U.S. patent application number 10/644187 was filed with the patent office on 2004-03-11 for alpha-amylase mutants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Bisgard-Frantzen, Henrik, Borchert, Torben Vedel, Svendsen, Allan.
Application Number | 20040048351 10/644187 |
Document ID | / |
Family ID | 27439333 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048351 |
Kind Code |
A1 |
Svendsen, Allan ; et
al. |
March 11, 2004 |
Alpha-amylase mutants
Abstract
The invention relates to a variant of a parent Termamyl-like
a-amylase, which variant has a-amylase activity and exhibits an
alteration in at least one of the following properties relative to
said parent a-amylase: substrate specificity, substrate binding,
substrate cleavage pattern, thermal stability, pH/activity profile,
pH/stability profile, stability towards oxidation, Ca.sup.2+
dependency and specific activity.
Inventors: |
Svendsen, Allan; (Birkerod,
DK) ; Borchert, Torben Vedel; (Jyllinge, DK) ;
Bisgard-Frantzen, Henrik; (Bagsvaerd, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27439333 |
Appl. No.: |
10/644187 |
Filed: |
August 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10644187 |
Aug 20, 2003 |
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10186042 |
Jun 28, 2002 |
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6642044 |
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10186042 |
Jun 28, 2002 |
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09672459 |
Sep 28, 2000 |
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6436888 |
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09672459 |
Sep 28, 2000 |
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09182859 |
Oct 29, 1998 |
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6143708 |
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09182859 |
Oct 29, 1998 |
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PCT/DK97/00197 |
Apr 30, 1997 |
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Current U.S.
Class: |
435/204 ;
510/226; 510/320 |
Current CPC
Class: |
C12P 19/02 20130101;
C11D 3/386 20130101; C12N 9/2417 20130101; C12P 19/14 20130101 |
Class at
Publication: |
435/204 ;
510/320; 510/226 |
International
Class: |
C12N 009/32; C11D
003/386 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1996 |
DK |
0515/96 |
Jun 28, 1996 |
DK |
0712/96 |
Jul 11, 1996 |
DK |
0775/96 |
Nov 8, 1996 |
DK |
1263/96 |
Claims
1. A variant of a parent Termamyl-like .alpha.-amylase, which
variant has .alpha.-amylase activity and exhibits an alteration in
at least one of the following properties relative to said parent
.alpha.-amylase: substrate specificity, substrate binding,
substrate cleavage pattern, thermal stability, pH/activity profile,
pH/stability profile, stability towards oxidation, Ca.sup.2+
dependency and specific activity; said variant comprising one or
more mutations corresponding to one or more of the following
mutations in the amino acid sequence shown in SEQ ID No. 2: the
mutations: A181E,D,Q,N,V; I201(bulkier amino acid), including
I201W,F,L; Y203Q; Q9K,L,E; F11R,K,E; E12Q; D100N,L;
V101H,R,K,D,E,F; V102A,T; I103H,K; N104R,K,D; H105R,K,D,E,W,F;
L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I; A232G,H,F,S,V; V233D;
K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E;
N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D; F284H; D285N,L;
V286R,K,H,D,E; Y290R,E,K; V312R,K,D,E; F323H; D325N; N326K,H,D,L;
H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D;
L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D; Q360K,R,E;
D365N; or G371D,R; substitutions at positions H68, H91, H247, R305,
K306, H382, K389, H405, H406, H450 or R483; the mutations: H140Y;
H142Y; H159Y; H140D+H142R; H140K+H142D; or H142Y+H156Y; deletion of
three amino acids within the partial sequence from T369 to I377,
including the deletions: K370*+G371*+D372* or D372*+S373*+Q374*;
replacement of the partial amino acid sequence from T369 to I377
with one of the following sequences (sequence number increasing
from left to right): I-P-T-H-S-V; I-P-T-H-G-V; I-P-Q-Y-N-I;
substitutions at positions R169 or R173, including R169I,L,F,T or
R173I,L,F,T; the mutations H156D; I201F; I212F; A209L,T; or V208I;
substitutions at positions N172, A181, N188, N190, H205, D207,
A209, A210, E211, Q264 or N265, including N172R,H,K; A181T; N188P;
N190L,F; H205C; D207Y; A209L,T,V; A210S; E211Q; Q264A,E,L,K,S,T;
N265A,S,T,Y; or Q264S+N265Y; the mutations: H156Y+A181T+A209V;
H156Y+A181T+N190F+A209V+Q264S; A1*+N2*+L3V+M15T+R23K+S-
29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A2 09V;
A1*+N2*+L3V+M15T+R23K+S29-
A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V; or
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1
90F+A209V+Q264S.
2. A variant according to claim 1, exhibiting increased stability
at low pH and low Ca.sup.2+ concentration relative to the parent
Termamyl-like .alpha.-amylase, and comprising mutations selected
from the following: H156Y+A181T+A209V;
H156Y+A181T+N190F+A209V+Q264S; A1*+N2*+L3V+M15T+R23K+S-
29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A2 09V;
A1*+N2*+L3V+M1ST+R23K+S29-
A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V;
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1
90F+A209V+Q264S.
3. A variant according to claim 1 or 2, wherein the parent
Termamyl-like .alpha.-amylase is selected from: the B.
licheniformis .alpha.-amylase having the sequence shown in SEQ ID
No. 2, the B. amyloliquefaciens .alpha.-amylase having the sequence
shown in SEQ ID No. 4, the B. stearothermophilus .alpha.-amylase
having the sequence shown in SEQ ID No. 6, the Bacillus strain NCIB
12512 .alpha.-amylase having the sequence shown in FIGS. 1 and 2,
the Bacillus strain NCIB 12513 .alpha.-amylase having the sequence
shown in FIG. 2, and the Bacillus sp. #707 .alpha.-amylase having
the sequence shown in FIG. 2.
4. A DNA construct comprising a DNA sequence encoding an
.alpha.-amylase variant according to any one of claims 1-3.
5. A recombinant expression vector which carries a DNA construct
according to claim 4.
6. A cell which is transformed with a DNA construct according to
claim 4 or a vector according to claim 5.
7. A cell according to claim 6, which is a microorganism.
8. A cell according to claim 6, which is a bacterium or a
fungus.
9. The cell according to claim 8, 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.
10. Use of an .alpha.-amylase variant according to any one of
claims 1-3 for washing and/or dishwashing.
11. Use of an .alpha.-amylase variant according to any one of
claims 1-3 for textile desizing.
12. Use of an .alpha.-amylase variant according to any of claims
1-3 for starch liquefaction.
13. A detergent additive comprising an .alpha.-amylase variant
according to any one of claims 1-3, optionally in the form of a
non-dusting granulate, stabilised liquid or protected enzyme.
14. A detergent additive according to claim 13 which contains
0.02-200 mg of enzyme protein/g of the additive.
15. A detergent additive according to claim 13 or 14, which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
16. A detergent composition comprising an .alpha.-amylase variant
according to any of claims 1-3.
17. A detergent composition according to claim 16 which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
18. A manual or automatic dishwashing detergent composition
comprising an .alpha.-amylase variant according to any one of
claims 1-3.
19. A dishwashing detergent composition according to claim 18 which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
20. A manual or automatic laundry washing composition comprising an
.alpha.-amylase variant according to any one of claims 1-3.
21. A laundry washing composition according to claim 20, which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, an amylolytic enzyme and/or a cellulase.
22. A composition comprising: (i) a mixture of the .alpha.-amylase
from B. licheniformis having the sequence shown in SEQ ID No. 2
with one or more variants according to the invention derived from
(as the parent Termamyl-like .alpha.-amylase) the B.
stearothermophilus .alpha.-amylase having the sequence shown in SEQ
ID No. 6; or (ii) a mixture of the .alpha.-amylase from B.
stearothermophilus having the sequence shown in SEQ ID No. 6 with
one or more variants according to the invention derived from one or
more other parent Termamyl-like .alpha.-amylases; or (iii) a
mixture of one or more variants according to the invention derived
from (as the parent Termamyl-like .alpha.-amylase) the B.
stearothermophilus .alpha.-amylase having the sequence shown in SEQ
ID No. 6 with one or more variants according to the invention
derived from one or more other parent Termamyl-like
.alpha.-amylases.
23. A method for generating a variant of a parent Termamyl-like
.alpha.-amylase, which variant exhibits increased stability at low
pH and at low calcium concentration 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 low pH and low
calcium concentration relative to the parent .alpha.-amylase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of Ser. No. 10/186,042, filed
on Jun. 27, 2002, which is a division of Ser. No. 09/672,459, filed
on Sep. 28, 2000 (now a U.S. Pat. No. 6,436,888), which is a
continuation of Ser. No. 09/182,859, filed on Oct. 29, 1998 (now
U.S. Pat. No. 6,143,708), which is a continuation of PCT/DK97/00197
filed Apr. 30, 1997 which claims priority under 35 U.S.C. 119 of
Danish applications 0515/96 filed Apr. 30, 1996, 0712/96 filed Jun.
28, 1996, 0775/96 filed Jul. 11, 1996, and 1263/96 filed Nov. 8,
1996, the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates, inter alia, to novel variants
(mutants) of parent Termamyl-like .alpha.-amylases, notably
variants exhibiting alterations in one or more properties (relative
to the parent) which are advantageous with respect to applications
of the variants in, 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, and there is a very extensive body of patent and
scientific literature relating to this industrially very important
class of enzymes.
[0004] Among more recent disclosures relating to .alpha.-amylases,
WO 96/23874 provides 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 comprising the amino acid sequence shown in SEQ ID
No. 4 herein and amino acids 301-483 of the C-terminal end of the
B. licheniformis .alpha.-amylase comprising the amino acid sequence
shown in SEQ ID No. 2 herein (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
[0005] As indicated above, the present invention relates, inter
alia, to novel .alpha.-amylolytic variants(mutants) of a
Termamyl-like .alpha.-amylase, in particular variants exhibiting
altered properties which are advantageous in connection with the
industrial processing of starch (starch liquefaction,
saccharification and the like).
[0006] Alterations in properties which may be achieved in mutants
of the invention are alterations in, e.g., substrate specificity,
substrate binding, substrate cleavage pattern, thermal stability,
pH/activity profile, pH/stability profile [such as increased
stability at low (e.g. pH<6, in particular pH<5) or high
(e.g. pH>9) pH values], stability towards oxidation, Ca.sup.2+
dependency, specific activity, and other properties of interest.
For instance, the alteration may result in a variant which, as
compared to the parent Termamyl-like .alpha.-amylase, has a reduced
Ca.sup.2+ dependency and/or an altered pH/activity profile.
[0007] The invention further relates, inter alia, 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, e.g. starch
liquefaction.
DETAILED DISCLOSURE OF THE INVENTION
[0008] The Termamyl-like .alpha.-amylase
[0009] 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. 2 (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. 4 and about 79% homologous with the B.
stearothermophilus .alpha.-amylase comprising the amino acid
sequence shown in SEQ ID No. 6. Further homologous .alpha.-amylases
include an .alpha.-amylase derived from a strain of the Bacillus
sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which
are described in detail in WO 95/26397, and the .alpha.-amylase
described by Tsukamoto et al., Biochemical and Biophysical Research
Communications, 151 (1988), pp. 25-31. 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.
(available from Genencor), and Keistase.TM. (available from
Daiwa).
[0010] 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".
[0011] 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. 2 herein. In other words, a
Termamyl-like .alpha.-amylase is an .alpha.-amylase which has the
amino acid sequence shown in SEQ ID No. 2, No. 4 or No. 6 herein,
or the amino acid sequence shown in SEQ ID No. 1 of WO 95/26397
(which amino acid sequence is shown in FIG. 1 and FIG. 2 herein) or
in SEQ ID No. 2 of WO 95/26397 (which amino acid sequence is shown
in FIG. 2 herein) or in Tsukamoto et al., 1988, (which amino acid
sequence is shown in FIG. 2 herein) or i) which displays at least
60%, such as at least 70%, e.g. at least 75%, or at least 80%, e.g.
at least 85%, at least 90% or at least 95% homology with at least
one of said amino acid sequences and/or ii) displays immunological
cross-reactivity with an antibody raised against at least one of
said .alpha.-amylases, and/or iii) is encoded by a DNA sequence
which hybridizes to the DNA sequences encoding the above-specified
.alpha.-amylases which are apparent from SEQ ID Nos. 1, 3 and 5 of
the present application (which encoding sequences encode the amino
acid sequences shown in SEQ ID Nos. 2, 4 and 6 herein,
respectively), from SEQ ID No. 4 of WO 95/26397 (which DNA
sequence, together with the stop codon TAA, is shown in FIG. 1
herein and encodes the amino acid sequence shown in FIG. 1 herein)
and from SEQ ID No. 5 of WO 95/26397, respectively.
[0012] In connection with property i), the "homology" may be
determined by use of any conventional algorithm, preferably by use
of the GAP progamme from the GCG package version 7.3 (June 1993)
using default values for GAP penalties [Genetic Computer Group
(1991) Programme Manual for the GCG Package, version 7, 575 Science
Drive, Madison, Wis., USA 53711].
[0013] Property ii) 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., 1989. 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. 2, 4 and 6, respectively, has
been found.
[0014] 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. Suitable conditions for testing hybridization involve
presoaking in 5.times.SSC and prehybridizing for 1 h 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
.mu.g of denatured sonicated calf thymus DNA, followed by
hybridization in the same solution supplemented with 100 .mu.M ATP
for 18 h at .about.40.degree. C., or other methods described by,
e.g., Sambrook et al., 1989.
[0015] 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.
[0016] Parent Hybrid .alpha.-amylases
[0017] The parent .alpha.-amylase 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.
[0018] 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.
[0019] Thus, the parent hybrid .alpha.-amylase may comprise a
combination of partial amino acid sequences deriving from at least
two Termamyl-like .alpha.-amylases, or from at least one
Termamyl-like and at least one non-Termamyl-like bacterial
.alpha.-amylase, or from at least one Termamyl-like and at least
one fungal .alpha.-amylase. The Termamyl-like .alpha.-amylase from
which a partial amino acid sequence derives may, e.g., be any of
those specific Termamyl-like .alpha.a-mylase referred to
herein.
[0020] 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, and may, e.g. comprise a) an
amino acid segment corresponding to the 37 N-terminal amino acid
residues of the B. amyloliquefaciens .alpha.-amylase having the
amino acid sequence shown in SEQ ID No. 4 and an amino acid segment
corresponding to the 445 C-terminal amino acid residues of the B.
licheniformis .alpha.-amylase having the amino acid sequence shown
in SEQ ID No. 2, or b) 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.
6 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. 2.
[0021] 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.
[0022] 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..
[0023] 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.
[0024] 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. 2.
[0025] Construction of Variants of the Invention
[0026] The construction of the variant of interest may be
accomplished by cultivating a microorganism comprising a DNA
sequence encoding the variant under conditions which are conducive
for producing the variant. The variant may then subsequently be
recovered from the resulting culture broth. This is described in
detail further below.
[0027] Altered Properties of Variants of the Invention
[0028] The following discusses the relationship between 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.
