U.S. patent application number 14/601871 was filed with the patent office on 2015-05-14 for alpha-amylase variants.
The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Carsten Andersen, Henrik Bisgaard-Frantzen, Soren Kjaerulff, Allan Svendsen.
Application Number | 20150132823 14/601871 |
Document ID | / |
Family ID | 34519637 |
Filed Date | 2015-05-14 |
United States Patent
Application |
20150132823 |
Kind Code |
A1 |
Svendsen; Allan ; et
al. |
May 14, 2015 |
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; (Bagsvaerd,
DK) ; Kjaerulff; Soren; (Bagsvaerd, DK) ;
Bisgaard-Frantzen; Henrik; (Bagsvaerd, DK) ;
Andersen; Carsten; (Bagsvaerd, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
BAGSVAERD |
|
DK |
|
|
Family ID: |
34519637 |
Appl. No.: |
14/601871 |
Filed: |
January 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13898146 |
May 20, 2013 |
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14601871 |
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13478512 |
May 23, 2012 |
8465957 |
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13898146 |
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13038991 |
Mar 2, 2011 |
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13478512 |
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12417453 |
Apr 2, 2009 |
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13038991 |
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11039565 |
Jan 19, 2005 |
7601527 |
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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 ;
435/252.31; 435/264; 435/320.1; 510/392 |
Current CPC
Class: |
C07H 21/04 20130101;
C12N 9/2417 20130101; C12N 9/2414 20130101; C11D 3/38609 20130101;
C11D 3/38618 20130101 |
Class at
Publication: |
435/202 ;
435/320.1; 435/252.31; 510/392; 435/264 |
International
Class: |
C12N 9/26 20060101
C12N009/26; C11D 3/386 20060101 C11D003/386 |
Claims
1. 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 hydrophobicity 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.
2. The variant according to claim 1, wherein one or more solvent
exposed amino acid residues on a concave surface with inwards bend
are altered to more hydrophobic amino acid residues.
3. The variant according to claim 1, wherein one or more solvent
exposed amino acid residues on a convex surface are altered to
increase the number of methyl groups in the sidechain.
4. A 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; wherein (a) the alteration(s) are
independently (i) an insertion of an amino acid downstream of the
amino acid which occupies the position, (ii) a deletion of the
amino acid which occupies the position, or (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.
5. The variant according to claim 1, wherein the parent
Termamyl-like .alpha.-amylase is any of the .alpha.-amylases
selected from the group depicted in SEQ ID NOS: 1, 2, 3, 4, 5, 6,
7, and 8.
6. The variant according to claim 1, wherein the parent
Termamyl-like .alpha.-amylase has an amino acid sequence which has
a degree of identity to SEQ ID NO: 4 of at least 65%, preferably
70%, more preferably at least 80%, 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 97%.
7. The variant according to claim 1, wherein the parent
.alpha.-amylase further has a mutation in one or more of the
following positions: K176, I201 and H205 (using the numbering in
SEQ ID NO: 4).
8. The variant according to claim 1, wherein the variant has
increased stability at pHs below 7.0 (acidic pH) and/or at low
calcium concentration and/or at temperatures in the range from 95
to 160.degree. C. (high temperatures) relative to the parent
.alpha.-amylase.
9. The variant according to claim 1, which variant has one or more
of the following substitutions: E376K, S417T, A420Q, R, S356A,
Y358F.
10. A DNA construct comprising a DNA sequence encoding an
.alpha.-amylase variant according to claim 1.
11. A recombinant expression vector which carries a DNA construct
according to claim 10.
12. A cell which is transformed with a DNA construct according to
claim 10.
13. A cell according to claim 12, which is a microorganism.
14. A cell according to claim 13, which is a bacterium or a
fungus.
