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro
Asn Asn Arg Asn 115 120 125Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu
Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr
Ser Asp Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Val
Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175Ile Tyr Lys
Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Ser
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200
205Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val
Lys His225 230 235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr
His Val Arg Asn Ala 245 250 255Thr Gly Lys Glu Met Phe Ala Val Ala
Glu Phe Trp Lys Asn Asp Leu 260 265 270Gly Ala Leu Glu Asn Tyr Leu
Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu
His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300Gly Asn Tyr
Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys305 310 315
320His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro
Leu Ala 340 345 350Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser
Val Pro Ala Met Lys Ala 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala
Arg Gln Asn Phe Ala Tyr Gly Thr385 390 395 400Gln His Asp Tyr Phe
Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr
Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly
Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440
445Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser
Val Ser465 470 475 480Ile Trp Val Lys Arg 4853514PRTBacillus
stearothermophilus 3Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr Leu1 5 10 15Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala
Asn Glu Ala Asn Asn 20 25 30Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp
Leu Pro Pro Ala Tyr Lys 35 40 45Gly Thr Ser Arg Ser Asp Val Gly Tyr
Gly Val Tyr Asp Leu Tyr Asp 50 55 60Leu Gly Glu Phe Asn Gln Lys Gly
Ala Val Arg Thr Lys Tyr Gly Thr65 70 75 80Lys Ala Gln Tyr Leu Gln
Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95Gln Val Tyr Ala Asp
Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110Thr Glu Trp
Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125Glu
Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135
140Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
His145 150 155 160Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu
Ser Arg Ile Tyr 165 170 175Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp
Trp Glu Val Asp Thr Glu 180 185 190Asn Gly Asn Tyr Asp Tyr Leu Met
Tyr Ala Asp Leu Asp Met Asp His 195 200 205Pro Glu Val Val Thr Glu
Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn 210 215 220Thr Thr Asn Ile
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys225 230 235 240Phe
Ser Phe Phe Pro Asp Trp Leu Ser Asp Val Arg Ser Gln Thr Gly 245 250
255Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270Leu His Asn Tyr Ile Met Lys Thr Asn Gly Thr Met Ser Leu
Phe Asp 275 280 285Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys
Ser Gly Gly Thr 290 295 300Phe Asp Met Arg Thr Leu Met Thr Asn Thr
Leu Met Lys Asp Gln Pro305 310 315 320Thr Leu Ala Val Thr Phe Val
Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335Ala Leu Gln Ser Trp
Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350Phe Ile Leu
Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365Tyr
Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375
380Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
His385 390 395 400Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr
Arg Glu Gly Val 405 410 415Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Trp Met Tyr Val
