U.S. patent application number 12/643104 was filed with the patent office on 2010-04-22 for amylolytic enzyme variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Carsten Andersen, Lars Beier, Joel Robert Cherry, Torben Peter Frandsen, Thomas Schafer, Allan Svendsen.
Application Number | 20100098804 12/643104 |
Document ID | / |
Family ID | 27439288 |
Filed Date | 2010-04-22 |
United States Patent
Application |
20100098804 |
Kind Code |
A1 |
Cherry; Joel Robert ; et
al. |
April 22, 2010 |
AMYLOLYTIC ENZYME VARIANTS
Abstract
The inventors have discovered some striking, and not previously
predicted structural similarities and differences between the
structure of Novamyl and the reported structures of CGTases, and
based on this they have constructed variants of maltogenic
alpha-amylase having CGTase activity and variants of CGTase having
maltogenic alpha-amylase activity. Further, on the basis of
sequence homology between Novamyl.RTM. and CGTases, the inventors
have constructed hybrid enzymes with one or more improvements to
specific properties of the parent enzymes, using recombinant DNA
methodology.
Inventors: |
Cherry; Joel Robert; (Davis,
CA) ; Svendsen; Allan; (Birkerod, DK) ;
Andersen; Carsten; (Vaerloese, DK) ; Beier; Lars;
(Lyngby, DK) ; Frandsen; Torben Peter;
(Frederiksberg C, DK) ; Schafer; Thomas; (Farum,
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: |
27439288 |
Appl. No.: |
12/643104 |
Filed: |
December 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10442558 |
May 21, 2003 |
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12643104 |
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10234266 |
Sep 4, 2002 |
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10442558 |
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09645707 |
Aug 24, 2000 |
6482622 |
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10234266 |
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PCT/DK99/00087 |
Feb 26, 1999 |
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09645707 |
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60077509 |
Mar 11, 1998 |
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60077795 |
Mar 12, 1998 |
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Current U.S.
Class: |
426/20 ; 435/193;
435/69.1; 435/71.1 |
Current CPC
Class: |
A21D 8/042 20130101;
C12N 9/2417 20130101 |
Class at
Publication: |
426/20 ; 435/193;
435/71.1; 435/69.1 |
International
Class: |
A21D 8/04 20060101
A21D008/04; C12N 9/10 20060101 C12N009/10; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 1998 |
DK |
1998 00269 |
Feb 27, 1998 |
DK |
1998 00273 |
Claims
1. A method for preparing a dough or baked product prepared from
the dough comprising adding to the dough a cyclodextrin
glucanotransferase variant which forms linear oligosaccharides when
acting on starch in an amount effective to retard the staling of
the baked product prepared from the dough.
2. The method of claim 1, wherein the cyclodextrin
glucanotransferase variant is derived from a strain of Bacillus,
Brevibacterium, Clostridium, Corynebacterium, Klebsiella,
Micrococcus, Thermoanaerobacter or Thermoanaerobacterium.
3. A method for producing a variant of a parent cyclodextrin
glucanotransferase, comprising modifying the amino acid sequence of
a parent cyclodextrin glucanotransferase by substituting, inserting
or deleting one or more amino acids of said amino acid sequence,
wherein said substitution is a substitution an amino acid residue
which is present in a corresponding position in the amino acid
sequence of amino acids 1 to 686 of SEQ ID NO:2 but which is not
present in the amino acid sequence of the parent cyclodextrin
glucanotransferase; wherein said insertion is an insertion of an
amino acid residue which is present in a corresponding position in
the amino acid sequence of amino acids 1 to 686 of SEQ ID NO:1 but
which is not present in the amino acid sequence of the parent
cyclodextrin glucanotransferase; and wherein said deletion is a
deletion of an amino acid residue which is present in the parent
cyclodextrin glucanotransferase but which is not present in the
amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2, and
wherein the cyclodextrin glucanotransferase variant forms linear
oligosaccharides when acting on starch.
4. The method of claim 3, wherein the parent cyclodextrin
glucanotransferase is from a strain of Bacillus, Brevibacterium,
Clostridium, Corynebacterium, Klebsiella, Micrococcus,
Thermoanaerobacter or Thermoanaerobacterium.
5. A cyclodextrin glucanotransferase variant prepared by the method
of claim 3.
6. A method for producing a variant of a parent cyclodextrin
glucanotransferase, comprising (a) modifying the amino acid
sequence of a parent cyclodextrin glucanotransferase by
substituting, inserting or deleting one or more amino acids of said
amino acid sequence, wherein said substitution is a substitution an
amino acid residue which is present in a corresponding position in
the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but
which is not present in the amino acid sequence of the parent
cyclodextrin glucanotransferase; wherein said insertion is an
insertion of an amino acid residue which is present in a
corresponding position in the amino acid sequence of amino acids 1
to 686 of SEQ ID NO: 2 but which is not present in the amino acid
sequence of the parent cyclodextrin glucanotransferase; and wherein
said deletion is a deletion of an amino acid residue which is
present in the parent cyclodextrin glucanotransferase but which is
not present in the amino acid sequence of amino acids 1 to 686 of
SEQ ID NO: 2, (b) testing the variant cyclodextrin
glucanotransferase for the ability to form linear oligosaccharides
when acting on starch; (c) producing the variant cyclodextrin
glucanotransferase by cultivating a host cell comprising a nucleic
acid sequence encoding the variant cyclodextrin glucanotransferase;
and (d) recovering the cyclodextrin glucanotransferase variant.
7. The method of claim 6, wherein the cyclodextrin
glucanotransferase is derived from a strain of Bacillus,
Brevibacterium, Clostridium, Corynebacterium, Klebsiella,
Micrococcus, Thermoanaerobacter or Thermoanaerobacterium.
8. A cyclodextrin glucanotransferase variant prepared by the method
of claim 6.
9. A method for producing a variant of a parent cyclodextrin
glucanotransferase, comprising: (a) cultivating a host cell
comprising a nucleic acid sequence encoding a variant of a
cyclodextrin glucanotransferase, wherein said cyclodextrin
glucanotransferase variant comprises and insertion, substitution or
deletion of one or more amino acids, wherein said substitution is a
substitution an amino acid residue which is present in a
corresponding position in the amino acid sequence of amino acids 1
to 686 of SEQ ID NO: 2 but which is not present in the amino acid
sequence of the parent cyclodextrin glucanotransferase; wherein
said insertion is an insertion of an amino acid residue which is
present in a corresponding position in the amino acid sequence of
amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in
the amino acid sequence of the parent cyclodextrin
glucanotransferase; and wherein said deletion is a deletion of an
amino acid residue which is present in the parent cyclodextrin
glucanotransferase but which is not present in the amino acid
sequence of amino acids 1 to 686 of SEQ ID NO: 2, and wherein the
cyclodextrin glucanotransferase variant forms linear
oligosaccharides when acting on starch. (b) recovering the
cyclodextrin glucanotransferase variant.
10. The method of claim 9, wherein the cyclodextrin
glucanotransferase is derived from a strain of Bacillus,
Brevibacterium, Clostridium, Corynebacterium, Klebsiella,
Micrococcus, Thermoanaerobacter or Thermoanaerobacterium.
11. A cyclodextrin glucanotransferase variant prepared by the
method of claim 9.
12. A method for producing a variant of a parent cyclodextrin
glucanotransferase, comprising: (a) cultivating a host cell
comprising a nucleic acid sequence encoding a variant of a
cyclodextrin glucanotransferase, wherein said cyclodextrin
glucanotransferase variant comprises and insertion, substitution or
deletion of one or more amino acids, wherein said substitution is a
substitution an amino acid residue which is present in a
corresponding position in the amino acid sequence of amino acids 1
to 686 of SEQ ID NO: 2 but which is not present in the amino acid
sequence of the parent cyclodextrin glucanotransferase; wherein
said insertion is an insertion of an amino acid residue which is
present in a corresponding position in the amino acid sequence of
amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in
the amino acid sequence of the parent cyclodextrin
glucanotransferase; and wherein said deletion is a deletion of an
amino acid residue which is present in the parent cyclodextrin
glucanotransferase but which is not present in the amino acid
sequence of amino acids 1 to 686 of SEQ ID NO: 2; (b) transforming
a host cell with the nucleic acid sequence encoding the variant;
(c) cultivating the transformed host cell to express the variant;
(d) testing the variant cyclodextrin glucanotransferase for the
ability to form linear oligosaccharides when acting on starch; (e)
producing the variant cyclodextrin glucanotransferase by
cultivating a host cell comprising a nucleic acid sequence encoding
the variant cyclodextrin glucanotransferase; (f) recovering the
cyclodextrin glucanotransferase variant.
13. The method of claim 12, wherein the cyclodextrin
glucanotransferase is derived from a strain of Bacillus,
Brevibacterium, Clostridium, Corynebacterium, Klebsiella,
Micrococcus, Thermoanaerobacter or Thermoanaerobacterium.
14. A cyclodextrin glucanotransferase variant prepared by the
method of claim 12.
15. An isolated polypeptide which: a) has an amino acid sequence
having at least 70% identity to a parent Bacillus or
Thermoanerobacter cyclodextrin glucanotransferase (CGTase); b)
comprises an amino acid modification compared to the parent CGTase
in a region corresponding to amino acids 190-194 of the amino acid
sequence shown in SEQ ID NO: 2, wherein the modification is
selected from the group consisting of: an insertion of DAGF (SEQ ID
NO: 28), an insertion of DPGF (SEQ ID NO: 29), an insertion of DPF;
an insertion of DPAAGF (SEQ ID NO: 30), an insertion of DPAAGGF
(SEQ ID NO: 31) and a substitution at a position corresponding to
T189 of SEQ ID NO: 2; and c) has the ability to form linear
oligosaccharides as an initial product when acting on starch.
16. The polypeptide of claim 15, wherein the amino acid
modification comprises an insertion of DAGF (SEQ ID NO: 28) in a
region corresponding to amino acids 190-194 of the amino acid
sequence shown in SEQ ID NO: 2.
17. The polypeptide of claim 15, wherein the amino acid
modification comprises an insertion of DPGF (SEQ ID NO: 29) in a
region corresponding to amino acids 190-194 of the amino acid
sequence shown in SEQ ID NO: 2.
18. The polypeptide of claim 15, wherein the amino acid
modification comprises an insertion of DPF in a region
corresponding to amino acids 190-194 of the amino acid sequence
shown in SEQ ID NO: 2.
19. The polypeptide of claim 15, wherein the amino acid
modification comprises an insertion of DPAAGF (SEQ ID NO: 30) in a
region corresponding to amino acids 190-194 of the amino acid
sequence shown in SEQ ID NO: 2.
20. The polypeptide of claim 15, wherein the amino acid
modification comprises an insertion of DPAAGGF (SEQ ID NO: 31) in a
region corresponding to amino acids 190-194 of the amino acid
sequence shown in SEQ ID NO: 2.
21. The polypeptide of claim 15, wherein the amino acid
modification comprises a substitution at a position corresponding
to T189 of SEQ ID NO: 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 10/442,558 filed on May 21, 2003 (pending) which is a
divisional of U.S. application Ser. No. 10/234,266 (now abandoned),
filed on Sep. 4, 2002, which is a divisional of U.S. application
Ser. No. 09/645,707, filed on Aug. 24, 2000 (now U.S. Pat. No.
6,482,622), which is a continuation of PCT/DK/99/00087, filed on
Feb. 26, 1999, and claims priority under 35 U.S.C. 119 of Danish
Application Nos. PA 1998 00269 and PA 1998 00273, both filed on
Feb. 27, 1998, and U.S. Provisional Application Nos. 60/077,509 and
60/077,795, filed on Mar. 11, 1998 and Mar. 12, 1998, respectively,
the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of converting a
maltogenic alpha-amylase into a cyclodextrin glucanotransferase
(CGTase) or vice versa or creating hybrids of the two. The
invention also relates to the variants made by the methods.
BACKGROUND OF THE INVENTION
[0003] Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) and
maltogenic alpha-amylase (EC 3.2.1.133) are two classes of
glycosylases that degrade starch by hydrolysis of the
.alpha.-(1,4)-glycosidic bonds, but the initial products are
predominantly cyclic for CGTases and linear for the maltogenic
alpha-amylase.
[0004] Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), also
designated cyclodextrin glucanotransferase or cyclodextrin
glycosyltransferase, abbreviated herein as CGTase, catalyses the
conversion of starch and similar substrates into cyclomaltodextrins
via an intramolecular transglycosylation reaction, thereby forming
cyclomaltodextrins (or CD) of various sizes. Commercially most
important are cyclodextrins of 6, 7 and 8 glucose units, termed
.alpha.-, .beta.- and .gamma.-cyclodextrins, respectively.