[0029] Decreased Ca.sup.2+ Dependency
[0030] It is highly desirable to be able to decrease the Ca.sup.2+
dependency of a Termamyl-like .alpha.-amylase. Accordingly, one
aspect of the invention relates to a variant of a parent
Termamyl-like .alpha.-amylase, which variant exhibits
.alpha.-amylase activity and has a decreased Ca.sup.2+ dependency
as compared to the parent .alpha.-amylase. Decreased Ca.sup.2+
dependency will in general have the functional consequence that the
variant exhibits a satisfactory amylolytic activity in the presence
of a lower concentration of calcium ion in the extraneous medium
than is necessary for the parent enzyme. It will further often have
the consequence that the variant is less sensitive than the parent
to calcium ion-depleting conditions such as those obtained in media
containing calcium-complexing agents (such as certain detergent
builders).
[0031] Decreased Ca.sup.2+ dependency of a variant of the invention
may advantageously be achieved, for example, by increasing the
Ca.sup.2+ binding affinity relative to that of the parent
Termamyl-like .alpha.-amylase; in other words the stronger the
binding of Ca.sup.2+ in the enzyme, the lower the Ca.sup.2+
dependency.
[0032] It may be mentioned here that WO 96/23874 states that amino
acid residues located within 10.ANG. from a sodium or calcium ion
are believed to be involved in, or of importance for, the Ca.sup.2+
binding capability of the enzyme, and that in this connection the
mutation N104D [of the B. licheniformis .alpha.-amylase having the
amino acid sequence shown in SEQ ID No. 2, or an equivalent (N to
D) mutation of an equivalent position in another Termamyl-like
.alpha.-amylase] is contemplated to be of particular interest with
respect to decreasing the Ca.sup.2+ dependency of a Termamyl-like
.alpha.-amylase.
[0033] Other mutations mentioned in WO 96/23874 as being of
possible importance in connection with Ca.sup.2+ dependency include
mutations which are contemplated therein to achieve increased
calcium binding (and/or thermostability of the enzyme) via
stabilization of the C-domain (as defined in WO 96/23874) of the
three-dimensional structure of a Termamyl-like .alpha.-amylase via
formation, for example, of cysteine bridges or salt bridges. Thus,
WO 96/23874 discloses that the C-domain of the B. licheniformis
.alpha.-amylase having the amino acid sequence shown in SEQ ID No.
2 may be stabilized by introduction of a cysteine bridge between
domain A and domain C (as defined in WO 96/23874) by introduction
of the following mutations:
[0034] A349C+I479C and/or L346C+I430C.
[0035] WO 96/23874 likewise discloses that a salt bridge may be
obtained by introduction of one or more of the following mutations
in the B. licheniformis .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 2:
[0036] N457D,E
[0037] N457D,E+K385R
[0038] F350D,E+I430R,K
[0039] F350D,E+I411R,K
[0040] and that the calcium site of Domain C may be stabilized by
replacing the amino acid residues H408 and/or G303 with any other
amino acid residue, in particular by introducing one of the
substitutions:
[0041] H408Q,E,N,D and/or G303N,D,Q,E
[0042] which are contemplated to provide better calcium binding or
protection from calcium depletion.
[0043] (similar mutations in equivalent positions of other
Termamyl-like .alpha.-amylases being encompassed hereby).
[0044] Other substitution mutations (relative to B. licheniformis
.alpha.-amylase, SEQ ID No. 2) which are disclosed in WO 96/23874
as being of apparent importance, inter alia, in the context of
reducing calcium dependency include the following: R23K, H156Y,
A181T, A209V, R214, G310D and P345 (or equivalent mutations in
equivalent positions in another Termamyl-like .alpha.-amylase).
[0045] In the context of the present invention, further
substitution mutations which appear to be of importance, inter
alia, in relation to reduction of calcium dependency include the
following mutations in Domain B (as defined in WO 96/23874):
[0046] A181E,D,Q,N,V (which appear to result in shielding of the
outermost Ca.sup.2+ binding site in the junction region between
Domain A and Domain B to some extent);
[0047] I201 (bulkier amino acid), e.g. I201W,F,L (which appear to
result in slight alterations in the geometry of the region in the
immediate vicinity of the Ca.sup.2+-Na.sup.+-Ca.sup.2+ binding
site(s) in the junction region between Domain A and Domain B, and
in the geometry and/or size of a nearby hole/cavity); and
[0048] Y203E,Q (which are believed to result in stronger binding of
the outermost Ca.sup.2+ ion in its binding site in the junction
region between Domain A and Domain B);
[0049] (or equivalent mutations in equivalent positions in another
Termamyl-like .alpha.-amylase).
[0050] Altered pH Optimum (Altered pH/Activity Profile)
[0051] WO 96/23874 discloses that it is contemplated to be possible
to change the pH optimum of a Termamyl-like .alpha.-amylase, or the
enzymatic activity thereof at a given pH, by changing the pKa of
the active site residues, and that this may be achieved, e.g., by
changing the electrostatic interaction or hydrophobic interaction
between functional groups of amino acid side chains of the amino
acid residue to be modified and of its close surroundings.
[0052] In the context of the present invention, it is believed on
the basis of electrostatic considerations [see, e.g., M. K. Gilson,
Current Opinion in Structural Biology 5 (1995) pp. 216-223; and B.
Honig and A. Nicholls, Science 268 (1995) pp. 1144-1149; and
references given therein] and hygroscopicity considerations in
relation to the three-dimensional structure of the Termamyl-like
.alpha.-amylase disclosed in WO 96/23874 that mutations of
relevance, inter alia, for altering (increasing or decreasing) the
pH optimum of a Termamyl-like .alpha.-amylase include the following
mutations or equivalents thereof [referring here to the sequence of
B. licheniformis .alpha.-amylase (SEQ ID NO 2)]:
[0053] Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T;
I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E;
L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E;
L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E;
L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E;
V312R,K,D,E; F323H; D325N; N326K,H,D,L; H327Q,N,E,D,F; Q330L,E;
G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E;
T337D,K; T338D,E; T339D; Q360K,R,E; D365N; G371D,R;
[0054] Increased Stability at Low (Acidic) pH
[0055] In the context of the present invention, mutations (amino
acid substitutions) of importance with respect to achieving
increased stability at low pH appear to include mutations
corresponding to the following mutations in the B. licheniformis
.alpha.-amylase having the amino acid sequence shown in SEQ ID No.
2:
[0056] mutations at positions H68, H91, H247, R305, K306, H382,
K389, H405, H406, H450 or R483;
[0057] the mutations:
[0058] H140Y;
[0059] H142Y;
[0060] H156Y;
[0061] H159Y;
[0062] H140D+H142R;
[0063] H140K+H142D; or
[0064] H142Y+H156Y
[0065] as well as combinations of any two or more of these
mutations.
[0066] Increased Thermostability and/or Altered Temperature Optimum
(Altered Temperature/Activity Profile)
[0067] A further aspect of the invention relates to a variant of a
parent Termamyl-like .alpha.-amylase, which variant is the result
of one or more amino acid residues having been deleted from,
substituted in or added to the parent .alpha.-amylase so as to
achieve increased thermostability of the variant.
[0068] In may be mentioned that in relation to achieving increased
thermostability, WO 96/23874 discloses that a particularly
interesting variant of a Termamyl-like .alpha.-amylase comprises a
mutation corresponding to one of the following mutations (using the
numbering of the B. licheniformis .alpha.-amylase amino acid
sequence shown in SEQ ID NO 2):
[0069] L61W,V,F;
[0070] Y62W;
[0071] F67W;
[0072] K106R,F,W;
[0073] G145F,W
[0074] I212F,L,W,Y,R,K;
[0075] S151 replaced with any other amino acid residue and in
particular with F,W,I or L;
[0076] R214W;
[0077] Y150R, K;
[0078] F143W; and/or
[0079] R146W.
[0080] WO 96/23874 further discloses in this connection that
mutations corresponding to one or more of the following mutations
in the B. licheniformis .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 2 are of interest in relation to
achieving increased thermostability relative to that of the parent
.alpha.-amylase:
[0081] L241I,F,Y,W; and/or
[0082] I236L,F,Y,W
[0083] L7F,I,W
[0084] V259F,I,L
[0085] F284W
[0086] F350W
[0087] F343W
[0088] L427F,L,W
[0089] V481,F,I,L,W
[0090] In the context of the present invention, it can be seen from
an alignment of the amino acid sequences of .alpha.-amylases from
various Bacillus species that B. licheniformis .alpha.-amylase and
B. amyloliquefaciens .alpha.-amylase both contain an "insertion" of
three amino acids relative to, e.g., B. stearothermophilus
.alpha.-amylase.
[0091] From a model of the structure of B. licheniformis
.alpha.-amylase built on the basis of the three-dimensional
structure of the Termamyl-like .alpha.-amylase disclosed in WO
96/23784 (vide supra), taking into account the homology of B.
licheniformis .alpha.-amylase to the Termamyl-like .alpha.-amylase
in question, it can be seen that the above-mentioned "insertion"
lies within a part of the structure denoted "loop 8" in WO
96/23784, making this loop bulkier in B. licheniformis
.alpha.-amylase than in the Termamyl-like .alpha.-amylase and
resulting in a loop that protrudes from the structure, thereby
possibly destabilizing the structure. It is therefore contemplated
that deletion of one or more amino acids in the region in question
in B. licheniformis or B. amyloliquefaciens .alpha.-amylase will
improve the thermostability of these .alpha.-amylases.
[0092] Especially interesting in this connection is deletion of
three amino acids within the partial sequence from T369 to I377
(referring to the amino acid sequence of B. licheniformis
.alpha.-amylase shown in SEQ ID No. 2), i.e. the partial sequence:
T369-K370-G371-D372-S373-Q374-R375-- E376-I377 (or the
corresponding partial sequence in B. amyloliquefaciens
.alpha.-amylase). In addition to such deletions, substitution of
one or more of the undeleted amino acids within the latter partial
sequence may also be advantageous.
[0093] Preferable deletions of three amino acids in the partial
sequence from T369 to I377 (in the B. licheniformis
.alpha.-amylase) are deletion of K370+G371+D372 (i.e.
K370*+G371*+D372*) or deletion of D372+S373+Q374 (i.e.
D372*+S373*+Q374*) (or equivalent deletions in the corresponding
partial sequence in B. amyloliquefaciens .alpha.-amylase).
[0094] Another type of mutation which would appear to be of value
in improving the thermostability of these .alpha.-amylases is
substitution (replacement) of the entire partial amino acid
sequence from T369 to I377 (referring to the sequence of the B.
licheniformis .alpha.-amylase) with one of the following partial
sequences of six amino acids (sequence numbering increasing from
left to right): I-P-T-H-S-V; I-P-T-H-G-V; and I-P-Q-Y-N-I (or one
of the same substitutions of the corresponding partial sequence in
B. amyloliquefaciens .alpha.-amylase).
[0095] Other mutations which can apparently be of some importance
in relation to achieving increased thermostability include amino
acid substitutions at the following positions (referring to SEQ ID
No. 2):
[0096] R169 (e.g. R169I,L,F,T);
[0097] R173 (especially R173I,L,F,T);
[0098] I201F;
[0099] I212F;
[0100] A209L,T; or
[0101] V208I
[0102] as well as combinations of any two or more of these
mutations.
[0103] Increased Thermostability at Acidic pH and/or at Low
Ca.sup.2+ Concentration
[0104] In the context of the invention, mutations which appear to
be of particular relevance in relation to obtaining variants
according to the invention having increased thermostability at
acidic pH (pH<7) and/or at low Ca.sup.2+ concentration include
mutations at the following positions (relative to B. licheniformis
.alpha.-amylase, SEQ ID No. 2):
[0105] H156, N172, A181, N188, N190, H205, D207, A209, A210, E211,
Q264, N265
[0106] It may be mentioned here that N and E amino acid residues,
respectively, at positions corresponding to N109 and E211,
respectively, in SEQ ID No. 2 constitute amino acid residues which
are conserved in numerous Termamyl-like .alpha.-amylases. Thus, for
example, the corresponding positions of these residues in the amino
acid sequences of a number of Termamyl-like .alpha.-amylases which
have already been mentioned (vide supra) are as follows:
1 Termamyl-like .alpha.-amylase N position E position B.
licheniformis (SEQ ID No. 2) N190 E211 B. amyloliquefaciens (SEQ ID
No. 4) N190 E211 B. stearothermophilus (SEQ ID No. 6) N193 E210
Bacillus NCIB 12512 (WO 95/26397) N195 E212 Bacillus NCIB 12513 (WO
95/26397) N195 E212 "Bacillus sp. #707" (Tsukamoto et al.) N195
E212
[0107] Mutations of these conserved amino acid residues appear to
be very important in relation to improving thermostability at
acidic pH and/or at low calcium concentration, and the following
mutations are of particular interest in this connection (with
reference to the numbering of the B. licheniformis amino acid
sequence shown in SEQ ID No. 2):
[0108] H156Y,D
[0109] N172R,H,K
[0110] A181T
[0111] N188P
[0112] N190L,F
[0113] H205C
[0114] D207Y
[0115] A209L,T,V
[0116] A210S
[0117] E211Q
[0118] Q264A,E,L,K,S,T
[0119] N265A,S,T,Y
[0120] as well as any combination of two or more of these
mutations.
[0121] An example of a particularly interesting double mutation in
this connection is Q264S+N265Y.
[0122] Altered Cleavage Pattern
[0123] In the starch liquefaction process it is desirable to use an
.alpha.-amylase which is capable of degrading the starch molecules
into long, branched oligosaccharides, rather than an
.alpha.-amylase which gives rise to formation of shorter, branched
oligosaccharides (like conventional Termamyl-like
.alpha.-amylases). Short, branched oligosaccharides (panose
precursors) are not hydrolyzed satisfactorily by pullulanases,
which are used after .alpha.-amylase treatment in the liquefaction
process, but before addition of a saccharifying amyloglucosidase
(glucoamylase). Thus, in the presence of panose precursors, the
product mixture present after the glucoamylase treatment contains a
significant proportion of short, branched, so-called limit-dextrin,
viz. the trisaccharide panose. The presence of panose lowers the
saccharification yield significantly and is thus undesirable.
[0124] Thus, one aim of the present invention is to arrive at a
mutant .alpha.-amylase having appropriately modified
starch-degradation characteristics but retaining the
thermostability of the parent Termamyl-like .alpha.-amylase.
[0125] It may be mentioned here that according to WO 96/23874,
variants comprising at least one of the following mutations are
expected to prevent cleavage close to the branching point:
[0126] V54L, I,F,Y,W,R,K,H,E,Q
[0127] D53L, I,F,Y,W
[0128] Y56W
[0129] Q333W
[0130] G57all possible amino acid residues
[0131] A52amino acid residues larger than A, e.g. A52W,Y,L,F,I.
[0132] Increased Specific Activity
[0133] In a further aspect of the present invention, important
mutations with respect to obtaining variants exhibiting increased
specific activity appear to include mutations corresponding to the
following mutations in the B. licheniformis .alpha.-amylase having
the amino acid sequence shown in SEQ ID No. 2:
[0134] mutations (amino acid substitutions) at positions S187
(especially S187D) or Q264 (e.g. Q264R,K,S);
[0135] mutations (substitutions) at position Y290 (especially
Y290E,K);
[0136] the mutation V54I;
[0137] as well as combinations of any two or more of the latter
mutations, or combinations of one, two or more of the latter
mutations with the following multiple mutation:
[0138] A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I
[0139] General Mutations in Variants of the Invention
[0140] 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.