Gly Lys Gln His Ala Gly Lys Val 435 440 445Phe Tyr Asp Leu Thr Gly
Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460Asp Gly Trp Gly
Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val
Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Trp Ser Ile Thr Thr 485 490
495Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510Ala Trp4483PRTBacillus licheniformis 4Ala Asn Leu Asn
Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15Asn Asp Gly
Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30Ala Glu
His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Thr
Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55
60Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65
70 75 80Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile
Asn 85 90 95Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp
Ala Thr 100 105 110Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp
Arg Asn Arg Val 115 120 125Ile Ser Gly Glu His Leu Ile Lys Ala Trp
Thr His Phe His Phe Pro 130 135 140Gly Arg Gly Ser Thr Tyr Ser Asp
Phe Lys Trp His Trp Tyr His Phe145 150 155 160Asp Gly Thr Asp Trp
Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly
Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190Tyr
Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200
205Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Arg Asp Trp Val Asn His Val Arg Glu Lys
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Asn Phe
Asn His Ser Val Phe Asp Val Pro Leu 275 280 285His Tyr Gln Phe His
Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Lys Leu
Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305 310 315
320Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro
Ala Leu Lys His Lys Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys
Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400Asp Tyr Phe Asp His
His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440
445Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser
Ile Tyr465 470 475 480Val Gln Arg5480PRTBacillus amyloliquefaciens
5Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp1 5
10 15Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser
Asp 20 25 30Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
Leu Ser 35 40 45Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp
Leu Gly Glu 50 55 60Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr Lys Ser Glu65 70 75 80Leu Gln Asp Ala Ile Gly Ser Leu His Ser
Arg Asn Val Gln Val Tyr 85 90 95Gly Asp Val Val Leu Asn His Lys Ala
Gly Ala Asp Ala Thr Glu Asp 100 105 110Val Thr Ala Val Glu Val Asn
Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125Glu Glu Tyr Gln Ile
Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140Gly Asn Thr
Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly145 150 155
160Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg
165 170 175Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn
Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp
His Pro Asp Val 195 200 205Val Ala Glu Thr Lys Lys Trp Gly Ile Trp
Tyr Ala Asn Glu Leu Ser 210 215 220Leu Asp Gly Phe Arg Ile Asp Ala
Ala Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Arg Asp Trp Val
Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val
Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270Tyr
Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280
285His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met
290 295 300Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu
Lys Ala305 310 315 320Val Thr Phe Val Glu Asn His Asp Thr Gln Pro
Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro
Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro
Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Ser
Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380Glu Pro Ile
Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His385 390 395
400Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp
Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn
Ala Gly Glu Thr 435 440 445Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp
Thr Val Lys Ile Gly Ser 450 455 460Asp Gly Trp Gly Glu Phe His Val
Asn Asp Gly Ser Val Ser Ile Tyr465 470 475 4806485PRTBacillus
species 6His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp Tyr1 5 10
15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser
20 25 30Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala
Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp
Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser
Leu Lys Asn Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn
His Lys Gly Gly Ala Asp 100 105 110Ala Thr Glu Met Val Arg Ala Val
Glu Val Asn Pro Asn Asn Arg Asn 115 120 125Gln Glu Val Thr Gly Glu
Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp 130 135 140Phe Pro Gly Arg
Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr145 150 155 160His
Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170
175Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile
Asp Met 195 200 205Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp
Gly Val Trp Tyr 210 215 220Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg
Ile Asp Ala Val Lys His225 230 235 240Ile Lys Tyr Ser Phe Thr Arg
Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255Thr Gly Lys Asn Met
Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270Gly Ala Ile
Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275 280 285Phe
Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295
300Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln
Arg305 310 315 320His Pro Ser His Ala Val Thr Phe Val Asp Asn His
Asp Ser Gln Pro 325 330 335Glu Glu Ala Leu Glu Ser Phe Val Glu Glu
Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu Thr Leu Thr Arg Glu
Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile
Pro Thr His Gly Val Pro Ala Met Arg Ser 370 375 380Lys Ile Asp Pro
Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys385 390 395 400Gln
Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410
415Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys
Ala Gly 435 440 445Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly
Thr Val Thr Ile 450 455 460Asn Ala Asp Gly Trp Gly Asn Phe Ser Val
Asn Gly Gly Ser Val Ser465 470 475 480Ile Trp Val Asn Lys
4857485PRTBacillus species 7His His Asn Gly Thr Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn
Arg Leu Arg Asp Asp Ala Ala 20 25 30Asn Leu Lys Ser Lys Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn Asp
Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn Gln
Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95Ile Gln Val
Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Gly
Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120
125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg
Gln Leu Gln Asn Lys 165 170 175Ile Tyr Lys Phe Arg Gly Thr Gly Lys
Ala Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val
Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr Asn Thr
Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235
240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp
Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Asn Ser Gly 290 295 300Gly Tyr Tyr Asp Met Arg Asn Ile
Leu Asn Gly Ser Val Val Gln Lys305 310 315 320His Pro Thr His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu Ala
Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350Tyr
Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr
Gly Thr385 390 395 400Gln His Asp Tyr Phe Asp His His Asp Ile Ile
Gly Trp Thr Arg Glu 405 410 415Gly Asn Ser Ser His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Asn Lys Trp
Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445Gln Val Trp Arg Asp
Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460Asn Ala Asp
Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475
480Val Trp Val Lys Gln 4858485PRTBacillus species 8His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys
Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn
Gly 85 90 95Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly
Ala Asp 100 105 110Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro
Asn Asn Arg Asn 115 120 125Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu
Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr
Ser Asp Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Val
Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175Ile Tyr Lys
Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Ser
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200
205Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr
210 215 220Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val
Lys His225 230 235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr
His Val Arg Asn Ala 245 250 255Thr Gly Lys Glu Met Phe Ala Val Ala
Glu Phe Trp Lys Asn Asp Leu 260 265 270Gly Ala Leu Glu Asn Tyr Leu
Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu
His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300Gly Asn Tyr
Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys305 310 315
320His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro
Leu Ala 340 345 350Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser
Val Pro Ala Met Lys Ala 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala
Arg Gln Asn Phe Ala Tyr Gly Thr385 390 395 400Gln His Asp Tyr Phe
Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr
Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly
Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440
445Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser
Val Ser465 470 475 480Ile Trp Val Lys Arg 48591455DNABacillus
species 9catcataatg gaacaaatgg tactatgatg caatatttcg aatggtattt
gccaaatgac 60gggaatcatt ggaacaggtt gagggatgac gcagctaact taaagagtaa
agggataaca 120gctgtatgga tcccacctgc atggaagggg acttcccaga
atgatgtagg ttatggagcc 180tatgatttat atgatcttgg agagtttaac
cagaagggga cggttcgtac aaaatatgga 240acacgcaacc agctacaggc
tgcggtgacc tctttaaaaa ataacggcat tcaggtatat 300ggtgatgtcg
tcatgaatca taaaggtgga gcagatggta cggaaattgt aaatgcggta
360gaagtgaatc ggagcaaccg aaaccaggaa acctcaggag agtatgcaat
agaagcgtgg 420acaaagtttg attttcctgg aagaggaaat aaccattcca
gctttaagtg gcgctggtat 480cattttgatg ggacagattg ggatcagtca
cgccagcttc aaaacaaaat atataaattc 540aggggaacag gcaaggcctg
ggactgggaa gtcgatacag agaatggcaa ctatgactat 600cttatgtatg
cagacgtgga tatggatcac ccagaagtaa tacatgaact tagaaactgg
660ggagtgtggt atacgaatac actgaacctt gatggattta gaatagatgc
agtgaaacat 720ataaaatata gctttacgag agattggctt acacatgtgc
gtaacaccac aggtaaacca 780atgtttgcag tggctgagtt ttggaaaaat
gaccttggtg caattgaaaa ctatttgaat 840aaaacaagtt ggaatcactc