[0005] CGTases are widely distributed and from several different
bacterial sources, including Bacillus, Brevibacterium, Clostridium,
Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter and
Thermoanaerobacterium have been extensively described in the
literature. A CGTase produced by Thermoanaerobacter sp. has been
reported in Norman B E, J rgensen S T; Denpun Kagaku 1992 39
99-106, and WO 89/03421, and the amino acid sequence has been
disclosed in WO 96/33267. The sequence of CGTases from
Thermoanaerobacterium thermosulfurigenes and from Bacillus
circulansis available on the Internet (SCOP or PDF home pages) as
pdf file 1CIU, and the sequence of a CGTase from B. circulans is
available as pdf file 1CDG.
[0006] Tachibana, Y., Journal of Fermentation and Bioengineering,
83 (6), 540-548 (1997) describes the cloning and expression of a
CGTase. Variants of CGTases have been described by Kim, Y. H.,
Biochemistry and Molecular Biology International, 41 (2), 227-234
(1997); Sin K-A, Journal of Biotechnology, 32 (3), 283-288 (1994);
D Penninga, Biochemistry, 34 (10), 3368-3376 (1995); and WO
96/33267.
[0007] Maltogenic alpha-amylase (glucan 1,4-a-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose
in the alpha-configuration, and is also able to hydrolyze
maltotriose as well as cyclodextrin.
[0008] A maltogenic alpha-amylase from Bacillus (EP 120 693) is
commercially available under the trade name Novamyl.RTM. (product
of Novo Nordisk NS, Denmark) and is widely used in the baking
industry as an anti-staling agent due to its ability to reduce
retrogradation of starch (WO 91 /04669).
[0009] The maltogenic alpha-amylase Novamyl.RTM. shares several
characteristics with cyclodextrin glucanotransferases (CGTases),
including sequence homology (Henrissat B, Bairoch A; Biochem. J.,
316, 695-696 (1996)) and formation of transglycosylation products
(Christophersen, C., et al., 1997, Starch, vol. 50, No. 1,
39-45).
BRIEF DESCRIPTION OF THE INVENTION
[0010] The inventors have discovered some striking, and not
previously predicted structural similarities and differences
between the structure of Novamyl and the reported structures of
CGTases, and based on this they have constructed variants of
maltogenic alpha-amylase having CGTase activity and variants of
CGTase having maltogenic alpha-amylase activity. Further, on the
basis of sequence homology between Novamyl.RTM. and CGTases, the
inventors have constructed hybrid enzymes with one or more
improvements to specific properties of the parent enzymes, using
recombinant DNA methodology.
[0011] Accordingly, the present invention provides a polypeptide
which:
[0012] a) has at least 70% identity to amino acids 1-686 of SEQ ID
NO: 1;
[0013] b) comprises an amino acid modification which is an
insertion, substitution or deletion compared to SEQ ID NO: 1 in a
region corresponding to amino acids 40-43, 78-85, 136-139, 173-180,
188-195 or 259-268; and
[0014] c) has the ability to form cyclodextrin when acting on
starch.
[0015] The invention also provides a polypeptide which:
[0016] a) has an amino acid sequence having at least 70% identity
to a parent cyclodextrin glucanotransferase (CGTase);
[0017] b) comprises an amino acid modification which is an
insertion, substitution or deletion compared to the parent CGTase
in a region corresponding to amino acids 40-43, 78-85, 136-139,
173-180, 188-195 or 259-268 of SEQ ID NO: 1; and
[0018] c) has the ability to form linear oligosaccharides when
acting on starch.
[0019] Further, the invention provides a method for constructing a
maltogenic alpha-amylase, comprising:
[0020] a) recombining DNA encoding a cyclodextrin
glucanotransferase (CGTase) and DNA encoding a maltogenic
alpha-amylase;
[0021] b) using the recombinant DNA to express a polypeptide;
and
[0022] c) testing the polypeptide to select a polypeptide having
the ability to form linear oligosaccharides when acting on
starch.
[0023] Finally, the invention provides a method of selecting DNA
encoding maltogenic alpha-amylase in a DNA pool, comprising:
[0024] a) amplifying DNA encoding maltogenic alpha-amylase by a
polymerase chain reaction (PCR) using primers encoding a partial
amino acid sequence of amino acids 1-686 of SEQ ID NO: 1,
preferably comprising at least 5 amino acid residues, preferably
comprising one or more of positions 188-196, more preferably
comprising positions 190-194,
[0025] b) cloning and expressing the amplified DNA, and
[0026] c) screening for maltogenic alpha-amylase activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows plasmid pCA31, described in Example 1.
[0028] FIG. 2 is a diagram showing the shuffling of Novamyl with
CGTases, as described in Example 3.
[0029] FIG. 3 is a diagram showing the selection of clones with
desired features by PCR, as described in Examples 2 and 3.
[0030] FIG. 4a-4b shows an alignment of the amino acid sequence of
Novamyl (1-686 of SEQ ID NO: 2) with the sequence of the CGTase
from Thermoanaerobacterium thermosulfurigenese (SEQ ID NO:32); the
CGTase from Thermoanaerobacter (SEQ ID NO: 33), and the CGTase from
Bacillus circulans (SEQ ID NO:34).
DETAILED DESCRIPTION OF THE INVENTION
[0031] Maltogenic alpha-amylase
[0032] The parent maltogenic alpha-amylase used in the invention is
an enzyme classified in EC 3.2.1.133. The enzymatic activity does
not require a non-reducing end on the substrate and the primary
enzymatic activity results in the degradation of amylopectin and
amylose to maltose and longer maltodextrins. It is able to
hydrolyze amylose and amylopectin to maltose in the
alpha-configuration, and is also able to hydrolyze maltotriose as
well as cyclodextrin.
[0033] A particularly preferred maltogenic alpha-amylase is the
amylase cloned from Bacillus as described in EP 120 693
(hereinafter referred to as Novamyl). Novamyl has the amino acid
sequence set forth in amino acids 1-686 of SEQ ID NO: 1. Novamyl is
encoded in the gene harbored in the Bacillus strain NCIB 11837
which has the nucleic acid sequence set forth in SEQ ID NO:1.
CGTase
[0034] The parent CGTase used in the invention is an enzyme
classified in EC 2.4.1.19. It may be from any source, e.g.
bacterial sources, including Bacillus, Brevibacterium, Clostridium,
Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter and
Thermoanaerobacterium.
[0035] The CGTase preferably has one or more of the following
characteristics:
[0036] i) an amino acid sequence having at least 50% identity to
amino acids 1-686 of SEQ ID NO: 1, preferably at least 60%;
[0037] ii) being encoded by a DNA sequence which hybridizes at
conditions described below to the DNA sequence set forth in SEQ ID
NO:1 or to the DNA sequence encoding Novamyl harbored in the
Bacillus strain NCIB 11837; and
[0038] iii) a catalytic binding site comprising amino acid residues
corresponding to D228, E256 and D329 as shown in the amino acid
sequence set forth in amino acids 1-686 of SEQ ID NO: 1.
Variants of CGTase
[0039] The CGTase variant of this invention has the ability to form
linear oligosaccharides when acting on starch. The starch
hydrolysis and the analysis of initial reaction products may be
carried out as described in an Example.
[0040] The CGTase variant has a modification of at least one amino
acid residue in a region corresponding to residues 40-43, 78-85,
136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1. Each
modification may be an insertion, a deletion or a substitution, of
one or more amino acid residues in the region indicated. The
modification of the parent CGTase is preferably such that the
resulting modified amino acid or amino acid sequence more closely
resembles the corresponding amino acid or structural region in
Novamyl. Thus, the modification may be an insertion of or a
substitution with an amino acid present at the corresponding
position of Novamyl, or a deletion of an amino acid not present at
the corresponding position of Novamyl.
[0041] The CGTase variant may particularly comprise an insertion
into a position corresponding to the region D190-F194 of Novamyl
(amino acid sequence shown in SEQ ID NO: 1). The insertion may
comprise 3-7 amino acids, particularly 4-6, e.g. 5 amino acids. The
insertion may be DPAGF(SEQ ID NO:27) as found in Novamyl or an
analogue thereof, e.g. with the first amino acid being negative,
the last one being aromatic, and the ones in between being
preferably P, A or G. The variant may further comprise a
substitution at the position corresponding to T189 of Novamyl with
a neutral amino acid which is less bulky than F, Y or W. Other
examples of insertions are DAGF(SEQ ID NO:28), DPGF(SEQ ID. NO:29),
DPF, DPAAGF(SEQ ID NO:30), and DPAAGGF(SEQ ID NO:31).
[0042] Modifications in the region 78-85 preferably include
deletion of 2-5 amino acids, e.g. 3 or 4. Preferably, any aromatic
amino acid in the region 83-85 should be deleted or substituted
with a non-aromatic.
[0043] Modifications in the region 259-268 preferably include
deletion of 1-3 amino acid, e.g. two. The region may be modified so
as to correspond to Novamyl
[0044] The CGTase variant may comprise further modifications in
other regions, e.g. regions corresponding to amino acids 37-39,
44-45, 135, 140-145, 181-186, 269-273, or 377-383 of Novamyl.
[0045] Additional modifications of the amino acid sequence may be
modeled on a second CGTase, i.e. an insertion of or substitution
with an amino acid found at a given position in the second CGTase,
or they may be made close to the substrate (less than 8 .ANG. from
the substrate, e.g. less than 5 .ANG. or less than 3 .ANG.) as
described in WO 96/33267.
[0046] The following are some examples of variants based on a
parent CGTase from Thermoanaerobacter (using B. circulans
numbering). Similar variants may be made from other CGTases.
L194F+*194aT+*194bD+*194cP+*194dA+*194eG+D196S
L87H+D89*+T91G+F91aY+G92*+G93*+S94*+L194F+*194aT+*194bD+*194cP+*194dA+*1-
94eG+D196S
*194aT+*194bD+*194cP+*194dA+*194eG+D196S
L87H+D89*+T91G+F91aY+G92*+G93*+S94*+*194aT+*194bD+*194cP+*194dA+*194eG+D-
196S
Y260F+L261
G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V
*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N264P+E-
265G+V266T+*266aA+*266bN+D267H+P268V
Variants of Novamyl
[0047] The Novamyl variant of this invention has the ability to
form cyclodextrin when acting on starch. The starch hydrolysis and
the analysis of reaction products may be carried out as described
in an Example.
[0048] The Novamyl variant has a modification of at least one amino
acid residue in the same regions described above for CGTase
variants. However, the modifications are preferably in the opposite
direction, i.e. such that the resulting modified amino acid or
amino acid sequence more closely resembles the corresponding amino
acid or structural region of a CGTase. Thus, the modification may
be an insertion of or a substitution with an amino acid present at
the corresponding position of a CGTase, or a deletion of an amino
acid not present at the corresponding position of a CGTase.
[0049] Preferred modifications include a deletion in the region
190-195, preferably the deletion .DELTA. (191-195) and/or a
substitution of amino acid 188 and/or 189, preferably F188L and/or
Y189Y.
Amino Acid Identity
[0050] For purposes of the present invention, the degree of
identity may be suitably determined according to the method
described in Needleman, S. B. and Wunsch, C. D., (1970), Journal of
Molecular Biology, 48, 443-45, with the following settings for
polypeptide sequence comparison: GAP creation penalty of 3.0 and
GAP extension penalty of 0.1. The determination may be done by
means of a computer program known such as GAP provided in the GCG
program package (Program Manual for the Wisconsin Package, Version
8, August 1994, Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711).
[0051] The variants of the invention have an amino acid identity
with the parent enzyme (Novamyl or CGTase) of at least 70%,
preferably at least 80%, e.g. at least 90%, particularly at least
95% or at least 98%.
Hybridization
[0052] The hybridization referred to above indicates that the
analogous DNA sequence hybridizes to the nucleotide probe
corresponding to the protein encoding part of the nucleic sequence
shown in SEQ ID NO:1, under at least low stringency conditions as
described in detail below.