[0141] 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.
[0142] 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. 2 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. 2 by an Arg.
[0143] It will be understood that the present invention encompasses
variants incorporating two or more of the above outlined
modifications.
[0144] Furthermore, it may be advantageous to introduce
point-mutations in any of the variants described herein.
[0145] Methods for Preparing .alpha.-amylase Variants
[0146] Several methods for introducing mutations into genes are
known in the art. After a brief discussion of the cloning of
.alpha.-amylase-encoding DNA sequences, methods for generating
mutations at specific sites within the .alpha.-amylase-encoding
sequence will be discussed.
[0147] Cloning a DNA Sequence Encoding an .alpha.-amylaseCloning a
DNA Sequence Encoding an .alpha.-amylaseCloning a DNA Sequence
Encoding an .alpha.-amylaseCloning a DNA Sequence Encoding an
.alpha.-amylase
[0148] 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.
[0149] 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.
[0150] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g. the
phosphoroamidite method described by S. L. Beaucage and M. H.
Caruthers (1981) or the method described by Matthes et al. (1984).
In the phosphoroamidite method, oligonucleotides are synthesized,
e.g. in an automatic DNA synthesizer, purified, annealed, ligated
and cloned in appropriate vectors.
[0151] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et
al. (1988).
[0152] Site-directed Mutagenesis
[0153] 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.
[0154] 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.
[0155] Random Mutagenesis
[0156] Random mutagenesis is suitably performed either as localized
or region-specific random mutagenesis in at least three parts of
the gene translating to the amino acid sequence in question, or
within the whole gene.
[0157] WO 96/23874 discloses that in connection with achieving
improved binding of a substrate (i.e. improved binding of a
carbohydrate species, such as amylose or amylopectin) by a
Termamyl-like .alpha.-amylase variant, modified (e.g. higher)
substrate specificity and/or modified (e.g. higher) specificity
with respect to cleavage (hydrolysis) of substrate, the following
codon positions for the amino acid sequence shown in SEQ ID NO 2
(or equivalent codon positions for another parent Termamyl-like
.alpha.-amylase in the context of the invention) appear to be
particularly appropriate for targetting:
[0158] 13-18
[0159] 50-56
[0160] 70-76
[0161] 102-109
[0162] 163-172
[0163] 189-199
[0164] 229-235
[0165] 360-364
[0166] 327-335
[0167] Improvement of Liquefaction Performance at Low pH and Low
Calcium Ion Concentration
[0168] For an .alpha.-amylase to be used in a starch liquefaction
process it is of particular interest that it be thermostable and
able to function at low pH and low calcium concentrations. In order
to improve these properties of a parent Termamyl-like
.alpha.-amylase, in particular the B. licheniformis .alpha.-amylase
or a variant or hybrid thereof, random mutagenesis (preferably by
use of doped or spiked oligonucleotide primers) followed by
appropriate selection of the resulting mutated enzymes may be
performed. The direction of selection of regions to randomize and
selection of doping are based primarily on stabilization of calcium
ions already present, and on improvement in residue/residue or
domain/domain electrostatic interactions at low pH. In addition,
the regions which have been shown to include positions important
for achieving good starch liquefaction performance may be
selected.
[0169] In order to prepare a variant of a parent Termamyl-like
.alpha.-amylase having the above properties, at least one of the
following regions may advantageously be subjected to random
mutagenesis (the numbering of the amino acid residues being as in
SEQ ID No. 2):
2 Region Residue Description I: 153-163 Calcium region between
domain A & B, also containing H156 II: 178-192 Calcium region
between domain A & B III: 193-214 Calcium region between domain
A & B, also containing A209 IV: 232-237 Calcium region between
domain A & B V: 297-308 Calcium region between domain A & C
VI: 403-409 Calcium region between domain A & C VII: 428-435
Calcium region between domain A & C VIII: 131-136 Region
containing H133 IX: 164-175 Region in contact with H133 region X:
262-278 Region containing Q264
[0170] Preferably, two, three or four of the above regions are
subjected to random mutagenesis in the construction of a novel
.alpha.-amylase variant of the invention. For instance, the
following combinations of regions are suitably subjected to random
mutagenesis:
[0171] VIII+IX
[0172] VIII+IX+II
[0173] II+III+IV
[0174] IV+I
[0175] Furthermore, it is preferred that the mutagenesis is carried
out by use of doped or spiked oligonucleotides. The doping is
preferably done so as to introduce amino acids contributing to
improved stability at low pH and reduced calcium dependency at low
pH of the resulting .alpha.-amylase variant. Furthermore, when
selecting the doping scheme, the possibility of introducing Asn and
Gln residues should generally be avoided, since Asn and Gln
residues in general are associated with instability at low pH.
Preferably, when a Pro residue can be inserted with potential
benefits (e.g. as assessed from protein-structural considerations),
the doping scheme is prepared to include a preference for
introduction of a Pro residue.
[0176] The parent Termamyl-like .alpha.-amylase to be subjected to
random mutagenesis according to the above principle may be any wild
type .alpha.-amylase or a variant thereof containing one or more
mutations. The parent may be a hybrid between at least two
.alpha.-amylases as explained in further detail herein. Preferably,
the parent .alpha.-amylase is a mutant of the B. licheniformis
.alpha.-amylase having the sequence shown in SEQ ID No. 2
containing at least one mutation, and preferably multiple
mutations. The parent .alpha.-amylase may alternatively be a hybrid
.alpha.-amylase which contains at least a part of the B.
licheniformis (SEQ ID No. 2) .alpha.-amylase. Specific examples of
parent .alpha.-amylases suited to mutagenesis according to the
above-described principles include: variants of the B.
licheniformis (SEQ ID No. 2) .alpha.-amylase which contain at least
one of, i.e. one, two, three, four or all five of, the mutations
H156Y, A181T, N190F, A209V and Q264S; hybrid .alpha.-amylases which
contain a part of the B. licheniformis (SEQ ID No. 2)
.alpha.-amylase, preferably a C-terminal part thereof, such as
amino acids 35-483 thereof, and a part of another Termamyl-like
.alpha.-amylase such as B. amyloliquefaciens (SEQ ID No. 4)
.alpha.-amylase, preferably an N-terminal part thereof such as the
first 38 amino acid residues thereof.
[0177] In relation to the above, a further aspect of the present
invention relates to a method for generating a variant of a parent
Termamyl-like .alpha.-amylase, which variant exhibits increased
stability at low pH and at low calcium concentration relative to
the parent, the method comprising:
[0178] (a) subjecting a DNA sequence encoding the parent
Termamyl-like .alpha.-amylase to random mutagenesis,
[0179] (b) expressing the mutated DNA sequence obtained in step (a)
in a host cell, and
[0180] (c) screening for host cells expressing a mutated
.alpha.-amylase which has increased stability at low pH and low
calcium concentration relative to the parent .alpha.-amylase.
[0181] Step (a) of the latter method of the invention is preferably
performed using doped primers, as described in the working examples
herein (vide infra).
[0182] Method of Performing Random Mutagenesis
[0183] The random mutagenesis of a DNA sequence encoding a parent
.alpha.-amylase to be performed in accordance with step a) of the
above-described method of the invention may conveniently be
performed by use of any method known in the art.
[0184] 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.
[0185] The mutagenizing agent may, e.g., be one which induces
transitions, transversions, inversions, scrambling, deletions,
and/or insertions.
[0186] Examples of a physical or chemical mutagenizing agent
suitable for the present purpose include ultraviolet (UV)
irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide
analogues.
[0187] 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.
[0188] When the mutagenesis is performed by the use of an
oligo-nucleotide, 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 amylolytic enzyme by any
published technique, using e.g. PCR, LCR or any DNA polymerase and
ligase.
[0189] 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 to
have a preference for the introduction of certain nucleotides, and
thereby a preference for introduction of one or more specific amino
acid residues. The doping may, e.g., be made so as to allow for the
introduction of 90% wild type and 10% mutations in each position.
An additional consideration in choice of doping scheme is genetic
as well as protein-structural constraints. The doping scheme may be
made by using the DOPE program (see the working examples herein)
which, inter alia, ensures that introduction of stop codons is
avoided.
[0190] When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent .alpha.-amylase
enzyme is subjected to PCR under conditions that increase the
misincorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol. 1, 1989, pp. 11-15).
[0191] A mutator strain of E. coli (Fowler et al., Molec. Gen.
Genet., 133, 1974, pp. 179-191), S. cereviseae or any other
microbial organism may be used for the random mutagenesis of the
DNA encoding the amylolytic enzyme by e.g. transforming a plasmid
containing the parent enzyme into the mutator strain, growing the
mutator strain with the plasmid and isolating the mutated plasmid
from the mutator strain. The mutated plasmid may subsequently be
transformed into the expression organism.
[0192] The DNA sequence to be mutagenized may conveniently be
present in a genomic or cDNA library prepared from an organism
expressing the parent amylolytic enzyme. 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 otherwise
exposed to the mutagenizing 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.
[0193] In some cases it may be convenient to amplify the mutated
DNA sequence prior to the expression step (b) or the screening step
(c) being performed. 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.
[0194] Subsequent to the incubation with or exposure to the
mutagenizing 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:
grampositive 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 gramnegative bacteria such as
E. coli.
[0195] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
[0196] Localized random mutagenesis: the random mutagenesis may
advantageously be 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.
[0197] The localized random mutagenesis is conveniently performed
by use of PCR-generated mutagenesis techniques as described above,
or any other suitable technique known in the art.
[0198] Alternatively, the DNA sequence encoding the part of the DNA
sequence to be modified may be isolated, e.g. by being inserted
into a suitable vector, and said part may subsequently be subjected
to mutagenesis by use of any of the mutagenesis methods discussed
above.
[0199] With respect to the screening step in the above-mentioned
method of the invention, this may conveniently performed by use of
an assay as described in connection with Example 2 herein.
[0200] With regard to screening in general, a filter assay based on
the following is generally applicable:
[0201] A microorganism capable of expressing the mutated amylolytic
enzyme of interest is incubated on a suitable medium and under
suitable conditions for the enzyme to be secreted, the medium being
provided with a double filter comprising a first protein-binding
filter and on top of that a second filter exhibiting a low protein
binding capability. The microorganism is located on the second
filter. Subsequent to the incubation, the first filter comprising
enzymes secreted from the microorganisms is separated from the
second filter comprising the microorganisms. The first filter is
subjected to screening for the desired enzymatic activity and the
corresponding microbial colonies present on the second filter are
identified.
[0202] The filter used for binding the enzymatic activity may be
any protein binding filter e.g. nylon or nitrocellulose. The
topfilter carrying the colonies of the expression organism may be
any filter that has no or low affinity for binding proteins e.g.
cellulose acetate or Durapore.TM.. The filter may be pretreated
with any of the conditions to be used for screening or may be
treated during the detection of enzymatic activity.
[0203] The enzymatic activity may be detected by a dye,
fluorescence, precipitation, pH indicator, IR-absorbance or any
other known technique for detection of enzymatic activity.
[0204] The detecting compound may be immobilized by any
immobilizing agent e.g. agarose, agar, gelatine, polyacrylamide,
starch, filter paper, cloth; or any combination of immobilizing
agents.
[0205] .alpha.-Amylase activity is detected by Cibacron Red
labelled amylopectin, which is immobilized on agarose. For
screening for variants with increased thermal and high-pH
stability, the filter with bound .alpha.-amylase variants is
incubated in a buffer at pH 10.5 and 600 or 65.degree. C. for a
specified time, rinsed briefly in deionized water and placed on the
amylopectin-agarose matrix for activity detection. Residual
activity is seen as lysis of Cibacron Red by amylopectin
degradation. The conditions are chosen to be such that activity due
to the .alpha.-amylase having the amino acid sequence shown in SEQ
ID No. 2 can barely be detected. Stabilized variants show, under
the same conditions, increased colour intensity due to increased
liberation of Cibacron Red.
[0206] For screening for variants with an activity optimum at a
lower temperature and/or over a broader temperature range, the
filter with bound variants is placed directly on the
amylopectin-Cibacron Red substrate plate and incubated at the
desired temperature (e.g. 4.degree. C., 10.degree. C. or 30.degree.
C.) for a specified time. After this time activity due to the
.alpha.-amylase having the amino acid sequence shown in SEQ ID No.
2 can barely be detected, whereas variants with optimum activity at
a lower temperature will show increase amylopectin lysis. Prior to
incubation onto the amylopectin matrix, incubation in all kinds of
desired media--e.g. solutions containing Ca.sup.2+, detergents,
EDTA or other relevant additives--can be carried out in order to
screen for changed dependency or for reaction of the variants in
question with such additives.
[0207] Testing of Variants of the Invention
[0208] The testing of variants of the invention may suitably be
performed by determining the starch-degrading activity of the
variant, for instance by growing host cells transformed with a DNA
sequence encoding a variant on a starch-containing agarose plate
and identifying starch-degrading host cells. Further testing as to
altered properties (including specific activity, substrate
specificity, cleavage pattern, thermoactivation, pH optimum, pH
dependency, temperature optimum, and any other parameter) may be
performed in accordance with methods known in the art.
[0209] Expression of .alpha.-amylase Variants
[0210] 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.
[0211] 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.
[0212] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA sequence
encoding an .alpha.-amylase variant of the invention, especially in
a bacterial host, are the promoter of the lac operon of E. coli,
the Streptomyces coelicolor agarase gene dagA promoters, the
promoters of the Bacillus licheniformis .alpha.-amylase gene
(amyL), the promoters of the Bacillus stearothermophilus maltogenic
amylase gene (amyM), the promoters of the Bacillus
amyloliquefaciens .alpha.-amylase (amyQ), the promoters of the
Bacillus subtilis xylA and xylB genes etc. For transcription in a
fungal host, examples of useful promoters are those derived from
the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, A. niger neutral .alpha.-amylase, A. niger
acid stable .alpha.-amylase, A. niger glucoamylase, Rhizomucor
miehei lipase, A. oryzae alkaline protease, A. oryzae triose
phosphate isomerase or A. nidulans acetamidase.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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. (1989)).
[0218] 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.
[0219] 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.
[0220] Examples of suitable bacteria are grampositive 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
gramnegative bacteria such as E. coli. The transformation of the
bacteria may, for instance, be effected by protoplast
transformation or by using competent cells in a manner known per
se.
[0221] The yeast organism may favourably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces
cerevisiae. The filamentous fungus may advantageously belong to a
species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus
niger. Fungal cells may be transformed by a process involving
protoplast formation and transformation of the protoplasts followed
by regeneration of the cell wall in a manner known per se. A
suitable procedure for transformation of Aspergillus host cells is
described in EP 238 023.
[0222] 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.
[0223] 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).
[0224] 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.
[0225] Industrial Applications
[0226] The .alpha.-amylase variants of this invention possesses
valuable properties allowing for a variety of industrial
applications. In particular, enzyme variants of the invention are
applicable as a component in washing, dishwashing and hard-surface
cleaning detergent compositions. Numerous variants are particularly
useful in the production of sweeteners and ethanol from starch,
and/or for textile desizing. Conditions for conventional
starch-conversion processes, including starch liquefaction and/or
saccharification processes, are described in, e.g., U.S. Pat. No.
3,912,590 and in EP patent publications Nos. 252,730 and
63,909.
