U.S. patent application number 13/660218 was filed with the patent office on 2013-03-07 for alpha-amylase mutants.
This patent application is currently assigned to NOVOZYMES A/S. The applicant listed for this patent is Novozymes A/S. Invention is credited to Henrik Bisgaard-Frantzen, Torben Vedel Borchert, Allan Svendsen.
Application Number | 20130059315 13/660218 |
Document ID | / |
Family ID | 27512749 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130059315 |
Kind Code |
A1 |
Svendsen; Allan ; et
al. |
March 7, 2013 |
Alpha-Amylase Mutants
Abstract
The present invention relates to a method of constructing a
variant of a parent Termamyl-like alpha-amylase, which variant has
alpha-amylase activity and at least one altered property as
compared to the parent alpha-amylase, comprises i) analyzing the
structure of the parent Termamyl-like alpha-amylase to identify at
least one amino acid residue or at least one structural part of the
Termamyl-like alpha-amylase structure, which amino acid residue or
structural part is believed to be of relevance for altering the
property of the parent Termamyl-like alpha-amylase (as evaluated on
the basis of structural or functional considerations), ii)
constructing a Termamyl-like alpha-amylase variant, which as
compared to the parent Termamyl-like alpha-amylase, has been
modified in the amino acid residue or structural part identified in
i) so as to alter the property, and, optionally, iii) testing the
resulting Termamyl-like alpha-amylase variant with respect to the
property in question.
Inventors: |
Svendsen; Allan; (Birkeroed,
DK) ; Bisgaard-Frantzen; Henrik; (Lyngby, DK)
; Borchert; Torben Vedel; (Copenhagen, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S; |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
27512749 |
Appl. No.: |
13/660218 |
Filed: |
October 25, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13161931 |
Jun 16, 2011 |
8323946 |
|
|
13660218 |
|
|
|
|
12100506 |
Apr 10, 2008 |
7993897 |
|
|
13161931 |
|
|
|
|
11064196 |
Feb 22, 2005 |
7378264 |
|
|
12100506 |
|
|
|
|
10184771 |
Jun 28, 2002 |
|
|
|
11064196 |
|
|
|
|
09636252 |
Aug 10, 2000 |
6440716 |
|
|
10184771 |
|
|
|
|
09327563 |
Jun 8, 1999 |
7115409 |
|
|
09636252 |
|
|
|
|
08683838 |
Jul 18, 1996 |
6022724 |
|
|
09327563 |
|
|
|
|
08600908 |
Feb 13, 1996 |
5989169 |
|
|
08683838 |
|
|
|
|
Current U.S.
Class: |
435/7.4 ;
435/202; 435/203; 435/22; 435/252.31; 435/254.11; 435/263; 435/264;
435/320.1; 435/99; 510/226; 510/320; 510/392 |
Current CPC
Class: |
C12N 9/2417 20130101;
C11D 3/386 20130101 |
Class at
Publication: |
435/7.4 ; 435/22;
435/202; 435/203; 435/320.1; 435/252.31; 435/254.11; 435/264;
435/263; 435/99; 510/392; 510/226; 510/320 |
International
Class: |
C12N 9/28 20060101
C12N009/28; C12Q 1/40 20060101 C12Q001/40; C12N 9/30 20060101
C12N009/30; C12N 15/63 20060101 C12N015/63; C11D 3/386 20060101
C11D003/386; C12N 1/15 20060101 C12N001/15; C12S 9/00 20060101
C12S009/00; C12S 11/00 20060101 C12S011/00; C12P 19/14 20060101
C12P019/14; G01N 33/573 20060101 G01N033/573; C12N 1/21 20060101
C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 3, 1995 |
DK |
0128/95 |
Oct 23, 1995 |
DK |
1192/95 |
Nov 10, 1995 |
DK |
1256/95 |
Claims
1. A method of constructing a variant of a parent Termamyl-like
alpha-amylase, which variant has alpha-amylase activity and at
least one altered property as compared to said parent
alpha-amylase, which method comprises i) analyzing the structure of
the parent Termamyl-like alpha-amylase to identify at least one
amino acid residue or at least one structural part of the
Termamyl-like alpha-amylase structure, which amino acid residue or
structural part is believed to be of relevance for altering said
property of the parent Termamyl-like alpha-amylase (as evaluated on
the basis of structural or functional considerations), ii)
constructing a Termamyl-like alpha-amylase variant, which as
compared to the parent Termamyl-like alpha-amylase, has been
modified in the amino acid residue or structural part identified in
i) so as to alter said property, and, optionally, iii) testing the
resulting Termamyl-like alpha-amylase variant with respect to said
property or properties.
2. The method according to claim 1, wherein the property to be
altered is selected from the group consisting of substrate
specificity, substrate binding, substrate cleavage pattern,
temperature stability, pH dependent activity, pH dependent
stability (especially increased stability at low (e.g., pH<6) or
high (e.g., pH>9) pH values), stability towards oxidation,
Ca.sup.2+-dependency and specific activity.
3. The method according to claim 1, wherein the property to be
altered is the calcium ion dependency and the structural part to be
modified is selected from the group consisting of the C domain, the
interface between the A and B domain, the interface between the A
and C domain, or the interaction to a calcium binding site of the
Termamyl-like alpha-amylase.
4. The method according to claim 1, wherein the property to be
altered is the substrate cleavage pattern and the structural part
to be modified is located within 10 .ANG. from an amino acid
residue of the substrate binding site.
5. A method of constructing a variant of a parent Termamyl-like
alpha-amylase, which variant has alpha-amylase activity and one or
more altered properties as compared to said parent alpha-amylase,
which method comprises i) comparing the three-dimensional structure
of the Termamyl-like alpha-amylase with the structure of a
non-Termamyl-like alpha-amylase, ii) identifying a part of the
Termamyl-like alpha-amylase structure which is different from the
non-Termamyl-like alpha-amylase structure and which from structural
or functional considerations is contemplated to be responsible for
differences in one or more properties of the Termamyl-like and
non-Termamyl-like alpha-amylase, iii) modifying the part of the
Termamyl-like alpha-amylase identified in ii) whereby a
Termamyl-like alpha-amylase variant is obtained, one or more
properties of which differ from the parent Termamyl-like
alpha-amylase, and optionally, iv) testing the resulting
Termamyl-like alpha-amylase variant with respect to said property
or properties.
6. The method according to claim 5, wherein, in step iii), the part
of the Termamyl-like alpha-amylase is modified so as to resemble
the corresponding part of the non-Termamyl-like alpha-amylase.
7. The method according to claim 5, wherein, in step iii), the
modification is accomplished by deleting one or more amino acid
residues of the part of the Termamyl-like alpha-amylase to be
modified; by replacing one or more amino acid residues of the part
of the Termamyl-like alpha-amylase to be modified with the amino
acid residues occupying corresponding positions in the
non-Termamyl-like alpha-amylase; or by insertion of one or more
amino acid residues present in the non-Termamyl-like alpha-amylase
into a corresponding position in the Termamyl-like
alpha-amylase.
8. The method according to claim 5, wherein the non-Termamyl-like
alpha-amylase structure is the structure of a fungal alpha-amylase
or a mammalian alpha-amylase.
9. The method according to claim 8, wherein the non-Termamyl-like
alpha-amylase is the Aspergillus oryzae TAKA alpha-amylase, the A.
niger acid alpha-amylase, the Bacillus subtilis alpha-amylase or
the pig pancreatic alpha-amylase.
10. The method according to claim 1, wherein the parent
Termamyl-like alpha-amylase is derived from a strain of
Bacillus.
11. The method according to claim 10, wherein the parent
alpha-amylase is derived from a strain of a B. licheniformis, B.
amyloliquefaciens, B. stearothermophilus or a strain from an
alkalophilic Bacillus sp. such as NCIB 12289, NCIB 12512 or NCIB
12513.
12. The method according to claim 1, wherein the parent
alpha-amylase is a hybrid alpha-amylase comprising a combination of
partial amino acid sequences derived from at least two
alpha-amylases, of which one is a Termamyl-like alpha-amylase and
the other(s) are, e.g., from a microbial and/or a mammalian
alpha-amylase.
13. The method according to claim 5, wherein the part of the parent
Termamyl-like alpha-amylase to be modified and identified in step
ii) is loop 1, loop 2, loop 3 and/or loop 8 of the parent
alpha-amylase.
14. A method of constructing a variant of a parent Termamyl-like
alpha-amylase, which has a decreased calcium ion dependency as
compared to said parent, which method comprises: i) identifying an
amino acid residue within 10 .ANG. from a Ca.sup.2+ binding site of
a Termamyl-like alpha-amylase in a model of the three-dimensional
structure of said alpha-amylase, which from structural or
functional considerations is believed to be responsible for a
non-optimal calcium ion interaction, ii) constructing a variant in
which said amino acid residue is replaced with another amino acid
residue which from structural or functional considerations is
believed to be important for establishing a higher Ca.sup.2+
binding affinity, and iii) testing the Ca.sup.2+ dependency of the
resulting Termamyl-like alpha-amylase variant.
15. A method of constructing a variant of a parent Termamyl-like
alpha-amylase which variant has alpha-amylase activity and an
altered pH dependent activity, which method comprises i) in a
three-dimensional structure of the Termamyl-like alpha-amylase in
question, identifying an amino acid residue within 15 .ANG. from an
active site residue, in particular 10 .ANG. from an active site
residue, which amino acid residue is contemplated to be involved in
electrostatic or hydrophobic interactions with an active site
residue, ii) replacing, in the structure, said amino acid residue
with an amino acid residue which changes the electrostatic and/or
hydrophobic surroundings of an active site residue and evaluating
the accommodation of the amino acid residue in the structure, iii)
optionally repeating step i) and/or ii) until an amino acid
replacement has been identified which is accommodated into the
structure, iv) constructing a Termamyl-like alpha-amylase variant
resulting from steps i), ii) and optionally iii) and testing the pH
dependent activity of said variant.
16. A method of increasing the thermostability and/or altering the
temperature optimum of a parent Termamyl-like alpha-amylase, which
method comprises i) identifying an internal hole or a crevice of
the parent Termamyl-like alpha-amylase in the three-dimensional
structure of said alpha-amylase, ii) replacing, in the structure,
one or more amino acid residues in the neighbourhood of the hole or
crevice identified in i) with another amino acid residue which from
structural or functional considerations is believed to increase the
hydrophobic interaction and to fill out or reduce the size of the
hole or crevice, iii) constructing a Termamyl-like alpha-amylase
variant resulting from step ii) and testing the thermostability
and/or temperature optimum of the variant.
17. A method of constructing a variant of a Termamyl-like
alpha-amylase which has a reduced ability to cleave a substrate
close to the branching point, which method comprises i) identifying
the substrate binding area of the parent Termamyl-like
alpha-amylase in a model of the three-dimensional structure of said
alpha-amylase, ii) replacing, in the model, one or more amino acid
residues of the substrate binding area of the cleft identified in
i), which is/are believed to be responsible for the cleavage
pattern of the parent alpha-amylase, with another amino acid
residue which from structural considerations is believed to result
in an altered substrate cleavage pattern, or deleting one or more
amino acid residues of the substrate binding area contemplated to
introduce favorable interactions to the substrate or adding one or
more amino acid residues to the substrate binding area contemplated
to introduce favorable interactions to the substrate, and iii)
constructing a Termamyl-like alpha-amylase variant resulting from
step ii) and testing the substrate cleavage pattern of the
variant.
18. The method according to claim 1, in which the alpha-amylase
variant is obtained 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.
19. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one amino acid residue of the parent
alpha-amylase, which is/are present in a fragment corresponding to
the amino acid fragment 44-57 of the amino acid sequence of SEQ ID
NO: 4, has been deleted or replaced with one or more amino acid
residues which is/are present in a fragment corresponding to the
amino acid fragment 66-84 of the amino acid sequence shown in SEQ
ID NO: 10, or in which one or more additional amino acid residues
has been added using the relevant part of SEQ ID NO: 10 or a
corresponding part of another Fungamyl-like alpha-amylase as a
template.
20. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80% sequence homology with the part of SEQ ID NO: 10
extending from residue Z to residue V of SEQ ID NO: 10, wherein X
is the amino acid residue occupying position 44, 45, 46, 47 or 48
of SEQ ID NO: 4, Y is the amino acid residue occupying position 51,
52, 53, 54, 55, 56 or 57 of SEQ ID NO: 4, Z is the amino acid
residue occupying position 66, 67, 68, 69 or 70 of SEQ ID NO: 10,
and V is the amino acid residue occupying position 78, 79, 80, 81,
82, 83 or 84 of SEQ ID NO: 10.
21. The variant according to claim 19, wherein X is the amino acid
residue occupying position 48 and Y the amino acid residue
occupying position 51 of SEQ ID NO: 4 and Z is the amino acid
residue occupying position 70 and V the amino acid residue
occupying position 78 in SEQ ID NO: 10.
22. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in an amino acid fragment
corresponding to the amino acid fragment 195-202 of the amino acid
sequence of SEQ ID NO: 4, has been deleted or replaced with one or
more of the amino acid residues which is/are present in an amino
acid fragment corresponding to the amino acid fragment 165-177 of
the amino acid sequence shown in SEQ ID NO: 10, or in which one or
more additional amino acid residues has been added using the
relevant part of SEQ ID NO: 10 or a corresponding part of another
Fungamyl-like alpha-amylase as a template.
23. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80%, such as 90% sequence homology with the part of SEQ ID
NO: 10 extending from residue Z to residue V of SEQ ID NO: 10,
wherein X is the amino acid occupying position 195 or 196 of SEQ ID
NO: 4, Y is the amino acid residue occupying position 198, 199,
200, 201, or 202 of SEQ ID NO: 4, Z is the amino acid residue
occupying position 165 or 166 of SEQ ID NO: 10, and V is the amino
acid residue occupying position 173, 174, 175, 176 or 177 of SEQ ID
NO: 10.
24. The variant according to claim 22, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 196-198 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 166-173 of
the amino acid sequence shown in SEQ ID NO: 10.
25. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in a fragment corresponding to
the amino acid fragment 117-185 of the amino acid sequence of SEQ
ID NO: 4, has/have been deleted or replaced with one or more of the
amino acid residues, which is/are present in an amino acid fragment
corresponding to the amino acid fragment 98-210 of the amino acid
sequence shown in SEQ ID NO: 10, or in which one or more additional
amino acid residues has been added using the relevant part of SEQ
ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
26. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80%, such as at least 90% sequence homology with the part
of SEQ ID NO: 10 extending from residue Z to residue V of SEQ ID
NO: 10, wherein X is the amino acid occupying position 117, 118,
119, 120 or 121 of SEQ ID NO: 4, Y is the amino acid occupying
position 181, 182, 183, 184 or 185 of SEQ ID NO: 4, Z is the amino
acid occupying position 98, 99, 100, 101, 102 of SEQ ID NO: 10, and
V is the amino acid occupying position 206, 207, 208, 209 or 210 of
SEQ ID NO: 10.
27. The variant according to claim 25, in which an amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 121-181 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 102-206 of
the amino acid sequence shown in SEQ ID NO: 10.
28. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in a fragment corresponding to
the amino acid fragment 117-181 of the amino acid sequence of SEQ
ID NO: 4, has/have been deleted or replaced with one or more of the
amino acid residues, which is/are present in an amino acid fragment
corresponding to the amino acid fragment to 98-206 of the amino
acid sequence shown in SEQ ID NO: 10, or in which one or more
additional amino acid residues has been added using the relevant
part of SEQ ID NO: 10 or a corresponding part of another
Fungamyl-like alpha-amylase as a template.
29. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80%, such as at least 90% sequence homology with the part
of SEQ ID NO: 10 extending from residue Z to residue V of SEQ ID
NO: 10, wherein X is the amino acid occupying position 117, 118,
119, 120 or 121 of SEQ ID NO: 4, Y is the amino acid occupying
position 174, 175, 176 or 177 of SEQ ID NO: 4, Z is the amino acid
occupying position 98, 99, 100, 101, 102 of SEQ ID NO: 10, and V is
the amino acid occupying position 199, 200, 201 or 202 of SEQ ID
NO: 10.
30. The variant according to claim 28, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 121-174 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 102-199 of
the amino acid sequence shown in SEQ ID NO: 10.
31. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in an amino acid fragment
corresponding to the amino acid fragment 12-19 of the amino acid
sequence of SEQ ID NO: 4, has/have been deleted or replaced with
one or more of the amino acid residues, which is/are present in an
amino acid fragment which corresponds to the amino acid fragment
28-42 of SEQ ID NO: 10, or in which one or more additional amino
acid residues has/have been inserted using the relevant part of SEQ
ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
32. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80%, such as at least 90% sequence homology with the part
of SEQ ID NO: 10 extending from residue Z to residue V of SEQ ID
NO: 10, wherein X is the amino acid occupying position 12, 13 or 14
of SEQ ID NO: 4, Y is the amino acid occupying position 15, 16, 17,
18 or 19 of SEQ ID NO: 4, Z is the amino acid occupying position
28, 29, 30, 31 or 32 of SEQ ID NO: 10, and V is an amino acid
residue corresponding to the amino acid occupying position 38, 39,
40, 41 or 42 of SEQ ID NO: 10.
33. The variant according to claim 31, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 14-15 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 32-38 of
the amino acid sequence shown in SEQ ID NO: 10.
34. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is present in a fragment corresponding to
amino acid residues 7-23 of the amino acid sequence of SEQ ID NO:
4, has/have been deleted or replaced with one or more amino acid
residues, which is/are present in an amino acid fragment
corresponding to amino acid residues 13-45 of the amino acid
sequence shown in SEQ ID NO: 10, or in which one or more additional
amino acid residues has/have been inserted using the relevant part
of SEQ ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
35. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 4, the said region having
at least 80%, such as at least 90% sequence homology with the part
of SEQ ID NO: 10 extending from residue Z to residue V of SEQ ID
NO: 10, wherein X is the amino acid occupying position 7 or 8 of
SEQ ID NO: 4, Y is the amino acid occupying position 18, 19, 20,
21, 22 or 23 of SEQ ID NO: 4, Z is the amino acid occupying
position 13 or 14 of SEQ ID NO: 10, and V is the amino acid
occupying position 40, 41, 42, 43, 44 or 45 of SEQ ID NO: 10.
36. The variant according to claim 34, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 8-18 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 14-40 of
the amino acid sequence shown in SEQ ID NO: 10.
37. A variant of a parent Termamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is present in a fragment corresponding to
amino acid residues 322-346 of the amino acid sequence of SEQ ID
NO: 2, has/have been deleted or replaced with one or more amino
acid residues, which is/are present in an amino acid fragment
corresponding to amino acid residues 291-313 of the amino acid
sequence shown in SEQ ID NO: 10, or in which one or more additional
amino acid residues has/have been inserted using the relevant part
of SEQ ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
38. A variant of a parent Termamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 2, the said region having
at least 80% sequence homology with the part of SEQ ID NO: 10
extending from residue Z to residue V of SEQ ID NO: 10, wherein X
is the amino acid occupying position 322, 323, 324 or 325 of SEQ ID
NO: 2, Y is the amino acid occupying position 343, 344, 345 or 346
of SEQ ID NO: 2, Z is the amino acid occupying position 291, 292,
293 or 294 of SEQ ID NO: 10, and V is the amino acid occupying
position 310, 311, 312 or 313 of SEQ ID NO: 10.
39. The variant according to claim 37, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 325-345 of SEQ D No. 2, has been replaced with the
amino acid fragment corresponding to amino acid residues 294-313 of
the amino acid sequence shown in SEQ ID NO: 10.
40. A variant of a parent Fungamyl-like alpha-amylase, in which
variant at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in an amino acid fragment
corresponding to amino acid residues 291-313 of the amino acid
sequence of SEQ ID NO: 10, has/have been deleted or replaced with
one or more of the amino acid residues, which is/are present in an
amino acid fragment corresponding to amino acid residues 98-210 of
the amino acid sequence shown in SEQ ID NO: 4, or in which one or
more additional amino acid residues has/have been inserted using
the relevant part of SEQ ID NO: 4 or a corresponding part of
another Termamyl-like alpha-amylase as a template.
41. A variant of a parent Fungamyl-like alpha-amylase, which
variant has a region which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent alpha-amylase, occupies the same position as the portion
from residue X to residue Y of SEQ ID NO: 10, the said region
having at least 80%, such as at least 90% sequence homology with
the part of SEQ ID NO: 10 extending from residue Z to residue V of
SEQ ID NO: 4, wherein X is the amino acid occupying position 117,
118, 119, 120 or 121 of SEQ ID NO: 10, Y is the amino acid
occupying position 181, 182, 183, 184 or 185 of SEQ ID NO: 10, Z is
the amino acid occupying position 98, 99, 100, 101 or 102 of SEQ ID
NO: 4, and V is the amino acid occupying position 206, 207, 208,
209 or 210 of SEQ ID NO: 4.
42. The variant according to claim 40, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 121-181 of SEQ ID NO: 10, has been replaced with the
amino acid fragment corresponding to amino acid residues 102-206 of
the amino acid sequence shown in SEQ ID NO: 4.
43. A variant according to claim 40, in which the amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 121-174 of SEQ ID NO: 10, has been replaced with the
amino acid fragment corresponding to amino acid residues 102-199 of
the amino acid sequence shown in SEQ ID NO: 4.
44. A variant of a parent Fungamyl-like alpha-amylase, in which an
amino acid fragment corresponding to amino acid residues 181-184 of
the amino acid sequence shown in SEQ ID NO: 10 has been
deleted.
45. A variant of a parent Termamyl-like alpha-amylase, which
exhibits alpha-amylase activity and which has a decreased Ca.sup.2+
dependency as compared to the parent alpha-amylase.
46. A variant according to claim 45, which comprises a mutation in
a position corresponding to at least one of the following positions
in SEQ ID NO 2: N104, G303, L346, A349, F350, K385, H408, I411,
I430, N457, or I479, in particular a mutation corresponding to
N104D; G303N,D,Q,E; L346C+I430C; A349C+I479C; F350D,E+I411R,K;
F350D,E+I430R,K; K385R+N457D,E; H408Q,E,N,D; and/or N457D,E.