[0053] Suitable experimental conditions for determining
hybridization at low stringency between a nucleotide probe and a
homologous DNA or RNA sequence involves presoaking of the filter
containing the DNA fragments or RNA to hybridize in 5.times. SSC
(sodium chloride/sodium citrate, Sambrook, J., Fritsch, E. J., and
Maniatis, T. (1989) Molecular cloning: a laboratory manual, Cold
Spring Harbor Laboratory Press, New York) for 10 min, and
prehybridization of the filter in a solution of 5.times. SSC,
5.times. Denhardt's solution (Sambrook, et al., op.cit.), 0.5% SDS
and 100 .mu.g/ml of denatured sonicated salmon sperm DNA (Sambrook,
et al., op.cit.), followed by hybridization in the same solution
containing a random-primed (Feinberg, A. P. and Vogelstein, B.
(1983) Anal. Biochem. 132:6-13), .sup.32P-dCTP-labeled (specific
activity>1.times.10.sup.9 cpm/.mu.g) probe for 12 hours at ca.
45.degree. C. The filter is then washed twice for 30 minutes in
2.times. SSC, 0.5% SDS at least 55.degree. C. (low stringency),
more preferably at least 60.degree. C. (medium stringency), more
preferably at least 65.degree. C. (medium/high stringency), more
preferably at least 70.degree. C. (high stringency), even more
preferably at least 75.degree. C. (very high stringency).
[0054] Molecules which hybridize to the oligonucleotide probe under
these conditions are detected by exposure to x-ray film.
Corresponding Amino Acids
[0055] Corresponding amino acids for the following 4 amino acid
sequences are shown in the alignment in FIG. 4a-4b which is based
on the three-dimensional structure of the sequences.
[0056] 1) Novamyl (amino acids 1-686 of SEQ ID NO: 2)
[0057] 2) CGTase from Thermoanaerobacterium thermosulfurigenes (pdf
file 1CIU) (SEQ ID NO: 32)
[0058] 3) CGTase from Thermoanaerobacter, described in WO 96/33267
(SEQ ID NO: 33)
[0059] 4) CGTase from Bacillus circulans (pdf file 1CDG) (SEQ ID
NO: 34)
[0060] Corresponding amino acid residues in other CGTases may be
found by aligning with one of the sequences in FIG. 4a-4b by to the
method described in Needleman (supra) using the same parameters,
e.g. by means of the GAP program (supra).
Nomenclature for Amino Acid Modifications
[0061] The nomenclature used herein for defining mutations is
essentially as described in WO 92/05249. Thus, F188L indicates a
substitution of the amino acid F (Phe) in position 188 with the
amino acid L (Leu). .DELTA. (191-195) or .DELTA. (191-195)
indicates a deletion of amino acids in positions 191-195. 192-A-193
indicates an insertion of A between amino acids 192 and 193. *194aT
indicates an insertion of T at the first position after 194. G92*
indicates a deletion of G at position 92.
Recombination of CGTase and Maltogenic Alpha-amylase
[0062] The present invention further relates to a method for
constructing a variant enzyme comprising Novamyl and one or more
parent CGTases, wherein said variant has at least one altered
property relative to Novamyl and said parent CGTases, which method
comprises:
[0063] i) generating DNA fragments encoding amino acid sequences
obtainable from Novamyl and said parent CGTases;
[0064] ii) constructing a hybrid variant which contains amino acid
sequences generated in step i) by in vivo or in vitro DNA
shuffling; and
[0065] iii) testing the resulting variant for said property.
[0066] The methods for generating DNA fragments referred to in step
i) of the method above are well known in the art and may include,
for example, treatment of a DNA sequence encoding an amino acid
sequence with a restriction enzyme, e.g., DNAse I.
[0067] The DNA shuffling referred to in step ii) in the method
above may be recombination, either in vivo or in vitro, of
nucleotide sequence fragment(s) between two or more polynucleotides
resulting in output polynucleotides (i.e., polynucleotides having
been subjected to a shuffling cycle) having a number of nucleotide
fragments exchanged, in comparison to the input polynucleotides
(i.e. starting point polynucleotides). Shuffling may be
accomplished either in vitro or in vivo by recombination within a
cell by methods described in the art (cf., Crameri, et al, 1997,
Nature Biotechnology Vol. 15:436-438).
[0068] In a preferred embodiment, at least one DNA fragment
obtainable from Novamyl in step i) of the method above encodes an
amino acid sequence, which is determined to be of relevance for
altering said property.
[0069] In a more preferred embodiment, a hybrid variant of a parent
CGTase is obtained by the above method comprising a modification of
at least one amino acid residue in the group consisting of amino
acid residues corresponding to residues 37 to 45, residues 135 to
145, residues 173 to 180, residues 189 to 196, residues 261 to 266,
residues 327 to 330, and residues 370 to 376 of SEQ ID NO: 1.
[0070] In another more preferred embodiment, a hybrid variant
comprising Novamyl and one or more parent CGTases is constructed by
the above method in which the amino acid sequence of
Asp190-Pro191-Ala192-Gly-193-Phe194 corresponding to the positions
in the amino acid sequence shown in SEQ ID NO: 1 is inserted into
the corresponding positions in said hybrid, wherein the
corresponding positions is determined on the basis of amino acid
sequence alignment.
[0071] In another more preferred embodiment, a hybrid variant
comprising Novamyl and one or more parent CGTase is obtained by the
above method in which the amino acid sequence of
Asp190-Pro191-Ala192-Gly193-Phe194-Ser195 corresponding to the
positions in the amino acid sequence shown in SEQ ID NO: 1 is
inserted into the corresponding positions in said hybrid, wherein
the corresponding positions is determined on the basis of amino
acid sequence alignment.
[0072] It is possible to use the unique active site loop to select
hybrid enzymes with maltogenic alpha-amylase activity from a
library of random recombinants. Thus, a maltogenic alpha-amylase
and a CGTase may be randomly recombined, e.g. by the DNA shuffling
method of Crameri A, et al., op.cit. Those resulting mutants
containing the Novamyl loop may be selected using PCR, e.g. as
described above in the Examples.
[0073] The property to be altered may be substrate specificity,
substrate binding, substrate cleavage pattern, specific activity of
cleavage, transglycosylation, and relative activity of
cyclization.
[0074] The DNA sequence encoding a parent CGTase to be used in the
methods of the invention may be isolated from any cell or
microorganism producing the CGTase in question using methods known
in the art.
Cloning a DNA Sequence Encoding a CGTase
[0075] The DNA sequence encoding a parent CGTase may be isolated
from any cell or microorganism producing the CGTase in question,
using various methods well known in the art, for example, from the
Bacillus strain NCIB 11837.
[0076] First, a genomic DNA and/or cDNA library should be
constructed using chromosomal DNA or messenger RNA from the
organism that produces the CGTase to be studied. Then, if the amino
acid sequence of the CGTase is known, homologous, labeled
oligonucleotide probes may be synthesized and used to identify
CGTase-encoding clones from a genomic library prepared from the
organism in question. Alternatively, a labeled oligonucleotide
probe containing sequences homologous to a known CGTase gene could
be used as a probe to identify CGTase-encoding clones, using
hybridization and washing conditions of lower stringency.
[0077] Another method for identifying CGTase-encoding clones
involves inserting fragments of genomic DNA into an expression
vector, such as a plasmid, transforming maltogenic alpha-amylase
negative bacteria with the resulting genomic DNA library, and then
plating the transformed bacteria onto agar containing a substrate
for maltogenic alpha-amylase, thereby allowing clones expressing
maltogenic alpha-amylase activity to be identified.
[0078] 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.
[0079] 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, wherein the fragments correspond to various
parts of the entire DNA sequence, in accordance with techniques
well known in the art. 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).
Random Mutagenesis
[0080] A general approach for modifying proteins and enzymes has
been based on random mutagenesis, for instance, as disclosed in
U.S. Pat. No. 4,894,331 and WO 93/01285. For instance, the random
mutagenesis may be performed by use of a suitable physical or
chemical mutagenizing agent, by use of a suitable oligonucleotide,
or by subjecting the DNA sequence to PCR generated mutagenesis.
Furthermore, the random mutagenesis may be performed by use of any
combination of these mutagenizing agents. The mutagenizing agent
may, e.g., be one which induces transitions, transversions,
inversions, scrambling, deletions, and/or insertions.
[0081] 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. 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.
[0082] When the mutagenesis is performed by the use of an
oligonucleotide, the oligonucleotide may be doped or spiked with
the three non-parent nucleotides during the synthesis of the
oligonucleotide at the positions which are to be changed. The
doping or spiking may be done so that codons for unwanted amino
acids are avoided. The doped or spiked oligonucleotide can be
incorporated into the DNA encoding the maltogenic alpha-amylase
enzyme by any published technique, using e.g. PCR, LCR or any DNA
polymerase and ligase as deemed appropriate.
[0083] Preferably, the doping is carried out using "constant random
doping", in which the percentage of wild-type and mutation in each
position is predefined. Furthermore, the doping may be directed
toward a preference for the introduction of certain nucleotides,
and thereby a preference for the introduction of one or more
specific amino acid residues. The doping may be made, e.g., so as
to allow for the introduction of 90% wild type and 10% mutations in
each position. An additional consideration in the choice of a
doping scheme is based on genetic as well as protein-structural
constraints. The doping scheme may be made by using the DOPE
program (cf., Tomandl, D. et al., 1997, Journal of Computer-Aided
Molecular Design 11:29-38; Jensen, L J, Andersen, K V, Svendsen, A,
and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702) which,
inter alia, ensures that introduction of stop codons is
avoided.
[0084] When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent CGTase 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).
[0085] 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 CGTase by, e.g., transforming a plasmid containing
the parent CGTase into the mutator strain, growing the mutator
strain with the plasmid and isolating the mutated plasmid from the
mutator strain. The mutated plasmid may be subsequently transformed
into the expression organism.
[0086] The DNA sequence to be mutagenized may be conveniently
present in a genomic or cDNA library prepared from an organism
expressing the parent CGTase. 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 mutagenising agent. The DNA to be mutagenized may
also be present in a host cell either by being integrated in the
genome of said cell or by being present on a vector harbored 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.
[0087] In some cases it may be convenient to amplify the mutated
DNA sequence prior to expression or screening. 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.
[0088] Subsequent to the incubation with or exposure to the
mutagenising agent, the mutated DNA is expressed by culturing a
suitable host cell carrying the DNA sequence under conditions
allowing expression to take place. The host cell used for this
purpose may be one which has been transformed with the mutated DNA
sequence, optionally present on a vector, or one which was carried
the DNA sequence encoding the parent enzyme during the mutagenesis
treatment. Examples of suitable host cells are the following: gram
positive bacteria such as Bacillus subtilis, Bacillus
licheniformis, Bacillus lentus, Bacillus brevis, Bacillus
stearothermophilus, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus
lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces
lividans or Streptomyces murinus; and gram negative bacteria such
as E. coli.
[0089] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
DNA Shuffling
[0090] Alternative methods for rapid preparation of modified
polypeptides may be prepared using methods of in vivo or in vitro
DNA shuffling wherein DNA shuffling is defined as recombination,
either in vivo or in vitro, of nucleotide sequence fragment(s)
between two or more polynucleotides resulting in output
polynucleotides (i.e., polynucleotides having been subjected to a
shuffling cycle) having a number of nucleotide fragments exchanged,
in comparison to the input polynucleotides (i.e. starting point
polynucleotides). Shuffling may be accomplished either in vitro or
in vivo by recombination within a cell by methods described in the
art.
[0091] For instance, Weber et al. (1983, Nucleic Acids Research,
vol. 11, 5661-5661) describe a method for modifying genes by in
vivo recombination between two homologous genes, wherein
recombinants were identified and isolated using a resistance
marker.
[0092] Pompon et al., (1989, Gene 83:15-24) describe a method for
shuffling gene domains of mammalian cytochrome P-450 by in vivo
recombination of partially homologous sequences in Saccharomyces
cereviseae by transforming Saccharomyces cereviseae with a
linearized plasmid with filled-in ends, and a DNA fragment being
partially homologous to the ends of said plasmid.
[0093] In WO 97/07205 a method is described whereby polypeptide
variants are prepared by shuffling different nucleotide sequences
of homologous DNA sequences by in vivo recombination using plasmid
DNA as template.
[0094] U.S. Pat. No. 5,093,257 (Genencor Int. Inc.) discloses a
method for producing hybrid polypeptides by in vivo recombination.
Hybrid DNA sequences are produced by forming a circular vector
comprising a replication sequence, a first DNA sequence encoding
the amino-terminal portion of the hybrid polypeptide, a second DNA
sequence encoding the carboxy-terminal portion of said hybrid
polypeptide. The circular vector is transformed into a rec positive
microorganism in which the circular vector is amplified. This
results in recombination of said circular vector mediated by the
naturally occurring recombination mechanism of the rec positive
microorganism, which include prokaryotes such as Bacillus and E.
coli, and eukaryotes such as Saccharomyces cereviseae.