15. The cell according to claim 14, which is a grampositive
bacterium such as Bacillus subtilis, Bacillus licheniformis,
Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus,
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans, Bacillus lautus or Bacillus
thuringiensis.
16. A detergent additive comprising an .alpha.-amylase variant
according to claim 1, optionally in the form of a non-dusting
granulate, stabilised liquid or protected enzyme.
17. A detergent additive according to claim 16 which contains
0.02-200 mg of enzyme protein/g of the additive.
18. A detergent additive according to claim 16, which additionally
comprises another enzyme such as a protease, a lipase, a
peroxidase, another amylolytic enzyme and/or a cellulase.
19. A detergent composition comprising an .alpha.-amylase variant
according to claim 1.
20. A detergent composition according to claim 19 which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
21. A manual or automatic dishwashing detergent composition
comprising an .alpha.-amylase variant according to claim 1.
22. A manual or automatic laundry washing composition comprising an
.alpha.-amylase variant according to claim 1.
23. A composition comprising: (i) a mixture of the .alpha.-amylase
from B. licheniformis having the sequence shown in SEQ ID NO: 4
with one or more variants according to claim 1 derived from (as the
parent Termamyl-like .alpha.-amylase) the B. stearothermophilus
.alpha.-amylase having the sequence shown in SEQ ID NO: 3; or (ii)
a mixture of the .alpha.-amylase from B. stearothermophilus having
the sequence shown in SEQ ID NO: 3 with one or more variants
according to claim 1 derived from one or more other parent
Termamyl-like .alpha.-amylases; or (iii) a mixture of one or more
variants according of claim 1 derived from (as the parent
Termamyl-like .alpha.-amylase) the B. stearothermophilus
.alpha.-amylase having the sequence shown in SEQ ID NO: 3 with one
or more variants according to the invention derived from one or
more other parent Termamyl-like .alpha.-amylases.
24. A method of using an .alpha.-amylase variant according to claim
1 or a composition according to claim 23 for washing and/or
dishwashing.
25. A method for generating a variant of a parent Termamyl-like
.alpha.-amylase, which variant exhibits increased stability at high
temperatures relative to the parent, the method comprising: (a)
subjecting a DNA sequence encoding the parent Termamyl-like
.alpha.-amylase to random mutagenesis, (b) expressing the mutated
DNA sequence obtained in step (a) in a host cell, and (c) screening
for host cells expressing a mutated .alpha.-amylase which has
increased stability at high temperatures relative to the parent
Termamyl-like .alpha.-amylase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/898,146 filed on May 20, 2013, which is a divisional of U.S.
application Ser. No. 13/478,512 filed on May 23, 2012, now U.S.
Pat. No. 8,465,957, which is a divisional of U.S. application Ser.
No. 13/038,991 filed on Mar. 2, 2011, now abandoned, which is a
divisional of U.S. application Ser. No. 12/417,453 filed on Apr. 2,
2009, now abandoned, which is a divisional of U.S. application Ser.
No. 11/039,565 filed Jan. 19, 2005, now U.S. Pat. No. 7,601,527,
which is a continuation of U.S. application Ser. No. 09/441,313,
filed Nov. 16, 1999, now U.S. Pat. No. 6,887,986, which is a
continuation-in-part of U.S. application Ser. No. 09/193,068 filed
on Nov. 16, 1998, now U.S. Pat. No. 6,197,565, 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 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. 252730 and 063909.
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 immmobilized 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: [0020] Original
amino acid(s):position(s):substituted amino acid(s)
[0021] According to this nomenclature, for instance the
substitution of alanine for asparagine in position 30 is shown as:
[0022] Asn30Ala or N30A a deletion of alanine in the same position
is shown as: [0023] Ala30* or A30* and insertion of an additional
amino acid residue, such as lysine, is shown as: [0024] Ala30AlaLys
or A30AK
[0025] A deletion of a consecutive stretch of amino acid residues,
such as amino acid residues 30-33, is indicated as (30-33)* or
A(A30-N33).