47. A variant of a parent Termamyl-like alpha-amylase which
exhibits a higher activity below the pH optimum than the parent
alpha-amylase, which variant comprises a mutation of an amino acid
residue corresponding to at least one of the following positions of
the B. licheniformis alpha-amylase (SEQ ID NO: 2): V102, I103,
L196, A232, I236, P331, Q333, E336, in particular at least one of
the following mutations: V102R,K,A,T,S,G; I103K,R; L196K,R;
A232T,S,G; I236K, R, N; P331R,K Q333R,K; and/or E336R,K.
48. A variant of a parent Termamyl-like alpha-amylase which
exhibits a higher activity above the pH optimum than the parent
alpha-amylase, which variant comprises a mutation of an amino acid
residue corresponding to at least one of the following positions of
the B. licheniformis alpha-amylase (SEQ ID NO: 2): Y273, H281
and/or N326, in particular one of the following mutations: Y273F,W;
H281F,I,L; and/or N326I,Y,F,L,V.
49. A variant of a parent Termamyl-like alpha-amylase which
exhibits alpha-amylase activity and which has an increased
thermostability and/or altered temperature optimum as compared to
the parent alpha-amylase, which variant comprises a mutation of an
amino acid residue corresponding to at least one of the following
positions of the B. licheniformis alpha-amylase (SEQ ID NO: 2): L7,
L61, Y62, F67, K106, F143, G145, R146, Y150, S151, I212, R214,
I236, L241, V259, F284, F343, F350, L427 and/or V481, in particular
at least one of the following mutations: L7F,I,W; L61W,V,F; Y62W;
F67W; K106R,F,W; F143W; G145F,W R146W; Y150R,K; S151 replaced with
any other amino acid residue and in particular with F,W,I or L;
I212F,L,W,Y,R,K; R214W; I236L,F,W,Y; L241I,F,Y,W; V259F,I,L; F284W;
F343W; F350W; L427F,L,W; and/or V481,F,I,L,W.
50. A variant of a parent Termamyl-like alpha-amylase, which
exhibits alpha-amylase activity and which has a reduced capability
of cleaving an oligo-saccharide substrate close to the branching
point as compared to the parent alpha-amylase, which variant
comprises a mutation of an amino acid residue corresponding to at
least one of the following positions of the B. licheniformis
alpha-amylase (SEQ ID NO: 2): D53, V54, Y56, G57 and/or Q333, in
particular at least one of the following mutations: D53L,I,F,Y,W;
V54L,I,F,Y,W,R,K,H,E,Q; Y56W; G57 to all possible amino acid
residues; and/or Q333W.
51. The variant according to claim 17, wherein one or more proline
residues present in the amino acid residues with which the parent
alpha-amylase is modified are replaced with a non-proline residue
such as alanine.
52. The variant according to claim 17, wherein one or more cysteine
residues present in the amino acid residues with which the parent
alpha-amylase is modified are replaced with a non-cysteine residue
such as alanine.
53. A DNA construct comprising a DNA sequence encoding an
alpha-amylase variant according to claim 17.
54. A recombinant expression vector which carries a DNA construct
according to claim 53.
55. A cell which is transformed with a DNA construct according to
claim 53.
56. A cell according to claim 55, which is a microorganism.
57. A cell according to claim 56, which is a bacterium or a
fungus.
58. The cell according to claim 57, which is a gram-positive
bacterium such as Bacillus subtilis, Bacillus licheniformis,
Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus,
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans, Bacillus lautus or Bacillus
thuringiensis.
59. Use of an alpha-amylase variant according to claim 17 for
washing and/or dishwashing.
60. Use of an alpha-amylase variant according to claim 17 for
desizing.
61. Use of an alpha-amylase variant according to claim 17 for
starch liquefaction.
62. A detergent additive comprising an alpha-amylase variant
according to claim 17, optionally in the form of a non-dusting
granulate, stabilised liquid or protected enzyme.
63. A detergent additive according to claim 62, which contains
0.02-200 mg of enzyme protein/g of the additive.
64. A detergent additive according to claim 62, which additionally
comprises another enzyme such as a protease, a lipase, a
peroxidase, another amylolytic enzyme and/or a cellulase.
65. A detergent composition comprising a surfactant and an
alpha-amylase variant according to claim 17.
66. A detergent composition according to claim 65, which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
67. A manual or automatic dishwashing detergent composition
comprising an alpha-amylase variant according to claim 17.
68. A dishwashing detergent composition according to claim 66,
which additionally comprises another enzyme such as a protease, a
lipase, a peroxidase, another amylolytic enzyme and/or a
cellulase.
69. A manual or automatic laundry washing composition comprising an
alpha-amylase variant according to claim 17.
70. A laundry washing composition according to claim 68, which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, an amylolytic enzyme and/or a cellulase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/161,931 filed on Jun. 16, 2011, now allowed, which is a
divisional of U.S. application Ser. No. 12/100,506 filed on Apr.
10, 2008, now U.S. Pat. No. 7,993,897, which is a divisional of
U.S. application Ser. No. 11/064,196 filed on Feb. 22, 2005, now
U.S. Pat. No. 7,378,264, which is a continuation of U.S.
application Ser. No. 10/184,771 filed on Jun. 28, 2002, now
abandoned, which is a continuation of U.S. application Ser. No.
09/636,252 filed on Aug. 10, 2000, now U.S. Pat. No. 6,440,716,
which is a continuation of U.S. application Ser. No. 09/327,563
filed on Jun. 8, 1999, now U.S. Pat. No. 7,115,409, which is
continuation of U.S. application Ser. No. 08/683,838 filed on Jul.
18, 1996, now U.S. Pat. No. 6,022,724, which is a
continuation-in-part of U.S. application Ser. No. 08/600,908 filed
on Feb. 13, 1996, now U.S. Pat. No. 5,989,169, which is a
continuation of international application no. PCT/DK96/00057 filed
on Feb. 5, 1996, which claims priority under 35 U.S.C. 119 of
Danish application nos. 0128/95, 1192/95, and 1256/95 filed on Feb.
3, 1995, Oct. 23, 1995, and Nov. 10, 1995, respectively, the
contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel method of designing
alpha-amylase mutants with predetermined properties, which method
is based on the hitherto unknown three-dimensional structure of
bacterial alpha-amylases.
BACKGROUND OF THE INVENTION
[0003] Alpha-amylases (alpha-1,4 glucan-4-glucanohydrolase, EC
3.2.1.1) constitute a group of enzymes which is capable of
hydrolyzing starch and other linear and branched 1,4-glucosidic
oligo- and polysaccharides. Almost all alpha-amylases studied have
a few conserved regions with approximately the same length and
spacing. One of these regions resembles the Ca.sup.2+ binding site
of calmodulin and the others are thought to be necessary for the
active centre and/or binding of the substrate.
[0004] While the amino acid sequence and thus primary structure of
a large number of alpha-amylases are known, it has proved very
difficult to determine the three-dimensional structure of all
alpha-amylases. The three-dimensional structure can be determined
by X-ray crystallographic analysis of alpha-amylase crystals, but
it has proven difficult to obtain alpha-amylase crystals suitable
for actually solving the structure.
[0005] Until now the three-dimensional structure of only a few
alpha-amylases have been determined at high resolution. These
include the structure of the Aspergillus oryzae TAKA alpha-amylase
(Swift et al., 1991), the Aspergillus niger acid amylase (Brady et
al., 1991), the structure of pig pancreatic alpha-amylase (Qian et
al., 1993), and the barley alpha-amylase (Kadziola et al., 1994, J.
Mol. Biol. 239: 104-121, Kadziola, thesis, Dept of Chemistry, U. of
Copenhagen, Denmark). Furthermore, the three-dimensional structure
of a Bacillus circulans cyclodextrin glycosyltransferase (CGTase)
is known (Klein et al., 1992; Lawson et al., 1994). The CGTase
catalyzes the same type of reactions as alpha-amylases and exhibits
some structural resemblance with alpha-amylases.
[0006] Furthermore, crystallization and preliminary X-ray studies
of B. subtilis alpha-amylases have been described (Chang et al.,
1992, and Mizuno et al., 1993). No final B. subtilis structure has
been reported. Analogously, the preparation of B. licheniformis
alpha-amylase crystals has been reported (Suzuki et al., 1990), but
no subsequent report on X-ray crystallographic analysis or
three-dimensional structure is available.
[0007] Several research teams have attempted to build
three-dimensional structures on the basis of the above known
alpha-amylase structures. For instance, Vihinen et al. (J. Biochem.
107: 267-272 (1990)), disclose the modelling (or computer
simulation) of a three-dimensional structure of the Bacillus
stearothermophilus alpha-amylase on the basis of the TAKA amylase
structure. The model was used to investigate hypothetical
structural consequences of various site-directed mutations of the
B. stearothermophilus alpha-amylase. MacGregor (1987) predicts the
presence of alpha-helices and beta-barrels in alpha-amylases from
different sources, including barley, pig pancreas and Bacillus
amyloliquefaciens on the basis of the known structure of the A.
oryzae TAKA alpha-amylase and secondary structure predicting
algorithms. Furthermore, the possible loops and subsites which may
be found to be present in, e.g., the B. amyloliquefaciens
alpha-amylase are predicted (based on a comparison with the A.
oryzae sequence and structure).
[0008] MacGregor (Starch/Starke 45(7): 232-237 (1993)) presents a
review of the relationship between the structure and activity of
alpha-amylase related enzymes.
[0009] Hitherto, no three-dimensional structure has been available
for the industrially important Bacillus alpha-amylases (which in
the present context are termed "Termamyl-like alpha-amylases"),
including the B. licheniformis, the B. amyloliquefaciens, and the
B. stearothermophilus alpha-amylase.
BRIEF DISCLOSURE OF THE INVENTION
[0010] The three-dimensional structure of a Termamyl-like bacterial
alpha-amylase has now been elucidated. On the basis of an analysis
of said structure it is possible to identify structural parts or
specific amino acid residues which from structural or functional
considerations appear to be important for conferring the various
properties to the Termamyl-like alpha-amylases. Furthermore, when
comparing the Termamyl-like alpha-amylase structure with known
structures of the fungal and mammalian alpha-amylases mentioned
above, it has been found that some similarities exist between the
structures, but also that some striking, and not previously
predicted structural differences between the alpha-amylases exist.
The present invention is based on these findings.
[0011] Accordingly, in a first aspect the invention relates to a
method of constructing a variant of a parent Termamyl-like
alpha-amylase, which variant has alpha-amylase activity and at
least one altered property as compared to said parent
alpha-amylase, which comprises:
[0012] i) analyzing the structure of the Termamyl-like
alpha-amylase with a view to identifying at least one amino acid
residue or at least one structural part of the Termamyl-like
alpha-amylase structure, which amino acid residue or structural
part is believed to be of relevance for altering said property of
the parent Termamyl-like alpha-amylase (as evaluated on the basis
of structural or functional considerations),
[0013] ii) constructing a Termamyl-like alpha-amylase variant,
which as compared to the parent Termamyl-like alpha-amylase, has
been modified in the amino acid residue or structural part
identified in i) so as to alter said property, and, optionally,
[0014] iii) testing the resulting Termamyl-like alpha-amylase
variant with respect to said property.
[0015] In a second aspect the present invention relates to a method
of constructing a variant of a parent Termamyl-like alpha-amylase,
which variant has alpha-amylase activity and one or more altered
properties as compared to said parent alpha-amylase, which method
comprises
[0016] i) comparing the three-dimensional structure of the
Termamyl-like alpha-amylase with the structure of a
non-Termamyl-like alpha-amylase,
[0017] ii) identifying a part of the Termamyl-like alpha-amylase
structure which is different from the non-Termamyl-like
alpha-amylase structure,
[0018] iii) modifying the part of the Termamyl-like alpha-amylase
identified in ii) whereby a Termamyl-like alpha-amylase variant is
obtained, one or more properties of which differ from the parent
Termamyl-like alpha-amylase, and optionally,
[0019] iv) testing the resulting Termamyl-like alpha-amylase
variant with respect to said property or properties.
[0020] In a third aspect the invention relates to a method of
constructing a variant of a parent non-Termamyl-like alpha-amylase,
which variant has alpha-amylase activity and one or more altered
properties as compared to said parent alpha-amylase, which method
comprises
[0021] i) comparing the three-dimensional structure of the
non-Termamyl-like alpha-amylase with the structure of a
Termamyl-like alpha-amylase,
[0022] ii) identifying a part of the non-Termamyl-like
alpha-amylase structure which is different from the Termamyl-like
alpha-amylase structure,
[0023] iii) modifying the part of the non-Termamyl-like
alpha-amylase identified in ii) whereby a non-Termamyl-like
alpha-amylase variant is obtained, one or more properties of which
differ from the parent non-Termamyl-like alpha-amylase, and
optionally,
[0024] iv) testing the resulting non-Termamyl-like alpha-amylase
variant with respect to said property or properties.
[0025] The property which may be altered by the above methods of
the present invention may, e.g., be substrate specificity,
substrate binding, substrate cleavage pattern, temperature
stability, pH dependent activity, pH dependent stability
(especially increased stability at low (e.g., pH<6, in
particular pH<5) or high (e.g., pH>9) pH values), stability
towards oxidation, Ca.sup.2+-dependency, specific activity, and
other properties of interest. For instance, the alteration may
result in a variant which, as compared to the parent Termamyl-like
alpha-amylase, has an increased specific activity at a given pH
and/or an altered substrate specificity.
[0026] In still further aspects the invention relates to variants
of a Termamyl-like alpha-amylase, DNA encoding such variants and
methods of preparing the variants. Finally, the invention relates
to the use of the variants for various industrial purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1-6 show schematic representations of the
three-dimensional structure of the polypeptide depicted in SEQ ID
NO:13.
[0028] FIG. 7 shows a schematic representation of the
three-dimensional structure of the B domain of the polypeptide
depicted in SEQ ID NO:13.
[0029] FIG. 8 shows the amino acid sequence of the polypeptide of
SEQ ID NO:12 and the nucleotide sequence encoding the
polypeptide.
[0030] FIG. 9 is a schematic representation of the plasmid
designated pDN1528.
[0031] FIG. 10 is a schematic representation of the plasmid
designated pJEN1.
DETAILED DISCLOSURE OF THE INVENTION
The Termamyl-Like Alpha-Amylase
[0032] It is well known that a number of alpha-amylases produced by
Bacillus spp. are highly homologous on the amino acid level. For
instance, the B. licheniformis alpha-amylase comprising the amino
acid sequence shown in SEQ ID NO: 2 (commercially available as
Termamyl.RTM.) has been found to be about 89% homologous with the
B. amyloliquefaciens alpha-amylase comprising the amino acid
sequence shown in SEQ ID NO: 4 and about 79% homologous with the B.
stearothermophilus alpha-amylase comprising the amino acid sequence
shown in SEQ ID NO: 6. Further homologous alpha-amylases include an
alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289,
NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in
detail in WO 95/26397, and the alpha-amylase described by Tsukamoto
et al., 1988, Biochemical and Biophysical Research Communications
151(1). Still other homologous alpha-amylases include the
alpha-amylase produced by the B. licheniformis described in EP 252
666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353
and WO 94/18314. Other commercial Termamyl-like B. licheniformis
alpha-amylases are Optitherm.RTM. and Takatherm.RTM. (available
from Solvay), Maxamyl.RTM. (available from Gist-brocades/Genencor),
Spezym AA.RTM. (available from Genencor), and Keistase.RTM.
(available from Daiwa).
[0033] Because of the substantial homology found between these
alpha-amylases, they are considered to belong to the same class of
alpha-amylases, namely the class of "Termamyl-like
alpha-amylases".
[0034] Accordingly, in the present context, the term "Termamyl-like
alpha-amylase" is intended to indicate an alpha-amylase which, on
the amino acid level, exhibits a substantial homology to
Termamyl.RTM., i.e., the B. licheniformis alpha-amylase SEQ ID NO:
2. In other words, a Termamyl-like alpha-amylase is an
alpha-amylase, which has the amino acid sequence shown in SEQ ID
NO: 2, 4 or 6 herein, or the amino acid sequence shown in SEQ ID
NO: 1 or 2 of WO 95/26397 or in Tsukamoto et al., 1988, or i) which
displays at least 60%, such as at least 70%, e.g., at least 75%, or
at least 80%, e.g., at least 85%, at least 90% or at least 95%
homology with at least one of said amino acid sequences and/or ii)
displays immunological cross-reactivity with an antibody raised
against at least one of said alpha-amylases, and/or iii) is encoded
by a DNA sequence which hybridizes to the DNA sequences encoding
the above specified alpha-amylases which are apparent from SEQ ID
NOS: 1, 3 and 5 of the present application, and SEQ ID NOS: 4 and 5
of WO 95/26397, respectively.
[0035] In connection with property i) the "homology" may be
determined by use of any conventional algorithm, preferably by use
of the GAP progamme from the GCG package version 7.3 (June 1993)
using default values for GAP penalties (Genetic Computer Group
(1991) Programme Manual for the GCG Package, version 7, 575 Science
Drive, Madison, Wis., USA 53711).
[0036] Property ii) of the alpha-amylase, i.e., the immunological
cross reactivity, may be assayed using an antibody raised against
or reactive with at least one epitope of the relevant Termamyl-like
alpha-amylase. The antibody, which may either be monoclonal or
polyclonal, may be produced by methods known in the art, e.g., as
described by Hudson et al., 1989. The immunological
cross-reactivity may be determined using assays known in the art,
examples of which are Western Blotting or radial immunodiffusion
assay. In this respect, immunological cross-reactivity between the
alpha-amylases having the amino acid sequences of SEQ ID NOS: 2, 4
and 6, respectively, has been found.
[0037] The oligonucleotide probe used in the characterization of
the Termamyl-like alpha-amylase in accordance with property iii)
above may suitably be prepared on the basis of the full or partial
nucleotide or amino acid sequence of the alpha-amylase in question.
Suitable conditions for testing hybridization involve presoaking in
5.times.SSC and prehybridizing for 1 hour at .about.40.degree. C.
in a solution of 20% formamide, 5.times.Denhardt's solution, 50 mM
sodium phosphate, pH 6.8, and 50 micrograms of denatured sonicated
calf thymus DNA, followed by hybridization in the same solution
supplemented with 100 micro-M ATP for 18 hours at .about.40.degree.
C., or other methods described by, e.g., Sambrook et al., 1989.
[0038] In the present context, "derived from" is intended not only
to indicate an alpha-amylase produced or producible by a strain of
the organism in question, but also an alpha-amylase encoded by a
DNA sequence isolated from such strain and produced in a host
organism transformed with said DNA sequence. Finally, the term is
intended to indicate an alpha-amylase which is encoded by a DNA
sequence of synthetic and/or cDNA origin and which has the
identifying characteristics of the alpha-amylase in question. The
term is also intended to indicate that the parent alpha-amylase may
be a variant of a naturally occurring alpha-amylase, i.e., a
variant which is the result of a modification (insertion,
substitution, deletion) of one or more amino acid residues of the
naturally occurring alpha-amylase.
Parent Hybrid Alpha-Amylases
[0039] The parent alpha-amylase (being a Termamyl-like or
non-Termamyl-like alpha-amylase) may be a hybrid alpha-amylase,
i.e., an alpha-amylase which comprises a combination of partial
amino acid sequences derived from at least two alpha-amylases.
[0040] The parent hybrid alpha-amylase may be one which on the
basis of amino acid homology and/or immunological cross-reactivity
and/or DNA hybridization (as defined above) can be determined to
belong to the Termamyl-like alpha-amylase family. In this case, the
hybrid alpha-amylase is typically composed of at least one part of
a Termamyl-like alpha-amylase and part(s) of one or more other
alpha-amylases selected from Termamyl-like alpha-amylases or
non-Termamyl-like alpha-amylases of microbial (bacterial or fungal)
and/or mammalian origin.
[0041] Thus, the parent hybrid alpha-amylase may comprise a
combination of at least two Termamyl-like alpha-amylases, or of at
least one Termamyl-like and at least one non-Termamyl-like
bacterial alpha-amylase, or of at least one Termamyl-like and at
least one fungal alpha-amylase. For instance, the parent
alpha-amylase comprises a C-terminal part of an alpha-amylase
derived from a strain of B. licheniformis and an N-terminal part of
an alpha-amylase derived from a strain of B. amyloliquefaciens or
from a strain of B. stearothermophilus. For instance, the parent
alpha-amylase comprises at least 430 amino acid residues of the
C-terminal part of the B. licheniformis alpha-amylase, and may,
e.g., comprise a) an amino acid segment corresponding to the 37
N-terminal amino acid residues of the B. amyloliquefaciens
alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4
and an amino acid segment corresponding to the 445 C-terminal amino
acid residues of the B. licheniformis alpha-amylase having the
amino acid sequence shown in SEQ ID NO: 2, or b) an amino acid
segment corresponding to the 68 N-terminal amino acid residues of
the B. stearothermophilus alpha-amylase having the amino acid
sequence shown in SEQ ID NO: 6 and an amino acid segment
corresponding to the 415 C-terminal amino acid residues of the B.
licheniformis alpha-amylase having the amino acid sequence shown in
SEQ ID NO: 2.
[0042] Analogously, the parent hybrid alpha-amylase may belong to a
non-Termamyl-like alpha-amylase family, e.g., the Fungamyl-like
alpha-amylase family. In that case the hybrid may comprise at least
one part of an alpha-amylase belonging to the non-Termamyl-like
alpha-amylase family in combination with one or more parts derived
from other alpha-amylases.
The Three-Dimensional Termamyl-Like Alpha-Amylase Structure
[0043] The Termamyl-like alpha-amylase which was used to elucidate
the three-dimensional structure forming the basis for the present
invention consists of the 300 N-terminal amino acids of the B.
amyloliquefaciens alpha-amylase (with the amino acid sequence shown
in SEQ ID NO: 4) and amino acids 301-483 of the C-terminal end of
the B. licheniformis alpha-amylase with the amino acid sequence SEQ
ID NO: 2. The bacterial alpha-amylase belongs to the "Termamyl-like
alpha-amylase family" and the present structure is believed to be
representative for the structure of any Termamyl-like
alpha-amylase.