[0095] One method for the shuffling of homologous DNA sequences has
been described by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci.
USA, Vol. 91, 10747-10751; Stemmer, (1994), Nature, vol. 370, 389-
391; Crameri A, Dawes G, Rodriguez E Jr , Silver S, Stemmer WPC
(1997) Nature Biotechnology Vol. 15 , No. 5 pp. 436-438). The
method concerns shuffling homologous DNA sequences by using in
vitro PCR techniques. Positive recombinant genes containing
shuffled DNA sequences are selected from a DNA library based on the
improved function of the expressed proteins.
[0096] The above method is also described in WO 95/22625 in
relation to a method for shuffling homologous DNA sequences. An
important step in the method described in WO 95/22625 is to cleave
the homologous template double-stranded polynucleotide into random
fragments of a desired size followed by homologously reassembling
of the fragments into full-length genes.
Site-directed Mutagenesis
[0097] Once a maltogenic 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 maltogenic alpha-amylase-encoding
sequence, is created in a vector carrying the maltogenic
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.
[0098] Another method of introducing mutations into a maltogenic
alpha-amylase-encoding DNA sequences is described in Nelson and
Long, Analytical Biochemistry 180, 1989, pp. 147-151. It involves a
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.
Localized Random Mutagenesis
[0099] The random mutagenesis may be advantageously localised to a
part of the parent CGTase 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.
[0100] The localized, or region-specific, random mutagenesis is
conveniently performed by use of PCR generated mutagenesis
techniques as described above or any other suitable technique known
in the art. Alternatively, the DNA sequence encoding the part of
the DNA sequence to be modified may be isolated, e.g., by insertion
into a suitable vector, and said part may be subsequently subjected
to mutagenesis by use of any of the mutagenesis methods discussed
above.
Expression of Maltogenic Alpha-amylase Variants
[0101] The construction of the variant of interest is accomplished
by cultivating a microorganism comprising a DNA sequence encoding
the variant under conditions which are conducive for producing the
variant, and optionally subsequently recovering the variant from
the resulting culture broth. This is described in detail further
below.
[0102] 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 the form of a
protein or polypeptide, 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.
[0103] The recombinant expression vector carrying the DNA sequence
encoding an maltogenic 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.
[0104] 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 a maltogenic 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.
[0105] 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 maltogenic alpha-amylase variant of the invention.
Termination and polyadenylation sequences may suitably be derived
from the same sources as the promoter.
[0106] 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.
[0107] 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 tetracycline 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.
[0108] 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.
[0109] The procedures used to ligate the DNA construct of the
invention encoding maltogenic 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, Sambroo, et al., op.cit.).
[0110] 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
a maltogenic 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.
[0111] 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.
[0112] Examples of suitable bacteria are gram positive bacteria
such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus,
Bacillus brevis, Bacillus stearothermophilus, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus
thuringiensis, or Streptomyces lividans or Streptomyces murinus, or
gram negative bacteria such as E. coli. The transformation of the
bacteria may, for instance, be effected by protoplast
transformation or by using competent cells in a manner known per
se.
[0113] The yeast organism may favorably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces
cereviseae. 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.
[0114] In a yet further aspect, the present invention relates to a
method of producing a maltogenic 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.
[0115] 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 maltogenic 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).
[0116] The maltogenic 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 sulfate, followed by the use of chromatographic procedures
such as ion exchange chromatography, affinity chromatography, or
the like.
Screening of Variants with Maltogenic Alpha-amylase Activity
[0117] Variants produced by any of the methods described above may
be tested, either prior to or after purification, for maltogenic
alpha-amylase activity, such as amylolytic activity, in a screening
assay which measures the ability of the variant to degrade starch.
The screening in step 10 in the above-mentioned random mutagenesis
method of the invention may be conveniently performed by use of a
filter assay based on the following procedure: A microorganism
capable of expressing the variant of interest is incubated on a
suitable medium and under suitable conditions for secretion of the
variant, the medium being covered with two filters comprising a
protein-binding filter placed under a second filter exhibiting a
low protein binding capability. The microorganism is grown on the
second, top filter. Subsequent to the incubation, the bottom
protein-binding filter comprising enzymes secreted from the
microorganism is separated from the second filter comprising the
microorganism. The protein-binding filter is then subjected to
screening for the desired enzymatic activity, and the corresponding
microbial colonies present on the second filter are identified. The
first filter used for binding the enzymatic activity may be any
protein-binding filter, e.g., nylon or nitrocellulose. The second
filter 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..
[0118] Screening consists of treating the first filter to which the
secreted protein is bound with a substrate that allows detection of
the amylolytic activity. 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. 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. For example,
amylolytic activity can be detected by Cibacron Red labeled
amylopectin, which is immobilized in agarose. Amylolytic activity
on this substrate produces zones on the plate with reduced red
color intensity.
[0119] To screen for variants with increased stability, the filter
with bound maltogenic alpha-amylase variants can be pretreated
prior to the detection step described above to inactivate variants
that do not have improved stability relative to the parent CGTase.
This inactivation step may consist of, but is not limited to,
incubation at elevated temperatures in the presence of a buffered
solution at any pH from pH 2 to 12, and/or in a buffer containing
another compound known or thought to contribute to altered
stability e.g., surfactants, EDTA, EGTA, wheat flour components, or
any other relevant additives. Filters so treated for a specified
time are then rinsed briefly in deionized water and placed on
plates for activity detection as described above. The conditions
are chosen such that stabilized variants show increased enzymatic
activity relative to the parent after incubation on the detection
media.
[0120] To screen for variants with altered thermostability, filters
with bound variants are incubated in buffer at a given pH (e.g., in
the range from pH 2-12) at an elevated temperature (e.g., in the
range from 50.degree.-110.degree. C.) for a time period (e.g., from
1-20 minutes) to inactivate nearly all of the parent CGTase, rinsed
in water, then placed directly on a detection plate containing
immobilized Cibacron Red labeled amylopectin and incubated until
activity is detectable. Similarly, pH dependent stability can be
screened for by adjusting the pH of the buffer in the above
inactivation step such that the parent CGTase is inactivated,
thereby allowing detection of only those variants with increased
stability at the pH in question. To screen for variants with
increased calcium-dependent stability calcium chelators, such as
ethylene glycol-bis(.beta.-aminoethyl ether) N,N,N',N'-tetraacetic
acid (EGTA), is added to the inactivation buffer at a concentration
such that the parent CGTase is inactivated under conditions further
defined, such as buffer pH, temperature or a specified length of
incubation.
[0121] The variants of the invention may be suitably tested by
assaying 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 as described above. Further testing in
regard to altered properties, including specific activity,
substrate specificity, cleavage pattern, thermoactivation,
thermostability, pH dependent activity or optimum, pH dependent
stability, temperature dependent activity or optimum,
transglycosylation activity, stability, and any other parameter of
interest, may be performed on purified variants in accordance with
methods known in the art as described below.
[0122] The maltogenic alpha-amylase activity of variants of the
invention towards linear maltodextrins and cyclodextrins may be
assayed by measuring the hydrolysis of maltotriose. Hydrolysis is
monitored by the formation of glucose using the GLU-kit (Boehringer
Mannheim, Indianapolis, Ind). Hydrolysis of longer maltodextrins,
such as malto-tetraose to -heptaose) and cyclodextrins is monitored
by the formation of free reducing ends which is measured
spectrophotometrically.
[0123] Alternatively, amylolytic activity can be assayed using the
Phadebas method (BioRad, Inc., Richmond, Calif.) in which the
substrate is a water-insoluble cross-linked starch polymer carrying
a blue dye (Phadebas Amylase Test) that is hydrolyzed by amylolytic
activity to form water-soluble blue fragments which can then be
quantitated spectrophotometrically.
[0124] In cases where the variants of the invention have been
altered in the substrate binding site, it may be desirable to
determine whether such variant is capable of performing a
transglycosylation reaction, which is described below in Example 1,
as is normally observed for CGTases.
[0125] Substrate specificity of maltogenic alpha-amylase variants
may be assayed by measuring the degree to which such enzymes are
capable of degrading starch that has been exhaustively treated with
the exoglycosylase .beta.-amylase. To screen for variants which
show patterns of degradation on such a substrate differing from the
patterns produced by the parent CGTase the following assay is
performed: .beta.-limit dextrin is prepared by incubating 25 ml 1%
amylopectin in Mcllvane buffer (48.5 mM citrate and 193 mM sodium
phosphate pH 5.0) with 24 .mu.g/ml .beta.-amylase overnight at
30.degree. C. Unhydrolysed amylopectin (i.e., .beta.-limit dextrin)
is precipitated with 1 volume 98% ethanol, washed and redissolved
in water. 1 ml .beta.-limit dextrin is incubated with 18 .mu.l
enzymes (at 2.2 mg/ml) and 100 .mu.l 0.2 M citrate-phosphate pH 5.0
for 2 hrs at 30.degree. C. and analysed by HPLC as described above.
Total hydrolysis of .beta.-limit dextrin is carried out in 2M HCl
at 95.degree. C. The concentration of reducing ends is measured by
methods known in the art.
INDUSTRIAL APPLICATIONS
[0126] The maltogenic alpha-amylase variants of the invention
possess valuable properties which may be advantageously used in
various industrial applications. In particular, the enzyme finds
potential application for retarding or preventing retrogradation,
and thus the staling, of starch based food common in the baking
industry.
[0127] The variant may be used for the preparation of bread and
other bread products in accordance with conventional techniques
known in the art.
[0128] It is believed that the modification of the starch fraction
by use of the present invention results in increased volume in
baked products and improved organoleptic qualities, such as flavor,
mouth feel, palatability, aroma and crust color.
[0129] The maltogenic alpha-amylase variant may be used as the only
enzyme or as a major enzymatic activity in combination with one or
more additional enzymes, such as xylanase, lipase, glucose oxidase
and other oxidoreductases, or an amylolytic enzyme.
[0130] The enzyme variants of the invention also find industrial
applicability as a component in washing, dishwashing and
hard-surface cleaning detergent compositions. Some variants are
particularly useful in a process for the manufacture of linear
oligosaccharides, or 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.
[0131] The variants of the invention also find application in
processes for the manufacture of cyclodextrins for various
industrial applications, particularly in the food, cosmetic,
chemical, agrochemical and pharmaceutical industries.
[0132] Therefore in another aspect the invention provides
maltogenic alpha-amylase variants for use in a process for the
manufacture of cyclodextrins, in particular .alpha.-, .beta.-,
.gamma.-, .delta.-, .epsilon.-, and/or .zeta.-cyclodextrins. In a
more preferred embodiment, the invention provides maltogenic
alpha-amylase variants for use in a process for the manufacture of
.alpha.-, .beta.- and .gamma.-cyclodextrins, or mixtures
hereof.
[0133] In yet another preferred embodiment, the variants of the
invention may be used for in situ generation of cyclodextrins. In
this way the variants of the invention may be added to a substrate
containing medium in which the enzyme variants are capable of
forming the desired cyclodextrins. This application is particularly
well suited for use in methods of producing baked products as
described above, in methods for stabilizing chemical products
during their manufacture, and in detergent compositions.
[0134] Cyclodextrins have an inclusion ability useful for
stabilization, solubilization, etc. Thus cyclodextrins can make
oxidizing and photolytic substances stable, volatile substances
non-volatile, poorly-soluble substances soluble, and odoriferous
substances odorless, etc. and thus are useful to encapsulate
perfumes, vitamins, dyes, pharmaceuticals, pesticides and
fungicides. Cyclodextrins are also capable of binding lipophilic
substances such as cholesterol, to remove them from egg yolk,
butter, etc.
[0135] Cyclodextrins also find utilization in products and
processes relating to plastics and rubber, where they have been
used for different purposes in plastic laminates, films, membranes,
etc. Also, cyclodextrins have been used for the manufacture of
biodegradable plastics.
EXAMPLES
[0136] The invention is further illustrated with reference to the
following examples which are not intended to be in any way limiting
to the scope of the invention as claimed.
Example 1
Construction of variants of Thermoanaerobacter CGTase with Altered
Substrate Specificity
[0137] This example describes the construction of CGTase variants
with modified substrate specificity. The variants are derived from
a parent Thermoanaerobacter sp. CGTase (i.e. the wild type),
obtained as described in WO 89/03421 and WO 96/33267.