[0227] Production of sweeteners from starch: 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 h. In order to ensure an optimal
enzyme stability under these conditions, 1 mM of calcium is added
(40 ppm free calcium ions).
[0228] 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.
[0229] 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.).
[0230] At least 3 enzymatic improvements of this process could be
envisaged. All three improvements could be seen as individual
benefits, but any combination (e.g. 1+2, 1+3, 2+3 or 1+2+3) could
be employed:
[0231] Improvement 1. Reduction of the Calcium Dependency of the
Liquefying Alpha-amylase.
[0232] Addition of free calcium is required to ensure adequately
high stability of the .alpha.-amylase, but free calcium strongly
inhibits the activity of the glucoseisomerase and needs to be
removed, by means of an expensive unit operation, to an extent
which reduces the level of free calcium to below 3-5 ppm. Cost
savings could be obtained if such an operation could be avoided and
the liquefaction process could be performed without addition of
free calcium ions.
[0233] 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.
[0234] Improvement 2. Reduction of Formation of Unwanted Maillard
Products
[0235] The extent of formation of unwanted Maillard products during
the liquefaction process is dependent on the pH. Low pH favours
reduced formation of Maillard products. It would thus be desirable
to be able to lower the process pH from around pH 6.0 to a value
around pH 4.5; unfortunately, all commonly known, thermostable
Termamyl-like .alpha.-amylases are not very stable at low pH (i.e.
pH<6.0) and their specific activity is generally low.
[0236] Achievement of the above-mentioned goal requires a
Termamyl-like .alpha.-amylase which is stable at low pH in the
range of 4.5-5.5 and at free calcium concentrations in the range of
0-40 ppm, and which maintains a high specific activity.
[0237] Improvement 3.
[0238] It has been reported previously (U.S. Pat. No. 5,234,823)
that when saccharifying with A. niger glucoamylase and B.
acidopullulyticus pullulanase, the presence of residual
.alpha.-amylase activity from the liquefaction process can lead to
lower yields of dextrose if the .alpha.-amylase is not inactivated
before the saccharification stage. This inactivation can typically
be carried out by adjusting the pH to below 4.3 at 95.degree. C.,
before lowering the temperature to 60.degree. C. for
saccharification.
[0239] The reason for this negative effect on dextrose yield is not
fully understood, but it is assumed that the liquefying
.alpha.-amylase (for example Termamyl.TM. 120 L from B.
licheniformis) generates "limit dextrins" (which are poor
substrates for B. acidopullulyticus pullulanase) by hydrolysing
1,4-.alpha.-glucosidic linkages close to and on both sides of the
branching points in amylopectin. Hydrolysis of these limit dextrins
by glucoamylase leads to a build-up of the trisaccharide panose,
which is only slowly hydrolysed by glucoamylase.
[0240] The development of a thermostable .alpha.-amylase which does
not suffer from this disadvantage would be a significant process
improvement, as no separate inactivation step would be
required.
[0241] If a Termamyl-like, low-pH-stable .alpha.-amylase is
developed, an alteration of the specificity could be an advantage
needed in combination with increased stability at low pH.
[0242] The methodology and principles of the present invention make
it possible to design and produce variants according to the
invention having required properties as outlined above. In this
connection, particularly interesting mutations are mutations in a
Termamyl-like .alpha.-amylase [for example Termamyl.TM. itself (B.
licheniformis .alpha.-amylase; SEQ ID No. 2); or a Termamyl-like
.alpha.-amylase having an N-terminal amino acid sequence (i.e. the
partial sequence up to the amino acid position corresponding to
position 35 in Termamyl.TM.) which is identical to that in B.
amyloliquefaciens .alpha.-amylase (SEQ ID No. 4), i.e. a
Termamyl-like .alpha.-amylase having the following N-terminal
sequence relative to amino acid sequence of Termamyl.TM.:
[0243] A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I, where
an asterisk (*) indicates deletion of the amino acid residue in
question] at positions corresponding to any of the following
positions in Termamyl.TM.:
[0244] H133
[0245] H156
[0246] A181
[0247] A209
[0248] G310
[0249] H450
[0250] V128
[0251] N104
[0252] V54
[0253] S187
[0254] H293
[0255] A294
[0256] (where each of the latter amino acid residues may be
replaced by any other amino acid residue, i.e. any other residue
chosen among A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W,
Y and V), as well as the following triple deletions:
[0257] K370*+G371*+D372*
[0258] D372*+S373*+Q374*
[0259] Particularly preferred substitutions at the above-indicated
positions are the following:
[0260] H133I
[0261] H156Y
[0262] A181T
[0263] A209V
[0264] G310D
[0265] H450Y
[0266] V128E
[0267] N104D
[0268] V54W,Y,F,I,L
[0269] S187D
[0270] H293Y
[0271] A294V.
[0272] Any combination of one or more (i.e. one, two, three, four .
. . etc.) of the above indicated mutations may appropriately be
effected in a Termamyl-like .alpha.-amylase in the context in
question, and particularly interesting variants of the invention in
the context of achieving one or more of the above-mentioned
improvements in relation to the starch liquefaction behaviour of
.alpha.-amylases include variants comprising combinations of
multiple mutations corresponding to the following combinations of
mutations in Termamy.TM. (SEQ ID No. 2) itself:
[0273]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A
294V+K370*+G371*+D372*;
[0274]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A
294V+D372*+S373*+Q374*;
[0275]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A
294V+K370*+G371*+D372*;
[0276]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A
294V+D372*+S373*+Q374*;
[0277]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A
294V+K370*+G371*+D372*;
[0278]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A
294V+D372*+S373*+Q374*;
[0279]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A
294V+K370*+G371*+D372*;
[0280]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A
294V+D372*+S373*+Q374*;
[0281]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A
294V+K370*+G371*+D372*;
[0282]
H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A
294V+D372*+S373*+Q374*;
[0283] Further interesting variants of the invention in this
context include variants comprising single or multiple mutations
corresponding to the following single or multiple mutations in
Termamyl.TM. itself:
[0284] mutations (amino acid substitutions) at positions N172 (e.g.
N172R,K), S187 (e.g. S187D), N188 (e.g. N188P), N190 (e.g.
N190L,F), H205 (e.g. H205C), D207 (e.g. D207Y), A210 (e.g. A210S),
Q264 (e.g. Q264S) or N265 (e.g. N265Y);
[0285] the following multiple mutations;
[0286] H156Y+A181T+A209V;
[0287] H156Y+A181T+N190F+A209V+Q264S
[0288]
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A20-
9V;
[0289]
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1
90F+A209V; or
[0290]
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1
90F+A209V+Q264S
[0291] as well as combinations of any two or more of the latter
single or multiple mutations.
[0292] As already indicated, numerous variants according to the
invention are particularly well suited for use in starch
conversion, e.g. in starch liquefaction. In this connection, a
further aspect of the present invention relates to compositions
comprising a mixture of:
[0293] (i) the .alpha.-amylase from B. licheniformis having the
sequence shown in SEQ ID No. 2 with one or more variants (mutant
.alpha.-amylases) according to the invention derived from (as the
parent Termamyl-like .alpha.-amylase) the B. stearothermophilus
.alpha.-amylase having the sequence shown in SEQ ID No. 6; or
[0294] (ii) the .alpha.-amylase from B. stearothermophilus having
the sequence shown in SEQ ID No. 6 with one or more variants
(mutant .alpha.-amylases) according to the invention derived from
one or more other parent Termamyl-like .alpha.-amylases (e.g. from
the B. licheniformis .alpha.-amylase having the sequence shown in
SEQ ID No. 2, or from one of the other parent Termamyl-like
.alpha.-amylases specifically referred to herein); or
[0295] (iii) one or more variants (mutant .alpha.-amylases)
according to the invention derived from (as the parent
Termamyl-like .alpha.-amylase) the B. stearothermophilus
.alpha.-amylase having the sequence shown in SEQ ID No. 6 with one
or more variants (mutant .alpha.-amylases) according to the
invention derived from one or more other parent Termamyl-like
.alpha.-amylases (e.g. from the B. licheniformis .alpha.-amylase
having the sequence shown in SEQ ID No. 2, or from one of the other
parent Termamyl-like .alpha.-amylases specifically referred to
herein).
[0296] Preferred mutations in a variant of B. stearothermophilus
.alpha.-amylase to be incorporated in such a mixture include
substitutions at N193 and/or at E210, and/or the pairwise deletions
R179*+G180* or I181*+G182* (using the numbering of the amino acid
sequence for this particular .alpha.-amylase).
[0297] Compositions of one of the latter types, containing B.
stearothermophilus (.alpha.-amylase or a variant thereof according
to the invention, appear to have great potential for use in starch
liquefaction. The ratio (expressed, e.g., in terms of mg of active
amylolytic protein per liter of liquid medium) between the
individual .alpha.-amylolytic components of a given mixture will
depend on the exact nature and properties of each component.
[0298] Detergent Compositions
[0299] 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.
[0300] 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.
[0301] .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.
[0302] The present invention is further described with reference to
the appended drawing, in which:
[0303] FIG. 1 shows the DNA sequence, together with the stop codon
TAA, encoding the Bacillus strain NCIB 12512 .alpha.-amylase
described in WO 95/26397, together with the amino acid sequence of
the encoded .alpha.-amylase (cf. FIG. 2).
[0304] FIG. 2 is an alignment of the amino acid sequences of four
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:
[0305] 1: the amino acid sequence of the Bacillus strain NCIB 12512
.alpha.-amylase described in WO 95/26397;
[0306] 2: the amino acid sequence of the Bacillus strain NCIB 12513
.alpha.-amylase described in WO 95/26397;
[0307] 3: the amino acid sequence of the B. stearothermophilus
.alpha.-amylase as shown in SEQ ID No. 6 herein;
[0308] 4: the amino acid sequence of the Bacillus sp. #707
[0309] .alpha.-amylase described by Tsukamoto et al. in Biochem.
Biophys. Res. Commun. 151 (1988), pp. 25-31.
[0310] The numbers on the extreme right of the figure give the
running total number of amino acids for each of the sequences in
question. Note that for the sequence numbered 3 (corresponding to
the sequence in SEQ ID No. 6), the alignment results in "gaps" at
the positions corresponding to amino acid No. 1 and No. 175,
respectively, in the sequences numbered 1, 2 and 4.
[0311] FIG. 3 illustrates the PCR strategy employed in Example 2
(vide infra).
MATERIALS AND METHODS
[0312] Construction of pSNK101
[0313] This E. coli/bacillus shuttle vector can be used to
introduce mutations without expression of .alpha.-amylase in E.
coli and then be modified in such way that the .alpha.-amylase is
active in Bacillus. The vector was constructed as follows: The
.alpha.-amylase gene in the pX vector (pDN1528 with the following
alterations within amyL: BAN(1-33), H156Y, A181T, N190F, A209V,
Q264S, the plasmid pDN1528 is further described in Example 1) was
inactivated by interruption in the PstI site in the 5'coding region
of the alph.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:
5'-gacctgcagtcaggcaacta-3' and the reverse primer:
5'-tagagtcgacctgcaggcat-3'. The PCR amplicon and the pX plasmid
containing the .alpha.-amylase gene were digested with PstI at
37.degree. C. for 2 hrs. The pX vector fragment and the E. coli
origin amplicon were ligated at room temperature. for 1 h and
transformed in E. coli by electrotransformation. The resulting
vector is designated pSnK101.
[0314] Fermentation and Purification of .alpha.-amylase
Variants
[0315] A B. subtilis strain harbouring the relevant expression
plasmid is streaked on a LB-agar plate with 15 .mu.g/ml
chloramphenicol from -80.degree. C. stock, and grown overnight at
37.degree. C. The colonies are transferred to 100 ml BPX media
supplemented with 15 .mu.g/ml chloramphenicol in a 500 ml shaking
flask.
3 Composition of BPX medium: 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
[0316] The culture is shaken at 37.degree. C. at 270 rpm for 5
days.
[0317] Cells and cell debris are removed from the fermentation
broth by centrifugation at 4500 rpm in 20-25 min. Afterwards the
supernatant is filtered to obtain a completely clear solution. The
filtrate is concentrated and washed on a UF-filter (10000 cut off
membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The
UF-filtrate is applied on a S-sepharose F.F. and elution is carried
out by step elution with 0.2M NaCl in the same buffer. The eluate
is dialysed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose
F.F. and eluted with a linear gradient from 0-0.3M NaCl over 6
column volumes. The fractions which contain the activity (measured
by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and
remaining color was removed by a treatment with 0.5% W/vol. active
coal in 5 min.
[0318] Assay for .alpha.-Amylase Activity
[0319] .alpha.-Amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-coloured starch polymer which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0320] 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.
[0321] 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.
[0322] General Method for Random Mutagenesis by Use of the DOPE
Program
[0323] The random mutagenesis may be carried out by the following
steps:
[0324] 1. Select regions of interest for modification in the parent
enzyme
[0325] 2. Decide on mutation sites and nonmutated sites in the
selected region
[0326] 3. Decide on which kind of mutations should be carried out,
e.g. with respect to the desired stability and/or performance of
the variant to be constructed
[0327] 4. Select structurally reasonable mutations.
[0328] 5. Adjust the residues selected by step 3 with regard to
step 4.
[0329] 6. Analyze by use of a suitable dope algoritm the nucleotide
distribution.
[0330] 7. If necessary, adjust the wanted residues to genetic code
realism (e.g. taking into account constraints resulting from the
genetic code (e.g. in order to avoid introduction of stop
codons))(the skilled person will be aware that some codon
combinations cannot be used in practice and will need to be
adapted)
[0331] 8. Make primers
[0332] 9. Perform random mutagenesis by use of the primers
[0333] 10. Select resulting .alpha.-amylase variants by screening
for the desired improved properties.
[0334] Suitable dope algorithms for use in step 6 are well known in
the art. One algorithm is described by Tomandl, D. et al., Journal
of Computer-Aided Molecular Design, 11 (1997), pp. 29-38). Another
algorithm, DOPE, is described in the following:
[0335] The Dope Program
[0336] The "DOPE" program is a computer algorithm useful to
optimize the nucleotide composition of a codon triplet in such a
way that it encodes an amino acid distribution which resembles most
the wanted amino acid distribution. In order to assess which of the
possible distributions is the most similar to the wanted amino acid
distribution, a scoring function is needed. In the "Dope" program
the following function was found to be suited: 1 s i = 1 N ( x i y
i ( 1 - x i ) 1 - y i y i y i ( 1 - y i ) 1 - y i ) w i ,
[0337] where the x.sub.i's are the obtained amounts of amino acids
and groups of amino acids as calculated by the program, y.sub.i's
are the wanted amounts of amino acids and groups of amino acids as
defined by the user of the program (e.g. specify which of the 20
amino acids or stop codons are wanted to be introduced, e.g. with a
certain percentage (e.g. 90% Ala, 3% Ile, 7% Val), and w.sub.i's
are assigned weight factors as defined by the user of the program
(e.g. depending on the importance of having a specific amino acid
residue inserted into the position in question). N is 21 plus the
number of amino acid groups as defined by the user of the program.
For purposes of this function 0.sup.0 is defined as being 1.