[0044] The structure of the alpha-amylase was solved in accordance
with the principle for X-ray crystallographic methods given in
"X-Ray Structure Determination", Stout, G. K. and Jensen, L. H.,
John Wiley & Sons, Inc. NY, 1989. The structural coordinates
for the solved crystal structure of the alpha-amylase at 2.2 .ANG.
resolution using the isomorphous replacement method are given in a
standard PDB format (Brookhaven Protein Data Base) in Appendix 1.
It is to be understood that Appendix 1 forms part of the present
application.
[0045] Amino acid residues of the enzyme are identified by
three-letter amino acid code (capitalized letters).
[0046] The alpha-amylase structure is made up of three globular
domains ordered A, B, and C with respect to sequence, which lie
approximately along a line in the order B, A, C. The domains can be
defined as being residues 1-103 and 206-395 for domain A, residues
104-205 for domain B, and residues 396-483 for domain C, the
numbers referring to the B. licheniformis alpha-amylase. This gives
rise to an elongated molecule, the longest axis being about 85
.ANG.. The widest point perpendicular to this axis is approximately
50 .ANG. and spans the central A domain. The active site residues
of the B. licheniformis alpha-amylase (SEQ ID NO: 2) are D323, D231
and E261.
Domain A
[0047] Domain A is the largest domain and contains the active site
(comprised of a cluster of three amino acid residues placed at the
bottom of a deep cleft in the enzyme's surface). Domain A of all
known alpha-amylase structures have the same overall fold, viz. the
(beta/alpha)8 barrel with 8 central beta strands (numbers 1-8) and
8 flanking alpha-helices. The beta-barrel is defined by McGregor
op. cit. The C-terminal end of beta-strand 1 is connected to helix
1 by a loop denoted loop 1 and an identical pattern is found for
the other loops. These loops show some variation in size and some
can be quite extensive.
[0048] The 8 central beta-strands in the (beta/alpha)8 barrel
superimpose well between the various known alpha-amylase
structures, and this part of the structure, including the close
surroundings of the active site located at the C-terminal end of
the beta-strands, show high similarity between the different
amylases.
[0049] The loops connecting beta-strands and alpha helices display
high variations between alpha amylases. These loops constitute the
structural context of the active site and the majority of the
contacts to the substrate is found among residues located in these
loops. Such important characteristics as substrate specificity,
substrate binding, pH/activity profile, starch cleavage pattern are
determined by the amino acids and the positions of same in these
loops.
[0050] The substantial differences between the Fungamyl-like
alpha-amylase structure and the structure of the Termamyl-like
alpha-amylase disclosed herein which are found in loops 1, 2, 3,
and 8 are visualized in the Figures.
Domain B
[0051] The Termamyl-like alpha-amylase structure has been found to
comprise a special domain structure in the A domain's loop 3, also
called domain B. The structure of the Termamyl-like alpha-amylase B
domain has never been seen before in any of the known alpha-amylase
or (beta/alpha)8-barrel proteins.
[0052] The domain B structure is a very compact domain having a
very high number of charged residues. The B domain arises as an
extension of the loop between strand 3 and helix 3 of domain A
(shown in FIG. 7) and contains a 5 stranded antiparallel beta-sheet
structure containing at least one long loop structure and having
the connectivity -1, +3, -1X, +2 (Richardson, 1981, Adv. Protein
Chem. 34: 167-339).
[0053] The first four strands of the B domain form two hairpin
loops which twist around each other like a pair of crossed fingers
(right-hand twist). The mainchain folds into a beta-strand which
connects two small beta-sheet structures. After making one turn in
one sheet it folds back and makes up a two stranded sheet in
contact with domain A and an internal hole in the alpha-amylase
structure. Then the mainchain folds up to a small sheet structure
nearly perpendicular to the first two sheets. Before entering the
helix 3 on top of the beta-strand 3, the approximately 24 last
amino acids in domain B form two calcium binding sites in the
contact region to domain A.
[0054] Domain B is connected with domain A by two peptide
stretches, which divide the domain-domain contact areas into two.
Domain B is in contact with Domain A by a calcium binding region
and an internally buried hole containing waters. Many types of
molecular contacts are present. Ionic interacting between acid and
basic amino acids is possible, these interactions are very
important for the general stability at high pH and for keeping the
calcium binding sites intact.
Domain C
[0055] Domain C is the C-terminal part of the protein consisting of
amino acids 394-483. Domain C is composed entirely of beta-strands
which forms a single 8-stranded sheet structure, which folds back
on itself, and thus may be described as a beta-sandwich structure.
The connectivity is +1, +1, +5, -3, +1, +1, -3 although strands 6
and 7 are only loosely connected. One part of the beta-sheet forms
the interface to domain A.
Ca-Binding and Na-Binding Sites
[0056] The structure of the Termamyl-like alpha-amylase is
remarkable in that it exhibits four calcium-binding sites and one
sodium-binding site. In other words four calcium ions and one
sodium ion are found to be present in the structure, although one
of the calcium ions displays very weak coordination. Two of the
calcium ions form part of a linear cluster of three ions, the
central ion being attributed to sodium, which lie at the junction
of the A and B domains.
[0057] The coordinating residues for the calcium ions between the A
and B domains are as follows (using the Pdb file nomenclature for
amino acid residues and atoms in the Pdb file found in Appendix 1
herein): For the calcium ion nearest to the active site (IUM 502 in
the pdb file), the backbone carbonyls from His235 and Asp194, the
sidechain atom OD1 from residues Asp194, Asn102 and Asp200, and one
water molecule WAT X3 (atom OW7). For the sodium ion (IUM 505), the
binding site includes atom OD2 from Asp194, Asp200, Asp183 and
Asp159, and a backbone carbonyl from Val201. The coordinates for
the other calcium ion between domains A and B are (IUM 501): atom
OD2 from Asp204 and Asp159, backbone carbonyl from Asp183 and
Ala181, atom OD1 from Asp202, and one water molecule WAT X7 (atom
OW7).
[0058] One calcium ion is located between the A and C domains,
another is located in the C domain. The first mentioned calcium
ion, which is also the one best coordinated (IUM 503) includes a
carbonyl backbone from Gly300, Tyr302 and His406, atom OD2/OD1 from
Asp430, atom OD1 from Asp407, and one water molecule WAT X6 (atom
OW7). The other and very weakly coordinated calcium site (IUM 504)
comprises 4 water molecules WAT X21 (atom OW8), X6 (atom OW6), X9
(atom OW0) and X28 (atom OW8), OE1/OE2 from Glu447 and OD1 from
Asn444.
Substrate-Binding Site
[0059] Without being limited to any theory it is presently believed
that favorable interactions between a substrate molecule and the
enzyme (such as hydrogen bonds and/or strong electrostatic
interaction) are found within a sphere of 4 .ANG. of the substrate,
when bound to the enzyme. The following residues of the B.
licheniformis alpha-amylase having the amino acid sequence shown in
SEQ ID NO: 2 are contemplated to be within a distance of 4 .ANG. of
the substrate and thus believed to be involved in interactions with
the substrate:
Trp13, Tyr14, Asn17, Asp18, Ser50, Gln51, Ala52, Asp53, Val54,
Gly55, Tyr56, Lys70, Arg74, Lys76, Val102, His105, Gly107, Gly108,
Ala109, Trp138, Thr163, Asp164, Trp165, Asn172, Glu189, Tyr193,
Leu196, Met197, Tyr198, Ala199, Arg229, Asp231, Ala232, Lys234,
His235, Glu261, Trp263, His327, Asp328, Gln333, Ser334, and
Leu335.
[0060] The amino acid residues of another Termamyl-like
alpha-amylase, which are contemplated to be within a distance of 4
.ANG. of the substrate, may easily be identified by aligning the
amino acid sequence SEQ ID NO: 2 with that of the other
Termamyl-like alpha-amylase and thereby identifying the positions
equivalent to those identified above.
Generality of Structure
[0061] Because of the high homology between the various
Termamyl-like alpha-amylases, the solved structure defined by the
coordinates of Appendix 1 is believed to be representative for the
structure of all Termamyl-like alpha-amylases. A model structure of
other Termamyl-like alpha-amylases may easily be built on the basis
of the coordinates given in Appendix 1 adapted to the alpha-amylase
in question by use of an alignment between the respective amino
acid sequences. The creation of a model structure is exemplified in
Example 1.
[0062] The above identified structurally characteristic parts of
the Termamyl-like alpha-amylase structure (Ca-binding site,
substrate binding site, loops, etc.) may easily be identified in
other Termamyl-like alpha-amylases on the basis of a model (or
solved) structure of the relevant Termamyl-like alpha-amylase or
simply on the basis of an alignment between the amino acid sequence
of the Termamyl-like alpha-amylase in question with that of the B.
licheniformis alpha-amylase used herein for identifying the amino
acid residues of the respective structural elements.
[0063] Furthermore, in connection with Termamyl-like variants of
the invention, which are defined by modification of specific amino
acid residues of a specific Termamyl-like alpha-amylase, it will be
understood that variants of another Termamyl-like alpha-amylase
modified in an equivalent position (as determined from the best
possible amino acid sequence alignment between the respective
sequences) are intended to be covered as well. Thus, irrespective
of whether an amino acid residue is identified herein for the
purpose of defining a structural part of a given alpha-amylase or
used for identifying a variant of the alpha-amylase, this amino
acid residue shall be considered as representing the equivalent
amino acid residue of any other Termamyl-like alpha-amylase.
Methods of the Invention for Design of Novel Alpha-Amylase
Variants
[0064] In the methods according to the first, second and third
aspects of the invention the terms "structure of a Termamyl-like
alpha-amylase" and "Termamyl-like alpha-amylase structure" are
intended to indicate the solved structure defined by the
coordinates presented in Appendix 1 or a model structure of a given
Termamyl-like alpha-amylase (such as the B. licheniformis
alpha-amylase) built on the basis of the solved structure.
[0065] In most cases the parent Termamyl-like alpha-amylase to be
modified in accordance with the present invention is different from
the alpha-amylase which was actually used for solving the structure
(Appendix 1). This means that the amino acid residue(s) or
structural part(s) identified in the solved structure (Appendix 1)
in step i) of the method according to the first, second or third
aspect of the invention must be translated into the corresponding
amino acid residue(s) or structural part(s) of the parent
Termamyl-like alpha-amylase in question. The "translation" is
conveniently performed on the basis of an amino acid sequence
alignment between the amino acid sequence of the Termamyl-like
alpha-amylase used for solving the structure and the amino acid
sequence of the parent Termamyl-like alpha-amylase in question.
[0066] The analysis or comparison performed in step i) of the
method according to the first, second and third aspect,
respectively, of the invention may be performed by use of any
suitable computer program capable of analyzing and/or comparing
protein structures, e.g., the computer program Insight, available
from Biosym Technologies, Inc. For instance, the basic principle of
structure comparison is that the three-dimensional structures to be
compared are superimposed on the basis of an alignment of secondary
structure elements (such as the central 8 beta-strands in the
barrel) and the parts differing between the structures can
subsequently easily be identified from the superimposed
structure.
[0067] The structural part which is identified in step i) of the
methods of the first, second and third aspects of the invention may
be composed of one amino acid residue. However, normally the
structural part comprises more than one amino acid residue,
typically constituting one of the above parts of the Termamyl-like
alpha-amylase structure such as one of the A, B, or C domains, an
interface between any of these domains, a calcium binding site, a
loop structure, the substrate binding site, or the like.
[0068] In the present context the term "structural or functional
considerations" is intended to indicate that modifications are made
on the basis of an analysis of the relevant structure or structural
part and its contemplated impact on the function of the enzyme.
Thus, an analysis of the structures of the various alpha-amylases,
which until now has been elucidated, optionally in combination with
an analysis of the functional differences between these
alpha-amylases, may be used for assigning certain properties of the
alpha-amylases to certain parts of the alpha-amylase structure or
to contemplate such relationship. For instance, differences in the
pattern or structure of loops surrounding the active site may
result in differences in access to the active site of the substrate
and thus differences in substrate specificity and/or cleavage
pattern. Furthermore, parts of a Termamyl-like alpha-amylase
involved in or contemplated to be involved in substrate binding
(and thus, e.g., specificity/cleavage pattern), calcium or sodium
ion binding (e.g., of importance for the calcium-dependency of the
enzyme), and the like has been identified (vide infra).
[0069] The modification of an amino acid residue or structural part
is typically accomplished by suitable modifications of a DNA
sequence encoding the parent enzyme in question. The term
"modified" as used in step ii) in the method according to the first
aspect of the invention is intended to have the following meaning:
When used in relation to an amino acid residue the term is intended
to mean replacement of the amino acid residue in question with
another amino acid residue. When used in relation to a structural
part, the term is intended to mean replacement of one or more amino
acid residues of said structural part, addition of one or more
amino acid residues to said part, or deletion of one or more amino
acid residues of said structural part.
[0070] 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.
First Aspect of the Invention
[0071] In a preferred embodiment of the method according to the
first aspect of the invention the property of the parent enzyme to
be modified is selected from calcium dependency, substrate binding,
cleavage pattern, pH dependent activity and the like. Specific
examples of how to change these properties of a parent
Termamyl-like alpha-amylase are given further below.
[0072] In another preferred embodiment the parent Termamyl-like
alpha-amylase to be modified is a B. licheniformis
alpha-amylase.
Second and Third Aspects of the Invention
[0073] One important advantage of the methods according to the
second and third aspects of the present invention is that it is
possible to adapt the structure (or a structural part) of a
Termamyl-like alpha-amylase to the structure (or structural part)
of a non-Termamyl-like alpha-amylase and vice versa. For instance,
having identified a loop structure of the non-Termamyl-like
alpha-amylase which is believed to be responsible for or
contributing to a particular property of the non-Termamyl-like
alpha-amylase it is possible to replace the corresponding structure
of the Termamyl-like alpha-amylase with said non-Termamyl-like
alpha-amylase structure--or if no corresponding structure exists in
the Termamyl-like alpha-amylase--to insert the structure into the
Termamyl-like alpha-amylase in such a manner that the resulting
variant Termamyl-like alpha-amylase, as far as the relevant part is
concerned, resembles the corresponding part of the
non-Termamyl-like alpha-amylase. When two or more parts of the
structure of the parent Termamyl-like alpha-amylase are modified so
as to resemble the corresponding parts of the non-Termamyl-like
alpha-amylase it is possible to increase the resemblance to the
non-Termamyl-like alpha-amylase of the Termamyl-like alpha-amylase
variant and thus to alter the properties of said variant in the
direction of those of said non-Termamyl-like alpha-amylase. Loop
modifications are discussed in much further detail further
below.
[0074] Typically, the modification to be performed in step iii) of
the method according to the second aspect of the invention is
accomplished by deleting one or more amino acid residues of the
part of the Termamyl-like alpha-amylase to be modified so as to
adapt the structure of said part of the parent alpha-amylase to the
corresponding part of the non-Termamyl-like alpha-amylase; by
replacing one or more amino acid residues of the part of the
Termamyl-like alpha-amylase to be modified with the amino acid
residues occupying corresponding positions in the non-Termamyl-like
alpha-amylase; or by insertion of one or more amino acid residues
present in the non-Termamyl-like alpha-amylase into a corresponding
position in the Termamyl-like alpha-amylase. For the method
according to the third aspect the modification is to be understood
analogously, performed on the non-Termamyl-like parent
alpha-amylase rather than the Termamyl-like alpha-amylase.
[0075] In step ii) of the method according to the second or third
aspect of the invention the part of the structure to be identified
is preferably one which in the folded enzyme is believed to be in
contact with the substrate (cf. the disclosure above in the section
entitled "Substrate-binding site") or involved in substrate
specificity and/or cleavage pattern, and/or one which is in contact
with one of the calcium or sodium ions and/or one, which is
contributing to the pH or temperature profile of the enzyme, or one
which otherwise, from structural or functional considerations, is
contemplated to be responsible for differences in one or more
properties of the Termamyl-like and non-Termamyl-like
alpha-amylase.
Non-Termamyl-Like Alpha-Amylase
[0076] The non-Termamyl-like alpha-amylase with which the
comparison is made in step i) of the method of the second aspect of
the invention and which is the parent alpha-amylase in the method
of the third aspect of the invention, may be any alpha-amylase,
which does not belong to the family of Termamyl-like alpha-amylases
(as defined above) and, which as a consequence thereof, has a
different three-dimensional structure. Furthermore, the
non-Termamyl-like alpha-amylase should be one which has, at the
time that the method is performed, an elucidated or contemplated
three-dimensional structure.
[0077] The non-Termamyl-like alpha-amylase may, e.g., be a fungal
alpha-amylase, a mammalian or a plant alpha-amylase or a bacterial
alpha-amylase (different from a Termamyl-like alpha-amylase).
Specific examples of such alpha-amylases include the Aspergillus
oryzae TAKA alpha-amylase, the A. niger acid alpha-amylase, the
Bacillus subtilis alpha-amylase, the porcine pancreatic
alpha-amylase and a barley alpha-amylase. All of these
alpha-amylases have elucidated structures which are clearly
different from the structure of the Termamyl-like alpha-amylase
shown herein.
[0078] The fungal alpha-amylases mentioned above, i.e., derived
from A. niger and A. oryzae, are highly homologous on the amino
acid level and generally considered to belong to the same family of
alpha-amylases. In the present disclosure, this family is termed
"Fungamyl-like alpha-amylase" and intends to indicate an
alpha-amylase which exhibits a high homology, i.e., more than 70%,
such as 80% homologous (as defined herein) to the fungal
alpha-amylase derived from Aspergillus oryzae, commercially
available as Fungamyl.RTM., and the A. niger alpha-amylase.
[0079] From the enclosed illustrations of the alpha-amylase
structure of a Termamyl-like alpha-amylase and a comparison of said
structure with the structure of a Fungamyl-like alpha-amylase it is
evident that major differences exist between the two structures. In
the method of the invention it is of particular interest to modify
parts of the parent Termamyl-like alpha-amylase, which belong to a
region with large differences to the Fungamyl-like alpha-amylase.
In particular, it is of interest to modify the parent Termamyl-like
alpha-amylase in one or more of the following loops: loop 1, loop
2, loop 3 and/or loop 8 of the parent alpha-amylase.
[0080] In the method of the third aspect of the invention it is of
particular interest to modify loop 1, loop 2, loop 3 and/or loop 8
of the parent non-Termamyl-like alpha-amylase to a closer
resemblance to the similar loops of a Termamyl-like alpha-amylase,
such as Termamyl.
[0081] In the following specific types of variants are described
which have been designed by use of the method of the invention.
Loop Modifications
[0082] In order to change the substrate specificity of the parent
alpha-amylase to be modified it is relevant to consider loop
modifications. For instance changing one or more of the loop
structures of the Termamyl-like alpha-amylase into a closer
resemblance with the corresponding loop structure(s) of a
non-Termamyl-like alpha-amylase (such as a Fungamyl-like
alpha-amylase) it is contemplated that it is possible to change the
substrate specificity in the direction of that of the non-Termamyl
alpha-amylase. In the following different types of loop
modifications of interest are listed. It will be understood that
the variants may have other changed properties in addition to the
modified substrate specificity. It will be understood that the
following modifications identified for a specific Termamyl-like
alpha-amylase are intended to include corresponding modifications
in other equivalent positions of other Termamyl-like
alpha-amylases. Furthermore, it will be understood that, normally,
the loop modification will comprise replacement of an entire loop
structure or a substantial part thereof in, e.g., the Termamyl-like
alpha-amylase, with the corresponding loop structure (or
substantial part thereof) in a non-Termamyl-like alpha-amylase.
Loop 2 Modifications
[0083] In one embodiment the invention relates to a variant of a
parent Termamyl-like alpha-amylase, in which variant at least one
amino acid residue of the parent alpha-amylase, which is/are
present in a fragment corresponding to the amino acid fragment
44-57 of the amino acid sequence of SEQ ID NO: 4, i.e., loop 2, has
been deleted or replaced with one or more amino acid residues which
is/are present in a fragment corresponding to the amino acid
fragment 66-84 of the amino acid sequence shown in SEQ ID NO: 10,
or in which one or more additional amino acid residues has been
added using the relevant part of SEQ ID NO: 10 or a corresponding
part of another Fungamyl-like alpha-amylase as a template.
[0084] The amino acid sequence shown in SEQ ID NO: 10 is the amino
acid sequence of the A. oryzae alpha-amylase, i.e., a Fungamyl-like
alpha-amylase. It will be understood that amino acid residues or
fragments found in corresponding positions in other alpha-amylases,
in particular Fungamyl-like alpha-amylases, may be used as a
template for the construction of the variant according to the
invention. The corresponding part in other homologous
alpha-amylases may easily be identified on the basis of a
comparison of the amino acid sequences and/or three-dimensional
structures of the respective alpha-amylases.
[0085] For instance, the variant may be one, which, when the amino
acid sequence of the variant is aligned most closely with the amino
acid sequence of the said parent alpha-amylase, occupies the same
position as the portion from residue X to residue Y of SEQ ID NO:
4, the said region having at least 80% such as at least 90%
sequence homology with the part of SEQ ID NO: 10 extending from
residue Z to residue V of SEQ ID NO: 10, wherein
[0086] X is the amino acid residue occupying position 44, 45, 46,
47 or 48 of SEQ ID NO: 4,
[0087] Y is the amino acid residue occupying position 51, 52, 53,
54, 55, 56 or 57 of SEQ ID NO: 4,
[0088] Z is the amino acid residue occupying position 66, 67, 68,
69 or 70 of SEQ ID NO: 10, and
[0089] V is the amino acid residue occupying position 78, 79, 80,
81, 82, 83 or 84 of SEQ ID NO: 10.
[0090] In other words, the variant may be one in which an amino
acid fragment X-Y of the parent alpha-amylase, which corresponds to
or is within the amino acid fragment 44-57 of SEQ ID NO: 4, has
been replaced with an amino acid fragment Z-V, which corresponds to
or is within the amino acid fragment 66-84 of the amino acid
sequence shown in SEQ ID NO: 10, in X, Y, Z and V have the meaning
indicated above.
[0091] A specific example of a variant according to this embodiment
is a variant of a parent Termamyl-like alpha-amylase, in which the
amino acid fragment of the parent alpha-amylase, which corresponds
to amino acid residues 48-51 of SEQ ID NO: 4, has been replaced
with an amino acid fragment corresponding to amino acid residues
70-78 of the amino acid sequence shown in SEQ ID NO: 10.