Bacterial Strains, Plasmids and Growth Conditions
[0138] Escherichia coli ME32 was used for recombinant DNA
manipulations. The variants were expressed in SHA273, a derivative
of Bacillus subtilis 168 which is apr.sup.-, npr.sup.-, amyE.sup.-,
amyR2.sup.- and prepared by methods known in the art. pCA31-wt is a
E. coli-B. subtilus shuttle vector harboring the parent
Thermoanaerobacter CGTase, shown in FIG. 1.
DNA Manipulations
[0139] DNA manipulations and transformation of E. coli were
essentially as described in Sambrook, J., Fritsch, E. J., and
Maniatis, T. (1989) Molecular cloning: a laboratory manual, Cold
Spring Harbor Laboratory Press, New York. B. subtilis was
transformed using methods known in the art.
Site-directed Mutagenesis
[0140] Mutant CGTase genes were constructed via SOE-PCR method
(Nelson and Long, op.cit.) using the Pwo DNA polymerase (Boehringer
Mannheim, Indianapolis, Ind.). The primary PCR reactions were
carried out with the mutagenesis primers 1 and 2 (SEQ ID NO: 3 and
4) plus an upstream or a downstream primer (SEQ ID NO: 5 or 6) on
the template strand, respectively. The reaction products were
subsequently used as template in a second PCR reaction together
with the upstream and downstream primers. The product of the last
reaction was digested with Styl and Spel, and exchanged with the
corresponding fragment (640 bp) from the vector pCA31-wt or
pCA31-.DELTA.(87-94)
(T-CGTase+L82H+D84*+T84bG+F84cY+G84d*+G85*+S86*). The resulting
variant plasmids were transformed into E. coli ME32 and vector DNA
was purified from E. coli colonies using the DNA-purification kit
from QIAGEN (Qiagen, Inc. Germany). The mutant vectors were finally
transformed into B. subtilis SHA273 for enzyme expression.
[0141] The degeneration of mutagenesis primer 2 (SEQ ID NO: 4)
containing A or C/G gave rise to two different amino acid
sequences. Thus, two variants of a parent CGTase were constructed.
Successful mutations resulted in restriction sites (Sac II) at
positions 7-12 of SEQ ID NO: 3 and positions 2-7 of SEQ ID NO: 4,
which allowed quick screening of transformants. Mutations were
verified by standard DNA sequencing techniques. The correctness of
the Styl-Spel fragment obtained by PCR was also confirmed by DNA
sequencing.
Production and Purification of CGTase Proteins
[0142] Enzymes were produced in transformed SHA273 cells grown in
shakeflasks at 30-37.degree. C. in 2*TY media containing 10 .mu.g/l
kanamycin. After 68-72 h of growth the culture was pelleted and the
supernatant separated from the cells by centrifugation. After
filtration through a 0.45 .mu.m nitrocellulose filter, the
supernatant was directly applied to an
.alpha.-cyclodextrin-sepharose-6FF affinity column (Monma et al.
1988 Biotechnol. Bioeng. 32, 404-407). After washing the column
with 10 mM sodium acetate (pH 5.5), the variants were eluted with
the same buffer supplemented with 1% (w/v) .alpha.-cyclodextrin.
Purity and molecular weight of the variants obtained were checked
by SDS-PAGE. Protein concentrations were determined by measuring
the absorption at 280 nm using a theoretical extinction coefficient
at 1.74 ml/mg.sup.-1/cm.sup.-1.
Enzyme Assays
[0143] All assays were performed at pH 6.0 and 37.degree. C. The
assay for cyclization activity was performed as described by
Penninga et al (1995, Biochemistry 34:3368-3376). Starch liquefying
activity was measured using the Phadebas Amylase Test kit
(Pharmacia NB, Sweden). Transglycosylation activity was assayed in
which 2.2 .mu.M of the variant was incubated with 200 mM
maltotriose at 40.degree. C. in 10 mM NaOAc pH 5.0 and 1 mM
CaCl.sub.2. At different time intervals, aliquots were analyzed by
HPLC to measure formation of different maltodextrins. Analytical
separations of maltodextrins were performed on a Dionex CarboPac
PA1-column connected to a Beckman Gold HPLC-system and a
pulsed-amperometric detector. The gradient was 0-600 mM NaOAc over
15 minutes in 0.1 M NaOH. Transglycosylation activity was detected
as an increase in the size of the from three to greater than three
glucose units covalently linked.
Example 2
Specific PCR Amplification Using Novamyl-specific PCR Primers
[0144] Comparison of Novamyl with CGTases reveals that it is thus
far unique in one structural feature: the insertion of a 5 amino
acid "loop" in domain A, residues 190 to 194 in the amino acid
sequence shown in SEQ ID NO: 1, that affects the enzyme structure
near the active site. It is therefore valuable to have a method of
obtaining variants with a similar active site structure, especially
with respect to the Novamyl active site loop. Here we describe such
a method using PCR primers specific to the Novamyl loop to amplify
from natural sources only those clones with this unique structural
feature.
Step 1
[0145] PCR Amplification of Glycosylases with Degenerate
Primers
[0146] Alignment of amino acid sequences for Novamyl with known
CGTases reveals regions of high homology that can be used to design
degenerate oligonucleotide primers for use in the PCR amplification
of CGTases from a mixed pool of genomic or cDNA. The resulting
fragments of a predicted size range can then be used as template
DNA in further PCR amplifications with Novamyl loop-specific
primers as described in Step 2.
[0147] Alignment of 10 amino acid sequences most related to Novamyl
was used to identify two regions of high local homology for the
design of degenerate primers: Primer 1 (SEQ ID NO: 7) corresponsing
to amino acids 88-93 from SEQ ID NO: 1 and Primer 2 (SEQ ID NO: 8)
corresponding to amino acids 417-412 from SEQ ID NO: 1.
[0148] Use of these primers on DNA fragments from bacterial sources
can, when used as primers in a PCR reaction under standard
conditions, amplify a DNA fragment approximately 1,000 basepairs in
length containing the central core of related glycosylases.
Resulting PCR fragments could then be used as templates in step 2,
as described below.
Step 2
[0149] PCR Amplification using Novamyl Loop-specific Primers (FIG.
3)
[0150] The following primer pair can be used, corresponding to the
degenerate translation of the amino acid sequence in the coding
(d3, SEQ ID 9) or noncoding (d4, SEQ ID NO: 10) DNA strand:
[0151] Primer d3 (SEQ ID NO: 9): degenerate sense primer
corresponding to amino acids 190-194 from SEQ ID NO: 1.
[0152] Primer d4 (SEQ ID NO: 10): degenerate anti-sense primer
corresponding to amino acids 194-190 from SEQ ID NO: 1.
[0153] Alternatively, it is possible to use the degenerate or exact
nucleotide sequence of Novamyl through eight amino acids that
contain the Novamyl sequence,
Phe188-Thr189-Asp190-Pro191-Ala192-Gly193-Phe194-Ser195 (loop
underlined) in both DNA strands as primers in a PCR reaction:
[0154] Primer d5 (SEQ ID NO: 11): degenerate sense primer
corresponding to amino acids 188-193 from SEQ ID NO: 1.
[0155] Primer d6 (SEQ ID NO: 12): degenerate anti-sense primer
corresponding to amino acids 195-190 from SEQ ID NO: 1.
[0156] Primer 7 (SEQ ID NO: 13): Exact Novamyl sense primer
corresponding to amino acids 188-193 from SEQ ID NO: 1.
[0157] Primer 8 (SEQ ID NO: 14): Exact Novamyl anti-sense primer
corresponding to amino acids 195-190 from SEQ ID NO: 1.
[0158] Using the PCR products of step 1 as templates in a PCR
reaction together with primer pairs 1 and d4, 2 and d4, 1 and d5, 2
and d6, 1 and 7, or 2 and 8, only those DNA sequences encoding the
Novamyl loop are expected to produce DNA fragments of approximately
the predicted size (FIG. 1, step 2.). Templates lacking the
loop-encoding DNA will not produce a product under standard PCR
conditions with an annealing temperature of 58.degree. C.
TABLE-US-00001 approximate size of Product Primer pair product A1 1
+ d4 321 A2 1 + d6 324 A3 1 + 8 324 B1 2 + d3 684 B2 2 + d5 690 B3
2 + 7 690
Step 3
Reconstruction of Full-length Fragments
[0159] Step 2 yields partial coding sequences of glycosylases that
contain the Novamyl loop at either the 3' (A fragments) or 5' ends
(B fragments) of the DNA fragments. Reassembly of longer clones
containing the loop will require the combining of the A and B
fragments by SOE-PCR methods known in the art.
Example 3
Conversion of CGTases into Novamyl-like Enzymes by Random
Recombination
[0160] In this example, the unique active site loop was used to
select hybrid enzymes with maltogenic alpha-amylase activity from a
library of random recombinants. In this method, Novamyl and the
cyclic maltodextrin glucosyl transferase (CGTase) from Bacillus
circulans, were randomly recombined by the DNA shuffling method of
Crameri A, et al., op.cit. Those resulting mutants containing the
Novamyl loop were selected using PCR as described above in Example
2.
Step 1
PCR Amplification and Shuffling of Novamyl and CGTase (FIG. 2)
[0161] Specific oligonucleotide primers specific for either the
Novamyl coding sequence or the CGTase coding sequenced were
designed as shown in SEQ ID NO: 15-20.
[0162] The entire Novamyl coding sequence (lacking the signal
sequence) was amplified using the Novamyl-specific primer pair #9
and #10 (SEQ ID NO: 15 and 16). Similarly, the mature CGTase coding
sequence was amplified using the CGTase-specific primer pair #11
and #12 (SEQ ID NO: 17 and 18). Both amplifications were performed
using the following reaction conditions: 100 .mu.M each primer, 0.2
mM each of dATP, dCTP, dGTP, and TTP, 2.5 U AmpliTaq polymerase
(Perkin Elmer, Inc.), and 1.times. concentration of the buffer
supplied by the manufacturer. PCR was performed in a Perkin Elmer
Thermocycler, model 2400, with the following conditions: 5 minutes
at 94 C. 25 cycles of 30 seconds at 94 C., 1 minute at 58 C, and 2
minutes at 72 C. followed by a final incubation of 7 minutes at
72.degree. C.
[0163] The resulting PCR products were then subjected to DNA
shuffling as described by Stemmer et al. Briefly, the two DNA
fragments were mixed in equimolar amounts and randomly digested
using DNase I treatment to generate gene fragments of between 50
and 500 bp. These gene fragments were then allowed to anneal to one
another and extend in a PCR reaction under low stringency
conditions, resulting in a re-assembling of an intact gene pool
containing the reassemble parental DNAs as well as chimeras between
the parents. Final amplification of shuffled products was performed
using the general primer pair #13 and #14 (SEQ ID NO: 19 and 20)
using the PCR conditions described above. Using these primers, all
full-length species, both parental and chimeric, were
amplified.
Step 2
PCR Amplification Using Novamyl Loop-specific Primers (FIG. 3)
[0164] Those genes within this mixture containing the Novamyl loop
were then selected for as described in Example 2 using the
loop-specific primers. In the first round of amplification, the 5'
and 3' ends of the genes containing the loop were amplified by
using the general primers in combination with the loop specific
primers # 7 and #8 (SEQ ID NO: 13 and 14) to amplify either the 5'
ends of the genes extending to the loop-encoding sequence #13 and
#8 (SEQ ID NO: 19 and 14) or the sequence extending from the loop
to the 3' ends of the genes #7 and #14 (SEQ ID NO: 13 and 20). As
described in Example 2, the resulting fragments were then assembled
using SOE PCR to create full-length genes. These products were then
selectively amplified using primer pairs lacking a Novamyl-specific
primer to produce only chimeras: #11, #10 and #12 (SEQ ID NO: 17,
16 and 18) or #10, #9 and #11 (SEQ ID NO: 16, 15 and 17). In this
way, only those clones that contained either the CGTase 5' end and
the Novamyl loop or the CGTase 3' end and the Novamyl loop were
selected.
[0165] The final PCR product were then digested with the enzymes
Xba I and Mlu I and inserted into a vector containing an intact
signal sequence. The resulting clones were transformed into
Bacillus, and the resulting polypeptides were sequenced.
[0166] 3 polypeptides obtained by this method were found to be
hybrids containing an N-terminal sequence from Novamyl and a
C-terminal sequence from the B. circulans CGTase as follows:
[0167] Novamyl amino acids 1-196+CGTase amino acids 198-685
[0168] Novamyl amino acids 1-230+CGTase amino acids 232-685
[0169] Novamyl amino acids 1-590+CGTase amino acids 596-685.