[0338] A Monte-Carlo algorithm (one example being the one described
by Valleau, J. P. & Whittington, S. G. (1977) A guide to Mont
Carlo for statistical mechanics: 1 Highways. In "Stastistical
Mechanics, Part A" Equlibrium Techniqeues ed. B. J. Berne, New
York: Plenum) is used for finding the maximum value of this
function. In each iteration the following steps are performed:
[0339] 1. A new random nucleotide composition is chosen for each
base, where the absolute difference between the current and the new
composition is smaller than or equal to d for each of the four
nucleotides G,A,T,C in all three positions of the codon (see below
for definition of d).
[0340] 2. The scores of the new composition and the current
composition are compared by the use of the function s as described
above. If the new score is higher or equal to the score of the
current composition, the new composition is kept and the current
composition is changed to the new one. If the new score is smaller,
the probability of keeping the new composition is
exp(1000(new_score-current_score)).
[0341] A cycle normally consists of 1000 iterations as described
above in which d is decreasing linearly from 1 to 0. One hundred or
more cycles are performed in an optimization process. The
nucleotide composition resulting in the highest score is finally
presented.
EXAMPLE 1
[0342] Construction of Termamyl.TM. Variants in Accordance with the
Invention
[0343] Termamyl (B. licheniformis .alpha.-amylase SEQ ID NO. 2) is
expressed in B. subtilis from a plasmid denoted pDN1528. This
plasmid contains the complete gene encoding Termamyl, amyL, the
expression of which is directed by its own promoter. Further, the
plasmid contains the origin of replication, ori, from plasmid
pUB110 and the cat gene from plasmid pC194 conferring resistance
towards chloramphenicol. pDN1528 is shown in FIG. 9 of WO
96/23874.
[0344] A specific mutagenesis vector containing a major part of the
coding region of SEQ ID NO 1 was prepared. The important features
of this vector, denoted pJeEN1, include an origin of replication
derived from the pUC plasmids, the cat gene conferring resistance
towards chloramphenicol, and a frameshift-containing version of the
bla gene, the wild type of which normally confers resistance
towards ampicillin (amp.sup.R phenotype). This mutated version
results in an amp.sup.S phenotype. The plasmid pJeEN1 is shown in
FIG. 10 of WO 96/23874, and the E. coli origin of replication, ori,
bla, cat, the 5'-truncated version of the Termamyl amylase gene,
and selected restriction sites are indicated on the plasmid.
[0345] Mutations are introduced in amyL by the method described by
Deng and Nickoloff (1992, Anal. Biochem. 200, pp. 81-88) except
that plasmids with the "selection primer" (primer #6616; see below)
incorporated are selected based on the amp.sup.R phenotype of
transformed E. coli cells harboring a plasmid with a repaired bla
gene, instead of employing the selection by restriction enzyme
digestion outlined by Deng and Nickoloff. Chemicals and enzymes
used for the mutagenesis were obtained from the Chameleon
mutagenesis kit from Stratagene (catalogue number 200509).
[0346] After verification of the DNA sequence in variant plasmids,
the truncated gene, containing the desired alteration, is subcloned
into pDN1528 as a PstI-EcoRI fragment and transformed into the
protease- and amylase-depleted Bacillus subtilis strain SHA273
(described in WO92/11357 and WO95/10603) in order to express the
variant enzyme.
[0347] The Termamyl variant V54W was constructed by the use of the
following mutagenesis primer (written 5' to 3', left to right):
[0348] PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG
[0349] The Termamyl variant A52W+V54W was constructed by the use of
the following mutagenesis primer (written 5' to 3', left to
right):
[0350] PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT CG
[0351] Primer #6616 (written 5' to 3', left to right; P denotes a
5' phosphate):
[0352] P CTG TGA CTG GTG AGT ACT CAA CCA AGT C
[0353] The Termamyl variant V54E was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0354] PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG
[0355] The Termamyl variant V54M was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0356] PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG
[0357] The Termamyl variant V54I was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0358] PGG TCG TAG GCA CCG TAG CCA ATA TCC GCT TG
[0359] The Termamyl variants Y290E and Y290K were constructed by
the use of the following mutagenesis primer (written 5'-3', left to
right):
[0360] PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA C
[0361] Y represent an equal mixture of C and T. The presence of a
codon encoding either Glutamate or Lysine in position 290 was
verified by DNA sequencing.
[0362] The Termamyl variant N190F was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0363] PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C
[0364] The Termamyl variant N188P+N190F was constructed by the use
of the following mutagenesis primer (written 5'-3', left to
right):
[0365] PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC
[0366] The Termamyl variant H140K+H142D was constructed by the use
of the following mutagenesis primer (written 5'-3', left to
right):
[0367] PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG
[0368] The Termamyl variant H156Y was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0369] PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG
[0370] The Termamyl variant A181T was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0371] PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC
[0372] The Termamyl variant A209V was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0373] PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC
[0374] The Termamyl variant Q264S was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0375] PCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC
[0376] The Termamyl variant S187D was constructed by the use of the
following mutagenesis primer (written 5'-3', left to right):
[0377] PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC
[0378] The Termamyl variant DELTA(K370-G371-D372) (i.e. deleted of
amino acid residues nos. 370, 371 and 372) was constructed by the
use of the following mutagenesis primer (written 5'-3', left to
right):
[0379] PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC
[0380] The Termamyl variant DELTA(D372-S373-Q374) was constructed
by the use of the following mutagenesis primer (written 5'-3', left
to right):
[0381] PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C
[0382] The Termamyl variants A181T and A209V were combined to
A181T+A209V by digesting the A181T containing pDN1528-like plasmid
(i.e. pDN1528 containing within amyL the mutation resulting in the
A181T alteration) and the A209V-containing pDN1528-like plasmid
(i.e. pDN1528 containing within amyL the mutation resulting in the
A209V alteration) with restriction enzyme ClaI which cuts the
pDN1528-like plasmids twice resulting in a fragment of 1116 bp and
the vector-part (i.e. contains the plasmid origin of replication)
of 3850 bp. The fragment containing the A209V mutation and the
vector part containing the A181T mutation were purified by QIAquick
gel extraction kit (purchased from QIAGEN) after separation on an
agarose gel. The fragment and the vector were ligated and
transformed into the protease and amylase depleted Bacillus
subtilis strain referred to above. Plasmid from amy+ (clearing
zones on starch containing agar-plates) and chloramphenicol
resistant transformants were analysed for the presence of both
mutations on the plasmid.
[0383] In a similar way as described above, H156Y and A209V were
combined utilizing restriction endonucleases Acc65I and EcoRI,
giving H156Y+A209V.
[0384] H156Y +A209V and A181T+A209V were combined into
H156Y+A181T+A209V by the use of restriction endonucleases Acc65I
and HindIII.
[0385] The 35 N-terminal residues of the mature part of Termamyl
variant H156Y+A181T+A209V were substituted by the 33 N-terminal
residues of the B. amyloliquefaciens .alpha.-amylase (SEQ ID NO 4)
(which in the present context is termed BAN) by a SOE-PCR approach
(Higuchi et al. 1988, Nucleic Acids Research 16:7351) as
follows:
4 Primer 19364 (sequence 5'-3'): CCT CAT TCT GCA GCA GCA GCC GTA
AAT GGC ACG CTG Primer 19362: CCA GAC GGC AGT AAT ACC GAT ATC CGA
TAA ATG TTC CG Primer 19363: CGG ATA TOG GTA TTA CTG CCG TCT GGA
TTC Primer 1C: CTC GTC CCA ATC GGT TCC GTC
[0386] A standard PCR, polymerase chain reaction, was carried out
using the Pwo thermostable polymerase from Boehringer Mannheim
according to the manufacturer's instructions and the temperature
cyclus: 5 minutes at 94.degree. C., 25 cycles of (94.degree. C. for
30 seconds, 50.degree. C. for 45 seconds, 72.degree. C. for 1
minute), 72.degree. C. for 10 minutes.
[0387] An approximately 130 bp fragment was amplified in a first
PCR denoted PCR1 with primers 19364 and 19362 on a DNA fragment
containing the gene encoding the B. amyloliquefaciens
.alpha.-amylase.
[0388] An approximately 400 bp fragment was amplified in another
PCR denoted PCR2 with primers 19363 and 1C on template pDN1528.
[0389] PCR1 and PCR2 were purified from an agarose gel and used as
templates in PCR3 with primers 19364 and 1C, which resulted in a
fragment of approximately 520 bp. This fragment thus contains one
part of DNA encoding the N-terminus from BAN fused to a part of DNA
encoding Termamyl from the 35th amino acid.
[0390] The 520 bp fragment was subcloned into a pDN1528-like
plasmid (containing the gene encoding Termamyl variant
H156Y+A181T+A209V) by digestion with restriction endonucleases PstI
and SacII, ligation and transformation of the B. subtilis strain as
previously described. The DNA sequence between restriction sites
PstI and SacII was verified by DNA sequencing in extracted plasmids
from amy+ and chloramphenicol resistant transformants.
[0391] The final construct containing the correct N-terminus from
BAN and H156Y+A181T+A209V was denoted
BAN(1-35)+H156Y+A181T+A209V.
[0392] N190F was combined with BAN(1-35)+H156Y+A181T+A209V giving
BAN(1-35)+H156Y+A181T+N190F+A209V by carrying out mutagenesis as
described above exept that the sequence of amyL in pJeEN1 was
substituted by the DNA sequence encoding Termamyl variant
BAN(1-35)+H156Y+A181T+A209V
[0393] Q264S was combined with BAN(1-35)+H156Y+A181T+A209V giving
BAN(1-35)+H156Y+A181T+A209V+Q264S by carrying out mutagenesis as
described above exept that the sequence of amyL in pJeEN was
substituted by the the DNA sequence encoding Termamyl variant
BAN(1-35)+H156Y+A181T+A- 209V
[0394] BAN(1-35)+H156Y+A181T+A209V+Q264S and
BAN(1-35)+H156Y+A181T+N190F+A- 209V were combined into
BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S utilizing restriction
endonucleases BsaHI (BsaHI site was introduced close to the A209V
mutation) and PstI.
EXAMPLE 2
[0395] Construction, by Localized Random, Doped Mutagenesis, of
Termamyl-like .alpha.-amylas Variants Having an Improved Stability
at Low pH and a Reduced Dependency on Calcium Ions for Stability
Compared to the Parent Enzyme
[0396] .alpha.-amylases are of great importance for the industrial
starch liquefaction process. The variant of the thermostable B.
licheniformis .alpha.-amylase consisting of amino acids 1-33 of the
B. amyloliquefaciens amylase (SEQ ID NO 4) fused to amino acids
36-483 of the B. licheniformis amylase (SEQ ID NO 2) and further
comprising the following mutations:
[0397] Y156, T181, F190, V209 and S264 (the construction of this
variant is described in Example 1)
[0398] has a very satisfactory stability at low pH and low calcium
concentrations. In an attempt to further improve the stability at
low pH and low calcium concentration of said .alpha.-amylase
variant random mutagenesis in preselected regions wase
performed.
5 The regions were: Region: Residue: I: Phe153-Thr163 II:
Gln178-Asn192 III: His205-Arg214 IV: Ala232-Asp237 and VIII:
Gly131-Lys136 IX: Asp164-Tyr175 X: Tyr262-Thr278 Region changed
Total % Mean % Number of residues I: 35 88 8 out of 11 II: 20 86 11
out of 15 III: 27 88 10 out of 10 IV: 34 92 11 out of 12 VIII: 39
86 6 out of 6 IX: 46 93 12 out of 12 X: 27 90 12 out of 13 VIII +
IX: 18 VIII + IX + II: 4 II + III + IV: 2 IV + I: 12
[0399] The numbers under Total % give the total number of wild-type
(wt) amino acids desired in a given region after doping. The number
is obtained by multiplication of the number of mutated positions
(e.g. 8 with respect to region I) by their respective wt. With
respect to region I the desired total % is
80*80*90*90*90*90*95*90/100=35%.
[0400] The Mean % is the mean doping level for the total number of
positions of the region in question (e.g. 11 positions with respect
to region I). For region I the mean % is calculated as follows:
80+80+90+90+90+90+95+90=705 divided by 11=88%
[0401] The DOPE software (see Materials and Methods) was used to
determine spiked codons for each suggested change in the seven
regions minimizing the amount of stop codons. The exact
distribution of nucleotides was calculated in the three positions
of the codon to give the suggested population of amino acid
changes. The doped regions were doped specifically in the indicated
positions to have a high chance of getting the desired residues,
but still allow other possibilities.
[0402] For instance, the original H156 in the wt sequence was
mutated into an Y, meaning a new codon, and then doped 10% for
other residues. That is the DNA sequence has the code for a Y
instead for a H. In position 156 the Tyr has been programmed to be
90% desired and other residues has been freely allowed. For some
positions it was not possible to create the suggested population of
amino acid residues because the genetic code restricted the
structurally and functionally desired residues. The resulting seven
doped oligonucleotides are shown in tables 1-7: with the wt
nucleotide and amino acid sequences and the distribution of
nucleotides for each doped position. All library primers were
synthesized as sense strands.
6TABLE 1 Library DASI (Phe153-Thr163) 153 154 155 156 157 158 159
160 161 162 163 Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly Thr Primer:
5'CGC GGC AGC ACA TAC AGC GAT T1T 2A3 TGG 45T TGG 67T 8AT TTT GAC
GGA A9C GAT TGG GAC GAG TCC CGA AAG3' Distribution of nucleotides
for each doped position. 1: 80% T, 20% A. 2: 96% A, 2% G, 2% C. 3:
98% A, 2% T. 4: 93% T, 4% G, 3% A. 5: 97% A, 3% G. 6: 98% T, 2% A.
7: 97% A, 3% C. 8: 90% C, 10% T. 9: 95% C, 5% A.
[0403]
7TABLE 2 Library DASh (Gln178-Asn192) 178 179 180 181 182 183 184
185 186 187 188 189 190 191 192 Gln Gly Lys Thr Trp Asp Trp Glu Val
Ser Asn Glu Phe Gly Asn Primer: 5'CTG AAC CGC ATC TAT AAG TTT 1A2
34T AAG 567 TGG GAT 89G GA10 GTT A11T 1213T GAA T1415 161718 AAC
TAT GAT TAT TTG ATG TAT3' Distribution of nucleotides for each
doped position. 1: 93% C, 7% A. 2: 84% G, 16% T. 3: 95% G, 5% A. 4:
95% G, 5% C. 5: 94% A, 6% G. 6: 95% C, 5% A. 7: 62% T, 38% G. 8:
87% T, 13% A. 9: 91% G, 9% C. 10: 92% G, 8% T. 11: 90% G, 5% A, 5%
C. 12: 88% A, 12% C. 13: 88% A, 12% C. 14: 93% T, 5% A, 2% C. 15:
97% T, 3% G. 16: 86% G, 14% A. 17: 89% G, 11% C. 18: 60% G, 40%
T.