[0026] 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: [0027] *36Asp or *36D for
insertion of an aspartic acid in position 36
[0028] Multiple mutations are separated by plus signs, i.e.: [0029]
Ala30Asp+Glu34Ser or A30D+E34S representing mutations in positions
30 and 34 substituting aspartic acid and serine for alanine and
glutamic acid, respectively. Multiple mutations may also be
separated as follows, i.e., meaning the same as the plus sign:
[0030] Ala30Asp/Glu34Ser or A30D/E34S
[0031] When one or more alternative amino acid residues may be
inserted in a given position it is indicated as
[0032] A30N, E or
[0033] A30N or A30E
[0034] 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.
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
[0035] 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,
[0036] 2: amylase (SEQ ID NO: 32)
3: SEQ ID NO: 1,
4: SEQ ID NO: 5,
5: SEQ ID NO: 4,
6: SEQ ID NO: 3.
[0037] FIG. 2 shows the PCR strategy used in Example 1.
DETAILED DISCLOSURE OF THE INVENTION
The Termamyl-Like Alpha-Amylase
[0038] 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., 1988, Biochemical and Biophysical Research Communications,
151: 25-31.
[0039] 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).
[0040] 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".
[0041] 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.
[0042] 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).
[0043] 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
[0044] 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: 149-155) and reverse threading (Huber and Torda,
1998, Protein Science 7(1): 142-149.
[0045] 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 (SEQ ID NO: 2) -- Y363 -- S419 -- Bac. WO 95/26397 (SEQ ID
NO: 1) -- Y363 -- -- --
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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
[0051] 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.
[0052] 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.
[0053] 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-amylase referred to herein.
[0054] 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.
[0055] 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.
[0056] 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..
[0057] 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.
[0058] 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
[0059] 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
[0060] 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 hydrophobicity 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.
[0061] In a preferred embodiment one or more solvent exposed amino
acid residues on a concave surface with inwards bend are altered to
more hydrophobic amino acid residues.
[0062] 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.
[0063] 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;
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;
[0070] In a preferred embodiment the substitution is S417T.
[0071] 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;
[0072] In a preferred embodiment the substitution is: A420Q, R.
[0073] 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.
[0074] 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.
[0075] In a preferred embodiment the substitution is Y358F.
[0076] In an embodiment of the invention a variant comprises one or
more of the following substitutions: E376K, S417T, A420Q, R, S356A,
Y358F.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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".
[0081] 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).
[0082] 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.
[0083] 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 concave 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.
[0084] Using the program CAST found on the internet at
sunrise.cbs.umn.edu/cast/version 1.0 (release February 1998)
(reference: Liang et al., 1998, Anatomy of protein Pockets and
Cavities: Measurements of binding site geometry and implications
for ligand design. Protein Science 7: 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):
[0085] Three types of interaction can be rationalized:
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.
[0086] 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.
[0087] Corresponding and other solvent exposed positions on the
surface of other Termamyl-like alpha-amylase may be identified
using the dssp program by Kabsch and Sander, 1983, Biopolymers 22:
2577-2637. The convex surfaces can be identified using the the
AACAVI program part from the WHATIF package (Vriend, 1990, Whatif
and drug design program. J. Mol. Graph. 8: 52-56. version
19980317).
[0088] In an embodiment of the invention a variant comprises one or
more of the following substitutions: E376K, S417T, A420Q, R, S356A,
Y358F.
[0089] 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.
[0090] 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;
[0091] 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
[0092] 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
[0093] 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.
[0094] 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.
[0095] 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.
[0096] It will be understood that the present invention encompasses
variants incorporating two or more of the above outlined
modifications.
[0097] 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
[0098] 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.
[0099] 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.
[0100] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g., the
phosphoroamidite method described by Beaucage and 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.