[0170] These results demonstrate that the method is effective for
generating and selecting hybrids containing the
Example 4
Construction of a Variant of Novamyl with CGTase Activity
[0171] A variant of Novamyl was constructed that has an altered
substrate specificity relative to the parent enzyme, in which the
variant has a CGTase-like transglycosylation/cyclization activity
not detectable in the parent enzyme. The variant differs from the
parent Novamyl with the amino acid sequence shown in amino acids
1-686 of SEQ ID NO: 1 in that residues 191-195 were removed, Phe188
was substituted with Leu and Thr189 was substituted with Tyr,
termed A (191-195)-F188L-T189Y. The variant was constructed by
sequence overlap extension PCR (SOE PCR) essentially as described
by Nelson and Long (op.cit.). SOE PCR consists of two primary PCRs
that produce two overlapping PCR fragments, both bearing the same
modification(s). In a second round of PCR, the two products from
the two primary PCRs are mixed without addition of template
DNA.
[0172] Oligonucleotide Primers Used in the Construction of A
(191-195)-F188L-T189Y:
[0173] Mutagenic Primer 1 (SEQ ID NO: 21) and Primer 2 (SEQ ID NO:
22). Positions 16-21 of SEQ ID NO: 21 and positions 4-9 of SEQ ID
NO: 22 are restriction sites.
[0174] The .DELTA. (191-195)-F188L-T189Y were obtained using
oligomers A82 (SEQ ID NO: 23) and B346 (SEQ ID NO: 24) as
end-primers.
[0175] DNA manipulations, transformation of Bacillus subtilis, and
purification of the resulting variant was performed as described
above in Example 4. The final purified variant was analysed for the
ability to form cyclodextrin from linear starch and compared to the
parent Novamyl enzyme as described below.
Detection of .beta.-cyclodextrin
[0176] The variant and the parent Novamyl enzyme were diluted with
10 mM citrate buffer pH 6.0 in order to obtain an equivalent
protein concentration prior to assay.
[0177] The cyclisation reaction mixture in a final volume of 1 ml
contained:
[0178] 0-50 .mu.l enzyme or variant diluted in 10 mM sodium citrate
buffer pH 6.0
[0179] 500 .mu.l 10(w/v) Paselli SA2 (AVEBE, Foxhol, The
Netherlands) dissolved in 10 mM citrate buffer pH 6.0 for a final
solution of 5%
[0180] The reaction mixture was pre-incubated in a 50.degree. C.
water bath for 10 min. before adding the enzyme or variant, and at
one-min time intervals a 100 .mu.l sample was put on ice for
further analysis.
[0181] .beta.-cyclodextrin was quantitated on the basis of
formation of a stable colourless inclusion complex with
phenolphthalein; thus, the colour of the solution decreases with as
the amount of .beta.-cyclodextrin detected increases.
[0182] To each of the 100 .mu.l samples from the cyclization
reaction 900 .mu.l of a working solution (3 ml of 3.75 mM
phenolphthalein added to 100 ml 0.2 M Na.sub.2CO.sub.3, pH 9.7) was
added, and the absorbance immediately read at 552 nm.
.beta.-cyclodextrin was quantitated on the basis of a calibration
curve prepared in a final volume of one-ml as follows:
[0183] 0-50 .mu.l 2 mM .beta.-cyclodextrin (0-100 nmol)
[0184] 50-0 .mu.l milli-Q water
[0185] 900 .mu.l working solution
[0186] 50 .mu.l 10% Paselli SA2
[0187] A new calibration curve was made for each new preparation of
Paselli SA2 solution.
[0188] The results of the cyclization assay are presented in the
table below as .beta.-cyclodextrin formation (mmol/mg enzyme) for
the variant .DELTA. (191-195)-F188L-T189Y and for the parent
enzyme, Novamyl:
TABLE-US-00002 Time (min) Variant Novamyl 0 0 0 2 160 0 3 230 0 4
240 0 5 320 0 6 380 0 7 390 0 8 500 0 10 680 0
[0189] The results clearly demonstrate that the variant, unlike the
parent Novamyl enzyme, can form .beta.-cyclodextrin.
Example 5
Construction of a CGTase Variant with Ability to Form Linear
Oligosaccharides
[0190] This example describes the construction of a CGTase variant
derived from a parent Thermoanaerobacter CGTase.
[0191] Mutant CGTase genes were constructed via SOE-PCR method as
described in Example 1. The primary PCR reactions were carried out
with the mutagenesis primers A91 (SEQ ID NO: 26) and A90 (SEQ ID
NO: 25) plus an upstream or a downstream primer (SEQ ID NO 5 or 6)
on the template strand, respectively. The product of the last
reaction was digested with Bst1107 I and Pst I, and exchanged with
the corresponding fragment (250 bp) from the vector pCA31-wt or
pCA31-(T-CGTase+F189L+*190D+*191P+*192A+*193G+*194F+D195S).
Successful mutations resulted in restriction sites (Xma I) at
positions 4-9 of A91 (SEQ ID NO: 26) and positions 11-16 of A90
(SEQ ID NO: 25), which allowed quick screening of transformants.
The following mutations were verified by standard DNA sequencing
techniques:
[0192] Y260F+L261
G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V
[0193]
*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N2-
64P+E265G+V266T+*266aA+*266bN+D267H+P268V
Example 6
Properties of CGTase Variant with Ability to Form Linear
Oligosaccharides
Inhibition of Starch Retrogradation
[0194] The first variant prepared in Example 5 was tested for its
ability to inhibit starch retrogradation was tested as follows:
[0195] 730 mg of 50% (w/w) amylopectin slurry in 0.1 M sodium
acetate, at a selected pH (3.7, 4.3 or 5.5) was mixed with 20 .mu.l
of an enzyme sample, and the mixture was incubated in a sealed
ampoule for 1 hour at 40.degree. C., followed by incubation at
100.degree. C. for 1 hour in order to gelatinize the samples. The
sample was then aged for 7 days at room temperature to allow
recrystallization of the amylopectin. A control without enzyme was
included.
[0196] After aging, DSC was performed on the sample by scanning
from 5.degree. C. to 95.degree. C. at a constant scan rate of
90.degree. C./hour. The area under the first endothermic peak in
the thermogram was taken to represent the amount of retrograded
amylopectin, and the relative inhibition of retrogradation was
taken as the area reduction (in %) relative to the control without
enzyme.
[0197] The result was a relative inhibition of 21%.
Reaction Pattern with Starch
[0198] The variant was compared with Novamyl and with
Thermoanaerobacter CGTase by determining the reaction products
formed after 24 hours incubation in 5% (w/v) amylopectin using 50
mM sodium acetate, 1 mM CaCl2, pH 5.0 at 50.degree. C. The reaction
products (in % by weight) were identified and quantitated using
HPLC.
TABLE-US-00003 Oligosaccharide Novamyl CGTase Variant G10 -- -- 0.7
G9 -- -- 1.5 G8 -- -- 2.4 G7 -- -- 1.9 G6/.alpha.-CD -- 53.9 23.1
G5 -- -- 6.1 G4 -- -- 8.1 G3/.gamma.-CD -- 12.0 14.5 G2 97.9 --
11.5 G1 2.1 -- 6.8 .beta.-CD 34.1 23.1
[0199] The results show clearly that whereas the parent CGTase
exclusively forms cyclodextrins, the reaction pattern of the
variant has been changed to form both cyclodextrins and linear
maltodextrins as initial products.
Sequence CWU 1
1
3412160DNABacillus speciesCDS(1)..(2157)mat_peptide(100)..() 1atg
aaa aag aaa acg ctt tct tta ttt gtg gga ctg atg ctc ctc atc 48Met
Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile -30 -25
-20ggt ctt ctg ttc agc ggt tct ctt ccg tac aat cca aac gcc gct gaa
96Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
-15 -10 -5gcc agc agt tcc gca agc gtc aaa ggg gac gtg att tac cag
att atc 144Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln
Ile Ile-1 1 5 10 15att gac cgg ttt tac gat ggg gac acg acg aac aac
aat cct gcc aaa 192Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn
Asn Pro Ala Lys 20 25 30agt tat gga ctt tac gat ccg acc aaa tcg aag
tgg aaa atg tat tgg 240Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys
Trp Lys Met Tyr Trp 35 40 45ggc ggg gat ctg gag ggg gtt cgt caa aaa
ctt cct tat ctt aaa cag 288Gly Gly Asp Leu Glu Gly Val Arg Gln Lys
Leu Pro Tyr Leu Lys Gln 50 55 60ctg ggc gta acg aca atc tgg ttg tcc
ccg gtt ttg gac aat ctg gat 336Leu Gly Val Thr Thr Ile Trp Leu Ser
Pro Val Leu Asp Asn Leu Asp 65 70 75aca ctg gcg ggc acc gat aac acg
ggc tat cac gga tac tgg acg cgc 384Thr Leu Ala Gly Thr Asp Asn Thr
Gly Tyr His Gly Tyr Trp Thr Arg80 85 90 95gat ttt aaa cag att gag
gaa cat ttc ggg aat tgg acc aca ttt gac 432Asp Phe Lys Gln Ile Glu
Glu His Phe Gly Asn Trp Thr Thr Phe Asp 100 105 110acg ttg gtc aat
gat gct cac caa aac gga atc aag gtg att gtc gac 480Thr Leu Val Asn
Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp 115 120 125ttt gtg
ccc aat cat tcg act cct ttt aag gca aac gat tcc acc ttt 528Phe Val
Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe 130 135
140gcg gaa ggc ggc gcc ctc tac aac aat gga acc tat atg ggc aat tat
576Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
145 150 155ttt gat gac gca aca aaa ggg tac ttc cac cat aat ggg gac
atc agc 624Phe Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp
Ile Ser160 165 170 175aac tgg gac gac cgg tac gag gcg caa tgg aaa
aac ttc acg gat cca 672Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys
Asn Phe Thr Asp Pro 180 185 190gcc ggt ttc tcg ctt gcc gat ttg tcg
cag gaa aat ggc acg att gct 720Ala Gly Phe Ser Leu Ala Asp Leu Ser
Gln Glu Asn Gly Thr Ile Ala 195 200 205caa tac ctg acc gat gcg gcg
gtt caa ttg gta gca cat gga gcg gat 768Gln Tyr Leu Thr Asp Ala Ala
Val Gln Leu Val Ala His Gly Ala Asp 210 215 220ggt ttg cgg att gat
gcg gtg aag cat ttt aat tcg ggg ttc tcc aaa 816Gly Leu Arg Ile Asp
Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys 225 230 235tcg ttg gcc
gat aaa ctg tac caa aag aaa gac att ttc ctg gtg ggg 864Ser Leu Ala
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly240 245 250
255gaa tgg tac gga gat gac ccc gga aca gcc aat cat ctg gaa aag gtc
912Glu Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val
260 265 270cgg tac gcc aac aac agc ggt gtc aat gtg ctg gat ttt gat