Loop 3 Modifications--Limited Alteration
[0092] In another embodiment the invention relates to a variant of
a parent Termamyl-like alpha-amylase, in which variant at least one
of the amino acid residues of the parent alpha-amylase, which
is/are present in an amino acid fragment corresponding to the amino
acid fragment 195-202 of the amino acid sequence of SEQ ID NO: 4,
has been deleted or replaced with one or more of the amino acid
residues which is/are present in an amino acid fragment
corresponding to the amino acid fragment 165-177 of the amino acid
sequence shown in SEQ ID NO: 10, or in which one or more additional
amino acid residues has been added using the relevant part of SEQ
ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
[0093] For instance, the variant may be one in which an amino acid
fragment X-Y of the parent alpha-amylase which corresponds to or is
within the amino acid fragment 195-202 of SEQ ID NO: 4, has been
replaced by an amino acid fragment Z-V, which corresponds to or is
within the amino acid fragment 165-177 of the amino acid sequence
shown in SEQ ID NO: 10, in which
[0094] X is an amino acid residue corresponding to the amino acid
occupying position 195 or 196 of SEQ ID NO: 4,
[0095] Y is an amino acid residue corresponding to the amino acid
occupying position 198, 199, 200, 201, or 202 of SEQ ID NO: 4,
[0096] Z is an amino acid residue corresponding to the amino acid
occupying position 165 or 166 of SEQ ID NO: 10, and
[0097] V is an amino acid residue corresponding to the amino acid
occupying position 173, 174, 175, 176 or 177 of SEQ ID NO: 10.
[0098] Expressed in another manner, the variant according to this
aspect may be one, which, when the amino acid sequence of variant
is aligned most closely with the amino acid sequence of the said
parent Termamyl-like alpha-amylase, occupies the same position as
the portion from residue X to residue Y of SEQ ID NO: 4, the said
region having at least 80%, such as 90% sequence homology with the
part of SEQ ID NO: 10 extending from residue Z to residue V of SEQ
ID NO: 10, the meaning of X, Y, Z and V being as identified
above.
[0099] A specific example of a variant according to this embodiment
is a variant of a parent Termamyl-like alpha-amylase, in which the
amino acid fragment of the parent alpha-amylase, which corresponds
to amino acid residues 196-198 of SEQ ID NO: 4, has been replaced
with the amino acid fragment corresponding to amino acid residues
166-173 of the amino acid sequence shown in SEQ ID NO: 10.
Loop 3 Modifications--Complete Domain B
[0100] In a further embodiment the invention relates to a variant
of a parent Termamyl-like alpha-amylase, in which variant at least
one of the amino acid residues of the parent alpha-amylase, which
is/are present in a fragment corresponding to the amino acid
fragment 117-185 of the amino acid sequence of SEQ ID NO: 4,
has/have been deleted or replaced with one or more of the amino
acid residues, which is/are present in an amino acid fragment
corresponding to the amino acid fragment 98-210 of the amino acid
sequence shown in SEQ ID NO: 10, or in which one or more additional
amino acid residues has been added using the relevant part of SEQ
ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
[0101] For instance, the variant may be one in which an amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 117-185 of SEQ ID NO: 4, has been
replaced with an amino acid fragment Z-V, which corresponds to or
is within the amino acid fragment 98-210 of the amino acid sequence
shown in SEQ ID NO: 10, in which variant
[0102] X is an amino acid residue corresponding to the amino acid
occupying position 117, 118, 119, 120 or 121 of SEQ ID NO: 4,
[0103] Y is an amino acid residue corresponding to the amino acid
occupying position 181, 182, 183, 184 or 185 of SEQ ID NO: 4,
[0104] Z is an amino acid residue corresponding to the amino acid
occupying position 98, 99, 100, 101, 102 of SEQ ID NO: 10, and
[0105] V is an amino acid residue corresponding to the amino acid
occupying position 206, 207, 208, 209 or 210 of SEQ ID NO: 10.
[0106] A specific example of a variant according to this embodiment
is a variant of a parent alpha-amylase, in which an amino acid
fragment of the parent alpha-amylase, which corresponds to amino
acid residues 121-181 of SEQ ID NO: 4, has been replaced with the
amino acid fragment corresponding to amino acid residues 102-206 of
the amino acid sequence shown in SEQ ID NO: 10.
[0107] In another embodiment the invention relates to a variant of
a parent Termamyl-like alpha-amylase, in which variant at least one
of the amino acid residues of the parent alpha-amylase, which
is/are present in a fragment corresponding to the amino acid
fragment 117-181 of the amino acid sequence of SEQ ID NO: 4,
has/have been deleted or replaced with one or more of the amino
acid residues, which is/are present in an amino acid fragment
corresponding to the amino acid fragment to 98-206 of the amino
acid sequence shown in SEQ ID NO: 10, or in which one or more
additional amino acid residues has been added using the relevant
part of SEQ ID NO: 10 or a corresponding part of another
Fungamyl-like alpha-amylase as a template.
[0108] For instance, the variant may be one in which the amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 117-177 if SEQ ID NO: 4, has/have
been replaced with an amino acid fragment Z-V, which corresponds to
or is within the amino acid fragment 98-202 of the amino acid
sequence shown in SEQ ID NO: 10, in which variant
[0109] X is an amino acid residue corresponding to the amino acid
occupying position 117, 118, 119, 120 or 121 of SEQ ID NO: 4,
[0110] Y is an amino acid residue corresponding to the amino acid
occupying position 174, 175, 176 or 177 of SEQ ID NO: 4,
[0111] Z is an amino acid residue corresponding to the amino acid
occupying position 98, 99, 100, 101, 102 of SEQ ID NO: 10, and
[0112] V is an amino acid residue corresponding to the amino acid
occupying position 199, 200, 201 or 202 of SEQ ID NO: 10.
[0113] A specific example of a variant according to this embodiment
of the invention is a variant, in which the amino acid fragment of
the parent alpha-amylase, which corresponds to amino acid residues
121-174 of SEQ ID NO: 4, has been replaced with the amino acid
fragment corresponding to amino acid residues 102-199 of the amino
acid sequence shown in SEQ ID NO: 10.
Loop 1 Modifications--Minimal Addition
[0114] In a further embodiment the present invention relates to a
variant of a parent Termamyl-like alpha-amylase, in which variant
at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in an amino acid fragment
corresponding to the amino acid fragment 12-19 of the amino acid
sequence of SEQ ID NO: 4, has/have been deleted or replaced with
one or more of the amino acid residues, which is/are present in an
amino acid fragment which corresponds to the amino acid fragment
28-42 of SEQ ID NO: 10, or in which one or more additional amino
acid residues has/have been inserted using the relevant part of SEQ
ID NO: 10 or a corresponding part of another Fungamyl-like
alpha-amylase as a template.
[0115] For instance, the variant may be one in which the amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 12-19 of SEQ ID NO: 4, has/have
been replaced with an amino acid fragment Z-V, which corresponds to
or is within the amino acid fragment 28-42 of the amino acid
sequence shown in SEQ ID NO: 10, in which variant
[0116] X is an amino acid residue corresponding to the amino acid
occupying position 12, 13 or 14 of SEQ ID NO: 4,
[0117] Y is an amino acid residue corresponding to the amino acid
occupying position 15, 16, 17, 18 or 19 of SEQ ID NO: 4,
[0118] Z is an amino acid residue corresponding to the amino acid
occupying position 28, 29, 30, 31 or 32 of SEQ ID NO: 10, and
[0119] V is an amino acid residue corresponding to the amino acid
occupying position 38, 39, 40, 41 or 42 of SEQ ID NO: 10.
[0120] A specific example of a variant according to this aspect of
the invention is a variant, in which the amino acid fragment of the
parent alpha-amylase, which corresponds to amino acid residues
14-15 of SEQ ID NO: 4, has been replaced with the amino acid
fragment corresponding to amino acid residues 32-38 of the amino
acid sequence shown in SEQ ID NO: 10.
Loop 1 Modifications--Complete Loop
[0121] In a further embodiment the invention relates to a variant
of a parent Termamyl-like alpha-amylase, in which variant at least
one of the amino acid residues of the parent alpha-amylase, which
is present in a fragment corresponding to amino acid residues 7-23
of the amino acid sequence of SEQ ID NO: 4, has/have been deleted
or replaced with one or more amino acid residues, which is/are
present in an amino acid fragment corresponding to amino acid
residues 13-45 of the amino acid sequence shown in SEQ ID NO: 10,
or in which one or more additional amino acid residues has/have
been inserted using the relevant part of SEQ ID NO: 10 or a
corresponding part of another Fungamyl-like alpha-amylase as a
template.
[0122] For instance, the variant may be one in which the amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 7-23 of SEQ ID NO: 4, has/have
been replaced with an amino acid fragment Z-V, which corresponds to
or is within the amino acid fragment 13-45 of the amino acid
sequence shown in SEQ ID NO: 10, in which variant
[0123] X is an amino acid residue corresponding to the amino acid
occupying position 7 or 8 of SEQ ID NO: 4,
[0124] Y is an amino acid residue corresponding to the amino acid
occupying position 18, 19, 20, 21, 22 or 23 of SEQ ID NO: 4,
[0125] Z is an amino acid residue corresponding to the amino acid
occupying position 13 or 14 of SEQ ID NO: 10, and
[0126] V is an amino acid residue corresponding to the amino acid
occupying position 40, 41, 42, 43, 44 or 45 of SEQ ID NO: 10.
[0127] A specific variant according to this embodiment is one in
which the amino acid fragment of the parent alpha-amylase, which
corresponds to amino acid residues 8-18 of SEQ ID NO: 4, has been
replaced with the amino acid fragment corresponding to amino acid
residues 14-40 of the amino acid sequence shown in SEQ ID NO:
10.
Loop 8 Modifications
[0128] In a further embodiment the invention relates to a variant
of a parent Termamyl-like alpha-amylase, in which variant at least
one of the amino acid residues of the parent alpha-amylase, which
is present in a fragment corresponding to amino acid residues
322-346 of the amino acid sequence of SEQ ID NO: 2, has/have been
deleted or replaced with one or more amino acid residues, which
is/are present in an amino acid fragment corresponding to amino
acid residues 291-313 of the amino acid sequence shown in SEQ ID
NO: 10, or in which one or more additional amino acid residues
has/have been inserted using the relevant part of SEQ ID NO: 10 or
a corresponding part of another Fungamyl-like alpha-amylase as a
template.
[0129] For instance, the variant may be one in which the amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 322-346 of SEQ ID NO: 2, has/have
been replaced with an amino acid fragment Z-V, which corresponds to
or is within the amino acid fragment 291-313 of the amino acid
sequence shown in SEQ ID NO: 10, in which variant
[0130] X is an amino acid residue corresponding to the amino acid
occupying position 322, 323, 324 or 325 of SEQ ID NO: 2,
[0131] Y is an amino acid residue corresponding to the amino acid
occupying position 343, 344, 345 or 346 of SEQ ID NO: 2,
[0132] Z is an amino acid residue corresponding to the amino acid
occupying position 291, 292, 293 or 294 of SEQ ID NO: 10, and
[0133] V is an amino acid residue corresponding to the amino acid
occupying position 310, 311, 312 or 313 of SEQ ID NO: 10.
[0134] A specific variant according to this aspect of the invention
is one in which the amino acid fragment of the parent
alpha-amylase, which corresponds to amino acid residues 325-345 of
SEQ D No. 2, has been replaced with the amino acid fragment
corresponding to amino acid residues 294-313 of the amino acid
sequence shown in SEQ ID NO: 10.
Ca.sup.2+ Dependency
[0135] It is highly desirable to be able to decrease the Ca.sup.2+
dependency of a Termamyl-like alpha-amylase. Accordingly, in a
further aspect the invention relates to a variant of a parent
Termamyl-like alpha-amylase, which exhibits alpha-amylase activity
and which has a decreased Ca.sup.2+ dependency as compared to the
parent alpha-amylase. The decreased Ca.sup.2+ dependency has the
functional result that the variant exhibits a satisfactory
amylolytic activity in the presence of a lower concentration of
calcium ion in the extraneous medium than is necessary for the
parent enzyme and, for example, therefore is less sensitive than
the parent to calcium ion-depleting conditions such as those
obtained in media containing calcium-complexing agents (such as
certain detergent builders).
[0136] The decreased Ca.sup.2+ dependency of the variant of the
invention may advantageously be achieved by increasing the
Ca.sup.2+ binding affinity of the parent Termamyl-like
alpha-amylase, in other words the stronger the Ca.sup.2+ binding of
the enzyme, the lower is the Ca.sup.2+ dependency.
[0137] It is presently believed that amino acid residues located
within 10 .ANG. from a sodium or calcium ion are involved in or are
of importance for the Ca.sup.2+ binding capability of the
enzyme.
[0138] Accordingly, the variant according to this aspect of the
invention is preferably one, which has been modified in one or more
amino acid residues present within 10 .ANG. from a calcium and/or
sodium ion identified in the three-dimensional Termamyl-like
alpha-amylase structure in such a manner that the affinity of the
alpha-amylase for calcium is increased.
[0139] The amino acid residues found within a distance of 10 .ANG.
from the Ca.sup.2+ binding sites of the B. licheniformis
alpha-amylase with the amino acid sequence SEQ ID NO: 2 were
determined as described in Example 2 and are as follows:
V102, I103, N104, H105, K106, R125, W155, W157, Y158, H159, F160,
D161, G162, T163, Y175, K176, F177, G178, K180, A181, W182, D183,
W184, E185, V186, S187, N192, Y193, D194, Y195, L196, M197, Y198,
A199, D200, I201, D202, Y203, D204, H205, P206, V208, A209, D231,
A232, V233, K234, H235, I236, K237, F238, F240, L241, A294, A295,
S296, T297, Q298, G299, G300, G301, Y302, D303, M304, R305, K306,
L307, W342, F343, L346, Q393, Y394, Y396, H405, H406, D407, I408,
V409, R413, E414, G415, D416, S417, V419, A420, N421, S422, G423,
L424, I428, T429, D430, G431, P432, V440, G441, R442, Q443, N444,
A445, G446, E447, T448, W449, I462, G475, Y480, V481, Q482,
R483.
[0140] In order to construct a variant according to this aspect of
the invention it is desirable to replace at least one of the above
mentioned amino acid residues (or an amino acid residue occupying
an equivalent position in another Termamyl-like alpha-amylase than
that defined by SEQ ID NO: 2), which is contemplated to be involved
in providing a non-optimal calcium binding, with any other amino
acid residue which improves the Ca.sup.2+ binding affinity of the
variant enzyme. In practice, the identification and subsequent
modification of the amino acid residue is performed by the
following method:
[0141] i) identifying an amino acid residue within 10 .ANG. from a
Ca.sup.2+ binding site of a Termamyl-like alpha-amylase structure,
which from structural or functional considerations is believed to
be responsible for a non-optimal calcium ion interaction,
[0142] ii) constructing a variant in which said amino acid residue
is replaced with another amino acid residue which from structural
or functional considerations is believed to be important for
establishing a higher Ca.sup.2+ binding affinity, and testing the
Ca.sup.2+ dependency of the resulting Termamyl-like alpha-amylase
variant.
[0143] In the present context, the term "non-optimal calcium ion
interaction" is intended to indicate that the amino acid residue in
question is selected on the basis of a presumption that
substituting said amino acid residue for another may improve a
calcium ion binding interaction of the enzyme. For instance, the
amino acid residue in question may be selected on the basis of one
or more of the following considerations: [0144] to obtain an
improved interaction between a calcium ion and an amino acid
residue located near to the surface of the enzyme (as identified
from the structure of the Termamyl-like alpha-amylase). For
instance, if the amino acid residue in question is exposed to a
surrounding solvent, it may be advantageous to increase the
shielding of said amino acid residue from the solvent so as to
provide for a stronger interaction between said amino acid residue
and a calcium ion. This can be achieved by replacing said residue
(or an amino acid residue in the vicinity of said residue
contributing to the shielding) by an amino acid residue which is
more bulky or otherwise results in an improved shielding effect.
[0145] to stabilize a calcium binding site, for instance by
stabilizing the structure of the Termamyl-like alpha-amylase (e.g.,
by stabilizing the contacts between the A, B and C domains or
stabilizing one or more of the domains as such). This may, e.g., be
achieved by providing for a better coordination to amino acid side
chains, which may, e.g., be obtained by replacing an N residue with
a D residue and/or a Q residue with an E residue (e.g., N104D),
e.g., within 10 .ANG., and preferably within 3 or 4 .ANG., of a
calcium binding site. [0146] to protect the calcium binding site or
to improve the coordination between the calcium ion and the calcium
binding site, e.g., by providing a stronger interaction between the
ion and the binding site.
[0147] Before actually constructing a Termamyl-like alpha-amylase
variant according to the above principles it may be convenient to
evaluate the contemplated amino acid modification by its
accommodation into the Termamyl-like alpha-amylase structure, e.g.,
into a model structure of the parent Termamyl-like
alpha-amylase.
[0148] Preferably, the amino acid residue to be modified is located
within 8 .ANG. of a Ca.sup.2+ binding site residue, such as within
5 .ANG., of such residue. The amino acid residues within 8 .ANG.
and 5 .ANG., respectively, may easily be identified by an analogous
method used for identifying amino acid residues within 10 .ANG.
(cf. Example 2).
[0149] The following mutation is contemplated to be of particular
interest with respect to decreasing the Ca.sup.2+ dependency of a
Termamyl-like alpha-amylase:
N104D (of the B. licheniformis alpha-amylase SEQ ID NO: 2, or an
equivalent (N to D) mutation of an equivalent position in another
Termamyl-like alpha-amylase).
[0150] In connection with substitutions of relevance for Ca.sup.2+
dependency, some other substitutions appear to be of importance in
stabilizing the enzyme conformation (for instance the domains A-B
and/or domains A-C interactions contributing to the overall
stability of the enzyme) in that they may, e.g., enhance the
strength of binding or retention of calcium ion or sodium ion at or
within a calcium or sodium binding site, respectively, within the
parent Termamyl-like alpha-amylase.
[0151] It is desirable to stabilize the C-domain in order to
increase the calcium stability and/or thermostability of the
enzyme. In this connection the stabilization may result in a
stabilization of the binding of calcium by the enzyme, and an
improved contact between the C-domain and the A-domain (of
importance for thermostability). The latter may be achieved by
introduction of cystein bridges, salt bridges or increase hydrogen,
hydrophobic and/or electrostatic interactions.
[0152] For instance, the C-domain of the B. licheniformis
alpha-amylase having the amino acid sequence shown in SEQ ID NO: 2
may be stabilized by introduction of a cystein bridge between
domain A and domain C, e.g., by introducing of the following
mutations:
L346C+I430C and/or A349C+I479C.
[0153] A salt bridge may be obtained by introduction of the
following mutations:
F350D,E+I411R,K
F350D,E+I430R,K
K385R+N457D,E
N457D,E
[0154] The calcium site of domain C may be stabilized by replacing
the amino acid residues H408 and/or G303 with any other amino acid
residue. Of particular interest are the following mutations:
G303N,D,Q,E and/or H408Q,E,N,D which are contemplated to provide a
better calcium binding or protection from calcium depletion.
[0155] Similar mutations may be introduced in equivalent positions
of other Termamyl-like alpha-amylases.
[0156] Other substitution mutations (relative to B. licheniformis
alpha-amylase, SEQ ID NO: 2) which appear to be of importance,
inter alia, in the context of reducing calcium dependency include
the following:
In Domain A:
R23K;
A209V; and
G310D;
In Domain B:
H156Y;
[0157] A181E,D,Q,N,V,T (which appear to result in shielding of the
outermost Ca.sup.2+ binding site in the junction region between
Domain A and Domain B to some extent); I201(bulkier amino acid),
e.g., I201W,F,L (which appear to result in slight alterations in
the geometry of the region in the immediate vicinity of the
Ca.sup.2+--Na.sup.+--Ca.sup.2+ binding site(s) in the junction
region between Domain A and Domain B, and in the geometry and/or
size of a nearby hole/cavity); and Y203E,Q (which are believed to
result in stronger binding of the outermost Ca.sup.2+ ion in its
binding site in the junction region between Domain A and Domain B);
(or equivalent mutations in equivalent positions in another
Termamyl-like alpha-amylase).
[0158] Substitutions of R214 and P345 of B. licheniformis
alpha-amylase, SEQ ID NO: 2 (or equivalent mutations in equivalent
positions in another Termamyl-like alpha-amylase) with other amino
acids may also be of importance in this connection.
Variants with Altered Activity at Higher/Lower pH
[0159] It is contemplated that it is possible to change the pH
optima of a Termamyl-like alpha-amylase or the enzymatic activity
at a given pH by changing the pKa of the active site residues. This
may be achieved, e.g., by changing the electrostatic interaction or
hydrophobic interaction between functional groups of amino acid
side chains of the amino acid residue to be modified and of its
close surroundings. This may, e.g., be accomplished by the
following method:
[0160] i) in a structure of the Termamyl-like alpha-amylase in
question to identifying an amino acid residue within 15 .ANG. from
an active site residue, in particular 10 .ANG. from an active site
residue, which amino acid residue is contemplated to be involved in
electrostatic or hydrophobic interactions with an active site
residue,
[0161] ii) replacing, in the structure, said amino acid residue
with an amino acid residue which changes the electrostatic and/or
hydrophobic surroundings of an active site residue and evaluating
the accommodation of the amino acid residue in the structure,
[0162] iii) optionally repeating step i) and/or ii) until an amino
acid replacement has been identified which is accommodated into the
structure,
[0163] iv) constructing a Termamyl-like alpha-amylase variant
resulting from steps i), ii) and optionally iii) and testing the pH
dependent enzymatic activity of interest of said variant.
[0164] In the above method it may be of particular relevance to add
a positively charged residue within 5 .ANG. of a glutamate (thereby
lowering the pKa of the glutamate from about 4.5 to 4), or to add a
negatively charged residue within 5 .ANG. of a glutamate (thereby
increasing the pKa to about 5), or to make similar modifications
within a distance of about 5 .ANG. of a histidine.