ggtgtttgat gttcctctcc actataattt gtacaatgca 900tctaatagcg
gtggttatta tgatatgaga aatattttaa atggttctgt ggtgcaaaaa
960catccaacac atgccgttac ttttgttgat aaccatgatt ctcagcccgg
ggaagcattg 1020gaatcctttg ttcaacaatg gtttaaacca cttgcatatg
cattggttct gacaagggaa 1080caaggttatc cttccgtatt ttatggggat
tactacggta tcccaaccca tggtgttccg 1140gctatgaaat ctaaaataga
ccctcttctg caggcacgtc aaacttttgc ctatggtacg 1200cagcatgatt
actttgatca tcatgatatt atcggttgga caagagaggg aaatagctcc
1260catccaaatt caggccttgc caccattatg tcagatggtc caggtggtaa
caaatggatg 1320tatgtgggga aaaataaagc gggacaagtt tggagagata
ttaccggaaa taggacaggc 1380accgtcacaa ttaatgcaga cggatggggt
aatttctctg ttaatggagg gtccgtttcg 1440gtttgggtga agcaa
1455101455DNABacillus species 10catcataatg ggacaaatgg gacgatgatg
caatactttg aatggcactt gcctaatgat 60gggaatcact ggaatagatt aagagatgat
gctagtaatc taagaaatag aggtataacc 120gctatttgga ttccgcctgc
ctggaaaggg acttcgcaaa atgatgtggg gtatggagcc 180tatgatcttt
atgatttagg ggaatttaat caaaagggga cggttcgtac taagtatggg
240acacgtagtc aattggagtc tgccatccat gctttaaaga ataatggcgt
tcaagtttat 300ggggatgtag tgatgaacca taaaggagga gctgatgcta
cagaaaacgt tcttgctgtc 360gaggtgaatc caaataaccg gaatcaagaa
atatctgggg actacacaat tgaggcttgg 420actaagtttg attttccagg
gaggggtaat acatactcag actttaaatg gcgttggtat 480catttcgatg
gtgtagattg ggatcaatca cgacaattcc aaaatcgtat ctacaaattc
540cgaggtgatg gtaaggcatg ggattgggaa gtagattcgg aaaatggaaa
ttatgattat 600ttaatgtatg cagatgtaga tatggatcat ccggaggtag
taaatgagct tagaagatgg 660ggagaatggt atacaaatac attaaatctt
gatggattta ggatcgatgc ggtgaagcat 720attaaatata gctttacacg
tgattggttg acccatgtaa gaaacgcaac gggaaaagaa 780atgtttgctg
ttgctgaatt ttggaaaaat gatttaggtg ccttggagaa ctatttaaat
840aaaacaaact ggaatcattc tgtctttgat gtcccccttc attataatct
ttataacgcg 900tcaaatagtg gaggcaacta tgacatggca aaacttctta
atggaacggt tgttcaaaag 960catccaatgc atgccgtaac ttttgtggat
aatcacgatt ctcaacctgg ggaatcatta 1020gaatcatttg tacaagaatg
gtttaagcca cttgcttatg cgcttatttt aacaagagaa 1080caaggctatc
cctctgtctt ctatggtgac tactatggaa ttccaacaca tagtgtccca
1140gcaatgaaag ccaagattga tccaatctta gaggcgcgtc aaaattttgc
atatggaaca 1200caacatgatt attttgacca tcataatata atcggatgga
cacgtgaagg aaataccacg 1260catcccaatt caggacttgc gactatcatg
tcggatgggc cagggggaga gaaatggatg 1320tacgtagggc aaaataaagc
aggtcaagtt tggcatgaca taactggaaa taaaccagga 1380acagttacga
tcaatgcaga tggatgggct aatttttcag taaatggagg atctgtttcc
1440atttgggtga aacga 1455111548DNABacillus stearothermophilus
11gccgcaccgt ttaacggcac catgatgcag tattttgaat ggtacttgcc ggatgatggc
60acgttatgga ccaaagtggc caatgaagcc aacaacttat ccagccttgg catcaccgct
120ctttggctgc cgcccgctta caaaggaaca agccgcagcg acgtagggta
cggagtatac 180gacttgtatg acctcggcga attcaatcaa aaagggaccg
tccgcacaaa atacggaaca 240aaagctcaat atcttcaagc cattcaagcc
gcccacgccg ctggaatgca agtgtacgcc 300gatgtcgtgt tcgaccataa
aggcggcgct gacggcacgg aatgggtgga cgccgtcgaa 360gtcaatccgt
ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg
420aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg
ctggtaccat 480tttgacggcg ttgattggga cgaaagccga aaattgagcc
gcatttacaa attccgcggc 540atcggcaaag cgtgggattg ggaagtagac
acggaaaacg gaaactatga ctacttaatg 600tatgccgacc ttgatatgga
tcatcccgaa gtcgtgaccg agctgaaaaa ctgggggaaa 660tggtatgtca
acacaacgaa cattgatggg ttccggcttg atgccgtcaa gcatattaag
720ttcagttttt ttcctgattg gttgtcgtat gtgcgttctc agactggcaa
gccgctattt 780accgtcgggg aatattggag ctatgacatc aacaagttgc
acaattacat tacgaaaaca 840gacggaacga tgtctttgtt tgatgccccg
ttacacaaca aattttatac cgcttccaaa 900tcagggggcg catttgatat