[0404]
8TABLE 3 Library DASIII (His205-Arg214) 205 206 207 208 209 210 211
212 213 214 His Pro Asp Val Val Ala Glu Ile Lys Arg Primer: 5'TAT
GCC GAC ATC GAT TAT GAC 12T 3CT 456 7TT 8910 1112T 13A14 15T16 A17A
1819A TGG GGC ACT TGG TAT GCC AAT 3' Distribution of nucleotides
for each doped position. 1: 89% C, 11% T. 2: 89% A, 11% G. 3: 95%
C, 2.5% T, 2.5% A. 4: 96% G, 1% A, 3% T. 5: 96% A, 4% C. 6: 98% T,
2% A. 7: 95% G, 2.5% A, 2.5% C. 8: 93% G, 7% A. 9: 96% T, 4% A. 10:
84% A, 16% G. 11: 81% G, 7% A, 7% T, 5% C. 12: 98% C, 2% A. 13: 96%
G, 4% C. 14: 94% G, 6% T. 15: 82% A, 18% T. 16: 50% A, 50% T. 17:
90% A, 10% G. 18: 70% A, 30% C. 19: 86% G, 14% A
[0405]
9TABLE 4 Library DASIV (Ala232-Asp243) 232 233 234 235 236 237 238
239 240 241 242 243 Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg Asp
Primer: 5'TTG GAC GGT TTC CGT CTT GAT 12T G3T AAA 456 7TT A8G T9T
1011T T12T 13T14 1516G GA17 TGG GTT AAT CAT GTC AGG GAA
Distribution of nucleotides for each doped position. 1: 93% G, 3.5%
A, 3.5% T. 2: 94% C, 4% T. 3: 94% T, 6% C. 4: 93% C, 2% T, 2% A, 3%
G. 5: 98% A, 2% T. 6: 98% T, 2% A. 7: 95% A, 5% C. 8: 94% A, 6% G.
9: 90% T, 10% A. 10: 89% T, 11% A. 11: 89% C, 11% A. 12: 95% T, 5%
A 13: 64% C, 33% T, 3% A. 14: 93% A, 7% T. 15: 90% A, 10% C. 16:
90% G, 5% A, 5% C 17: 90% T, 10% A
[0406]
10TABLE 5 Library DASVIII (Gly131-Lys136) 131 132 133 134 135 136
Gly Glu His Leu Ile lys Primer: 5'GCT GAC CGC AAC CGC GTA ATT TCA
123 GA4 56T 78A 9TA A10G GCC TGG ACA CAT TTT CAT TTT 3'
Distribution of nucleotides for each doped position. 1: 91% G, 9%
A. 2: 87% G, 13% C. 3: 90% T, 10% G. 4: 90% G, 10% T. 5: 85% C, 8%
T, 7% A. 6: 89% A, 9% T, 2% C. 7: 88% T, 12% A 8: 88% T, 11% C, 1%
G 9: 92% A, 8% T 10: 93% A, 7% G
[0407]
11TABLE 6 Library DASIX (Asp164-Tyr175) 164 165 166 167 168 169 170
171 172 173 174 175 Asp Trp Asp Glu Ser Arg lys Leu Asn Arg Ile Tyr
Primer: 5'TGG TAC CAT TTT GAC GGA ACC GAT TGG 1A2 GAG 3CG CGA A4G
56A A7T A8G 9 1011 T12T AAG TTT CAA GGA AAG GCT TGG 3' Distribution
of nucleotides for each doped position. 1: 94% G, 6% A. 2: 96% T,
4% G. 3: 92% T, 4% A, 4% G. 4: 95% A, 5% G. 5: 93% C, 7% A. 6: 92%
T, 8% A. 7: 90% A, 5% G, 5% C. 8: 90% G, 10% A. 9: 92% A, 6% G, 2%
T. 10: 92% T, 8% A. 11: 50% T, 50% C. 12: 96% A, 4% T.
[0408]
12TABLE 7 Library DASX (Tyr262-Asn278) 262 263 264 265 266 267 268
269 270 271 272 273 374 275 276 277 278 Tyr Trp Ser Asn Asp Leu Gly
Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Primer: 5'GAA ATG TTT ACG
GTA GCT GAA T1T TGG 234 56T 7A8 91011 1213T 1415T 16T17 0A18 Al9T
T2OT 21T22 A23C A24G ACA 25AT TTT AAT CAT TCA GTG TTT GAC3'
Distribution of nucleotides for each doped position. 1: 95% A, 5%
T. 2: 97% A, 3% G. 3: 95% G, 2.5% A, 2.5% C. 4: 94% T, 6.2% G. 5:
97% A, 3% T. 6: 94% A, 3% G, 3% C. 7: 95% G, 5% A. 8: 95% T, 5% A.
9: 52% T, 45% C, 3% A. 10: 96% T, 4% C. 11: 60% A, 40% G. 12: 90%
G, 10% A. 13: 94% G, 6% C. 14: 81% G, 8% A, 8% T, 3% C. 15: 98% C,
2% T. 16: 90% C, 10% A. 17: 50% G, 50% T. 18: 90% A, 10% T. 19: 90%
A, 5% G, 5% C. 20: 95% A, 5% T. 21: 91% T,9% A. 22: 92% A, 8% G.
23: 94% A, 3% G, 3% C. 24: 93% G, 7% A. 25: 90% A, 10% G.
[0409] Random Mutagenesis
[0410] The spiked oligonucleotides apparent from Tables 1-7 (which
by a common term is designated FDAS in FIG. 3) and reverse primers
RDAS for each region and specific B. licheniformis primers covering
the SacII and the SalI sites are used to generate
PCR-library-fragments by the overlap extension method (Horton et
al., Gene, 77 (1989), pp. 61-68) with an overlap of 21 bp. FIG. 3
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 Bacillus.
[0411] Screening
[0412] The seven libraries may be screened in the low pH and the
low calcium filter assays described below.
[0413] Low pH Filter Assay
[0414] Bacillus libraries are plated on a sandwich of cellulose
acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and
nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell,
Dassel, Germany) on TY agar plates with 10 .mu.g/ml chloramphenicol
at 37.degree. C. for at least 21 hrs. The cellulose acetate layer
is located on the TY agar plate.
[0415] 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 80.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 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 hrs. 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.
[0416] Low Calcium Filter Assay
[0417] The assay is performed in the same way as the low pH filter
assay with the following modifications: The filter with bound
proteins is incubated at 95.degree. C., pH 6.0 for 1 h. with
different EDTA concentrations (0.001 mM-100 mM).
[0418] The following variants were obtained by the above method
(BAN designates B. amyloliquefaciens .alpha.-amylase):
[0419] * BAN/Termamyl hybrid
*+H156Y+A181T+N190F+A209V+Q264S+E211Q
[0420] .cndot. BAN/Termamyl hybrid
*+H156Y+A181T+N190F+A209V+Q264S+H205C+D- 207Y+A210S
[0421] The mutations indicated in bold were introduced by the
random mutagenesis method. The stability data for these variants
appear from Table 11 in Example 3.
[0422] In an analogous manner to that described above, random
mutagenesis of the above identified seven regions are performed on
the parent B. licheniformis .alpha.-amylase (SEQ ID NO 2). The
doping scheme is determined analogously to that used above.
EXAMPLE 3
[0423] Measurement of the Calcium- and pH-dependent Stability
[0424] Normally, the industrial liquefaction process runs using pH
6.0-6.2 as liquefaction pH and an addition of 40 ppm free calcium
in order to improve the stability at 95.degree. C.-105.degree. C.
Some of the herein proposed substitutions have been made in order
to improve the stability at
[0425] 1. lower pH than pH 6.2 and/or
[0426] 2. at free calcium levels lower than 40 ppm free
calcium.
[0427] Three different methods have been used to measure the
improvements in stability obtained by the different substitutions
in Termamyl:
[0428] 1. One assay which measures the stability at slightly
reduced pH, pH 5.5, in the presence of 40ppm free calcium.
(thereby, the improvement of stability at low pH is measured). 10
.mu.g of the variant were incubated under the following conditions:
A 0.1 M acetate solution, pH adjusted to pH 5.5, containing 40 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.
[0429] 2. Another assay which measure the stability in the absence
of free calcium and where the pH is maintained at pH 6.2. This
assay measures the decrease in calcium sensitivity:
[0430] 10 .mu.g of the variant were incubated under the following
conditions: A 0.1 M acetate solution, pH adjusted to pH 6.2,
containing 5% w/w common corn starch (free of calcium). Incubation
was made in a water bath at 95.degree. C. for 30 minutes.
[0431] 1. A third assay wherein the conditions of assays nos. 1 and
2 have been combined. This assay measures the stability in the
absence of calcium and at low pH (pH 5.5).
[0432] 2. A fourth assay similar to no.3. where the pH has been
further reduced to pH 5.0
[0433] Stability Determination
[0434] All the stability trials 1, 2, 3 and 4 have been made using
the same set up. The method was:
[0435] The enzyme was incubated under the relevant conditions
(1-4). Samples were taken at 0, 5, 10, 15 and 30 minutes and
diluted 25 times (same dilution for all taken samples) in assay
buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity was
measured using the Phadebas assay (Pharmacia) under standard
conditions pH 7.3, 37.degree. C.
[0436] The activity measured before incubation (0 minutes) was used
as reference (100%). The decline in percent was calculated as a
function of the incubation time. The table shows the residual
activity after 30 minutes of incubation.
[0437] Specific Activity Determination.
[0438] The specific activity was determined using the Phadebas
assay (Pharmacia) as activity/mg enzyme. The activity was
determined using the .alpha.-amylase assay described in the
Materials and Methods section herein.
[0439] Results:
13TABLE 8 Stability method no. 1./Low pH stability improvement
residual activity Specific activity, after 30 min. of 37.degree.
C., pH 7.3. Percent Variant incubation relative to Termamyl
Termamyl wt. 5% 100% H156Y 15% 100% A209V 18% 100% Q264S 30% 130%
H156Y + A181T + 39% 100% A209V H133Y + H156Y + 58% 85% A1B1T +
A209V + H293Y + A294V + G310D + H450Y BAN/Termamyl 48% 115% hybrid*
+ H156Y + A181T + A209V
[0440]
14TABLE 9 Stability method no. 2./decreased calcium sensitivity
residual activity Specific activity, after 30 min. of 37.degree.
C., pH 7.3. Percent Variant incubation relative to Termamyl
Termamyl wt. 52% -- H156Y 70% -- A209V 75% -- A181T 57% 115% N190F
34% 140% N190F + N188P 57% 190%
[0441]
15TABLE 10 Stability method no. 3./Low pH stability improvement +
decreased calcium sensitivity residual activity Specific activity,
after 30 min. of 37.degree. C., pH 7.3 Variant incubation Units/mg
enzyme Termamyl wt. 3% -- BAN/Termamyl hybrid* + 20% -- H156Y +
A181T + A209V Q264S 5% -- H140K + H142D 5% 115% V128E 50%** 115%
BAN/Termamyl hybrid* + 39% 170% H156Y + A181T + N190F + A209V
BAN/Termamyl hybrid* + 29% 175% H156Y + A181T + A209V + Q264S
BAN/Termamyl hybrid* + 57% 210% H156Y + A181T + N190F + A209V +
Q264S *BAN(B. amyloliquefaciens .alpha.-amylase (SEQ ID NO
4)/Termamyl (B. licheniformis .alpha.-amylase (SEQ ID NO 2) PCR
hybrid. First 33 N-terminal aminoacids are BAN and the rest 36-483
are Termamyl (the construction of the variant is described in
Example 1). **Measured after 5 min. of incubation. Compared to
Termamyl wt. Which under same conditions shows 36% residual
activity.
[0442]
16TABLE 11 Stability method no. 4./Low pH stability improvement (pH
5.0) + decreased calcium sensitivity residual activity Specific
activity, after 30 min. of 37.degree. C., pH 7.3 Variant incubation
units/mg enzyme BAN/Termamyl hybrid* + 9% 210% H156Y + A181T +
N190F + A209V + Q264S BAN/Termamyl hybrid* + 28% 160% H156Y + A181T
+ N190F + A209V + Q264S + E211Q BAN/Termamyl hybrid* + 33% 130%
H156Y + A181T + N190F + A209V + Q264S + H205C + D207Y + A210S *as
indicated in relation to Table 10
[0443] The variants in the above Table 11 were constructed by means
of the localized random mutagenesis described in Example 2.
EXAMPLE 5
[0444] .alpha.-amylase Stability at Low pH and High Temperature
[0445] This example summarises the stability results of variants
characterised by a fluorimetric assay at 70.degree. C. under two
different conditions, (1) pH 4.5 and 1 mM CaCl.sub.2 and (2) pH 6.2
and 10 .mu.M CaCl.sub.2.
[0446] Description of Method
[0447] All fluorescence experiments were performed on a
Perkin-Elmer LS-50 luminescence spectrometer using a 4-cuvette
holder. The temperature was controlled by a circulating water-bath
and measured directly in the cuvette using a Noronix Digital
Thermometer (model NTD 100). During measurements, thorough mixing
of reagents in the cuvette was ensured using magnetic stirrers
operating at high stirring rate. The cuvettes were capped with
teflon-lids to minimize evaporation.
[0448] Intrinsic protein-fluorescence (due to Trp side-chains) was
monitored by excitation at 280 nm and emission at 350 nm.
Slit-widths were 5 nm.
[0449] During kinetic measurements, 4 reactions were monitored in
parallel. Data was collected in the Wavelength Programme dialogue,
allowing automatic data-collection over a prolonged period (e.g.
over an hour).
[0450] Unfolding was carried out at 70.degree. C. Unfolding
conditions were
[0451] (1) 50 mM NaOAc pH 4.5 and 1 mM CaCl.sub.2
[0452] (2) 50 mM NaOAc pH 6.2 and 10 .mu.M CaCl.sub.2.
[0453] Protein concentration was 5 .mu.g/ml and glycerol was at
0.5% w/v (from protein stock solution).
[0454] Note: There was some variation from day to day in the
absolute value of the unfolding half times due to slight
temperature variations (occasioned by e.g. different amounts of
water in the water bath). However, Termamyl was always included as
one of the four enzymes analyzed in each experiment, in effect
making it an internal standard. Unfolding rates relative to this
internal standard were satisfactorily reproducible (performed in
triplicate). Data analysis was carried out using GraphPad Prism
software. At pH 4.5, unfolding data could be fitted very
satisfactorily to a single-exponential decay with drift:
F(t)=A*exp(-ln(2)*t/t.sub.1/2)+drift*t+offset (1)
[0455] where F is the measured fluorescence, A is the amplitude of
the unfolding, t is time and t.sub.1/2 is the half-time of
unfolding.
[0456] At pH 6.2, unfolding was more complex (involving an initial
lag phase), and data could not be fitted to eq. 1. Instead, the
time taken for the fluorescence signal to decay to 50% of the
initial signal was used as an apparent t.sub.1/2.
[0457] From these half-times, the change in free energy of
unfolding relative to that of Termamyl could be calculated as
follows:
DDG=R*T*ln(t.sub.1/2.sup.mutant/t.sub.1/2.sup.Termamyl) (2)
[0458] where R is the universal gas constant and T is the
temperature (the value of R*T is 0.5919, giving a DDG value in
kcal/mol).
[0459] By converting data to DDG values, the
destabilizing/stabilizing effects of different mutations can be
compared directly and examined for additivity
(DDG.sub.1+2=DDG.sub.1+DDG.sub.2) and synergy
(DDG.sub.1+2>DDG.sub.1+DDG.sub.2) where DDG.sub.1+2 is the
energy-effect of introducing mutations 1 and 2.
[0460] Results
[0461] Unfolding of amylases at low pH and high temperature may be
followed by the decay in Trp-fluorescence. At pH 4.5 and 1 mM
CaCl.sub.2, all amylases unfold fairly rapidly.