[0165] On the basis of electrostatic considerations [see, e.g.,
Gilson, 1995, Current Opinion in Structural Biology 5: 216-223; and
Honig and Nicholls, 1995, Science 268: 1144-1149; and references
given therein] and hygroscopicity considerations in relation to the
three dimensional structure of a Termamyl-like alpha-amylase
disclosed herein, mutations of relevance, inter alia, for changing
(increasing or decreasing) the pH optimum of a Termamyl-like
alpha-amylase are believed to include the following mutations or
equivalents thereof [referring here to the sequence of B.
licheniformis alpha-amylase (SEQ ID NO: 2)]:
Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T;
I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E;
L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E;
L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E;
L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E;
V312R,K,D,E; F323H; D325N; N326K,H,D,L; H327Q,N,E,D,F; Q330L,E;
G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E;
T337D,K; T338D,E; T339D; Q360K,R,E; D365N; G371D,R.
[0166] In a further aspect the invention relates to a variant of a
Termamyl-like alpha-amylase which exhibits a higher activity at a
lower pH (e.g., compared to the pH optimum) than the parent
alpha-amylase. In particular, the variant comprises a mutation of
an amino acid residue corresponding to at least one of the
following positions of the B. licheniformis alpha-amylase (SEQ ID
NO: 2):
V102, I103, L196, A232, I236, P331, Q333, E336.
[0167] The following mutations are of particular interest:
V102R,K,A,T,S,G;
I103K,R;
L196K,R;
A232T,S,G;
I236K,R,N;
P331R,K
Q333R,K
E336R,K
[0168] or any combination of two or more of these variants or any
combination of one or more of these variants with any of the other
variants disclosed herein.
[0169] In a still further aspect the invention relates to a variant
of a Termamyl-like alpha-amylase which has a higher activity at a
higher pH than the parent alpha-amylase. In particular, the variant
comprises a mutation of an amino acid residue corresponding to at
least one of the following positions of the B. licheniformis
alpha-amylase (SEQ ID NO: 2):
Y273, H281, N326
[0170] In particular, the variant comprises a mutation
corresponding to at least one of the following mutations of the B.
licheniformis alpha-amylase (SEQ ID NO: 2):
Y273F,W
H281F,I,L
N326I,Y,F,L,V
[0171] or any combination of two or more of these variants or any
combination of one or more of these variants with any of the other
variants disclosed herein.
[0172] A mutation which appears to be importance in relation to the
specific activity of variants of the invention is a mutation
corresponding to the substitution S187D in B. licheniformis
alpha-amylase (SEQ ID NO: 2).
Variants with Increased Thermostability and/or Altered Temperature
Optimum
[0173] In a further desired aspect the invention relates to a
variant of a parent Termamyl-like alpha-amylase, which variant is
the result of one or more amino acid residues having been deleted
from, replaced or added to the parent alpha-amylase so as to obtain
an increased thermostability of the variant.
[0174] The Termamyl-like alpha-amylase structure contains a number
of unique internal holes, which may contain water, and a number of
crevices. In order to increase the thermostability of the
alpha-amylase it may be desirable to reduce the number of holes and
crevices (or reduce the size of the holes or crevices), e.g., by
introducing one or more hydrophobic contacts, preferably achieved
by introducing bulkier residues, in the vicinity or surroundings of
the hole. For instance, the amino acid residues to be modified are
those which are involved in the formation of the hole.
[0175] Accordingly, in a further aspect the present invention
relates to a method of increasing the thermostability and/or
altering the temperature optimum of a parent Termamyl-like
alpha-amylase, which method comprises
[0176] i) identifying an internal hole or a crevice of the parent
Termamyl-like alpha-amylase in the three-dimensional structure of
said alpha-amylase,
[0177] ii) replacing, in the structure, one or more amino acid
residues in the neighborhood of the hole or crevice identified in
i) with another amino acid residue which from structural or
functional considerations is believed to increase the hydrophobic
interaction and to fill out or reduce the size of the hole or
crevice,
[0178] iii) constructing a Termamyl-like alpha-amylase variant
resulting from step ii) and testing the thermostability and/or
temperature optimum of the variant.
[0179] The structure used for identifying the hole or crevice of
the parent Termamyl-like alpha-amylase may be the structure
identified in Appendix 1 or a model structure of the parent
Termamyl-like alpha-amylase built thereon.
[0180] It will be understood that the hole or crevice is identified
by the amino acid residues surrounding the hole/crevice, and that
modification of said amino acid residues are of importance for
filling or reducing the size of the hole/crevice. The particular
amino acid residues referred to below are those which in crystal
structure have been found to flank the hole/crevice in
question.
[0181] In order to fill (completely or partly) a major hole located
between domain A and B, mutation to any other amino acid residue of
an amino acid residue corresponding to one or more of the following
residues of the B. licheniformis alpha-amylase (SEQ ID NO: 2) is
contemplated:
L61, Y62, F67, K106, F143, G145, R146, Y150, S151, I212, R214.
[0182] Of particular interest is a mutation to a more bulky amino
acid residue than the amino acid residue of the parent enzyme.
[0183] Of particular interest is a variant of a Termamyl-like
alpha-amylase which comprises a mutation corresponding to the
following mutations (using the numbering of B. licheniformis
alpha-amylase (SEQ ID NO: 2):
L61W,V,F;
Y62W;
F67W;
K106R,F,W;
F143W;
G145F,W
R146W;
Y150R,K;
[0184] S151 replaced with any other amino acid residue and in
particular with F,W,I or L; I212F,L,W,Y,R,K; and/or
R214W.
[0185] In order to fill a hole in the vicinity of the active site
mutation to any other amino acid residue of an amino acid residue
corresponding to one or more of the following residues of the B.
licheniformis alpha-amylase (SEQ ID NO: 2) is contemplated:
I236,
L241.
[0186] Of interest is a mutation to a more bulky amino acid
residue.
[0187] Of particular interest is a variant of a Termamyl-like
alpha-amylase which comprises a mutation corresponding to one or
more of the following mutations in the B. licheniformis
alpha-amylase:
I236L,F,W,Y; and/or
L241I,F,Y,W.
[0188] In order to fill a hole in the vicinity of the active site
mutation to any other amino acid residue of an amino acid residue
corresponding to one or more of the following residues of the B.
licheniformis alpha-amylase (SEQ ID NO: 2) is contemplated:
L7, V259, F284
[0189] Of interest is a mutation to a more bulky amino acid
residue.
[0190] Of particular interest is a variant of a Termamyl-like
alpha-amylase which comprises a mutation corresponding to one or
more of the following mutations in the B. licheniformis
alpha-amylase:
L7F,I,W
V259F,I,L
F284W.
[0191] In order to fill a hole in the vicinity of the active site
mutation to any other amino acid residue of an amino acid residue
corresponding to one or more of the following residues of the B.
licheniformis alpha-amylase (SEQ ID NO: 2) is contemplated:
F343, F350.
[0192] Of interest is a mutation to a more bulky amino acid
residue.
[0193] Of particular interest is a variant of a Termamyl-like
alpha-amylase which comprises a mutation corresponding to one or
more of the following mutations in the B. licheniformis
alpha-amylase:
F343W
F350W.
[0194] In order to fill a hole in the vicinity of the active site
mutation to any other amino acid residue of an amino acid residue
corresponding to one or more of the following residues of the B.
licheniformis alpha-amylase (SEQ ID NO: 2) is contemplated:
L427, V481
[0195] Of interest is a mutation to a more bulky amino acid
residue.
[0196] Of particular interest is a variant of a Termamyl-like
alpha-amylase which comprises a mutation corresponding to one or
more of the following mutations in the B. licheniformis
alpha-amylase:
L427F,L,W
V481,F,I,L,W.
[0197] It can be seen from an alignment of the amino acid sequences
of alpha-amylases from various Bacillus species that B.
licheniformis alpha-amylase and B. amyloliquefaciens alpha-amylase
both contain an "insertion" of three amino acids relative to, e.g.,
B. stearothermophilus alpha-amylase.
[0198] From a model of the structure of B. licheniformis
alpha-amylase built on the basis of the three-dimensional structure
of the Termamyl-like alpha-amylase disclosed herein (vide supra),
taking into account the homology of B. licheniformis alpha-amylase
to the Termamyl-like alpha-amylase in question, it can be seen that
the above-mentioned "insertion" lies within loop 8 (vide supra),
making this loop bulkier in B. licheniformis alpha-amylase than in
the Termamyl-like alpha-amylase and resulting in a loop that
protrudes from the structure, thereby possibly destabilizing the
structure. It is therefore contemplated that deletion of one or
more amino acids in the region in question in B. licheniformis or
B. amyloliquefaciens alpha-amylase will improve the thermostability
of these alpha-amylases.
[0199] Especially interesting in this connection is deletion of
three amino acids within the partial sequence from T369 to I377
(referring to the sequence of B. licheniformis alpha-amylase),
i.e., the partial sequence:
T369-K370-G371-D372-5373-Q374-R375-E376-I377 (or the corresponding
partial sequence in B. amyloliquefaciens alpha-amylase). In
addition to such deletions, substitution of one or more of the
undeleted amino acids within the latter partial sequence may also
be advantageous.
[0200] Preferable deletions of three amino acids in the partial
sequence from T369 to 1377 (in B. licheniformis alpha-amylase) are
deletion of K370+G371+D372 (i.e., K370*+G371*+D372*) or deletion of
D372+S373+Q374 (i.e., D372*+S373*+Q374*) (or equivalent deletions
in the corresponding partial sequence in B. amyloliquefaciens
alpha-amylase).
[0201] Another type of mutation which would appear to be of value
in improving the thermostability of these alpha-amylases is
substitution (replacement) of the entire partial amino acid
sequence from T369 to I377 (referring to the sequence of B.
licheniformis alpha-amylase) with one of the following partial
sequences of six amino acids (sequence numbering increasing from
left to right): I-P-T-H-S-V; I-P-T-H-G-V; and 1-P-Q-Y-N-I (or one
of the same substitutions of the corresponding partial sequence in
B. amyloliquefaciens alpha-amylase).
Variants with an Altered Cleavage Pattern
[0202] In the starch liquefaction process it is desirable to use an
alpha-amylase which is capable of degrading the starch molecules
into long branched oligo saccharides (like, e.g., the Fungamyl-like
alpha-amylases) rather than shorter branched oligo saccharides
(like conventional Termamyl-like alpha-amylases). The resulting
very small branched oligosaccharides (panose precursors) cannot be
hydrolyzed properly by pullulanases, which in the liquefaction
process are used after the alpha-amylases and before the
amyloglucosidases. Thus, in the presence of panose precursors the
action of amylo-glucoamylase ends up with a high degree of the
small branched limiting-dextrin, the trisaccharide panose. The
presence of panose lowers the saccharification yield significantly
and is thus undesirable.
[0203] Thus, one aim of the present invention is to change the
degradation characteristics of a Termamyl-like alpha-amylase to
that of a Fungamyl-like alpha-amylase without at the same time
reducing the thermostability of the Termamyl-like
alpha-amylase.
[0204] Accordingly, in a further aspect the invention relates to a
variant of a Termamyl-like alpha-amylase which has a reduced
ability to cleave a substrate close to the branching point.
[0205] The variant may suitably be constructed by a method which
comprises
[0206] i) identifying the substrate binding area of the parent
Termamyl-like alpha-amylase in a model of the three-dimensional
structure of said alpha-amylase, (e.g., within a sphere of 4 .ANG.
from the substrate binding site (as defined in the section above
entitled "Substrate Binding Site"),
[0207] ii) replacing, in the model, one or more amino acid residues
of the substrate binding area of the cleft identified in i), which
is/are believed to be responsible for the cleavage pattern of the
parent alpha-amylase, with another amino acid residue which from
structural considerations is believed to result in an altered
substrate cleavage pattern, or deleting one or more amino acid
residues of the substrate binding area contemplated to introduce
favorable interactions to the substrate or adding one or more amino
acid residues to the substrate binding area contemplated to
introduce favorable interactions to the substrate, and
[0208] iii) constructing a Termamyl-like alpha-amylase variant
resulting from step ii) and testing the substrate cleavage pattern
of the variant.
[0209] Of particular interest is a variant which cleaves an
amylopectin substrate, from the reducing end, more than one glucose
unit from the branching point, preferably more than two or three
glucose units from the branching point, i.e., at a further distance
from the branching point than that obtained by use of a wild type
B. licheniformis alpha-amylase.
[0210] Residues of particular interest in connection with this
aspect of the invention correspond to the following residues of the
B. licheniformis alpha-amylase (SEQ ID NO: 2): D53, V54, Y56, G57,
Q333, and the variants according to this aspect preferably
comprises a mutation in one or more of these residues.
[0211] In particular, the variant comprises at least one of the
following mutations, which are expected to prevent cleavage close
to the branching point:
A52 amino acid residues larger than A, e.g., A52W,Y,L,F,I
D53L,I,F,Y,W,
V54L,I,F,Y,W,R,K,H,E,Q,
Y56W,
[0212] G57 all possible amino acid residues,
Q333W.
Variants of a Fungal Alpha-Amylase
[0213] In a still further embodiment the invention relates to a
variant of a parent Fungamyl-like alpha-amylase, in which variant
at least one of the amino acid residues of the parent
alpha-amylase, which is/are present in an amino acid fragment
corresponding to amino acid residues 291-313 of the amino acid
sequence of SEQ ID NO: 10, has/have been deleted or replaced with
one or more of the amino acid residues, which is/are present in an
amino acid fragment corresponding to amino acid residues 98-210 of
the amino acid sequence shown in SEQ ID NO: 4, or in which one or
more additional amino acid residues has/have been inserted using
the relevant part of SEQ ID NO: 4 or a corresponding part of
another Termamyl-like alpha-amylase as a template.
[0214] For instance, the variant may be one in which the amino acid
fragment X-Y of the parent alpha-amylase, which corresponds to or
is within the amino acid fragment 117-185 of SEQ ID NO: 10,
has/have been replaced with an amino acid fragment Z-V, which
corresponds to or is within the amino acid fragment 98-210 of the
amino acid sequence shown in SEQ ID NO: 4, in which variant
[0215] X is an amino acid residue corresponding to the amino acid
occupying position 117, 118, 119, 120 or 121 of SEQ ID NO: 10,
[0216] Y is an amino acid residue corresponding to the amino acid
occupying position 181, 182, 183, 184 or 185 of SEQ ID NO: 10,
[0217] Z is an amino acid residue corresponding to the amino acid
occupying position 98, 99, 100, 101 or 102 of SEQ ID NO: 4, and
[0218] V is an amino acid residue corresponding to the amino acid
occupying position 206, 207, 208, 209 or 210 of SEQ ID NO: 4.
[0219] A specific example of a variant according to this aspect of
the invention is one in which the amino acid fragment of the parent
alpha-amylase, which corresponds to amino acid residues 121-181 of
SEQ ID NO: 10, has been replaced with the amino acid fragment
corresponding to amino acid residues 102-206 of the amino acid
sequence shown in SEQ ID NO: 4.
[0220] Another example of a variant according to this aspect of the
invention is one in which the amino acid fragment of the parent
alpha-amylase, which corresponds to amino acid residues 121-174 of
SEQ ID NO: 10, has been replaced with the amino acid fragment
corresponding to amino acid residues 102-199 of the amino acid
sequence shown in SEQ ID NO: 4.
[0221] In a further embodiment the invention relates to a variant
of a parent Fungamyl-like alpha-amylase, in which an amino acid
fragment corresponding to amino acid residues 181-184 of the amino
acid sequence shown in SEQ ID NO: 10 has been deleted.
General Mutations in Variants of the Invention
[0222] It may be preferred that the variant of the invention or
prepared in accordance with the method of the invention comprises
one or more modifications in addition to those outlined above.
Thus, it may be advantageous that one or more proline residues
present in the part of the alpha-amylase variant having been
modified is/are replaced with a non-proline residue which may be
any of the possible, naturally occurring non-proline residues, and
which preferably is an alanine, glycine, serine, threonine, valine
or leucine.
[0223] Analogously, it may be preferred that one or more cysteine
residues present in the amino acid residues with which the parent
alpha-amylase is modified are replaced with a non-cysteine residues
such as serine, alanine, threonine, glycine, valine or leucine.
[0224] Furthermore, the variant of the invention may, as the only
modification or in combination with any of the above outlined
modifications, be modified so that one or more Asp and/or Glu
present in an amino acid fragment corresponding to the amino acid
sequence fragment 185-209 of SEQ ID NO: 2 is/are replaced by an Asn
and/or Gln, respectively. Also of interest is the replacement of
one or more of the Lys residues present in the Termamyl-like
alpha-amylase by an Arg residue.
[0225] It will be understood that in accordance with the present
invention variants may be prepared which carry two or more of the
above outlined modifications. For instance, variants may be
prepared which comprises a modification in the loop 1 and loop 2
region, a modification in loop 2 and limited loop 3, a modification
in loop 1, loop 2, loop 3 and loop 8, etc.
[0226] Furthermore, it may be advantageous to introduce
point-mutations in any of the variants described herein.
Methods of Preparing Alpha-Amylase Variants
[0227] Several methods for introducing mutations into genes are
known in the art. After a brief discussion of the cloning of
alpha-amylase-encoding DNA sequences, methods for generating
mutations at specific sites within the alpha-amylase-encoding
sequence will be discussed.
Cloning a DNA Sequence Encoding an Alpha-Amylase
[0228] The DNA sequence encoding a parent alpha-amylase may be
isolated from any cell or microorganism producing the alpha-amylase
in question, using various methods well known in the art. First, a
genomic DNA and/or cDNA library should be constructed using
chromosomal DNA or messenger RNA from the organism that produces
the alpha-amylase to be studied. Then, if the amino acid sequence
of the alpha-amylase is known, homologous, labelled oligonucleotide
probes may be synthesized and used to identify
alpha-amylase-encoding clones from a genomic library prepared from
the organism in question. Alternatively, a labelled oligonucleotide
probe containing sequences homologous to a known alpha-amylase gene
could be used as a probe to identify alpha-amylase-encoding clones,
using hybridization and washing conditions of lower stringency.
[0229] Yet another method for identifying alpha-amylase-encoding
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming
alpha-amylase-negative bacteria with the resulting genomic DNA
library, and then plating the transformed bacteria onto agar
containing a substrate for alpha-amylase, thereby allowing clones
expressing the alpha-amylase to be identified.
[0230] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g., the
phosphoroamidite method described by Beaucage and Caruthers (1981)
or the method described by Matthes et al. (1984). In the
phosphoroamidite method, oligonucleotides are synthesized, e.g., in
an automatic DNA synthesizer, purified, annealed, ligated and
cloned in appropriate vectors.
[0231] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or Saiki et al.
(1988).
Site-Directed Mutagenesis
[0232] Once an alpha-amylase-encoding DNA sequence has been
isolated, and desirable sites for mutation identified, mutations
may be introduced using synthetic oligonucleotides. These
oligonucleotides contain nucleotide sequences flanking the desired
mutation sites; mutant nucleotides are inserted during
oligonucleotide synthesis. In a specific method, a single-stranded
gap of DNA, bridging the alpha-amylase-encoding sequence, is
created in a vector carrying the alpha-amylase gene. Then the
synthetic nucleotide, bearing the desired mutation, is annealed to
a homologous portion of the single-stranded DNA. The remaining gap
is then filled in with DNA polymerase I (Klenow fragment) and the
construct is ligated using T4 ligase. A specific example of this
method is described in Morinaga et al. (1984). U.S. Pat. No.
4,760,025 discloses the introduction of oligonucleotides encoding
multiple mutations by performing minor alterations of the cassette.
However, an even greater variety of mutations can be introduced at
any one time by the Morinaga method, because a multitude of
oligonucleotides, of various lengths, can be introduced.
[0233] Another method of introducing mutations into
alpha-amylase-encoding DNA sequences is described in Nelson and
Long (1989). It involves the 3-step generation of a PCR fragment
containing the desired mutation introduced by using a chemically
synthesized DNA strand as one of the primers in the PCR reactions.
From the PCR-generated fragment, a DNA fragment carrying the
mutation may be isolated by cleavage with restriction endonucleases
and reinserted into an expression plasmid.
Random Mutagenesis
[0234] Random mutagenesis is suitably performed either as localized
or region-specific random mutagenesis in at least three parts of
the gene translating to the amino acid sequence shown in question,
or within the whole gene.
[0235] For region-specific random mutagenesis with a view to
improving the thermal stability of a parent Termamyl-like
alpha-amylase, codon positions corresponding to the following amino
acid residues of the B. licheniformis alpha-amylase (SEQ ID NO: 2)
may appropriately be targeted:
To Improve the Stability of the Calcium Site Between Domain A and
C
T297-L308
F403-V409
I428-A435
To Improve the Stability Between Domain A and B:
H156-T163
D180-D204
A232-F238
[0236] With a view to achieving improved binding of a substrate
(i.e., improved binding of a carbohydrate species, such as amylose
or amylopectin) by a Termamyl-like alpha-amylase variant, modified
(e.g., higher) substrate specificity and/or modified (e.g., higher)
specificity with respect to cleavage (hydrolysis) of substrate, it
appears that the following codon positions for the amino acid
sequence shown in SEQ ID NO: 2 (or equivalent codon positions for
another parent Termamyl-like alpha-amylase in the context of the
invention) may particularly appropriately be targeted:
13-18 50-56 70-76 102-109 163-172 189-199 229-235 327-335
360-364
[0237] The random mutagenesis of a DNA sequence encoding a parent
alpha-amylase to be performed in accordance with step a) of the
above-described method of the invention may conveniently be
performed by use of any method known in the art.
[0238] 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.
[0239] The mutagenizing agent may, e.g., be one which induces
transitions, transversions, inversions, scrambling, deletions,
and/or insertions.
[0240] 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.
[0241] 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.
[0242] 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 amylolytic enzyme by any
published technique, using, e.g., PCR, LCR or any DNA polymerase
and ligase.
[0243] When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent alpha-amylase enzyme
is subjected to PCR under conditions that increase the
misincorporation of nucleotides (Deshler, 1992; Leung et al., 1989,
Technique 1: 11-15).
[0244] A mutator strain of E. coli (Fowler et al., 1974, Molec.