ctc aac 960Arg Tyr Ala Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp
Leu Asn 275 280 285acg gtg att cga aat gtg ttc ggc aca ttt acg caa
acg atg tac gat 1008Thr Val Ile Arg Asn Val Phe Gly Thr Phe Thr Gln
Thr Met Tyr Asp 290 295 300ctt aac aat atg gtg aac caa acg ggg aac
gag tac aaa tac aaa gaa 1056Leu Asn Asn Met Val Asn Gln Thr Gly Asn
Glu Tyr Lys Tyr Lys Glu 305 310 315aat cta atc aca ttt atc gat aac
cat gat atg tca aga ttt ctt tcg 1104Asn Leu Ile Thr Phe Ile Asp Asn
His Asp Met Ser Arg Phe Leu Ser320 325 330 335gta aat tcg aac aag
gcg aat ttg cac cag gcg ctt gct ttc att ctc 1152Val Asn Ser Asn Lys
Ala Asn Leu His Gln Ala Leu Ala Phe Ile Leu 340 345 350act tcg cgg
ggt acg ccc tcc atc tat tat gga acc gaa caa tac atg 1200Thr Ser Arg
Gly Thr Pro Ser Ile Tyr Tyr Gly Thr Glu Gln Tyr Met 355 360 365gca
ggc ggc aat gac ccg tac aac cgg ggg atg atg ccg gcg ttt gat 1248Ala
Gly Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp 370 375
380acg aca acc acc gcc ttt aaa gag gtg tca act ctg gcg ggg ttg cgc
1296Thr Thr Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg
385 390 395agg aac aat gcg gcg atc cag tac ggc acc acc acc cag cgt
tgg atc 1344Arg Asn Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg
Trp Ile400 405 410 415aac aat gat gtt tac att tat gaa cgg aaa ttt
ttc aac gat gtc gtg 1392Asn Asn Asp Val Tyr Ile Tyr Glu Arg Lys Phe
Phe Asn Asp Val Val 420 425 430ttg gtg gcc atc aat cga aac acg caa
tcc tcc tat tcg att tcc ggt 1440Leu Val Ala Ile Asn Arg Asn Thr Gln
Ser Ser Tyr Ser Ile Ser Gly 435 440 445ttg cag acg gcc ttg cca aat
ggc agc tat gcg gat tat ctg tca ggg 1488Leu Gln Thr Ala Leu Pro Asn
Gly Ser Tyr Ala Asp Tyr Leu Ser Gly 450 455 460ctg ttg ggg ggg aac
ggg att tcc gtt tcc aat gga agt gtc gct tcg 1536Leu Leu Gly Gly Asn
Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser 465 470 475ttc acg ctt
gcg cct gga gcc gtg tct gtt tgg cag tac agc aca tcc 1584Phe Thr Leu
Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser480 485 490
495gct tca gcg ccg caa atc gga tcg gtt gct cca aat atg ggg att ccg
1632Ala Ser Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro
500 505 510ggt aat gtg gtc acg atc gac ggg aaa ggt ttt ggg acg acg
cag gga 1680Gly Asn Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr
Gln Gly 515 520 525acc gtg aca ttt ggc gga gtg aca gcg act gtg aaa
tcc tgg aca tcc 1728Thr Val Thr Phe Gly Gly Val Thr Ala Thr Val Lys
Ser Trp Thr Ser 530 535 540aat cgg att gaa gtg tac gtt ccc aac atg
gcc gcc ggg ctg acc gat 1776Asn Arg Ile Glu Val Tyr Val Pro Asn Met
Ala Ala Gly Leu Thr Asp 545 550 555gtg aaa gtc acc gcg ggt gga gtt
tcc agc aat ctg tat tct tac aat 1824Val Lys Val Thr Ala Gly Gly Val
Ser Ser Asn Leu Tyr Ser Tyr Asn560 565 570 575att ttg agt gga acg
cag aca tcg gtt gtg ttt act gtg aaa agt gcg 1872Ile Leu Ser Gly Thr
Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala 580 585 590cct ccg acc
aac ctg ggg gat aag att tac ctg acg ggc aac ata ccg 1920Pro Pro Thr
Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro 595 600 605gaa
ttg ggg aat tgg agc acg gat acg agc gga gcc gtt aac aat gcg 1968Glu
Leu Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala 610 615
620caa ggg ccc ctg ctc gcg ccc aat tat ccg gat tgg ttt tat gta ttc
2016Gln Gly Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe
625 630 635agc gtt cca gca gga aag acg att caa ttc aag ttc ttc atc
aag cgt 2064Ser Val Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile
Lys Arg640 645 650 655gcg gat gga acg att caa tgg gag aat ggt tcg
aac cac gtg gcc aca 2112Ala Asp Gly Thr Ile Gln Trp Glu Asn Gly Ser
Asn His Val Ala Thr 660 665 670act ccc acg ggt gca acc ggt aac att
act gtt acg tgg caa aac tag 2160Thr Pro Thr Gly Ala Thr Gly Asn Ile
Thr Val Thr Trp Gln Asn 675 680 6852719PRTBacillus species 2Met Lys
Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile -30 -25
-20Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
-15 -10 -5Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln
Ile Ile-1 1 5 10 15Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn
Asn Pro Ala Lys 20 25 30Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys
Trp Lys Met Tyr Trp 35 40 45Gly Gly Asp Leu Glu Gly Val Arg Gln Lys
Leu Pro Tyr Leu Lys Gln 50 55 60Leu Gly Val Thr Thr Ile Trp Leu Ser
Pro Val Leu Asp Asn Leu Asp 65 70 75Thr Leu Ala Gly Thr Asp Asn Thr
Gly Tyr His Gly Tyr Trp Thr Arg80 85 90 95Asp Phe Lys Gln Ile Glu
Glu His Phe Gly Asn Trp Thr Thr Phe Asp 100 105 110Thr Leu Val Asn
Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp 115 120 125Phe Val
Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe 130 135
140Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
145 150 155Phe Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp
Ile Ser160 165 170 175Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys
Asn Phe Thr Asp Pro 180 185 190Ala Gly Phe Ser Leu Ala Asp Leu Ser
Gln Glu Asn Gly Thr Ile Ala 195 200 205Gln Tyr Leu Thr Asp Ala Ala
Val Gln Leu Val Ala His Gly Ala Asp 210 215 220Gly Leu Arg Ile Asp
Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys 225 230 235Ser Leu Ala
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly240 245 250
255Glu Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val
260 265 270Arg Tyr Ala Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp
Leu Asn 275 280 285Thr Val Ile Arg Asn Val Phe Gly Thr Phe Thr Gln
Thr Met Tyr Asp 290 295 300Leu Asn Asn Met Val Asn Gln Thr Gly Asn
Glu Tyr Lys Tyr Lys Glu 305 310 315Asn Leu Ile Thr Phe Ile Asp Asn
His Asp Met Ser Arg Phe Leu Ser320 325 330 335Val Asn Ser Asn Lys
Ala Asn Leu His Gln Ala Leu Ala Phe Ile Leu 340 345 350Thr Ser Arg
Gly Thr Pro Ser Ile Tyr Tyr Gly Thr Glu Gln Tyr Met 355 360 365Ala
Gly Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp 370 375
380Thr Thr Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg
385 390 395Arg Asn Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg
Trp Ile400 405 410 415Asn Asn Asp Val Tyr Ile Tyr Glu Arg Lys Phe
Phe Asn Asp Val Val 420 425 430Leu Val Ala Ile Asn Arg Asn Thr Gln
Ser Ser Tyr Ser Ile Ser Gly 435 440 445Leu Gln Thr Ala Leu Pro Asn
Gly Ser Tyr Ala Asp Tyr Leu Ser Gly 450 455 460Leu Leu Gly Gly Asn
Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser 465 470 475Phe Thr Leu
Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser480 485 490
495Ala Ser Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro
500 505 510Gly Asn Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr
Gln Gly 515 520 525Thr Val Thr Phe Gly Gly Val Thr Ala Thr Val Lys
Ser Trp Thr Ser 530 535 540Asn Arg Ile Glu Val Tyr Val Pro Asn Met
Ala Ala Gly Leu Thr Asp 545 550 555Val Lys Val Thr Ala Gly Gly Val
Ser Ser Asn Leu Tyr Ser Tyr Asn560 565 570 575Ile Leu Ser Gly Thr
Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala 580 585 590Pro Pro Thr
Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro 595 600 605Glu
Leu Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala 610 615
620Gln Gly Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe
625 630 635Ser Val Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile
Lys Arg640 645 650 655Ala Asp Gly Thr Ile Gln Trp Glu Asn Gly Ser
Asn His Val Ala Thr 660 665 670Thr Pro Thr Gly Ala Thr Gly Asn Ile
Thr Val Thr Trp Gln Asn 675 680 685339DNAArtificial
SequenceMutagenisis Primer 1 3ccgatcccgc gggattctca ttagcagatt
tagatcagc 39432DNAArtificial SequenceMutagenesis primer 2
4cccgcgggat cggtaanatt acggtaaatt cc 32524DNAArtificial
SequenceUpstream Primer 5tattataagg ggctccatta cctg
24624DNAArtificial SequenceDownstream Primer 6cggatacttc agtttccaat
gttg 24718DNAArtificial SequencePrimer 1 7ksctatcayg ghtactgg
18818DNAArtificial SequencePrimer 2 8macrtcrttr ttkatcca
18915DNAArtificial SequencePrimer D3 9gayccngcng gntty
151015DNAArtificial SequencePrimer D4 10raanccngcn ggrtc
151117DNAArtificial SequencePrimer D5 11ttyacngayc cngcngg
171217DNAArtificial SequencePrimer D6 12ccngcnggrt cngtraa
171317DNAArtificial SequencePrimer 7 13ttcacggatc cagccgg
171417DNAArtificial SequencePrimer 8 14ccggctggat ccgtgaa
171558DNAArtificial SequenceNovamyl 5' primer #9 15gattacgcca
agcttctaga tgcctgcagc agcagccgta agcagttccg caagcgtc
581642DNAArtificial SequenceNovamyl 3' primer #10 16aacactaagc
tttggacgcg tatccatttc tttgacgttc ca 421758DNAArtificial
SequenceCGTase 5' primer #11 17gattacgcca agcttctaga tgcctgcagc
agcagccgta gcaccggata cttcagtt 581842DNAArtificial SequenceCGTase
3' primer #12 18aacactaagc tttggacgcg tagacaagtt gtagaagaag gt
421918DNAArtificial SequenceGeneral 5' primer #13 19gattacgcca
agcttcta 182021DNAArtificial SequenceGeneral 3' primer # 14
20aacactaagc tttggacgcg t 212136DNAArtificial SequenceMutasgenesis
primer 1 21cttgtacgat cttgcagatc tgtcgcagga aaatgg
362241DNAArtificial SequenceMutagenesis primer 2 22gacagatctg
caagatcgta caagtttctt cattgcgcct c 412320DNAArtificial SequenceA82
Oligomer 23ggggatctgg agggggttcg 202422DNAArtificial SequenceB346
oligomer 24tttgtactcg ttccccgttt gg 222542DNAArtificial
SequencePrimer A90 25ggttggcagt cccgggatcg tctccaaacc actcgccaaa tg
422641DNAArtificial SequencePrimer A91 26gatcccggga ctgccaacca
tgtaaataat acgtattttg c 41275PRTArtificial SequenceSynthetic
Construct 27Asp Pro Ala Gly Phe1 5284PRTArtificial
SequenceSynthetic Construct 28Asp Ala Gly Phe1294PRTArtificial
SequenceSynthetic Construct 29Asp Pro Gly Phe1306PRTArtificial
SequenceSynthetic Construct 30Asp Pro Ala Ala Gly Phe1
5317PRTArtificial SequenceSynthetic Construct 31Asp Pro Ala Ala Gly
Gly Phe1 532683PRTThermoanaerobacterium thermosulfurigenes 32Ala
Ser Asp Thr Ala Val Ser Asn Val Val Asn Tyr Ser Thr Asp Val1 5 10
15Ile Tyr Gln Ile Val Thr Asp Arg Phe Val Asp Gly Asn Thr Ser Asn
20 25 30Asn Pro Thr Gly Asp Leu Tyr Asp Pro Thr His Thr Ser Leu Lys