[0462] The unfolding data at pH 4.5 fit better to a
double-exponential equation than to a single-exponential equation.
However, since the second phase is very slow, it is approximated by
a linear drift (equation 1). Unfolding at pH 6.2 and 10 .mu.M
CaCl.sub.2 at 70.degree. C. is much less rapid than at pH 4.5
despite the low [Ca.sup.2+]. Unfolding is far from complete within
an hour and it is not possible to fit the data to a
single-exponential equation. Instead, the time taken for the
fluorescence signal to decay to 50% of the initial signal is used
as an apparent t.sub.1/2.
[0463] Results of the fluorescence assay are presented in Table
12.
17TABLE 12 Summary of data for unfolding of Termamyl variants pH
4.5 and pH 6.2 at 70.degree. C. At pH 45 t.sub.1/2.sup.Termamyl =
200 s; at pH 6.2, t.sub.1/2.sup.Termamyl = 2800 s. DDG =
-RT*ln(t.sub.1/2.sup.Termam- yl/t.sub.1/2.sup.mutant) pH 4.5, 1 mM
CaCl.sub.2 pH 6.2, 10 .mu.M CaCl.sub.2 DDG t.sub.1/2/ DDG
t.sub.1/2/ Mutation (kcal/mol) t.sub.1/2.sup.Termamyl (kcal/mol)
t.sub.1/2.sup.Termamyl Wildtype 0 1.0 0 1.0 A209V -0.36 1.85 -0.72
3.39 H133Y + A209V -0.77 3.67 -0.61 2.78 H156Y 0.06 0.90 -0.10 1.18
A181T -0.06 1.10 -0.28 1.60 A181T + A209V -0.44 2.09 < -1
(73%).sup.a >5 S187D 0.37 0.54 0.19 0.72 H450Y -0.49 2.29 0.15
0.78 L270D -0.35 1.8 -0.10 1.2 A181T + H156Y -0.17 1.34 -0.62 2.84
H133I -0.33 1.75 -0.42 2.02 H133Y + H156Y + -0.96 5.10 < -1
(58%).sup.a >5 A181T + A209V + H293Y + A294V + G310D + H450Y
V128E -0.10 1.2 -0.25 1.5 H156Y + A181T + -0.32 1.71 -0.49 2.30
A209V H156Y + A181T + -0.42 2.05 -0.63 2.92 A209V + H450Y H156Y +
A181T + -0.81 3.9 < -1 (65%).sup.a >5 A209V + H450Y + H133I
H156Y + A181T + -0.70 3.3 < -1 (77%).sup.a >5 A209V + H133I
Q264S -0.26 1.6 -0.14 1.3 H156Y + A181T + -0.43 2.1 -0.82 4.0 A209V
+ Delta (1, 2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S +
E3GD + H37I Q264S + N265Y -0.33 1.8 -0.07 1.1 Q264S + N265Y + -1.07
6.1 -0.67 3.1 N190F Q264S + N265Y + -1.66 16.5 > -1 (82%).sup.a
>5 N190F + H133I + A209V H156Y + A181T + -0.30 1.7 < -1
(66%).sup.a >5 A209V + Delta (1, 2) + L3V + M15T + R23K + S31A +
A32E + Y33H + A35S + E36D + H37I + N190F H156Y + A181T + -0.43 2.1
-0.86 4.3 A209V + Delta (1, 2) + L3V + M15T + R23K + S31A + A32E +
Y33H + A35S + E36D + H37I + Q264S H156Y + A181T + -0.36 1.8 < -1
(76%).sup.a >5 A209V + Delta (1, 2) + L3V + M15T + R23K + S31A +
A32E + Y33H + A35S + E36D + H37I Q264S + N190F H156Y + A181T + -1.3
8.6 < -1 (66%).sup.a >5 A209V + N190F + Q264S .sup.aThe
percentage indicates the level to which the initial fluorescence
level had declined in the course of 3 hours at 70.degree. C. The
slow decline is indicative of high stability.
EXAMPLE 5
[0464] .alpha.-amylase Variants with Increased Specific
Activity
[0465] This example summarises the results of variants
characterised by having increased specific activity compared to
Termamyl wt. The presence of these substitutions either in
combination with each other or as single substitutions added to
stabilising substitutions increases the specific activity of the
resulting variant. The specific activity was determined using the
.alpha.-amylase (Phadebas) assay described in the Materials and
Methods where the activity/mg enzyme was determined. The activity
was determined using the following description where the pH was
7.3, temperature 37.degree. C. and testing time 15 min. and buffer
as defined.
18 SPEC. ACTIVITY (PHADEBAS ASSAY), INDEX MUTATION RELATIVE TO
TERMAMYL WT. S187D 260% V541 160% BAN/Termamyl hybrid: 140%
(.DELTA.(1, 2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S +
E36D + H37I) .DELTA.(D372 + S373 + Q374) 125% .DELTA.(K370 + G371 +
D372) 125% BAN/Termamyl hybrid: 360% (.DELTA.(1, 2) + L3V + M15T +
R23K + S31A + A32E + Y33H + A35S + E36D + H37I + .DELTA.(D372 +
S373 + Q374) + V54I + S187D Q2645 130% Y290E 155% Y290K 140% N190F
150%
EXAMPLE 6
[0466] Testing of Specificity Variants (Saccharification)
[0467] It has been reported previously (U.S. Pat. No. 5,234,823)
that, when saccharifying with glucoamylase and pullulanase, the
presence of residual .alpha.-amylase activity arising from the
liquefaction process, can lead to lower yields of glucose, if the
.alpha.-amylase is not inactivated before the saccharification
stage. This inactivation can be typically carried out by adjusting
the pH to below 4.3 at 95.degree. C., before lowering the
temperature to 60.degree. C. for saccharification.
[0468] The reason for this negative effect on glucose yield is not
fully understood, but it is assumed that the liquefying
.alpha.-amylase (for example Termamyl 120 L from B. licheniformis)
generates "limit dextrins" (which are poor substrates for
pullulanase), by hydrolysing 1,4-alpha-glucosidic linkages close to
and on both sides of the branching points in amylopectin.
Hydrolysis of these limit dextrins by glucoamylase leads to a build
up of the trisaccharide panose, which is only slowly hydrolysed by
glucoamylase.
[0469] The development of a thermostable .alpha.-amylase, which
does not suffer from this disadvantage would be a significant
improvement, as no separate inactivation step would be
required.
[0470] A number of B. licheniformis .alpha.-amylase variants, with
altered specificity, were evaluated by saccharifying a DE 10
Maltodextrin substrate with A. niger glucoamylase and B.
acidopullulyticus pullulanase under conditions where the variant
amylase was active.
[0471] The saccharification reactions were monitored by taking
samples at 24 hour intervals and analysing them by HPLC. The
standard reaction conditions were:
19 Substrate concentration 28.2% w/w Temperature 60.degree. C.
Initial pH (at 60.degree. C.) 4.7 Enzyme dosage Glucoamylase 0.18
AG/g DS Pullulanase 0.06 PUN/g DS .alpha.-amylase 60 NU/g DS
[0472] The following enzymes were used:
20 Glucoamylase: AMG (Novo Nordisk) 153 AG/g Pullulanase: Promozyme
(Novo Nordisk) 295 PUN/g .alpha.-amylase: Termamyl (Novo Nordisk)
135 KNU/g V54Y 313 KNU/g A52W 5,116 NU/ml D53E 3,280 NU/ml D53W 599
NU/ml A52W + V54Y 134 NU/ml
[0473] The mutations listed in the .alpha.-amylase list above are
used to indicate variants of the B. licheniformis .alpha.-amylase
(SEQ ID NO 2) (Termamyl) which has been modified by the indicated
mutation(s).
[0474] Substrates for saccharification were prepared by dissolving
230 g DE 10 spray-dried maltodextrin, prepared from common corn
starch, in 460 ml boiling deionized water and adjusting the dry
substance to approximately 30% w/w. The pH was adjusted to 4.7
(measured at 60.degree. C.) and aliquots of substrate corresponding
to 15 g dry weight, were transferred to 50 ml blue cap glass
flasks.
[0475] The flasks were then placed in a shaking water bath
equilibrated at 60.degree. C., and the enzymes added. The pH was
readjusted to 4.7 where necessary. 2 ml samples were taken
periodically, the pH adjusted to about 3.0, and then heated in a
boiling water bath for 15 minutes to inactivate the enzymes. After
cooling, the samples were treated with approximately 0.1 g mixed
bed ion exchange resin (BIO-Rad 501 X8 (D)) for 30 minutes on a
rotary mixer to remove salts and soluble N. After filtration, the
carbohydrate composition was determined by HPLC. After 72 hours,
the following results were obtained:
21 Added .alpha.-amylase % DP.sub.1 % DP.sub.2 % DP.sub.3 %
DP.sub.4+ None (control) 96.59 2.2 0.3 1.0 V54Y 96.5 2.2 0.4 0.9
A52W + V54Y 96.4 2.2 0.5 0.9 Termamyl 96.3 2.1 0.8 0.8
[0476] Compared with the control (no active .alpha.-amylase present
during liquefaction), the presence of active .alpha.-amylase
variants V54Y and A52W+V54Y did not lead to elevated panose levels
(DP.sub.3).
[0477] If these .alpha.-amylase variants are used for starch
liquefaction, it will not be necessary to inactivate the enzyme
before the commencement of saccharification.
EXAMPLE 7
[0478] Evaluation of B. licheniformis Variants Under Simulated
Liquefaction Conditions
[0479] The standard process for industrial starch liquefaction
comprises two stages, normally referred to as primary and secondary
liquefaction. In the first stage, a 30-40% w/w starch slurry at pH
5.5-6.0, to which has been added a thermostable alpha-amylase from
B. licheniformis or B. stearothermophillus, is heated to
105-110.degree. C. in a jet cooker where live steam is injected
into the starch stream. After a holding time of 5-10 minutes under
pressure at this temperature, the liquefied starch is flash cooled
to about 95.degree. C. and held at that temperature for 60-120
minutes.
[0480] In order to evaluate small quantities of enzyme on a
laboratory scale the following test method was used:
[0481] 10 g aliquots of a suspension of common corn starch
(Cerestar GL 3406) in deionized water (approx. 30% w/w) are weighed
out into 100 ml conical flasks (Schott GL 125) which are fitted
with tight fitting screw caps. The pH, calcium level and enzyme
dosage in the suspension can be varied.
[0482] 4 flasks are used for each different set of experimental
conditions. The flasks are placed in a shaking oil-bath (Heto VS
01) maintained at 105.degree. C. After a period of 7 minutes, cold
oil is poured into the bath to lower the temperature to 95.degree.
C. For each experimental series, flasks are removed after 20, 40,
60 and 90 minutes and immediately cooled under running water. One
drop of 1N HCL is added to each flask to inactivate the enzyme. The
reaction is monitored by measuring the DE (reducing sugar content
expressed as glucose) using the Neocuproine method.
[0483] The details of this method can be found in "Determination of
reducing sugar with improved precision. Dygert, Li, Florida and
Thomas, Anal Biochem, 13, 368 (1965).
[0484] The following DEs were recorded after 90 minutes
22 pH 6.0 5 ppm calcium added 10.9 mg enzyme protein/g starch
Hybrid + H156Y + Hybrid + H156Y + Hybrid + A181T + H156Y + A181T +
H156Y + Hybrid + A209V + A181T + A209V + A181T + H156Y + Q264S +
H156Y + N190F + A209V + N190F + A209V + A181T + Q264S + N265Y +
A181T + I.D. no. Termamyl Q264S N190F Q264S Q264S A209V Q264S N265Y
N190F A209V 045-96 16.0 16.9 13.2 038-96 6.5 13.9 035-96 15.2 12.9
9.9 033-96 6.7 7.2 12.1 031-96 4.5 7.0 8.8 12.5 029-96 4.0 8.7 5.2
7.7 039-96 14.9 16.3 14.4 pH 6.0 40 ppm calcium added 10.9 mg
enzyme protein/g starch I.D. no. Termamyl 045-96 12.6 007-97 12.1
pH 5.5 5 ppm calcium added 10.9 mg enzyme protein/g starch Hybrid +
H156Y + Hybrid + H156Y + A181T + A209V + H156Y + A181T + A181T +
A209V + I.D. no N190F + Q264S A209V + N190F N190F + Q264S 001-97
14.8 15.2 12.6 Hybrid = BAN/Termamyl PCR hybrid as described in
Example 3
REFERENCES CITED
[0485] Klein, C., et al., Biochemistry 1992, 31, 8740-8746,
[0486] Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,
[0487] Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238,
[0488] Larson, S. B., J. Mol. Biol. (1994) 235, 1560-1584,
[0489] Lawson, C. L., J. Mol. Biol. (1994) 236, 590-600,
[0490] Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,
[0491] Brady, R. L., et al., Acta Crystallogr. sect. B, 47,
527-535,
[0492] Swift, H. J., et al., Acta Crystallogr. sect. B, 47,
535-544
[0493] A. Kadziola, Ph.D. Thesis: "An alph.alpha.-amylase from
Barley and its Complex with a Substrate Analogue Inhibitor Studied
by X-ray Crystallography", Department of Chemistry University of
Copenhagen 1993
[0494] MacGregor, E. A., Food Hydrocolloids, 1987, Vol. 1, No. 5-6,
p.
[0495] B. Diderichsen and L. Christiansen, Cloning of a maltogenic
.alpha.-amylase from Bacillus stearothermophilus, FEMS Microbiol.
letters: 56: pp. 53-60 (1988)
[0496] Hudson et al., Practical Immunology, Third edition (1989),
Blackwell Scientific Publications,
[0497] Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor, 1989
[0498] S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22,
1981, pp. 1859-1869
[0499] Matthes et al., The EMBO J. 3, 1984, pp. 801-805.
[0500] R. K. Saiki et al., Science 239, 1988, pp. 487-491.
[0501] Morinaga et al., (1984, Biotechnology 2:646-639)
[0502] Nelson and Long, Analytical Biochemistry 180, 1989, pp.
147-151
[0503] Hunkapiller et al., 1984, Nature 310:105-111
[0504] R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general
method of in vitro preparation and specific mutagenesis of DNA
fragments: study of protein and DNA interactions. Nucl. Acids Res.
16:7351-7367.
[0505] Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.
[0506] Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.
[0507] S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp.
1680-1682.
[0508] Boel et al., 1990, Biochemistry 29, pp. 6244-6249.