Gen. Genet. 133: 179-191), S. cereviseae or any other microbial
organism may be used for the random mutagenesis of the DNA encoding
the amylolytic enzyme by, e.g., transforming a plasmid containing
the parent enzyme into the mutator strain, growing the mutator
strain with the plasmid and isolating the mutated plasmid from the
mutator strain. The mutated plasmid may subsequently be transformed
into the expression organism.
[0245] The DNA sequence to be mutagenized may conveniently be
present in a genomic or cDNA library prepared from an organism
expressing the parent amylolytic enzyme. Alternatively, the DNA
sequence may be present on a suitable vector such as a plasmid or a
bacteriophage, which as such may be incubated with or otherwise
exposed to the mutagenizing agent. The DNA to be mutagenized may
also be present in a host cell either by being integrated in the
genome of said cell or by being present on a vector harboured in
the cell. Finally, the DNA to be mutagenized may be in isolated
form. It will be understood that the DNA sequence to be subjected
to random mutagenesis is preferably a cDNA or a genomic DNA
sequence.
[0246] In some cases it may be convenient to amplify the mutated
DNA sequence prior to the expression step (b) or the screening step
(c) being performed. Such amplification may be performed in
accordance with methods known in the art, the presently preferred
method being PCR-generated amplification using oligonucleotide
primers prepared on the basis of the DNA or amino acid sequence of
the parent enzyme.
[0247] Subsequent to the incubation with or exposure to the
mutagenizing agent, the mutated DNA is expressed by culturing a
suitable host cell carrying the DNA sequence under conditions
allowing expression to take place. The host cell used for this
purpose may be one which has been transformed with the mutated DNA
sequence, optionally present on a vector, or one which was carried
the DNA sequence encoding the parent enzyme during the mutagenesis
treatment. Examples of suitable host cells are the following:
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.
[0248] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
Localized random mutagenesis: the random mutagenesis may
advantageously be localized to a part of the parent alpha-amylase
in question. This may, e.g., be advantageous when certain regions
of the enzyme have been identified to be of particular importance
for a given property of the enzyme, and when modified are expected
to result in a variant having improved properties. Such regions may
normally be identified when the tertiary structure of the parent
enzyme has been elucidated and related to the function of the
enzyme.
[0249] The localized random mutagenesis is conveniently performed
by use of PCR-generated mutagenesis techniques as described above
or any other suitable technique known in the art.
[0250] Alternatively, the DNA sequence encoding the part of the DNA
sequence to be modified may be isolated, e.g., by being inserted
into a suitable vector, and said part may subsequently be subjected
to mutagenesis by use of any of the mutagenesis methods discussed
above.
[0251] With respect to the screening step in the above-mentioned
method of the invention, this may conveniently performed by use of
a filter assay based on the following principle:
[0252] A microorganism capable of expressing the mutated amylolytic
enzyme of interest is incubated on a suitable medium and under
suitable conditions for the enzyme to be secreted, the medium being
provided with a double filter comprising a first protein-binding
filter and on top of that a second filter exhibiting a low protein
binding capability. The microorganism is located on the second
filter. Subsequent to the incubation, the first filter comprising
enzymes secreted from the microorganisms is separated from the
second filter comprising the microorganisms. The first filter is
subjected to screening for the desired enzymatic activity and the
corresponding microbial colonies present on the second filter are
identified.
[0253] The filter used for binding the enzymatic activity may be
any protein binding filter, e.g., nylon or nitrocellulose. The
topfilter carrying the colonies of the expression organism may be
any filter that has no or low affinity for binding proteins, e.g.,
cellulose acetate or Durapore.TM.. The filter may be pretreated
with any of the conditions to be used for screening or may be
treated during the detection of enzymatic activity.
[0254] 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.
[0255] 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.
[0256] Alpha-amylase activity is detected by Cibacron Red labelled
amylopectin, which is immobilized on agarose. For screening for
variants with increased thermal and high-pH stability, the filter
with bound alpha-amylase variants is incubated in a buffer at pH
10.5 and 60 or 65.degree. C. for a specified time, rinsed briefly
in deionized water and placed on the amylopectin-agarose matrix for
activity detection. Residual activity is seen as lysis of Cibacron
Red by amylopectin degradation. The conditions are chosen to be
such that activity due to the alpha-amylase having the amino acid
sequence shown in SEQ ID NO: 1 can barely be detected. Stabilized
variants show, under the same conditions, increased colour
intensity due to increased liberation of Cibacron Red.
[0257] For screening for variants with an activity optimum at a
lower temperature and/or over a broader temperature range, the
filter with bound variants is placed directly on the
amylopectin-Cibacron Red substrate plate and incubated at the
desired temperature (e.g., 4.degree. C., 10.degree. C. or
30.degree. C.) for a specified time. After this time activity due
to the alpha-amylase having the amino acid sequence shown in SEQ ID
NO: 1 can barely be detected, whereas variants with optimum
activity at a lower temperature will show increase amylopectin
lysis. Prior to incubation onto the amylopectin matrix, incubation
in all kinds of desired media--e.g., solutions containing
Ca.sup.2+, detergents, EDTA or other relevant additives--can be
carried out in order to screen for changed dependency or for
reaction of the variants in question with such additives.
Testing of Variants of the Invention
[0258] The testing of variants of the invention may suitably be
performed by determining the starch-degrading activity of the
variant, for instance by growing host cells transformed with a DNA
sequence encoding a variant on a starch-containing agarose plate
and identifying starch-degrading host cells. Further testing as to
altered properties (including specific activity, substrate
specificity, cleavage pattern, thermoactivation, pH optimum, pH
dependency, temperature optimum, and any other parameter) may be
performed in accordance with methods known in the art.
Expression of Alpha-Amylase Variants
[0259] According to the invention, a DNA sequence encoding the
variant produced by methods described above, or by any alternative
methods known in the art, can be expressed, in enzyme form, using
an expression vector which typically includes control sequences
encoding a promoter, operator, ribosome binding site, translation
initiation signal, and, optionally, a repressor gene or various
activator genes.
[0260] The recombinant expression vector carrying the DNA sequence
encoding an alpha-amylase variant of the invention may be any
vector which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e., a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid, a bacteriophage or an
extrachromosomal element, minichromosome or an artificial
chromosome. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0261] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA sequence
encoding an alpha-amylase variant of the invention, especially in a
bacterial host, are the promoter of the lac operon of E. coli, the
Streptomyces coelicolor agarase gene dagA promoters, the promoters
of the Bacillus licheniformis alpha-amylase gene (amyL), the
promoters of the Bacillus stearothermophilus maltogenic amylase
gene (amyM), the promoters of the Bacillus amyloliquefaciens
alpha-amylase (amyQ), the promoters of the Bacillus subtilis xyIA
and xyIB genes etc. For transcription in a fungal host, examples of
useful promoters are those derived from the gene encoding A. oryzae
TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger
neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger
glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline
protease, A. oryzae triose phosphate isomerase or A. nidulans
acetamidase.
[0262] The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes,
polyadenylation sequences operably connected to the DNA sequence
encoding the alpha-amylase variant of the invention. Termination
and polyadenylation sequences may suitably be derived from the same
sources as the promoter.
[0263] 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.
[0264] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the host cell,
such as the dal genes from B. subtilis or B. licheniformis, or one
which confers antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracyclin resistance. Furthermore, the vector
may comprise Aspergillus selection markers such as amdS, argB, niaD
and sC, a marker giving rise to hygromycin resistance, or the
selection may be accomplished by co-transformation, e.g., as
described in WO 91/17243.
[0265] 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.
[0266] The procedures used to ligate the DNA construct of the
invention encoding an alpha-amylase variant, the promoter,
terminator and other elements, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al. (1989)).
[0267] The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant production of
an alpha-amylase variant of the invention. The cell may be
transformed with the DNA construct of the invention encoding the
variant, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is generally
considered to be an advantage as the DNA sequence is more likely to
be stably maintained in the cell. Integration of the DNA constructs
into the host chromosome may be performed according to conventional
methods, e.g., by homologous or heterologous recombination.
Alternatively, the cell may be transformed with an expression
vector as described above in connection with the different types of
host cells.
[0268] 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.
[0269] 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.
[0270] The yeast organism may favorably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces
cerevisiae. The filamentous fungus may advantageously belong to a
species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus
niger. Fungal cells may be transformed by a process involving
protoplast formation and transformation of the protoplasts followed
by regeneration of the cell wall in a manner known per se. A
suitable procedure for transformation of Aspergillus host cells is
described in EP 238 023.
[0271] In a yet further aspect, the present invention relates to a
method of producing an alpha-amylase variant of the invention,
which method comprises cultivating a host cell as described above
under conditions conducive to the production of the variant and
recovering the variant from the cells and/or culture medium.
[0272] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of the alpha-amylase variant of the
invention. Suitable media are available from commercial suppliers
or may be prepared according to published recipes (e.g., as
described in catalogues of the American Type Culture
Collection).
[0273] The alpha-amylase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such as
ion exchange chromatography, affinity chromatography, or the
like.
INDUSTRIAL APPLICATIONS
[0274] The alpha-amylase variants of this invention possess
valuable properties allowing for various industrial applications.
In particular the enzyme variants finds potential applications as a
component in washing, dishwashing and hard surface cleaning
detergent compositions, but it may also be useful in the production
of sweeteners and ethanol from starch and for textile desizing.
Conditions for conventional starch converting processes and
liquefaction and/or saccharification processes are described in for
instance U.S. Pat. No. 3,912,590 and EP patent publications Nos.
252,730 and 63,909.
Production of sweeteners from starch: A "traditional" process for
conversion of starch to fructose syrups normally consists of three
consecutive enzymatic processes, viz. a liquefaction process
followed by a saccharification process and an isomerization
process. During the liquefaction process, starch is degraded to
dextrins by an alpha-amylase (e.g., Termamyl.TM.) at pH values
between 5.5 and 6.2 and at temperatures of 95-160.degree. C. for a
period of approximately 2 hours. In order to ensure an optimal
enzyme stability under these conditions, 1 mM of calcium is added
(40 ppm free calcium ions).
[0275] After the liquefaction process the dextrins are converted
into dextrose by addition of a glucoamylase (e.g., AMG.TM.) and a
debranching enzyme, such as an isoamylase or a pullulanase (e.g.,
Promozyme.TM.). Before this step the pH is reduced to a value below
4.5, maintaining the high temperature (above 95.degree. C.), and
the liquefying alpha-amylase activity is denatured. The temperature
is lowered to 60.degree. C., and glucoamylase and debranching
enzyme are added. The saccharification process proceeds for 24-72
hours.
[0276] After the saccharification process the pH is increased to a
value in the range of 6-8, preferably pH 7.5, and the calcium is
removed by ion exchange. The dextrose syrup is then converted into
high fructose syrup using, e.g., an immobilized glucose isomerase
(such as Sweetzyme.TM.)
[0277] At least 3 enzymatic improvements of this process could be
obtained. All three improvements could be seen as individual
benefits, but any combination (e.g., 1+2, 1+3, 2+3 or 1+2+3) could
be employed:
Improvement 1. Reduction of the Calcium Dependency of the
Liquefying Alpha-Amylase.
[0278] Addition of free calcium is required to ensure adequately
high stability of the alpha-amylase, but free calcium strongly
inhibits the activity of the glucose isomerase and needs to be
removed, by means of an expensive unit operation, to an extent
which reduces the level of free calcium to below 3-5 ppm. Cost
savings could be obtained if such an operation could be avoided and
the liquefaction process could be performed without addition of
free calcium ions.
[0279] To achieve that, a less calcium-dependent Termamyl-like
alpha-amylase which is stable and highly active at low
concentrations of free calcium (<40 ppm) is required. Such a
Termamyl-like alpha-amylase should have a pH optimum at a pH in the
range of 4.5-6.5, preferably in the range of 4.5-5.5.
Improvement 2. Reduction of Formation of Unwanted Maillard
Products
[0280] The extent of formation of unwanted Maillard products during
the liquefaction process is dependent on the pH. Low pH favors
reduced formation of Maillard products. It would thus be desirable
to be able to lower the process pH from around pH 6.0 to a value
around pH 4.5; unfortunately, all commonly known, thermostable
Termamyl-like alpha-amylases are not very stable at low pH (i.e.,
pH<6.0) and their specific activity is generally low.
[0281] Achievement of the above-mentioned goal requires a
Termamyl-like alpha-amylase which is stable at low pH in the range
of 4.5-5.5 and at free calcium concentrations in the range of 0-40
ppm, and which maintains a high specific activity.
Improvement 3.
[0282] It has been reported previously (U.S. Pat. No. 5,234,823)
that when saccharifying with A. niger glucoamylase and B.
acidopullulyticus pullulanase, the presence of residual
alpha-amylase activity from the liquefaction process can lead to
lower yields of dextrose if the alpha-amylase is not inactivated
before the saccharification stage. This inactivation can typically
be carried out by adjusting the pH to below 4.3 at 95.degree. C.,
before lowering the temperature to 60.degree. C. for
saccharification.
[0283] The reason for this negative effect on dextrose yield is not
fully understood, but it is assumed that the liquefying
alpha-amylase (for example Termamyl.TM. 120 L from B.
licheniformis) generates "limit dextrins" (which are poor
substrates for B. acidopullulyticus pullulanase) by hydrolyzing
1,4-alpha-glucosidic linkages close to and on both sides of the
branching points in amylopectin. Hydrolysis of these limit dextrins
by glucoamylase leads to a build-up of the trisaccharide panose,
which is only slowly hydrolyzed by glucoamylase.
[0284] The development of a thermostable alpha-amylase which does
not suffer from this disadvantage would be a significant process
improvement, as no separate inactivation step would be
required.
[0285] If a Termamyl-like, low-pH-stable alpha-amylase is
developed, an alteration of the specificity could be an advantage
needed in combination with increased stability at low pH.
[0286] The methodology and principles of the present invention make
it possible to design and produce variants according to the
invention having required properties as outlined above. In this
connection, mutations in a Termamyl-like alpha-amylase [for example
Termamyl itself (B. licheniformis alpha-amylase; SEQ ID NO: 2); or
a Termamyl-like alpha-amylase having an N-terminal amino acid
sequence (i.e., the partial sequence up to the amino acid position
corresponding to position 35 in Termamyl) which is identical to
that in B. amyloliquefaciens alpha-amylase (SEQ ID NO: 4), i.e., a
Termamyl-like alpha-amylase having the following N-terminal
sequence relative to amino acid sequence of Termamyl:
A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H351, where an
asterisk (*) indicates deletion of the amino acid residue in
question] at positions corresponding to any of the following
positions in Termamyl are particularly interesting:
V54
N104
V128
H133
H156
A181
S187
A209
G310
H293
A294
H450
[0287] (where each of the latter amino acid residues may be
replaced by any other amino acid residue, i.e., any other residue
chosen among A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W,
Y and V), as well as the following triple deletions:
K370*+G371*+D372*
D372*+S373*+Q374*
[0288] Particularly preferred substitutions at the above-indicated
positions are the following:
V54W,Y,F,I,L
N104D
V128E
H133I
H156Y
A181T
S187D
A209V
H293Y
A294V
G310D
H450Y.
[0289] Any combination of one or more (i.e., one, two, three, four
etc.) of the above indicated mutations may appropriately be
effected in a Termamyl-like alpha-amylase in the context in
question, and particularly interesting variants of the invention in
the context of achieving one or more of the above-mentioned
improvements in relation to the starch liquefaction behavior of
alpha-amylases include variants comprising combinations of multiple
mutations corresponding to the following combinations of mutations
in Termamyl itself:
V54F+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G37-
1*+D372*+H450Y;
V54F+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S37-
3*+Q374*+H450Y;
V541+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G37-
1*+D372*+H450Y;
V541+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S37-
3*+Q374*+H450Y;
V54L+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G37-
1*+D372*+H450Y;
V54L+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S37-
3*+Q374*+H450Y;
V54W+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G37-
1*+D372*+H450Y;
V54W+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S37-
3*+Q374*+H450Y;
V54Y+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+K370*+G37-
1*+D372*+H450Y;
V54Y+N104D+V128E+H133I+H156Y+A181T+S187D+A209V+H293Y+A294V+G310D+D372*+S37-
3*+Q374*+H450Y.
Detergent Compositions
[0290] According to the invention, the alpha-amylase may typically
be a component of a detergent composition. As such, it may be
included in the detergent composition in the form of a non-dusting
granulate, a stabilized liquid, or a protected enzyme. Non-dusting
granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.
4,106,991 and 4,661,452 (both to Novo Industri A/S) and may
optionally be coated by methods known in the art. Examples of waxy
coating materials are poly(ethylene oxide) products
(polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000,
ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;
ethoxylated fatty alcohols in which the alcohol contains from 12 to
20 carbon atoms and in which there are 15 to 80 ethylene oxide
units; fatty alcohols; fatty acids; and mono- and di- and
triglycerides of fatty acids. Examples of film-forming coating
materials suitable for application by fluid bed techniques are
given in patent GB 1483591. Liquid enzyme preparations may, for
instance, be stabilized by adding a polyol such as propylene
glycol, a sugar or sugar alcohol, lactic acid or boric acid
according to established methods. Other enzyme stabilizers are well
known in the art. Protected enzymes may be prepared according to
the method disclosed in EP 238,216.
[0291] The detergent composition of the invention may be in any
convenient form, e.g., as powder, granules, paste or liquid. A
liquid detergent may be aqueous, typically containing up to 70% of
water and 0-30% of organic solvent, or nonaqueous.
[0292] The detergent composition comprises one or more surfactants,
each of which may be anionic, nonionic, cationic, or zwitterionic.
The detergent will usually contain 0-50% of anionic surfactant such
as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS),
alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate
(AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty
acid methyl esters, alkyl- or alkenylsuccinic acid or soap. It may
also contain 0-40% of nonionic surfactant such as alcohol
ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,
nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide
(e.g., as described in WO 92/06154).
[0293] The detergent composition may additionally comprise one or
more other enzymes, such as lipase, cutinase, protease, cellulase,
peroxidase, e.g., laccase.
[0294] The detergent may contain 1-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, citrate, nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTMPA), alkyl- or
alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,
SKS-6 from Hoechst). The detergent may also be unbuilt, i.e.,
essentially free of detergent builder.
[0295] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),
polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA),
polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers and lauryl methacrylate/acrylic acid copolymers.
[0296] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate
(NOBS). Alternatively, the bleaching system may comprise peroxy
acids of, e.g., the amide, imide, or sulfone type.
[0297] The enzymes of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative as,
e.g., an aromatic borate ester, and the composition may be
formulated as described in, e.g., WO 92/19709 and WO 92/19708.
[0298] The detergent may also contain other conventional detergent
ingredients such as, e.g., fabric conditioners including clays,
foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil redeposition agents, dyes,
bactericides, optical brighteners, or perfume.
[0299] The pH (measured in aqueous solution at use concentration)
will usually be neutral or alkaline, e.g., 7-11.