[0101] 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 Saiki et al.
(1988).
Site-Directed Mutagenesis
[0102] 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.
[0103] 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
[0104] 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.
[0105] 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.
[0106] 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:
[0107] (a) subjecting a DNA sequence encoding the parent
alpha-amylase to random mutagenesis,
[0108] (b) expressing the mutated DNA sequence obtained in step (a)
in a host cell, and
[0109] (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.
[0110] Step (a) of the above method of the invention is preferably
performed using doped primers.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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
mis-incorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol. 1, 1989, pp. 11-15).
[0116] A mutator strain of E. coli (Fowler et al., 1974, Molec.
Gen. Genet. 133: 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.
[0117] 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.
[0118] 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.
[0119] 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, Streptomyces
lividans or Streptomyces murinus; and gram-negative bacteria such
as E. coli.
[0120] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
Localized Random Mutagenesis
[0121] 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.
[0122] 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
[0123] 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 NS).
Expression of Alpha-Amylase Variants of the Invention
[0124] 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.
[0125] 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.
[0126] 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 xyIA
and xyIB 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.
[0127] The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes,
polyadenylation 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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).
[0132] 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.
[0133] 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.
[0134] Examples of suitable bacteria are 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, or Streptomyces lividans or Streptomyces murinus, or
gram-negative 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.
[0135] 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 238023.
[0136] 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.
[0137] 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).
[0138] 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
[0139] The alpha-amylase variants of this invention possess
valuable properties allowing for a variety of industrial
applications. An enzyme variant of the invention is 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:
[0140] 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).
[0141] 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.
[0142] 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 immmobilized glucose isomerase
(such as Sweetzyme.TM.).
[0143] At least 1 enzymatic improvement 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 glucose isomerase 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.
[0144] 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
[0145] 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.
[0146] 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.
[0147] 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:
[0148] 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
[0149] 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 Pstl 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
Pstl 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.
[0150] 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 Pstl 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
Pstl 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
[0151] 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 micrograms/ml
chloramphenicol at 37.degree. C. for at least 21 hours. The
cellulose acetate layer is located on the TY agar plate.
[0152] 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
[0153] 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
[0154] 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.
[0155] The colonies are transferred to 100 ml BPX media
supplemented with 15 micrograms/ml chloramphenicol in a 500 ml
shaking flask.
Composition of BPX Medium:
TABLE-US-00002 [0156] 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
[0157] The culture is shaken at 37.degree. C. at 270 rpm for 5
days.
[0158] 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 an S-sepharose F.F. and elution is
carried out by step elution with 0.2 M 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
[0159] All the stability trials are made using the same set up. The
method is:
[0160] 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 (0.1 M
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.
[0161] 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)
[0162] One Kilo alpah-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
0.0043M Reaction time 7-20 minutes Temperature 37.degree. C. pH
5.6
[0163] Detailed description of Novo Nordisk's analytical method (AF
9) is available on request.
Specific Activity Determination
Assay for Alpha-Amylase Activity
[0164] 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
tableted.
[0165] 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.
[0166] 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
[0167] 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.
[0168] 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
[0169] 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)
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
[0170] 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 Eagl 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 Eagl sites in the LE174
sequence are used to generate PCR-library-fragments by the overlap
extension method (Horton et al., 1989, Gene 77: 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
[0171] The six libraries were screened in the low pH filter assays
described in the "Material and Methods" section above.
[0172] All variants listed in the table in Example 2 below was
prepared as described in Example 1.
Example 2
Measurement of Stability
[0173] 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.
[0174] 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.
[0175] 10 micrograms 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:
[0176] 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 +
I201F + WITH K176R + K176R + I201F + Minutes of K176R + I201F +
H205N + E376K + I201F + H205N + H205N + S356A + Incubation H205N
A420R S417T + 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.