[0509]
Sequence CWU 1
1
37 1 1920 DNA Bacillus licheniformis 1 cggaagattg gaagtacaaa
aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa
aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120
agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag
180 aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc
ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtac ttgttaaaaa
ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaa ggggaggaga
atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatt
tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gcaaatctta
atgggacgct gatgcagtat tttgaatggt acatgcccaa tgacggccaa 480
cattggaggc gtttgcaaaa cgactcggca tatttggctg aacacggtat tactgccgtc
540 tggattcccc cggcatataa gggaacgagc caagcggatg tgggctacgg
tgcttacgac 600 ctttatgatt taggggagtt tcatcaaaaa gggacggttc
ggacaaagta cggcacaaaa 660 ggagagctgc aatctgcgat caaaagtctt
cattcccgcg acattaacgt ttacggggat 720 gtggtcatca accacaaagg
cggcgctgat gcgaccgaag atgtaaccgc ggttgaagtc 780 gatcccgctg
accgcaaccg cgtaatttca ggagaacacc taattaaagc ctggacacat 840
tttcattttc cggggcgcgg cagcacatac agcgatttta aatggcattg gtaccatttt
900 gacggaaccg attgggacga gtcccgaaag ctgaaccgca tctataagtt
tcaaggaaag 960 gcttgggatt gggaagtttc caatgaaaac ggcaactatg
attatttgat gtatgccgac 1020 atcgattatg accatcctga tgtcgcagca
gaaattaaga gatggggcac ttggtatgcc 1080 aatgaactgc aattggacgg
tttccgtctt gatgctgtca aacacattaa attttctttt 1140 ttgcgggatt
gggttaatca tgtcagggaa aaaacgggga aggaaatgtt tacggtagct 1200
gaatattggc agaatgactt gggcgcgctg gaaaactatt tgaacaaaac aaattttaat
1260 cattcagtgt ttgacgtgcc gcttcattat cagttccatg ctgcatcgac
acagggaggc 1320 ggctatgata tgaggaaatt gctgaacggt acggtcgttt
ccaagcatcc gttgaaatcg 1380 gttacatttg tcgataacca tgatacacag
ccggggcaat cgcttgagtc gactgtccaa 1440 acatggttta agccgcttgc
ttacgctttt attctcacaa gggaatctgg ataccctcag 1500 gttttctacg
gggatatgta cgggacgaaa ggagactccc agcgcgaaat tcctgccttg 1560
aaacacaaaa ttgaaccgat cttaaaagcg agaaaacagt atgcgtacgg agcacagcat
1620 gattatttcg accaccatga cattgtcggc tggacaaggg aaggcgacag
ctcggttgca 1680 aattcaggtt tggcggcatt aataacagac ggacccggtg
gggcaaagcg aatgtatgtc 1740 ggccggcaaa acgccggtga gacatggcat
gacattaccg gaaaccgttc ggagccggtt 1800 gtcatcaatt cggaaggctg
gggagagttt cacgtaaacg gcgggtcggt ttcaatttat 1860 gttcaaagat
agaagagcag agaggacgga tttcctgaag gaaatccgtt tttttatttt 1920 2 483
PRT Bacillus licheniformis 2 Ala 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 3 2084 DNA Bacillus
amyloliquefaciens 3 gccccgcaca tacgaaaaga ctggctgaaa acattgagcc
tttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa
gaagaagacc ataaaaatac cttgtctgtc 120 atcagacagg gtatttttta
tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180 ggggggttgt
tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240
agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc
300 acgctgttat ttgtcagttt gccgattaca aaaacatcag ccgtaaatgg
cacgctgatg 360 cagtattttg aatggtatac gccgaacgac ggccagcatt
ggaaacgatt gcagaatgat 420 gcggaacatt tatcggatat cggaatcact
gccgtctgga ttcctcccgc atacaaagga 480 ttgagccaat ccgataacgg
atacggacct tatgatttgt atgatttagg agaattccag 540 caaaaaggga
cggtcagaac gaaatacggc acaaaatcag agcttcaaga tgcgatcggc 600
tcactgcatt cccggaacgt ccaagtatac ggagatgtgg ttttgaatca taaggctggt
660 gctgatgcaa cagaagatgt aactgccgtc gaagtcaatc cggccaatag
aaatcaggaa 720 acttcggagg aatatcaaat caaagcgtgg acggattttc
gttttccggg ccgtggaaac 780 acgtacagtg attttaaatg gcattggtat
catttcgacg gagcggactg ggatgaatcc 840 cggaagatca gccgcatctt
taagtttcgt ggggaaggaa aagcgtggga ttgggaagta 900 tcaagtgaaa
acggcaacta tgactattta atgtatgctg atgttgacta cgaccaccct 960
gatgtcgtgg cagagacaaa aaaatggggt atctggtatg cgaatgaact gtcattagac
1020 ggcttccgta ttgatgccgc caaacatatt aaattttcat ttctgcgtga
ttgggttcag 1080 gcggtcagac aggcgacggg aaaagaaatg tttacggttg
cggagtattg gcagaataat 1140 gccgggaaac tcgaaaacta cttgaataaa
acaagcttta atcaatccgt gtttgatgtt 1200 ccgcttcatt tcaatttaca
ggcggcttcc tcacaaggag gcggatatga tatgaggcgt 1260 ttgctggacg
gtaccgttgt gtccaggcat ccggaaaagg cggttacatt tgttgaaaat 1320
catgacacac agccgggaca gtcattggaa tcgacagtcc aaacttggtt taaaccgctt
1380 gcatacgcct ttattttgac aagagaatcc ggttatcctc aggtgttcta
tggggatatg 1440 tacgggacaa aagggacatc gccaaaggaa attccctcac
tgaaagataa tatagagccg 1500 attttaaaag cgcgtaagga gtacgcatac
gggccccagc acgattatat tgaccacccg 1560 gatgtgatcg gatggacgag
ggaaggtgac agctccgccg ccaaatcagg tttggccgct 1620 ttaatcacgg
acggacccgg cggatcaaag cggatgtatg ccggcctgaa aaatgccggc 1680
gagacatggt atgacataac gggcaaccgt tcagatactg taaaaatcgg atctgacggc
1740 tggggagagt ttcatgtaaa cgatgggtcc gtctccattt atgttcagaa
ataaggtaat 1800 aaaaaaacac ctccaagctg agtgcgggta tcagcttgga
ggtgcgttta ttttttcagc 1860 cgtatgacaa ggtcggcatc aggtgtgaca
aatacggtat gctggctgtc ataggtgaca 1920 aatccgggtt ttgcgccgtt
tggctttttc acatgtctga tttttgtata atcaacaggc 1980 acggagccgg
aatctttcgc cttggaaaaa taagcggcga tcgtagctgc ttccaatatg 2040
gattgttcat cgggatcgct gcttttaatc acaacgtggg atcc 2084 4 480 PRT
Bacillus amyloliquefaciens 4 Val 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 5 1814 DNA Bacillus stearothermohilus 5
aaattcgata ttgaaaacga ttacaaataa aaattataat agacgtaaac gttcgagggt
60 ttgctccctt tttactcttt ttatgcaatc gtttccctta attttttgga
agccaaaccg 120 tcgaatgtaa catttgatta agggggaagg gcattgtgct
aacgtttcac cgcatcattc 180 gaaaaggatg gatgttcctg ctcgcgtttt
tgctcactgt ctcgctgttc tgcccaacag 240 gacagcccgc caaggctgcc
gcaccgttta acggcaccat gatgcagtat tttgaatggt 300 acttgccgga
tgatggcacg ttatggacca aagtggccaa tgaagccaac aacttatcca 360
gccttggcat caccgctctt tggctgccgc ccgcttacaa aggaacaagc cgcagcgacg
420 tagggtacgg agtatacgac ttgtatgacc tcggcgaatt caatcaaaaa
gggaccgtcc 480 gcacaaaata cggaacaaaa gctcaatatc ttcaagccat
tcaagccgcc cacgccgctg 540 gaatgcaagt gtacgccgat gtcgtgttcg
accataaagg cggcgctgac ggcacggaat 600 gggtggacgc cgtcgaagtc
aatccgtccg accgcaacca agaaatctcg ggcacctatc 660 aaatccaagc
atggacgaaa tttgattttc ccgggcgggg caacacctac tccagcttta 720
agtggcgctg gtaccatttt gacggcgttg attgggacga aagccgaaaa ttgagccgca
780 tttacaaatt ccgcggcatc ggcaaagcgt gggattggga agtagacacg
gaaaacggaa 840 actatgacta cttaatgtat gccgaccttg atatggatca
tcccgaagtc gtgaccgagc 900 tgaaaaactg ggggaaatgg tatgtcaaca
caacgaacat tgatgggttc cggcttgatg 960 ccgtcaagca tattaagttc
agtttttttc ctgattggtt gtcgtatgtg cgttctcaga 1020 ctggcaagcc
gctatttacc gtcggggaat attggagcta tgacatcaac aagttgcaca 1080
attacattac gaaaacagac ggaacgatgt ctttgtttga tgccccgtta cacaacaaat
1140 tttataccgc ttccaaatca gggggcgcat ttgatatgcg cacgttaatg
accaatactc 1200 tcatgaaaga tcaaccgaca ttggccgtca ccttcgttga
taatcatgac accgaacccg 1260 gccaagcgct gcagtcatgg gtcgacccat
ggttcaaacc gttggcttac gcctttattc 1320 taactcggca ggaaggatac
ccgtgcgtct tttatggtga ctattatggc attccacaat 1380 ataacattcc
ttcgctgaaa agcaaaatcg atccgctcct catcgcgcgc agggattatg 1440
cttacggaac gcaacatgat tatcttgatc actccgacat catcgggtgg acaagggaag
1500 ggggcactga aaaaccagga tccggactgg ccgcactgat caccgatggg
ccgggaggaa 1560 gcaaatggat gtacgttggc aaacaacacg ctggaaaagt
gttctatgac cttaccggca 1620 accggagtga caccgtcacc atcaacagtg
atggatgggg ggaattcaaa gtcaatggcg 1680 gttcggtttc ggtttgggtt
cctagaaaaa cgaccgtttc taccatcgct cggccgatca 1740 caacccgacc
gtggactggt gaattcgtcc gttggaccga accacggttg gtggcatggc 1800
cttgatgcct gcga 1814 6 514 PRT Bacillus stearothermophilus 6 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 Thr 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
Asn 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 Tyr 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 Thr Lys Thr Asp 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 Ala 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
Gly 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 Arg Pro Ile Thr Thr 485 490 495 Arg Pro
Trp Thr Gly Glu Phe
Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp 7 478 PRT
Bacillus licheniformis 7 Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser
Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Ala Arg Thr Asp Gly
Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Ala Asp Gln Lys Tyr Cys
Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45 Leu Asp Tyr Ile
Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60 Val Thr
Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65 70 75 80
Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85
90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly
Met 100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr
Asp Gly Ala 115 120 125 Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro
Phe Ser Ser Gln Asp 130 135 140 Tyr Phe His Pro Phe Cys Phe Ile Gln
Asn Tyr Glu Asp Gln Thr Gln 145 150 155 160 Val Glu Asp Cys Trp Leu
Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175 Asp Thr Thr Lys
Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190 Ser Leu
Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205
Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210
215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr
Cys 225 230 235 240 Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr
Pro Ile Tyr Tyr 245 250 255 Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser
Gly Ser Met Asp Asp Leu 260 265 270 Tyr Asn Met Ile Asn Thr Val Lys
Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 Leu Gly Thr Phe Val Glu
Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300 Thr Asn Asp Ile
Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310 315 320 Asn
Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330
335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr
340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn
Ala Ile 355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val
Thr Tyr Lys Asn 370 375 380 Trp Pro Ile Tyr Lys Asp Asp Ile Thr Ile
Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln Ile Val Thr
Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415 Asp Ser Tyr Thr Leu
Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430 Gln Leu Thr
Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445 Gly
Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455
460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser 465 470
475 8 20 DNA Bacillus amyloliquefaciens 8 gacctgcagt caggcaacta 20
9 20 DNA Bacillus amyloliquefaciens 9 tagagtcgac ctgcaggcat 20 10
31 DNA Bacillus licheniformis 10 ggtcgtaggc accgtagccc caatccgctt g
31 11 36 DNA Bacillus licheniformis 11 ggtcgtaggc accgtagccc
caatcccatt ggctcg 36 12 28 DNA Bacillus licheniformis 12 ctgtgactgg
tgagtactca accaagtc 28 13 31 DNA Bacillus licheniformis 13
ggtcgtaggc accgtagccc tcatccgctt g 31 14 31 DNA Bacillus
licheniformis 14 ggtcgtaggc accgtagccc atatccgctt g 31 15 31 DNA
Bacillus licheniformis 15 ggtcgtaggc accgtagcca atatccgctt g 31 16
36 DNA Bacillus licheniformis 16 gcagcatgga actgctyatg aagaggcacg
tcaaac 36 17 30 DNA Bacillus licheniformis 17 catagttgcc gaattcattg
gaaacttccc 30 18 34 DNA Bacillus licheniformis 18 catagttgcc
gaattcaggg gaaacttccc aatc 34 19 41 DNA Bacillus licheniformis 19
ccgcgccccg ggaaatcaaa ttttgtccag gctttaatta g 41 20 32 DNA Bacillus
licheniformis 20 caaaatggta ccaataccac ttaaaatcgc tg 32 21 29 DNA
Bacillus licheniformis 21 cttcccaatc ccaagtcttc ccttgaaac 29 22 36
DNA Bacillus licheniformis 22 cttaatttct gctacgacgt caggatggtc
ataatc 36 23 38 DNA Bacillus licheniformis 23 cgcccaagtc attcgaccag
tactcagcta ccgtaaac 38 24 29 DNA Bacillus licheniformis 24
gccgttttca ttgtcgactt cccaatccc 29 25 35 DNA Bacillus licheniformis
25 ggaatttcgc gctgactagt cccgtacata tcccc 35 26 36 DNA Bacillus
licheniformis 26 ggcaggaatt tcgcgacctt tcgtcccgta catatc 36 27 36
DNA Bacillus amyloliquefaciens 27 cctcattctg cagcagcagc cgtaaatggc
acgctg 36 28 38 DNA Bacillus amyloliquefaciens 28 ccagacggca
gtaataccga tatccgataa atgttccg 38 29 30 DNA Bacillus
amyloliquefaciens 29 cggatatcgg tattactgcc gtctggattc 30 30 21 DNA
Bacillus amyloliquefaciens 30 ctcgtcccaa tcggttccgt c 21 31 75 DNA
Bacillus licheniformis 31 cgcggcagca catacagcga ttwtvawtgg
drttggwmty attttgacgg aamcgattgg 60 gacgagtccc gaaag 75 32 84 DNA
Bacillus licheniformis 32 ctgaaccgca tctataagtt tmakrstaag
rmktgggatw sggakgttav tmmtgaathk 60 rskaactatg attatttgat gtat 84
33 72 DNA Bacillus licheniformis misc_feature (0)...(0) N at 37 is
81% G, 7% A, 7% T, 5% C 33 tatgccgaca tcgattatga cyrthctdmw
vttrwrnmts akwtwaramr atggggcact 60 tggtatgcca at 72 34 78 DNA
Bacillus licheniformis misc_feature (0)...(0) N at 31 is 93% C, 2%
T, 2% A, 3% G 34 ttggacggtt tccgtcttga tdytgytaaa nwwmttargt
wtwmttwtht wmvggawtgg 60 gttaatcatg tcagggaa 78 35 63 DNA Bacillus
licheniformis 35 gctgaccgca accgcgtaat ttcarskgak hhtwbawtaa
rggcctggac acattttcat 60 ttt 63 36 78 DNA Bacillus licheniformis 36
tggtaccatt ttgacggaac cgattggrak gagdcgcgaa rgmwaavtar gdwytwtaag
60 tttcaaggaa aggcttgg 78 37 93 DNA Bacillus licheniformis
misc_feature (0)...(0) N at 43 is 81% G, 8% T, 3% C 37 gaaatgttta
cggtagctga atwttggrvk wvtrawhyrr stnytmtkga wavttwtwtr 60
avcargacar attttaatca ttcagtgttt gac 93
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