[0300] Particular forms of detergent compositions within the scope
of the invention include:
1) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00001 Linear alkylbenzenesulfonate (calculated as acid)
7-12% Alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 EO) or
1-4% alkyl sulfate (e.g., C.sub.16-18) Alcohol ethoxylate (e.g.,
C.sub.14-15 alcohol, 7 EO) 5-9% Sodium carbonate (as
Na.sub.2CO.sub.3) 14-20% Soluble silicate (as Na.sub.2O,
2SiO.sub.2) 2-6% Zeolite (as NaAlSiO.sub.4) 15-22% Sodium sulfate
(as Na.sub.2SO.sub.4) 0-6% Sodium citrate/citric acid (as
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) 0-15% Sodium
perborate (as NaBO.sub.3.cndot.H.sub.2O) 11-18% TAED 2-6%
Carboxymethylcellulose 0-2% Polymers (e.g., maleic/acrylic acid
copolymer, PVP, 0-3% PEG) Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., suds suppressors,
perfume, 0-5% optical brightener, photobleach)
2) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00002 Linear alkylbenzenesulfonate (calculated as acid)
6-11% Alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 EO 1-3%
or alkyl sulfate (e.g., C.sub.16-18) Alcohol ethoxylate (e.g.,
C.sub.14-15 alcohol, 7 EO) 5-9% Sodium carbonate (as
Na.sub.2CO.sub.3) 15-21% Soluble silicate (as Na.sub.2O,
2SiO.sub.2) 1-4% Zeolite (as NaA1SiO.sub.4) 24-34% Sodium sulfate
(as Na.sub.2SO.sub.4) 4-10% Sodium citrate/citric acid (as
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) 0-15%
Carboxymethylcellulose 0-2% Polymers (e.g., maleic/acrylic acid
copolymer, 1-6% PVP, PEG) Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., suds suppressors,
perfume) 0-5%
3) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00003 Linear alkylbenzenesulfonate (calculated as acid)
5-9% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) 7-14%
Soap as fatty acid (e.g., C.sub.16-22 fatty acid) 1-3% Sodium
carbonate (as Na.sub.2CO.sub.3) 10-17% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 3-9% Zeolite (as NaAlSiO.sub.4) 23-33%
Sodium sulfate (as Na.sub.2SO.sub.4) 0-4% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 8-16% TAED 2-8% Phosphonate (e.g.,
EDTMPA) 0-1% Carboxymethylcellulose 0-2% Polymers (e.g.,
maleic/acrylic acid copolymer, 0-3% PVP, PEG) Enzymes (calculated
as pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., suds
suppressors, perfume, 0-5% optical brightener)
4) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00004 Linear alkylbenzenesulfonate (calculated as acid)
8-12% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) 10-25%
Sodium carbonate (as Na.sub.2CO.sub.3) 14-22% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 1-5% Zeolite (as NaAlSiO.sub.4) 25-35%
Sodium sulfate (as Na.sub.2SO.sub.4) 0-10% Carboxymethylcellulose
0-2% Polymers (e.g., maleic/acrylic acid copolymer, 1-3% PVP, PEG)
Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., suds suppressors, perfume) 0-5%
5) An aqueous liquid detergent composition comprising
TABLE-US-00005 Linear alkylbenzenesulfonate (calculated as acid)
15-21% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO or
12-18% C.sub.12-15 alcohol, 5 EO) Soap as fatty acid (e.g., oleic
acid) 3-13% Alkenylsuccinic acid (C.sub.12-14) 0-13% Aminoethanol
8-18% Citric acid 2-8% Phosphonate 0-3% Polymers (e.g., PVP, PEG)
0-3% Borate (as B.sub.4O.sub.7) 0-2% Ethanol 0-3% Propylene glycol
8-14% Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., dispersants, suds suppressors, 0-5% perfume,
optical brightener)
6) An aqueous structured liquid detergent composition
comprising
TABLE-US-00006 Linear alkylbenzenesulfonate (calculated as acid)
15-21% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, 3-9% or
C.sub.12-15alcohol, 5 EO) Soap as fatty acid (e.g., oleic acid)
3-10% Zeolite (as NaAlSiO.sub.4) 14-22% Potassium citrate 9-18%
Borate (as B.sub.4O.sub.7) 0-2% Carboxymethylcellulose 0-2%
Polymers (e.g., PEG, PVP) 0-3% Anchoring polymers such as, e.g.,
lauryl methacrylate/ 0-3% acrylic acid copolymer; molar ratio 25:1;
MW 3800 Glycerol 0-5% Enzymes (calculated as pure enzyme protein)
0.0001-0.1% Minor ingredients (e.g., dispersants, suds suppressors,
0-5% perfume, optical brighteners)
7) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00007 Fatty alcohol sulfate 5-10% Ethoxylated fatty acid
monoethanolamide 3-9% Soap as fatty acid 0-3% Sodium carbonate (as
Na.sub.2CO.sub.3) 5-10% Soluble silicate (as Na.sub.2O, 2SiO.sub.2)
1-4% Zeolite (as NaAlSiO.sub.4) 20-40% Sodium sulfate (as
Na.sub.2SO.sub.4) 2-8% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 12-18% TAED 2-7% Polymers (e.g.,
maleic/acrylic acid copolymer, PEG) 1-5% Enzymes (calculated as
pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., optical
brightener, suds 0-5% suppressors, perfume)
8) A detergent composition formulated as a granulate comprising
TABLE-US-00008 Linear alkylbenzenesulfonate (calculated as acid)
8-14% Ethoxylated fatty acid monoethanolamide 5-11% Soap as fatty
acid 0-3% Sodium carbonate (as Na.sub.2CO.sub.3) 4-10% Soluble
silicate (as Na.sub.2O, 2SiO.sub.2) 1-4% Zeolite (as NaAlSiO.sub.4)
30-50% Sodium sulfate (as Na.sub.2SO.sub.4) 3-11% Sodium citrate
(as C.sub.6H.sub.5Na.sub.3O.sub.7) 5-12% Polymers (e.g., PVP,
maleic/acrylic acid copolymer, 1-5% PEG) Enzymes (calculated as
pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., suds
suppressors, perfume) 0-5%
9) A detergent composition formulated as a granulate comprising
TABLE-US-00009 Linear alkylbenzenesulfonate (calculated as acid)
6-12% Nonionic surfactant 1-4% Soap as fatty acid 2-6% Sodium
carbonate (as Na.sub.2CO.sub.3) 14-22% Zeolite (as NaAlSiO.sub.4)
18-32% Sodium sulfate (as Na.sub.2SO.sub.4) 5-20% Sodium citrate
(as C.sub.6H.sub.5Na.sub.3O.sub.7) 3-8% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 4-9% Bleach activator (e.g., NOBS or
TAED) 1-5% Carboxymethylcellulose 0-2% Polymers (e.g.,
polycarboxylate or PEG) 1-5% Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., optical brightener,
perfume) 0-5%
10) An aqueous liquid detergent composition comprising
TABLE-US-00010 Linear alkylbenzenesulfonate (calculated as acid)
15-23% Alcohol ethoxysulfate (e.g., C.sub.12-15 alcohol, 2-3 EO)
8-15% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or 3-9%
C.sub.12-15alcohol, 5 EO) Soap as fatty acid (e.g., lauric acid)
0-3% Aminoethanol 1-5% Sodium citrate 5-10% Hydrotrope (e.g.,
sodium toluensulfonate) 2-6% Borate (as B.sub.4O.sub.7) 0-2%
Carboxymethylcellulose 0-1% Ethanol 1-3% Propylene glycol 2-5%
Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., polymers, dispersants, 0-5% perfume, optical
brighteners)
11) An aqueous liquid detergent composition comprising
TABLE-US-00011 Linear alkylbenzenesulfonate (calculated as acid)
20-32% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or
6-12% C.sub.12-15 alcohol, 5 EO) Aminoethanol 2-6% Citric acid
8-14% Borate (as B.sub.4O.sub.7) 1-3% Polymer (e.g., maleic/acrylic
acid copolymer, 0-3% anchoring polymer such as, e.g., lauryl
methacrylate/ acrylic acid copolymer) Glycerol 3-8% Enzymes
(calculated as pure enzyme protein) 0.0001-0.1% Minor ingredients
(e.g., hydrotropes, dispersants, 0-5% perfume, optical
brighteners)
12) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00012 Anionic surfactant (linear alkylbenzenesulfonate,
alkyl 25-40% sulfate, alpha-olefinsulfonate, alpha-sulfo fatty acid
methyl esters, alkanesulfonates, soap) Nonionic surfactant (e.g.,
alcohol ethoxylate) 1-10% Sodium carbonate (as Na.sub.2CO.sub.3)
8-25% Soluble silicates (as Na.sub.2O, 2SiO.sub.2) 5-15% Sodium
sulfate (as Na.sub.2SO.sub.4) 0-5% Zeolite (as NaAlSiO.sub.4)
15-28% Sodium perborate (as NaBO.sub.3.cndot.4H.sub.2O) 0-20%
Bleach activator (TAED or NOBS) 0-5% Enzymes (calculated as pure
enzyme protein) 0.0001-0.1% Minor ingredients (e.g., perfume,
optical brighteners) 0-3%
13) Detergent compositions as described in compositions 1-12
wherein all or part of the linear alkylbenzenesulfonate is replaced
by (C.sub.12-C.sub.18) alkyl sulfate. 14) A detergent composition
formulated as a granulate having a bulk density of at least 600 g/l
comprising
TABLE-US-00013 (C.sub.12-C.sub.18) alkyl sulfate 9-15% Alcohol
ethoxylate 3-6% Polyhydroxy alkyl fatty acid amide 1-5% Zeolite (as
NaAlSiO.sub.4) 10-20% Layered disilicate (e.g., SK56 from Hoechst)
10-20% Sodium carbonate (as Na.sub.2CO.sub.3) 3-12% Soluble
silicate (as Na.sub.2O, 2SiO.sub.2) 0-6% Sodium citrate 4-8% Sodium
percarbonate 13-22% TAED 3-8% Polymers (e.g., polycarboxylates and
PVP) 0-5% Enzymes (calculated as pure enzyme protein) 0.0001-0.1%
Minor ingredients (e.g., optical brightener, photo 0-5% bleach,
perfume, suds suppressors)
15) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00014 (C.sub.12-C.sub.18) alkyl sulfate 4-8% Alcohol
ethoxylate 11-15% Soap 1-4% Zeolite MAP or zeolite A 35-45% Sodium
carbonate (as Na.sub.2CO.sub.3) 2-8% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 0-4% Sodium percarbonate 13-22% TAED 1-8%
Carboxymethyl cellulose 0-3% Polymers (e.g., polycarboxylates and
PVP) 0-3% Enzymes (calculated as pure enzyme protein) 0.0001-0.1%
Minor ingredients (e.g., optical brightener, 0-3% phosphonate,
perfume)
16) Detergent formulations as described in 1)-15) which contain a
stabilized or encapsulated peracid, either as an additional
component or as a substitute for already specified bleach systems.
17) Detergent compositions as described in 1), 3), 7), 9) and 12)
wherein perborate is replaced by percarbonate. 18) Detergent
compositions as described in 1), 3), 7), 9), 12), 14) and 15) which
additionally contain a manganese catalyst. The manganese catalyst
may, e.g., be one of the compounds described in "Efficient
manganese catalysts for low-temperature bleaching", Nature 369:
637-639 (1994). 19) Detergent composition formulated as a
nonaqueous detergent liquid comprising a liquid nonionic surfactant
such as, e.g., linear alkoxylated primary alcohol, a builder system
(e.g., phosphate), enzyme and alkali. The detergent may also
comprise anionic surfactant and/or a bleach system.
[0301] The alpha-amylase variant of the invention may be
incorporated in concentrations conventionally employed in
detergents. It is at present contemplated that, in the detergent
composition of the invention, the alpha-amylase may be added in an
amount corresponding to 0.00001-1 mg (calculated as pure enzyme
protein) of alpha-amylase per liter of wash liquor.
Dishwashing Composition
[0302] The dishwashing detergent composition comprises a surfactant
which may be anionic, non-ionic, cationic, amphoteric or a mixture
of these types. The detergent will contain 0-90% of non-ionic
surfactant such as low- to non-foaming ethoxylated propoxylated
straight-chain alcohols.
[0303] The detergent composition may contain detergent builder
salts of inorganic and/or organic types. The detergent builders may
be subdivided into phosphorus-containing and
non-phosphorus-containing types. The detergent composition usually
contains 1-90% of detergent builders.
[0304] Examples of phosphorus-containing inorganic alkaline
detergent builders, when present, include the water-soluble salts
especially alkali metal pyrophosphates, orthophosphates, and
polyphosphates. An example of phosphorus-containing organic
alkaline detergent builder, when present, includes the
water-soluble salts of phosphonates. Examples of
non-phosphorus-containing inorganic builders, when present, include
water-soluble alkali metal carbonates, borates and silicates as
well as the various types of water-insoluble crystalline or
amorphous alumino silicates of which zeolites are the best-known
representatives.
[0305] Examples of suitable organic builders include the alkali
metal, ammonium and substituted ammonium, citrates, succinates,
malonates, fatty acid sulphonates, carboxymetoxy succinates,
ammonium polyacetates, carboxylates, polycarboxylates,
aminopolycarboxylates, polyacetyl carboxylates and
polyhydroxsulphonates.
[0306] Other suitable organic builders include the higher molecular
weight polymers and co-polymers known to have builder properties,
for example appropriate polyacrylic acid, polymaleic and
polyacrylic/polymaleic acid copolymers and their salts.
[0307] The dishwashing detergent composition may contain bleaching
agents of the chlorine/bromine-type or the oxygen-type. Examples of
inorganic chlorine/bromine-type bleaches are lithium, sodium or
calcium hypochlorite and hypobromite as well as chlorinated
trisodium phosphate. Examples of organic chlorine/bromine-type
bleaches are heterocyclic N-bromo and N-chloro imides such as
trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and
dichloroisocyanuric acids, and salts thereof with
water-solubilizing cations such as potassium and sodium. Hydantoin
compounds are also suitable.
[0308] The oxygen bleaches are preferred, for example in the form
of an inorganic persalt, preferably with a bleach precursor or as a
peroxy acid compound. Typical examples of suitable peroxy bleach
compounds are alkali metal perborates, both tetrahydrates and
monohydrates, alkali metal percarbonates, persilicates and
perphosphates. Preferred activator materials are TAED and glycerol
triacetate.
[0309] The dishwashing detergent composition of the invention may
be stabilized using conventional stabilizing agents for the
enzyme(s), e.g., a polyol such as, e.g., propylene glycol, a sugar
or a sugar alcohol, lactic acid, boric acid, or a boric acid
derivative, e.g., an aromatic borate ester.
[0310] The dishwashing detergent composition of the invention may
also contain other conventional detergent ingredients, e.g.,
deflocculant material, filler material, foam depressors,
anti-corrosion agents, soil-suspending agents, sequestering agents,
anti-soil redeposition agents, dehydrating agents, dyes,
bactericides, fluorescers, thickeners and perfumes.
[0311] Finally, the alpha-amylase variant of the invention may be
used in conventional dishwashing detergents, e.g., in any of the
detergents described in any of the following patent publications:
EP 518719, EP 518720, EP 518721, EP 516553, EP 516554, EP 516555,
GB 2200132, DE 3741617, DE 3727911, DE 4212166, DE 4137470, DE
3833047, WO 93/17089, DE 4205071, WO 52/09680, WO 93/18129, WO
93/04153, WO 92/06157, WO 92/08777, EP 429124, WO 93/21299, U.S.
Pat. No. 5,141,664, EP 561452, EP 561446, GB 2234980, WO 93/03129,
EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, U.S. Pat.
No. 5,112,518, EP 318204, EP 318279, EP 271155, EP 271156, EP
346136, GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197,
U.S. Pat. No. 5,240,632.
EXAMPLES
Example 1
Example on Homology Building of TERM
[0312] The overall homology of the B. licheniformis alpha-amylase
(in the following referred to as TERM) to other Termamyl-like
alpha-amylases is high and the percent similarity is extremely
high. The similarity calculated for TERM to BSG (the B.
stearothermophilus alpha-amylase with SEQ ID NO: 6), and BAN (the
B. amyloliquefaciens alpha-amylase with SEQ ID NO: 4) using the
University of Wisconsin Genetics Computer Group's program GCG gave
89% and 78%, respectively. TERM has a deletion of 2 residues
between residue G180 and K181 compared to BAN and BSG. BSG has a
deletion of 3 residues between G371 and I372 in comparison with BAN
and TERM. Further BSG has a C-terminal extension of more than 20
residues compared to BAN and TERM. BAN has 2 residues less and TERM
has one residue less in the N-terminal compared to BSG.
[0313] The structure of the B. licheniformis (TERM) and of the B.
amyloliquefaciens alpha-amylase (BAN), respectively, was model
built on the structure disclosed in Appendix 1 herein. The
structure of other Termamyl-like alpha-amylases (e.g., those
disclosed herein) may be built analogously.
[0314] In comparison with the alpha-amylase used for elucidating
the present structure, TERM differs in that it lacks two residues
around 178-182. In order to compensate for this in the model
structure, the HOMOLOGY program from BIOSYM was used to substitute
the residues in equivalent positions in the structure (not only
structurally conserved regions) except for the deletion point. A
peptide bond was established between G179(G177) and K180(K180) in
TERM(BAN). The close structural relationship between the solved
structure and the model structure (and thus the validity of the
latter) is indicated by the presence of only very few atoms found
to be too close together in the model.
[0315] To this very rough structure of TERM was then added all
waters (605) and ions (4 calcium and 1 sodium) from the solved
structure (Appendix 1) at the same coordinates as for said solved
structure using the INSIGHT program. This could be done with only
few overlaps--in other words with a very nice fit. This model
structure were then minimized using 200 steps of Steepest descent
and 600 steps of Conjugated gradient (see Brooks et al., 1983, J.
Computational Chemistry 4: 187-217). The minimized structure was
then subjected to molecular dynamics, 5 ps heating followed by up
to 200 ps equilibration but more than 35 ps. The dynamics as run
with the Verlet algorithm and the equilibration temperature 300K
were kept using the Behrendsen coupling to a waterbath (Berendsen
et. al., 1984, J. Chemical Physics 81: 3684-3690). Rotations and
translations were removed every picosecond. The potential energy
became stable after approximately 35 ps equilibration. A mean
dynamics structure was extracted and can be used for further
analysis.
Example 2
Determination of Residues within 10 .ANG. from the Ions Present in
the Solved Structure
[0316] The coordinates of Appendix 1 are read into the INSIGHT
program provided by BIOSYM technologies. The spatial coordinates
are presented showing the bonds between the atoms. The ions are
presented as well as the water atoms. The program package part of
creating subset is used to create a 10 .ANG. subset around the
calcium and sodium ions in the structure using the command ZONE.
All residues having an atom within the 10 .ANG. are compiled and
written out by the LIST MOLECULE command. By giving the ions the
name ium in the coordinate file a 10 .ANG. sphere around all atoms
called ium is compiled. The specific residues identified in this
manner are given further above in the section entitled "Ca.sup.2+
dependency".
Example 3
Determination of Cavities in the Solved Structure (Appendix 1)
[0317] The solved structure exhibits many internal holes and
cavities. When analyzing for such cavities the Connolly program is
normally used (Lee and Richards, 1971, J. Mol. Biol. 55: 379-400).
The program uses a probe with radius to search the external and
internal surface of the protein. The smallest hole observable in
this way has the probe radius.
[0318] To analyze the solved structure a modified version of the
Connolly program included in the program of INSIGHT were used.
First the water molecules and the ions were removed by unmerging
these atoms from the solved structure. By using the command
MOLECULE SURFACE SOLVENT the solvent accessible surface area were
calculated for all atoms and residues using a probe radius of 1.4
.ANG., and displayed on the graphics screen together with the model
of the solved structure. The internal cavities where then seen as
dot surfaces with no connections to external surface.
[0319] Mutant suggestions for filling out the holes are given in
the specification (in the section entitled "Variants with increased
thermostability and/or altered temperature optimum"). By using the
homology build structures or/and the sequence alignment mutations
for the homologous structures of TERM and BSG and BAN can be
made.
Example 4
Construction of Termamyl.TM. Variants in Accordance with the
Invention
[0320] Termamyl (SEQ ID NO: 2) is expressed in B. subtilis from a
plasmid denoted pDN1528. This plasmid contains the complete gene
encoding Termamyl, amyL, the expression of which is directed by its
own promoter. Further, the plasmid contains the origin of
replication, ori, from plasmid pUB110 and the cat gene from plasmid
pC194 conferring resistance towards chloramphenicol. pDN1528 is
shown in FIG. 9.
[0321] A specific mutagenesis vector containing a major part of the
coding region of SEQ ID NO: 1 was prepared. The important features
of this vector, denoted pJeEN1, include an origin of replication
derived from the pUC plasmids, the cat gene conferring resistance
towards chloramphenicol, and a frameshift-containing version of the
bla gene, the wild type of which normally confers resistance
towards ampicillin (amp.sup.R phenotype). This mutated version
results in an amp.sup.S phenotype. The plasmid pJeEN1 is shown in
FIG. 10, and the E. coli origin of replication, on, bla, cat, the
5'-truncated version of the Termamyl amylase gene, and selected
restriction sites are indicated on the plasmid.
[0322] Mutations are introduced in amyL by the method described by
Deng and Nickoloff (1992, Anal. Biochem. 200: 81-88) except that
plasmids with the "selection primer" (primer #6616; see below)
incorporated are selected based on the amp.sup.R phenotype of
transformed E. coli cells harboring a plasmid with a repaired bla
gene, instead of employing the selection by restriction enzyme
digestion outlined by Deng and Nickoloff. Chemicals and enzymes
used for the mutagenesis were obtained from the Chameleon.TM.
mutagenesis kit from Stratagene (catalogue number 200509).
[0323] After verification of the DNA sequence in variant plasmids,
the truncated gene, containing the desired alteration, is subcloned
into pDN1528 as a PstI-EcoRI fragment and transformed into a
protease- and amylase-depleted Bacillus subtilis strain in order to
express the variant enzyme.
[0324] The Termamyl variant V54W was constructed by the use of the
following mutagenesis primer (written 5' to 3', left to right):
TABLE-US-00015 PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG
[0325] The Termamyl variant A52W+V54W was constructed by the use of
the following mutagenesis primer (written 5' to 3', left to
right):
TABLE-US-00016 PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT
CG
[0326] Primer #6616 (written 5' to 3', left to right; P denotes a
5' phosphate):
TABLE-US-00017 P CTG TGA CTG GTG AGT ACT CAA CCA AGT C
Example 5
Saccharification in the Presence of "Residual" Alpha-Amylase
Activity
[0327] Two appropriate Termamyl variants with altered specificity
were evaluated by saccharifying a DE 10 (DE=dextrose equivalent)
maltodextrin substrate with A. niger glucoamylase and B.
acidopullulyticus pullulanase under conditions where the variant
amylase was active.
Saccharification: Substrates for saccharification were prepared by
dissolving 230 g DE 10 spray-dried maltodextrin, prepared from
common corn starch, in 460 ml boiling deionized water and adjusting
the dry substance (DS) content to approximately 30% w/w. The pH was
adjusted to 4.7 (measured at 60.degree. C.) and aliquots of
substrate corresponding to 15 g dry weight were transferred to 50
ml blue cap glass flasks.
[0328] The flasks were then placed in a shaking water bath
equilibrated at 60.degree. C., and the enzymes added. The pH was
readjusted to 4.7 where necessary.
[0329] The following enzymes were used:
Glucoamylase: AMG.TM. (Novo Nordisk A/S); dosage 0.18 AG/g DS
Pullulanase: Promozyme.TM. (Novo Nordisk A/S);
[0330] dosage 0.06 PUN/g DS Alpha-amylases: Termamyl.TM. (Novo
Nordisk A/S); dosage 60 NU/g DS [0331] Termamyl variant V54W;
dosage 60 NU/g DS [0332] Termamyl variant A52W+V54W; dosage 60 NU/g
DS
[0333] 2 ml samples were taken periodically. The pH of each sample
was adjusted to about 3.0, and the sample was then heated in a
boiling water bath for 15 minutes to inactivate the enzymes. After
cooling, the samples were treated with approximately 0.1 g
mixed-bed ion exchange resin (BIO-Rad 501-X (D)) for 30 minutes on
a rotary mixer and then filtered. The carbohydrate composition of
each sample was determined by HPLC. The following results were
obtained after 72 hours [DP.sub.n denotes a dextrose (D-glucose)
oligomer with n glucose units]:
TABLE-US-00018 alpha-amylase % DP.sub.1 % DP.sub.2 % DP.sub.3 %
DP.sub.4 None (control) 95.9 2.8 0.4 1.0 A52W + V54W 95.9 2.8 0.4
0.8 V54W 96.0 2.9 0.4 0.8 Termamyl .TM. 95.6 2.8 0.8 0.8
[0334] It can be seen from the above results that compared with the
control (no alpha-amylase activity present during liquefaction),
the presence of alpha-amylase activity from variants V54W and
A52W+V54W did not lead to elevated panose (DP.sub.3) levels. In
contrast, Termamyl alpha-amylase activity resulted in higher levels
of panose and a subsequent loss of D-glucose (DP.sub.1) yield.