[0177] 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 Specific
activity determined: 13,400 NU/mg LE174 with the following
substitutions: K176R+I201F+H205N+E376K+A420R: Specific activity
determined: 14,770 NU/mg LE174 with the following substitutions:
K176R+I201F+H205N+S417T+A420Q: Specific activity determined: 16,670
NU/mg LE174 with the following substitutions:
K176R+I201F+H205N+S356A+Y358F: Specific activity determined: 15,300
NU/mg
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527-535 (1991), [0185] Swift et al., Acta Crystallogr. sect. B,
Vol. 47, pp. 535-544 (1988) [0186] 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, Food
Hydrocolloids, Vol. 1, No. 5-6, pp. 407-411 (1987) [0188]
Diderichsen and Christiansen, Cloning of a maltogenic alpha-amylase
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6244-6249 (1990).
Sequence CWU 1
1
321485PRTBacillus 1His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg
Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100
105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg
Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr
Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe
Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp
Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly
Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly
Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His
Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220
Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225
230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg
Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp
Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr
Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn
Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp Met Arg
Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Thr
His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly
Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345
350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met
Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe
Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp
Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn
Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 Gln Val Trp
Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn
Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470
475 480 Val Trp Val Lys Gln 485 2485PRTBacillus 2His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro
Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35
40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu
Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala
Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp Val Val Met
Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala
Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly
Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly
Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165
170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val
Asp 180 185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp
Val Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg
Trp Gly Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe
Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr
Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu
Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala
Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285
Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290
295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln
Lys 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His
Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu
Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu
Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile
Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro
Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln
His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410
415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys
Ala Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly
Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val
Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485
3514PRTBacillus stearothermophilus 3 Ala Ala Pro Phe Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr
Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser
Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly
Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55
60 Leu Gly Glu Phe Asn Gln Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr
65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala
Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly
Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly
Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys
Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185
190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205 Pro Glu Val Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr
Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser
Asp Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly
Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile
Met Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu
His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Thr 290 295 300 Phe
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310
315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly
Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu
Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile
Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp
His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu
Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435
440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val
Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile
Ala Trp Ser Ile Thr Thr 485 490 495 Arg Pro Trp Thr Asp Glu Phe Val
Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp 4483PRTBacillus
licheniformis 4Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp
Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn
Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp
Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu
Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val
Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105
110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His
Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His
Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp
Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met
Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu
Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230
235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu
Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala
Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val
Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr
Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr
Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355
360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys
Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly
Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly
Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu
Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met
Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile
Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475
480 Val Gln Arg 5480PRTBacillus amyloliquefaciens 5Val Asn Gly Thr
Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln
His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35
40 45 Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly
Glu 50 55 60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
Lys Ser Glu 65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg
Asn Val Gln Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala
Gly Ala Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn
Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile
Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr
Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165
170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly
Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His
Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr
Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala
Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln
Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala
Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu
Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290
295 300 Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys
Ala 305 310 315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln
Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys
Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr
Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr
Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro
Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395
400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp
Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn
Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp
Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val