gcgcacgtta atgaccaata ctctcatgaa agatcaaccg 960acattggccg
tcaccttcgt tgataatcat gacaccgaac ccggccaagc gctgcagtca
1020tgggtcgacc catggttcaa accgttggct tacgccttta ttctaactcg
gcaggaagga 1080tacccgtgcg tcttttatgg tgactattat ggcattccac
aatataacat tccttcgctg 1140aaaagcaaaa tcgatccgct cctcatcgcg
cgcagggatt atgcttacgg aacgcaacat 1200gattatcttg atcactccga
catcatcggg tggacaaggg aagggggcac tgaaaaacca 1260ggatccggac
tggccgcact gatcaccgat gggccgggag gaagcaaatg gatgtacgtt
1320ggcaaacaac acgctggaaa agtgttctat gaccttaccg gcaaccggag
tgacaccgtc 1380accatcaaca gtgatggatg gggggaattc aaagtcaatg
gcggttcggt ttcggtttgg 1440gttcctagaa aaacgaccgt ttctaccatc
gctcggccga tcacaacccg accgtggact 1500ggtgaattcg tccgttggac
cgaaccacgg ttggtggcat ggccttga 1548121920DNABacillus
licheniformismisc_feature(421)..(1872)CDS 12cggaagattg gaagtacaaa
aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60gagacggaaa aatcgtctta
atgcacgata tttatgcaac gttcgcagat gctgctgaag 120agattattaa
aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag
180aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc
ggcgcttttc 240ttttggaaga aaatataggg aaaatggtac ttgttaaaaa
ttcggaatat ttatacaaca 300tcatatgttt cacattgaaa ggggaggaga
atcatgaaac aacaaaaacg gctttacgcc 360cgattgctga cgctgttatt
tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420gcaaatctta
atgggacgct gatgcagtat tttgaatggt acatgcccaa tgacggccaa
480cattggaggc gtttgcaaaa cgactcggca tatttggctg aacacggtat
tactgccgtc 540tggattcccc cggcatataa gggaacgagc caagcggatg
tgggctacgg tgcttacgac 600ctttatgatt taggggagtt tcatcaaaaa
gggacggttc ggacaaagta cggcacaaaa 660ggagagctgc aatctgcgat
caaaagtctt cattcccgcg acattaacgt ttacggggat 720gtggtcatca
accacaaagg cggcgctgat gcgaccgaag atgtaaccgc ggttgaagtc
780gatcccgctg accgcaaccg cgtaatttca ggagaacacc taattaaagc
ctggacacat 840tttcattttc cggggcgcgg cagcacatac agcgatttta
aatggcattg gtaccatttt 900gacggaaccg attgggacga gtcccgaaag
ctgaaccgca tctataagtt tcaaggaaag 960gcttgggatt gggaagtttc
caatgaaaac ggcaactatg attatttgat gtatgccgac 1020atcgattatg
accatcctga tgtcgcagca gaaattaaga gatggggcac ttggtatgcc
1080aatgaactgc aattggacgg tttccgtctt gatgctgtca aacacattaa
attttctttt 1140ttgcgggatt gggttaatca tgtcagggaa aaaacgggga
aggaaatgtt tacggtagct 1200gaatattggc agaatgactt gggcgcgctg
gaaaactatt tgaacaaaac aaattttaat 1260cattcagtgt ttgacgtgcc
gcttcattat cagttccatg ctgcatcgac acagggaggc 1320ggctatgata
tgaggaaatt gctgaacggt acggtcgttt ccaagcatcc gttgaaatcg
1380gttacatttg tcgataacca tgatacacag ccggggcaat cgcttgagtc
gactgtccaa 1440acatggttta agccgcttgc ttacgctttt attctcacaa
gggaatctgg ataccctcag 1500gttttctacg gggatatgta cgggacgaaa
ggagactccc agcgcgaaat tcctgccttg
1560aaacacaaaa ttgaaccgat cttaaaagcg agaaaacagt atgcgtacgg
agcacagcat 1620gattatttcg accaccatga cattgtcggc tggacaaggg
aaggcgacag ctcggttgca 1680aattcaggtt tggcggcatt aataacagac
ggacccggtg gggcaaagcg aatgtatgtc 1740ggccggcaaa acgccggtga
gacatggcat gacattaccg gaaaccgttc ggagccggtt 1800gtcatcaatt
cggaaggctg gggagagttt cacgtaaacg gcgggtcggt ttcaatttat
1860gttcaaagat agaagagcag agaggacgga tttcctgaag gaaatccgtt
tttttatttt 1920131455DNABacillus species 13catcataatg gaacaaatgg
tactatgatg caatatttcg aatggtattt gccaaatgac 60gggaatcatt ggaacaggtt
gagggatgac gcagctaact taaagagtaa agggataaca 120gctgtatgga
tcccacctgc atggaagggg acttcccaga atgatgtagg ttatggagcc
180tatgatttat atgatcttgg agagtttaac cagaagggga cggttcgtac
aaaatatgga 240acacgcaacc agctacaggc tgcggtgacc tctttaaaaa
ataacggcat tcaggtatat 300ggtgatgtcg tcatgaatca taaaggtgga
gcagatggta cggaaattgt aaatgcggta 360gaagtgaatc ggagcaaccg
aaaccaggaa acctcaggag agtatgcaat agaagcgtgg 420acaaagtttg