Lys 35 40 45Tyr Phe Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn
Asp Gly 50 55 60Tyr Leu Thr Gly Met Gly Val Thr Ala Ile Trp Ile Ser
Gln Pro Val65 70 75 80Glu Asn Ile Tyr Ala Val Leu Pro Asp Ser Thr
Phe Gly Gly Ser Thr 85 90 95Ser Tyr His Gly Tyr Trp Ala Arg Asp Phe
Lys Arg Thr Asn Pro Tyr 100 105 110Phe Gly Ser Phe Thr Asp Phe Gln
Asn Leu Ile Asn Thr Ala His Ala 115 120 125His Asn Ile Lys Val Ile
Ile Asp Phe Ala Pro Asn His Thr Ser Pro 130 135
140Ala Ser Glu Thr Asp Pro Thr Tyr Ala Glu Asn Gly Arg Leu Tyr
Asp145 150 155 160Asn Gly Thr Leu Leu Gly Gly Tyr Thr Asn Asp Thr
Asn Gly Tyr Phe 165 170 175His His Tyr Gly Gly Thr Asp Phe Ser Ser
Tyr Glu Asp Gly Ile Tyr 180 185 190Arg Asn Leu Phe Asp Leu Ala Asp
Leu Asn Gln Gln Asn Ser Thr Ile 195 200 205Asp Ser Tyr Leu Lys Ser
Ala Ile Lys Val Trp Leu Asp Met Gly Ile 210 215 220Asp Gly Ile Arg
Leu Asp Ala Val Lys His Met Pro Phe Gly Trp Gln225 230 235 240Lys
Asn Phe Met Asp Ser Ile Leu Ser Tyr Arg Pro Val Phe Thr Phe 245 250
255Gly Glu Trp Phe Leu Gly Thr Asn Glu Ile Asp Val Asn Asn Thr Tyr
260 265 270Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe
Ser Gln 275 280 285Lys Val Arg Gln Val Phe Arg Asp Asn Thr Asp Thr
Met Tyr Gly Leu 290 295 300Asp Ser Met Ile Gln Ser Thr Ala Ser Asp
Tyr Asn Phe Ile Asn Asp305 310 315 320Met Val Thr Phe Ile Asp Asn
His Asp Met Asp Arg Phe Tyr Asn Gly 325 330 335Gly Ser Thr Arg Pro
Val Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser 340 345 350Arg Gly Val
Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Thr Gly 355 360 365Asn
Gly Asp Pro Tyr Asn Arg Ala Met Met Thr Ser Phe Asn Thr Ser 370 375
380Thr Thr Ala Tyr Asn Val Ile Lys Lys Leu Ala Pro Leu Arg Lys
Ser385 390 395 400Asn Pro Ala Ile Ala Tyr Gly Thr Thr Gln Gln Arg
Trp Ile Asn Asn 405 410 415Asp Val Tyr Ile Tyr Glu Arg Lys Phe Gly
Asn Asn Val Ala Leu Val 420 425 430Ala Ile Asn Arg Asn Leu Ser Thr
Ser Tyr Asn Ile Thr Gly Leu Tyr 435 440 445Thr Ala Leu Pro Ala Gly
Thr Tyr Thr Asp Val Leu Gly Gly Leu Leu 450 455 460Asn Gly Asn Ser
Ile Ser Val Ala Ser Asp Gly Ser Val Thr Pro Phe465 470 475 480Thr
Leu Ser Ala Gly Glu Val Ala Val Trp Gln Tyr Val Ser Ser Ser 485 490
495Asn Ser Pro Leu Ile Gly His Val Gly Pro Thr Met Thr Lys Ala Gly
500 505 510Gln Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Thr Thr Ser
Gly Gln 515 520 525Val Leu Phe Gly Ser Thr Ala Gly Thr Ile Val Ser
Trp Asp Asp Thr 530 535 540Glu Val Lys Val Lys Val Pro Ser Val Thr
Pro Gly Lys Tyr Asn Ile545 550 555 560Ser Leu Lys Thr Ser Ser Gly
Ala Thr Ser Asn Thr Tyr Asn Asn Ile 565 570 575Asn Ile Leu Thr Gly
Asn Gln Ile Cys Val Arg Phe Val Val Asn Asn 580 585 590Ala Ser Thr
Val Tyr Gly Glu Asn Val Tyr Leu Thr Gly Asn Val Ala 595 600 605Glu
Leu Gly Asn Trp Asp Thr Ser Lys Ala Ile Gly Pro Met Phe Asn 610 615
620Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser Val
Pro625 630 635 640Ala Gly Thr Thr Ile Gln Phe Lys Phe Ile Lys Lys
Asn Gly Asn Thr 645 650 655Ile Thr Trp Glu Gly Gly Ser Asn His Thr
Tyr Thr Val Pro Ser Ser 660 665 670Ser Thr Gly Thr Val Ile Val Asn
Trp Gln Gln 675 68033683PRTThermoanaerobacter 33Ala Pro Asp Thr Ser
Val Ser Asn Val Val Asn Tyr Ser Thr Asp Val1 5 10 15Ile Tyr Gln Ile
Val Thr Asp Arg Phe Leu Asp Gly Asn Pro Ser Asn 20 25 30Asn Pro Thr
Gly Asp Leu Tyr Asp Pro Thr His Thr Ser Leu Lys Lys 35 40 45Tyr Phe
Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly 50 55 60Tyr
Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val65 70 75
80Glu Asn Ile Tyr Ala Val Leu Pro Asp Ser Thr Phe Gly Gly Ser Thr
85 90 95Ser Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro
Phe 100 105 110Phe Gly Ser Phe Thr Asp Phe Gln Asn Leu Ile Ala Thr
Ala His Ala 115 120 125His Asn Ile Lys Val Ile Ile Asp Phe Ala Pro
Asn His Thr Ser Pro 130 135 140Ala Ser Glu Thr Asp Pro Thr Tyr Gly
Glu Asn Gly Arg Leu Tyr Asp145 150 155 160Asn Gly Val Leu Leu Gly
Gly Tyr Thr Asn Asp Thr Asn Gly Tyr Phe 165 170 175His His Tyr Gly
Gly Thr Asn Phe Ser Ser Tyr Glu Asp Gly Ile Tyr 180 185 190Arg Asn
Leu Phe Asp Leu Ala Asp Leu Asp Gln Gln Asn Ser Thr Ile 195 200
205Asp Ser Tyr Leu Lys Ala Ala Ile Lys Leu Trp Leu Asp Met Gly Ile
210 215 220Asp Gly Ile Arg Met Asp Ala Val Lys His Met Ala Phe Gly
Trp Gln225 230 235 240Lys Asn Phe Met Asp Ser Ile Leu Ser Tyr Arg
Pro Val Phe Thr Phe 245 250 255Gly Glu Trp Tyr Leu Gly Thr Asn Glu
Val Asp Pro Asn Asn Thr Tyr 260 265 270Phe Ala Asn Glu Ser Gly Met
Ser Leu Leu Asp Phe Arg Phe Ala Gln 275 280 285Lys Val Arg Gln Val
Phe Arg Asp Asn Thr Asp Thr Met Tyr Gly Leu 290 295 300Asp Ser Met
Ile Gln Ser Thr Ala Ala Asp Tyr Asn Phe Ile Asn Asp305 310 315
320Met Val Thr Phe Ile Asp Asn His Asp Met Asp Arg Phe Tyr Thr Gly
325 330 335Gly Ser Thr Arg Pro Val Glu Gln Ala Leu Ala Phe Thr Leu
Thr Ser 340 345 350Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln
Tyr Met Thr Gly 355 360 365Asn Gly Asp Pro Tyr Asn Arg Ala Met Met
Thr Ser Phe Asp Thr Thr 370 375 380Thr Thr Ala Tyr Asn Val Ile Lys
Lys Leu Ala Pro Leu Arg Lys Ser385 390 395 400Asn Pro Ala Ile Ala
Tyr Gly Thr Gln Lys Gln Arg Trp Ile Asn Asn 405 410 415Asp Val Tyr
Ile Tyr Glu Arg Gln Phe Gly Asn Asn Val Ala Leu Val 420 425 430Ala
Ile Asn Arg Asn Leu Ser Thr Ser Tyr Tyr Ile Thr Gly Leu Tyr 435 440
445Thr Ala Leu Pro Ala Gly Thr Tyr Ser Asp Met Leu Gly Gly Leu Leu
450 455 460Asn Gly Ser Ser Ile Thr Val Ser Ser Asn Gly Ser Val Thr
Pro Phe465 470 475 480Thr Leu Ala Pro Gly Glu Val Ala Val Trp Gln
Tyr Val Ser Thr Thr 485 490 495Asn Pro Pro Leu Ile Gly His Val Gly
Pro Thr Met Thr Lys Ala Gly 500 505 510Gln Thr Ile Thr Ile Asp Gly
Arg Gly Phe Gly Thr Thr Ala Gly Gln 515 520 525Val Leu Phe Gly Thr
Thr Pro Ala Thr Ile Val Ser Trp Glu Asp Thr 530 535 540Glu Val Lys
Val Lys Val Pro Ala Leu Thr Pro Gly Lys Tyr Asn Ile545 550 555
560Thr Leu Lys Thr Ala Ser Gly Val Thr Ser Asn Ser Tyr Asn Asn Ile
565 570 575Asn Val Leu Thr Gly Asn Gln Val Cys Val Arg Phe Val Val
Asn Asn 580 585 590Ala Thr Thr Val Trp Gly Glu Asn Val Tyr Leu Thr
Gly Asn Val Ala 595 600 605Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala
Ile Gly Pro Met Phe Asn 610 615 620Gln Val Val Tyr Gln Tyr Pro Thr
Trp Tyr Tyr Asp Val Ser Val Pro625 630 635 640Ala Gly Thr Thr Ile
Glu Phe Lys Phe Ile Lys Lys Asn Gly Ser Thr 645 650 655Val Thr Trp
Glu Gly Gly Tyr Asn His Val Tyr Thr Thr Pro Thr Ser 660 665 670Gly
Thr Ala Thr Val Ile Val Asp Trp Gln Pro 675 68034686PRTBacillus
circulans 34Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr
Asp Val1 5 10 15Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn
Pro Ala Asn 20 25 30Asn Pro Thr Gly Ala Ala Phe Asp Gly Thr Cys Thr
Asn Leu Arg Leu 35 40 45Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn
Lys Ile Asn Asp Gly 50 55 60Tyr Leu Thr Gly Met Gly Val Thr Ala Ile
Trp Ile Ser Gln Pro Val65 70 75 80Glu Asn Ile Tyr Ser Ile Ile Asn
Tyr Ser Gly Val Asn Asn Thr Ala 85 90 95Tyr His Gly Tyr Trp Ala Arg
Asp Phe Lys Lys Thr Asn Pro Ala Tyr 100 105 110Gly Thr Ile Ala Asp
Phe Gln Asn Leu Ile Ala Ala Ala His Ala Lys 115 120 125Asn Ile Lys
Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala 130 135 140Ser
Ser Asp Gln Pro Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn145 150
155 160Gly Thr Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe
His 165 170 175His Asn Gly Gly Thr Asp Phe Ser Thr Thr Glu Asn Gly
Ile Tyr Lys 180 185 190Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn
Asn Ser Thr Val Asp 195 200 205Val Tyr Leu Lys Asp Ala Ile Lys Met
Trp Leu Asp Leu Gly Ile Asp 210 215 220Gly Ile Arg Met Asp Ala Val
Lys His Met Pro Phe Gly Trp Gln Lys225 230 235 240Ser Phe Met Ala
Ala Val Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly 245 250 255Glu Trp
Phe Leu Gly Val Asn Glu Val Ser Pro Glu Asn His Lys Phe 260 265
270Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys
275 280 285Val Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly
Leu Lys 290 295 300Ala Met Leu Glu Gly Ser Ala Ala Asp Tyr Ala Gln
Val Asp Asp Gln305 310 315 320Val Thr Phe Ile Asp Asn His Asp Met
Glu Arg Phe His Ala Ser Asn 325 330 335Ala Asn Arg Arg Lys Leu Glu
Gln Ala Leu Ala Phe Thr Leu Thr Ser 340 345 350Arg Gly Val Pro Ala
Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ser Gly 355 360 365Gly Thr Asp
Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Ser 370 375 380Thr
Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Cys385 390
395 400Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn
Asn 405 410 415Asp Val Leu Ile Tyr Glu Arg Lys Phe Gly Ser Asn Val
Ala Val Val 420 425 430Ala Val Asn Arg Asn Leu Asn Ala Pro Ala Ser
Ile Ser Gly Leu Val 435 440 445Thr Ser Leu Pro Gln Gly Ser Tyr Asn
Asp Val Leu Gly Gly Leu Leu 450 455 460Asn Gly Asn Thr Leu Ser Val
Gly Ser Gly Gly Ala Ala Ser Asn Phe465 470 475 480Thr Leu Ala Ala
Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Ala Thr 485 490 495Ala Thr
Pro Thr Ile Gly His Val Gly Pro Met Met Ala Lys Pro Gly 500 505
510Val Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Ser Ser Lys Gly Thr
515 520 525Val Tyr Phe Gly Thr Thr Ala Val Ser Gly Ala Asp Ile Thr
Ser Trp 530 535 540Glu Asp Thr Gln Ile Lys Val Lys Ile Pro Ala Val
Ala Gly Gly Asn545 550 555 560Tyr Asn Ile Lys Val Ala Asn Ala Ala
Gly Thr Ala Ser Asn Val Tyr 565 570 575Asp Asn Phe Glu Val Leu Ser
Gly Asp Gln Val Ser Val Arg Phe Val 580 585 590Val Asn Asn Ala Thr
Thr Ala Leu Gly Gln Asn Val Tyr Leu Thr Gly 595 600 605Ser Val Ser
Glu Leu Gly Asn Trp Asp Pro Ala Lys Ala Ile Gly Pro 610 615 620Met
Tyr Asn Gln Val Val Tyr Gln Tyr Pro Asn Trp Tyr Tyr Asp Val625 630
635 640Ser Val Pro Ala Gly Lys Thr Ile Glu Phe Lys Phe Leu Lys Lys
Gln 645 650 655Gly Ser Thr Val Thr Trp Glu Gly Gly Ser Asn His Thr
Phe Thr Ala 660 665 670Pro Ser Ser Gly Thr Ala Thr Ile Asn Val Asn
Trp Gln Pro 675 680 685
* * * * *