Asn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 6485PRTBacillus
6His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1
5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala
Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro
Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Gln Ala
Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly
Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu
Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln
Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp 130 135
140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu
Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp
Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu
Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn
Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly
Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys
Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255
Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260
265 270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser
Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser
Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly
Thr Val Val Gln Arg 305 310 315 320 His Pro Ser His Ala Val Thr Phe
Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser
Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr
Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp
Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser 370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys 385
390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr
Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr
Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Ser Lys Trp Met Phe Val
Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Ser Asp Ile Thr Gly
Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly
Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val
Asn Lys 485 7485PRTBacillus 7His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His
Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys
Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr
Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70
75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn
Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly
Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn
Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile
Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn
His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly
Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr
Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195
200 205 Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp
Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala
Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu
Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val
Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr
Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro
Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr
Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315
320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro
Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly
Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala
Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe
Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ser
Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly
Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440
445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser
Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 8485PRTBacillus
8His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1
5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala
Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro
Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu Ser
Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly
Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu
Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln
Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135
140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe
Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp
Asp Trp Glu Val Asp 180 185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Val Asn
Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn
Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys
Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250 255
Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260
265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser
Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser
Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly
Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val Thr Phe
Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser
Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile
Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp
Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380
Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385
390 395 400 Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr
Arg Glu 405 410 415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr
Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val
Gly Gln Asn Lys Ala Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly
Asn Lys Pro Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala
Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val
Lys Arg 485 91455DNABacillus 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 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 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 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 15gcgttttgcc ggccgacata
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nncaaacctg aatt
7416122DNAArtificial sequencePrimer 16gcgttttgcc ggccgacata
cattcgcttt gccccaccgg gtccgtctgt tattaatgcc 60gcnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnngccgac aatgtcatgg 120tg
1221778DNAArtificial sequencePrimer 17gtcgccttcc cttgtccann
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60gtacgcatac tgttttct
781820DNAArtificial sequencePrimer 18tggacaaggg aaggcgacag
201981DNAArtificial sequencePrimer RSERV 19taagatcggt tcaattttnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60cccgtacata tccccgtaga
a 812018DNAArtificial sequencePrimer 20aaaattgaac cgatctta
1821107DNAArtificial sequencePrimer FSERVII 21ttccatgctg catcgacaca
gggaggcggc tatgatatga ggaaattgct gaannnnnnn 60nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnntgtcg ataacca 1072218DNAArtificial sequencePrimer
22tgtcgatgca gcatggaa 182380DNAArtificial sequencePrimer FSERIX
23gtccaaacat ggtttaagcc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
60nntcaggttt tctacgggga 802420DNAArtificial sequencePrimer
24ggcttaaacc atgtttggac 202524DNAArtificial sequencePrimer
25cgattgctga cgctgttatt tgcg 242625DNAArtificial sequencePrimer
26ctatctttga acataaattg aaacc 252720DNAArtificial sequencePrimer
27gacctgcagt caggcaacta 202820DNAArtificial sequencePrimer
28tagagtcgac ctgcaggcat 202920DNAArtificial sequenceForward Primer2
29gacctgcagt caggcaacta 203020DNAArtificial sequencePrimer
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 208432400PRTBacillus 32Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp Tyr Leu Pro 1 5 10 15 Asn Asp Gly Asn His
Trp Asn Arg Leu Arg Ser Asp Ala Ser Asn Leu 20 25 30 Lys Asp Lys
Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp Lys Gly 35 40 45 Ala
Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55
60 Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Arg
65 70 75 80 Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly
Ile Gln 85 90 95 Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly
Ala Asp Ala Thr 100 105 110 Glu Met Val Arg Ala Val Glu Val Asn Pro
Asn Asn Arg Asn Gln Glu 115 120 125 Val Ser Gly Glu Tyr Thr Ile Glu
Ala Trp Thr Lys Phe Asp Phe Pro 130 135 140 Gly Arg Gly Asn Thr His
Ser Asn Phe Lys Trp Arg Trp Tyr His Phe 145 150 155 160 Asp Gly Val
Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg Ile Tyr 165 170 175 Lys
Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu 180 185
190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His
195 200 205 Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
Thr Asn 210 215 220 Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val
Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Ser Ile
His Val Arg Ser Ala Thr Gly 245 250 255 Lys Asn Met Phe Ala Val Ala
Glu Phe Trp Lys Asn Asp Leu Gly Ala 260 265 270 Ile Glu Asn Tyr Leu
Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu
His Tyr Asn Phe Tyr Asn Ala Ser Lys Ser Gly Gly Asn 290 295 300 Tyr
Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg His Pro 305 310
315 320 Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu
Glu 325 330 335 Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu
Ala Tyr Ala 340 345 350 Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser
Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val
Pro Ala Met Lys Ser Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg
Gln Lys Tyr Ala Tyr Gly Arg Gln Asn 385 390 395 400
* * * * *