attttcctgg aagaggaaat aaccattcca gctttaagtg gcgctggtat
480cattttgatg ggacagattg ggatcagtca cgccagcttc aaaacaaaat
atataaattc 540aggggaacag gcaaggcctg ggactgggaa gtcgatacag
agaatggcaa ctatgactat 600cttatgtatg cagacgtgga tatggatcac
ccagaagtaa tacatgaact tagaaactgg 660ggagtgtggt atacgaatac
actgaacctt gatggattta gaatagatgc agtgaaacat 720ataaaatata
gctttacgag agattggctt acacatgtgc gtaacaccac aggtaaacca
780atgtttgcag tggctgagtt ttggaaaaat gaccttggtg caattgaaaa
ctatttgaat 840aaaacaagtt ggaatcactc ggtgtttgat gttcctctcc
actataattt gtacaatgca 900tctaatagcg gtggttatta tgatatgaga
aatattttaa atggttctgt ggtgcaaaaa 960catccaacac atgccgttac
ttttgttgat aaccatgatt ctcagcccgg ggaagcattg 1020gaatcctttg
ttcaacaatg gtttaaacca cttgcatatg cattggttct gacaagggaa
1080caaggttatc cttccgtatt ttatggggat tactacggta tcccaaccca
tggtgttccg 1140gctatgaaat ctaaaataga ccctcttctg caggcacgtc
aaacttttgc ctatggtacg 1200cagcatgatt actttgatca tcatgatatt
atcggttgga caagagaggg aaatagctcc 1260catccaaatt caggccttgc
caccattatg tcagatggtc caggtggtaa caaatggatg 1320tatgtgggga
aaaataaagc gggacaagtt tggagagata ttaccggaaa taggacaggc
1380accgtcacaa ttaatgcaga cggatggggt aatttctctg ttaatggagg
gtccgtttcg 1440gtttgggtga agcaa 1455141455DNABacillus species
14catcataatg ggacaaatgg gacgatgatg caatactttg aatggcactt gcctaatgat
60gggaatcact ggaatagatt aagagatgat gctagtaatc taagaaatag aggtataacc
120gctatttgga ttccgcctgc ctggaaaggg acttcgcaaa atgatgtggg
gtatggagcc 180tatgatcttt atgatttagg ggaatttaat caaaagggga
cggttcgtac taagtatggg 240acacgtagtc aattggagtc tgccatccat
gctttaaaga ataatggcgt tcaagtttat 300ggggatgtag tgatgaacca
taaaggagga gctgatgcta cagaaaacgt tcttgctgtc 360gaggtgaatc
caaataaccg gaatcaagaa atatctgggg actacacaat tgaggcttgg
420actaagtttg attttccagg gaggggtaat acatactcag actttaaatg
gcgttggtat 480catttcgatg gtgtagattg ggatcaatca cgacaattcc
aaaatcgtat ctacaaattc 540cgaggtgatg gtaaggcatg ggattgggaa
gtagattcgg aaaatggaaa ttatgattat 600ttaatgtatg cagatgtaga
tatggatcat ccggaggtag taaatgagct tagaagatgg 660ggagaatggt
atacaaatac attaaatctt gatggattta ggatcgatgc ggtgaagcat
720attaaatata gctttacacg tgattggttg acccatgtaa gaaacgcaac
gggaaaagaa 780atgtttgctg ttgctgaatt ttggaaaaat gatttaggtg
ccttggagaa ctatttaaat 840aaaacaaact ggaatcattc tgtctttgat
gtcccccttc attataatct ttataacgcg 900tcaaatagtg gaggcaacta
tgacatggca aaacttctta atggaacggt tgttcaaaag 960catccaatgc
atgccgtaac ttttgtggat aatcacgatt ctcaacctgg ggaatcatta
1020gaatcatttg tacaagaatg gtttaagcca cttgcttatg cgcttatttt
aacaagagaa 1080caaggctatc cctctgtctt ctatggtgac tactatggaa
ttccaacaca tagtgtccca 1140gcaatgaaag ccaagattga tccaatctta
gaggcgcgtc aaaattttgc atatggaaca 1200caacatgatt attttgacca
tcataatata atcggatgga cacgtgaagg aaataccacg 1260catcccaatt
caggacttgc gactatcatg tcggatgggc cagggggaga gaaatggatg
1320tacgtagggc aaaataaagc aggtcaagtt tggcatgaca taactggaaa
taaaccagga 1380acagttacga tcaatgcaga tggatgggct aatttttcag
taaatggagg atctgtttcc 1440atttgggtga aacga 14551574DNAArtificial
sequencePrimer RSERI 15gcgttttgcc ggccgacata nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 60nncaaacctg aatt 7416122DNAArtificial
sequencePrimer RSERII 16gcgttttgcc ggccgacata cattcgcttt gccccaccgg
gtccgtctgt tattaatgcc 60gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnngccgac aatgtcatgg 120tg 1221778DNAArtificial sequencePrimer
RSERIII 17gtcgccttcc cttgtccann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60gtacgcatac tgttttct 781820DNAArtificial sequencePrimer
FSERIII 18tggacaaggg aaggcgacag 201981DNAArtificial sequencePrimer
RSERV 19taagatcggt tcaattttnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60cccgtacata tccccgtaga a 812018DNAArtificial
sequencePrimer FSERV 20aaaattgaac cgatctta 1821107DNAArtificial
sequencePrimer FSERVII 21ttccatgctg catcgacaca gggaggcggc
tatgatatga ggaaattgct gaannnnnnn 60nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnntgtcg ataacca 1072218DNAArtificial sequencePrimer RSERVII
22tgtcgatgca gcatggaa 182380DNAArtificial sequencePrimer FSERIX
23gtccaaacat ggtttaagcc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
60nntcaggttt tctacgggga 802420DNAArtificial sequencePrimer RSERIX
24ggcttaaacc atgtttggac 202524DNAArtificial sequencePrimer 1B
25cgattgctga cgctgttatt tgcg 242625DNAArtificial sequencePrimer #63
26ctatctttga acataaattg aaacc 252720DNAArtificial sequenceForward
Primer1 27gacctgcagt caggcaacta 202820DNAArtificial sequenceReverse
Primer1 28tagagtcgac ctgcaggcat 202920DNAArtificial sequenceForward
Primer2 29gacctgcagt caggcaacta 203020DNAArtificial sequenceReverse
Primer2 30tagagtcgac ctgcaggcat 20312084DNABacillus
amyloliquefaciensmisc_feature(343)..(1794)CDS 31gccccgcaca
tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60ctgaagaagt
ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc
120atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat
aaggaataaa 180ggggggttgt tattatttta ctgatatgta aaatataatt
tgtataagaa aatgagaggg 240agaggaaaca tgattcaaaa acgaaagcgg
acagtttcgt tcagacttgt gcttatgtgc 300acgctgttat ttgtcagttt
gccgattaca aaaacatcag ccgtaaatgg cacgctgatg 360cagtattttg
aatggtatac gccgaacgac ggccagcatt ggaaacgatt gcagaatgat
420gcggaacatt tatcggatat cggaatcact gccgtctgga ttcctcccgc
atacaaagga 480ttgagccaat ccgataacgg atacggacct tatgatttgt
atgatttagg agaattccag 540caaaaaggga cggtcagaac gaaatacggc
acaaaatcag agcttcaaga tgcgatcggc 600tcactgcatt cccggaacgt
ccaagtatac ggagatgtgg ttttgaatca taaggctggt 660gctgatgcaa
cagaagatgt aactgccgtc gaagtcaatc cggccaatag aaatcaggaa
720acttcggagg aatatcaaat caaagcgtgg acggattttc gttttccggg
ccgtggaaac 780acgtacagtg attttaaatg gcattggtat catttcgacg
gagcggactg ggatgaatcc 840cggaagatca gccgcatctt taagtttcgt
ggggaaggaa aagcgtggga ttgggaagta 900tcaagtgaaa acggcaacta
tgactattta atgtatgctg atgttgacta cgaccaccct 960gatgtcgtgg
cagagacaaa aaaatggggt atctggtatg cgaatgaact gtcattagac
1020ggcttccgta ttgatgccgc caaacatatt aaattttcat ttctgcgtga
ttgggttcag 1080gcggtcagac aggcgacggg aaaagaaatg tttacggttg
cggagtattg gcagaataat 1140gccgggaaac tcgaaaacta cttgaataaa
acaagcttta atcaatccgt gtttgatgtt 1200ccgcttcatt tcaatttaca
ggcggcttcc tcacaaggag gcggatatga tatgaggcgt 1260ttgctggacg
gtaccgttgt gtccaggcat ccggaaaagg cggttacatt tgttgaaaat
1320catgacacac agccgggaca gtcattggaa tcgacagtcc aaacttggtt
taaaccgctt 1380gcatacgcct ttattttgac aagagaatcc ggttatcctc
aggtgttcta tggggatatg 1440tacgggacaa aagggacatc gccaaaggaa
attccctcac tgaaagataa tatagagccg 1500attttaaaag cgcgtaagga
gtacgcatac gggccccagc acgattatat tgaccacccg 1560gatgtgatcg
gatggacgag ggaaggtgac agctccgccg ccaaatcagg tttggccgct
1620ttaatcacgg acggacccgg cggatcaaag cggatgtatg ccggcctgaa
aaatgccggc 1680gagacatggt atgacataac gggcaaccgt tcagatactg
taaaaatcgg atctgacggc 1740tggggagagt ttcatgtaaa cgatgggtcc
gtctccattt atgttcagaa ataaggtaat 1800aaaaaaacac ctccaagctg
agtgcgggta tcagcttgga ggtgcgttta ttttttcagc 1860cgtatgacaa
ggtcggcatc aggtgtgaca aatacggtat gctggctgtc ataggtgaca
1920aatccgggtt ttgcgccgtt tggctttttc acatgtctga tttttgtata
atcaacaggc 1980acggagccgg aatctttcgc cttggaaaaa taagcggcga
tcgtagctgc ttccaatatg 2040gattgttcat cgggatcgct gcttttaatc
acaacgtggg atcc 2084
* * * * *
References