[0335] Thus, if alpha-amylase variant A52W+V54W or V54W is used for
starch liquefaction, it will not be necessary to inactivate the
residual alpha-amylase activity before the commencement of
saccharification.
Example 6
Calcium-Binding Affinity of Alpha-Amylase Variants of the
Invention
[0336] Unfolding of amylases by exposure to heat or to denaturants
such as guanidine hydrochloride is accompanied by a decrease in
fluorescence. Loss of calcium ions leads to unfolding, and the
affinity of alpha-amylases for calcium can be measured by
fluorescence measurements before and after incubation of each
alpha-amylase (e.g., at a concentration of 10 micrograms/ml) in a
buffer (e.g., 50 mM HEPES, pH 7) with different concentrations of
calcium (e.g., in the range of 1 .mu.M-100 mM) or of EGTA (e.g., in
the range of 1-1000 .mu.M)
[EGTA=1,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid] for a
sufficiently long period of time (such as 22 hours at 55.degree.
C.).
[0337] The measured fluorescence F is composed of contributions
form the folded and unfolded forms of the enzyme. The following
equation can be derived to describe the dependence of F on calcium
concentration ([Ca]):
F=[Ca]/(K.sub.diss+[Ca])(.alpha..sub.N-.beta..sub.N
log([Ca]))+K.sub.diss/(K.sub.diss+[Ca])(.alpha..sub.U-.beta..sub.U
log([Ca]))
where .alpha..sub.N is the fluorescence of the native (folded) form
of the enzyme, .beta..sub.N is the linear dependence of
.alpha..sub.N on the logarithm of the calcium concentration (as
observed experimentally), .alpha..sub.U is the fluorescence of the
unfolded form and .beta..sub.U is the linear dependence of
.alpha..sub.U on the logarithm of the calcium concentration.
K.sub.diss is the apparent calcium-binding constant for an
equilibrium process as follows:
[0338] K.sub.diss
N-CaU+Ca (N=native enzyme; U=unfolded enzyme)
[0339] In fact, unfolding proceeds extremely slowly and is
irreversible. The rate of unfolding is a dependent on calcium
concentration, and the dependency for a given alpha-amylase
provides a measure of the Ca-binding affinity of the enzyme. By
defining a standard set of reaction conditions (e.g., 22 hours at
55.degree. C.), a meaningful comparison of K.sub.diss for different
alpha-amylases can be made. The calcium dissociation curves for
alpha-amylases in general can be fitted to the equation above,
allowing determination of the corresponding values of
K.sub.diss.
[0340] The following values for K.sub.diss were obtained for a
parent Termamyl-like alpha-amylase having the amino acid sequence
shown in SEQ ID NO: 1 of WO 95/26397 and for the indicated variant
thereof according to the invention:
TABLE-US-00019 Alpha-Amylase K.sub.diss (mol/l) T183* + G184* +
L351C + M430C 1.7 (.+-.0.5) .times. 10.sup.-3 Parent 3.5 (.+-.1.1)
.times. 10.sup.-1
[0341] It is apparent from the above that the calcium-binding
affinity of the variant in question binds calcium significantly
more strongly than the parent, and thereby has a correspondingly
lower calcium dependency than the parent.
REFERENCES CITED
[0342] Beaucage and Caruthers, 1981, Tetrahedron Letters 22:
1859-1869. [0343] Boel et al., 1990, Biochemistry 29: 6244-6249.
[0344] Brady et al., Acta Crystallogr. sect. B, 47: 527-535. [0345]
Chang et al., 1993, J. Mol. Biol. 229: 235-238. [0346] Diderichsen
and Christiansen, 1988, "Cloning of a maltogenic alpha-amylase from
Bacillus stearothermophilus", FEMS Microbiol. Letters 56: 53-60.
[0347] Dubnau et al., 1971, J. Mol. Biol. 56: 209-221. [0348]
Erlich, 1977, Proc. Natl. Acad. Sci. 74: 1680-1682. [0349] Gryczan
et al., 1978, J. Bacteriol. 134: 318-329. [0350] Higuchi et al.,
1988, "A general method of in vitro preparation and specific
mutagenesis of DNA fragments: study of protein and DNA
interactions", Nucl. Acids Res. 16: 7351-7367. [0351] Hudson et
al., Practical Immunology, Third edition (1989), Blackwell
Scientific Publications, Hunkapiller et al., 1984, Nature 310:
105-111. [0352] Kadziola, Ph.D. Thesis: "An alpha-amylase from
Barley and its Complex with a Substrate Analogue Inhibitor Studied
by X-ray Crystallography", Department of Chemistry University of
Copenhagen (1993). [0353] Klein et al., 1992, Biochemistry 31:
8740-8746 [0354] Larson, 1994, J. Mol. Biol. 235: 1560-1584. [0355]
Lawson, 1994, J. Mol. Biol. 236: 590-600. [0356] MacGregor, 1987,
Food Hydrocolloids 1(5-6): [0357] Matthes et al., 1984, The EMBO J.
3: 801-805. [0358] Mizuno et al., 1993, J. Mol. Biol. 234:
1282-1283. [0359] Morinaga et al., 1984, Biotechnology 2: 646-639.
[0360] Nelson and Long, 1989, Analytical Biochemistry 180: 147-151.
[0361] Qian et al., 1993, J. Mol. Biol. 231: 785-799. [0362] Saiki
et al., 1988, Science 239: 487-491. [0363] Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
(1989). [0364] Swift et al., Acta Crystallogr. sect. B, 47:
535-544.
Sequence CWU 1
1
1311920DNABacillus
licheniformisCDS(334)..(1869)sig_peptide(334)..(420)mat_peptide(421)..(18-
69) 1cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg
agccatgcgg 60gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat
gctgctgaag 120agattattaa aaagctgaaa gcaaaaggct atcaattggt
aactgtatct cagcttgaag 180aagtgaagaa gcagagaggc tattgaataa
atgagtagaa gcgccatatc ggcgcttttc 240ttttggaaga aaatataggg
aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300tcatatgttt
cacattgaaa ggggaggaga atc atg aaa caa caa aaa cgg ctt 354 Met Lys
Gln Gln Lys Arg Leu -25 tac gcc cga ttg ctg acg ctg tta ttt gcg ctc
atc ttc ttg ctg cct 402Tyr Ala Arg Leu Leu Thr Leu Leu Phe Ala Leu
Ile Phe Leu Leu Pro -20 -15 -10 cat tct gca gca gcg gcg gca aat ctt
aat ggg acg ctg atg cag tat 450His Ser Ala Ala Ala Ala Ala Asn Leu
Asn Gly Thr Leu Met Gln Tyr -5 -1 1 5 10 ttt gaa tgg tac atg ccc
aat gac ggc caa cat tgg agg cgt ttg caa 498Phe Glu Trp Tyr Met Pro
Asn Asp Gly Gln His Trp Arg Arg Leu Gln 15 20 25 aac gac tcg gca
tat ttg gct gaa cac ggt att act gcc gtc tgg att 546Asn Asp Ser Ala
Tyr Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile 30 35 40 ccc ccg
gca tat aag gga acg agc caa gcg gat gtg ggc tac ggt gct 594Pro Pro
Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala 45 50 55
tac gac ctt tat gat tta ggg gag ttt cat caa aaa ggg acg gtt cgg
642Tyr Asp Leu Tyr Asp Leu Gly Glu Phe His Gln Lys Gly Thr Val Arg
60 65 70 aca aag tac ggc aca aaa gga gag ctg caa tct gcg atc aaa
agt ctt 690Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gln Ser Ala Ile Lys
Ser Leu 75 80 85 90 cat tcc cgc gac att aac gtt tac ggg gat gtg gtc
atc aac cac aaa 738His Ser Arg Asp Ile Asn Val Tyr Gly Asp Val Val
Ile Asn His Lys 95 100 105 ggc ggc gct gat gcg acc gaa gat gta acc
gcg gtt gaa gtc gat ccc 786Gly Gly Ala Asp Ala Thr Glu Asp Val Thr
Ala Val Glu Val Asp Pro 110 115 120 gct gac cgc aac cgc gta att tca
gga gaa cac cta att aaa gcc tgg 834Ala Asp Arg Asn Arg Val Ile Ser
Gly Glu His Leu Ile Lys Ala Trp 125 130 135 aca cat ttt cat ttt ccg
ggg cgc ggc agc aca tac agc gat ttt aaa 882Thr His Phe His Phe Pro
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys 140 145 150 tgg cat tgg tac
cat ttt gac gga acc gat tgg gac gag tcc cga aag 930Trp His Trp Tyr
His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys 155 160 165 170 ctg
aac cgc atc tat aag ttt caa gga aag gct tgg gat tgg gaa gtt 978Leu
Asn Arg Ile Tyr Lys Phe Gln Gly Lys Ala Trp Asp Trp Glu Val 175 180
185 tcc aat gaa aac ggc aac tat gat tat ttg atg tat gcc gac atc gat
1026Ser Asn Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp
190 195 200 tat gac cat cct gat gtc gca gca gaa att aag aga tgg ggc
act tgg 1074Tyr Asp His Pro Asp Val Ala Ala Glu Ile Lys Arg Trp Gly
Thr Trp 205 210 215 tat gcc aat gaa ctg caa ttg gac ggt ttc cgt ctt
gat gct gtc aaa 1122Tyr Ala Asn Glu Leu Gln Leu Asp Gly Phe Arg Leu
Asp Ala Val Lys 220 225 230 cac att aaa ttt tct ttt ttg cgg gat tgg
gtt aat cat gtc agg gaa 1170His Ile Lys Phe Ser Phe Leu Arg Asp Trp
Val Asn His Val Arg Glu 235 240 245 250 aaa acg ggg aag gaa atg ttt
acg gta gct gaa tat tgg cag aat gac 1218Lys Thr Gly Lys Glu Met Phe
Thr Val Ala Glu Tyr Trp Gln Asn Asp 255 260 265 ttg ggc gcg ctg gaa
aac tat ttg aac aaa aca aat ttt aat cat tca 1266Leu Gly Ala Leu Glu
Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser 270 275 280 gtg ttt gac
gtg ccg ctt cat tat cag ttc cat gct gca tcg aca cag 1314Val Phe Asp
Val Pro Leu His Tyr Gln Phe His Ala Ala Ser Thr Gln 285 290 295 gga
ggc ggc tat gat atg agg aaa ttg ctg aac ggt acg gtc gtt tcc 1362Gly
Gly Gly Tyr Asp Met Arg Lys Leu Leu Asn Gly Thr Val Val Ser 300 305
310 aag cat ccg ttg aaa tcg gtt aca ttt gtc gat aac cat gat aca cag
1410Lys His Pro Leu Lys Ser Val Thr Phe Val Asp Asn His Asp Thr Gln
315 320 325 330 ccg ggg caa tcg ctt gag tcg act gtc caa aca tgg ttt
aag ccg ctt 1458Pro Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe
Lys Pro Leu 335 340 345 gct tac gct ttt att ctc aca agg gaa tct gga
tac cct cag gtt ttc 1506Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly
Tyr Pro Gln Val Phe 350 355 360 tac ggg gat atg tac ggg acg aaa gga
gac tcc cag cgc gaa att cct 1554Tyr Gly Asp Met Tyr Gly Thr Lys Gly
Asp Ser Gln Arg Glu Ile Pro 365 370 375 gcc ttg aaa cac aaa att gaa
ccg atc tta aaa gcg aga aaa cag tat 1602Ala Leu Lys His Lys Ile Glu
Pro Ile Leu Lys Ala Arg Lys Gln Tyr 380 385 390 gcg tac gga gca cag
cat gat tat ttc gac cac cat gac att gtc ggc 1650Ala Tyr Gly Ala Gln
His Asp Tyr Phe Asp His His Asp Ile Val Gly 395 400 405 410 tgg aca
agg gaa ggc gac agc tcg gtt gca aat tca ggt ttg gcg gca 1698Trp Thr
Arg Glu Gly Asp Ser Ser Val Ala Asn Ser Gly Leu Ala Ala 415 420 425
tta ata aca gac gga ccc ggt ggg gca aag cga atg tat gtc ggc cgg
1746Leu Ile Thr Asp Gly Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg
430 435 440 caa aac gcc ggt gag aca tgg cat gac att acc gga aac cgt
tcg gag 1794Gln Asn Ala Gly Glu Thr Trp His Asp Ile Thr Gly Asn Arg
Ser Glu 445 450 455 ccg gtt gtc atc aat tcg gaa ggc tgg gga gag ttt
cac gta aac ggc 1842Pro Val Val Ile Asn Ser Glu Gly Trp Gly Glu Phe
His Val Asn Gly 460 465 470 ggg tcg gtt tca att tat gtt caa aga
tagaagagca gagaggacgg 1889Gly Ser Val Ser Ile Tyr Val Gln Arg 475
480 atttcctgaa ggaaatccgt ttttttattt t 19202512PRTBacillus
licheniformis 2Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr
Leu Leu Phe -25 -20 -15 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala
Ala Ala Ala Asn Leu -10 -5 -1 1 Asn Gly Thr Leu Met Gln Tyr Phe Glu
Trp Tyr Met Pro Asn Asp Gly 5 10 15 Gln His Trp Arg Arg Leu Gln Asn
Asp Ser Ala Tyr Leu Ala Glu His 20 25 30 35 Gly Ile Thr Ala Val Trp
Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln 40 45 50 Ala Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe 55 60 65 His Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu 70 75 80
Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr Gly 85
90 95 Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp
Val 100 105 110 115 Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg
Val Ile Ser Gly 120 125 130 Glu His Leu Ile Lys Ala Trp Thr His Phe
His Phe Pro Gly Arg Gly 135 140 145 Ser Thr Tyr Ser Asp Phe Lys Trp
His Trp Tyr His Phe Asp Gly Thr 150 155 160 Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile Tyr Lys Phe Gln Gly 165 170 175 Lys Ala Trp Asp
Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp Tyr 180 185 190 195 Leu
Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala Ala Glu 200 205
210 Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp Gly
215 220 225 Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu
Arg Asp 230 235 240 Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu
Met Phe Thr Val 245 250 255 Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala
Leu Glu Asn Tyr Leu Asn 260 265 270 275 Lys Thr Asn Phe Asn His Ser
Val Phe Asp Val Pro Leu His Tyr Gln 280 285 290 Phe His Ala Ala Ser
Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys Leu 295 300 305 Leu Asn Gly
Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr Phe 310 315 320 Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val 325 330
335 Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu
340 345 350 355 Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
Thr Lys Gly 360 365 370 Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His
Lys Ile Glu Pro Ile 375 380 385 Leu Lys Ala Arg Lys Gln Tyr Ala Tyr
Gly Ala Gln His Asp Tyr Phe 390 395 400 Asp His His Asp Ile Val Gly
Trp Thr Arg Glu Gly Asp Ser Ser Val 405 410 415 Ala Asn Ser Gly Leu
Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ala 420 425 430 435 Lys Arg
Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His Asp 440 445 450
Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly Trp 455
460 465 Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln
Arg 470 475 480 32084DNABacillus
amyloliquefaciensCDS(250)..(1791)sig_peptide(250)..(342)mat_peptide(343).-
.(1791) 3gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact
gatgatttgg 60ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac
cttgtctgtc 120atcagacagg gtatttttta tgctgtccag actgtccgct
gtgtaaaaat aaggaataaa 180ggggggttgt tattatttta ctgatatgta
aaatataatt tgtataagaa aatgagaggg 240agaggaaac atg att caa aaa cga
aag cgg aca gtt tcg ttc aga ctt gtg 291 Met Ile Gln Lys Arg Lys Arg
Thr Val Ser Phe Arg Leu Val -30 -25 -20 ctt atg tgc acg ctg tta ttt
gtc agt ttg ccg att aca aaa aca tca 339Leu Met Cys Thr Leu Leu Phe
Val Ser Leu Pro Ile Thr Lys Thr Ser -15 -10 -5 gcc gta aat ggc acg
ctg atg cag tat ttt gaa tgg tat acg ccg aac 387Ala Val Asn Gly Thr
Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn -1 1 5 10 15 gac ggc
cag cat tgg aaa cga ttg cag aat gat gcg gaa cat tta tcg 435Asp Gly
Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser 20 25 30
gat atc gga atc act gcc gtc tgg att cct ccc gca tac aaa gga ttg
483Asp Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu
35 40 45 agc caa tcc gat aac gga tac gga cct tat gat ttg tat gat
tta gga 531Ser Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp
Leu Gly 50 55 60 gaa ttc cag caa aaa ggg acg gtc aga acg aaa tac
ggc aca aaa tca 579Glu Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys Ser 65 70 75 gag ctt caa gat gcg atc ggc tca ctg cat
tcc cgg aac gtc caa gta 627Glu Leu Gln Asp Ala Ile Gly Ser Leu His
Ser Arg Asn Val Gln Val 80 85 90 95 tac gga gat gtg gtt ttg aat cat
aag gct ggt gct gat gca aca gaa 675Tyr Gly Asp Val Val Leu Asn His
Lys Ala Gly Ala Asp Ala Thr Glu 100 105 110 gat gta act gcc gtc gaa
gtc aat ccg gcc aat aga aat cag gaa act 723Asp Val Thr Ala Val Glu
Val Asn Pro Ala Asn Arg Asn Gln Glu Thr 115 120 125 tcg gag gaa tat
caa atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc 771Ser Glu Glu Tyr
Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly 130 135 140 cgt gga
aac acg tac agt gat ttt aaa tgg cat tgg tat cat ttc gac 819Arg Gly
Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp 145 150 155
gga gcg gac tgg gat gaa tcc cgg aag atc agc cgc atc ttt aag ttt
867Gly Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe
160 165 170 175 cgt ggg gaa gga aaa gcg tgg gat tgg gaa gta tca agt
gaa aac ggc 915Arg Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser
Glu Asn Gly 180 185 190 aac tat gac tat tta atg tat gct gat gtt gac
tac gac cac cct gat 963Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp
Tyr Asp His Pro Asp 195 200 205 gtc gtg gca gag aca aaa aaa tgg ggt
atc tgg tat gcg aat gaa ctg 1011Val Val Ala Glu Thr Lys Lys Trp Gly
Ile Trp Tyr Ala Asn Glu Leu 210 215 220 tca tta gac ggc ttc cgt att
gat gcc gcc aaa cat att aaa ttt tca 1059Ser Leu Asp Gly Phe Arg Ile
Asp Ala Ala Lys His Ile Lys Phe Ser 225 230 235 ttt ctg cgt gat tgg
gtt cag gcg gtc aga cag gcg acg gga aaa gaa 1107Phe Leu Arg Asp Trp
Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu 240 245 250 255 atg ttt
acg gtt gcg gag tat tgg cag aat aat gcc ggg aaa ctc gaa 1155Met Phe
Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu 260 265 270
aac tac ttg aat aaa aca agc ttt aat caa tcc gtg ttt gat gtt ccg
1203Asn Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro
275 280 285 ctt cat ttc aat tta cag gcg gct tcc tca caa gga ggc gga
tat gat 1251Leu His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly
Tyr Asp 290 295 300 atg agg cgt ttg ctg gac ggt acc gtt gtg tcc agg
cat ccg gaa aag 1299Met Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg
His Pro Glu Lys 305 310 315 gcg gtt aca ttt gtt gaa aat cat gac aca
cag ccg gga cag tca ttg 1347Ala Val Thr Phe Val Glu Asn His Asp Thr
Gln Pro Gly Gln Ser Leu 320 325 330 335 gaa tcg aca gtc caa act tgg
ttt aaa ccg ctt gca tac gcc ttt att 1395Glu Ser Thr Val Gln Thr Trp
Phe Lys Pro Leu Ala Tyr Ala Phe Ile 340 345 350 ttg aca aga gaa tcc
ggt tat cct cag gtg ttc tat ggg gat atg tac 1443Leu Thr Arg Glu Ser
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr 355 360 365 ggg aca aaa
ggg aca tcg cca aag gaa att ccc tca ctg aaa gat aat 1491Gly Thr Lys
Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn 370 375 380 ata
gag ccg att tta aaa gcg cgt aag gag tac gca tac ggg ccc cag 1539Ile
Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln 385 390
395 cac gat tat att gac cac ccg gat gtg atc gga tgg acg agg gaa
ggt
1587His Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly
400 405 410 415 gac agc tcc gcc gcc aaa tca ggt ttg gcc gct tta atc
acg gac gga 1635Asp Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile
Thr Asp Gly 420 425 430 ccc ggc gga tca aag cgg atg tat gcc ggc ctg
aaa aat gcc ggc gag 1683Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu
Lys Asn Ala Gly Glu 435 440 445 aca tgg tat gac ata acg ggc aac cgt
tca gat act gta aaa atc gga 1731Thr Trp Tyr Asp Ile Thr Gly Asn Arg
Ser Asp Thr Val Lys Ile Gly 450 455 460 tct gac ggc tgg gga gag ttt
cat gta aac gat ggg tcc gtc tcc att 1779Ser Asp Gly Trp Gly Glu Phe
His Val Asn Asp Gly Ser Val Ser Ile 465 470 475 tat gtt cag aaa
taaggtaata aaaaaacacc tccaagctga gtgcgggtat 1831Tyr Val Gln Lys 480
cagcttggag gtgcgtttat tttttcagcc gtatgacaag gtcggcatca ggtgtgacaa
1891atacggtatg ctggctgtca taggtgacaa atccgggttt tgcgccgttt
ggctttttca 1951catgtctgat ttttgtataa tcaacaggca cggagccgga
atctttcgcc ttggaaaaat 2011aagcggcgat cgtagctgct tccaatatgg
attgttcatc gggatcgctg cttttaatca 2071caacgtggga tcc
20844514PRTBacillus amyloliquefaciens 4Met Ile Gln Lys Arg Lys Arg
Thr Val Ser Phe Arg Leu Val Leu Met -30 -25 -20 Cys Thr Leu Leu Phe
Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val -15 -10 -5 -1 1 Asn Gly
Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly 5 10 15 Gln
His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile 20 25
30 Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln
35 40 45 Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly
Glu Phe 50 55 60 65 Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
Lys Ser Glu Leu 70 75 80 Gln Asp Ala Ile Gly Ser Leu His Ser Arg
Asn Val Gln Val Tyr Gly 85 90 95 Asp Val Val Leu Asn His Lys Ala
Gly Ala Asp Ala Thr Glu Asp Val 100 105 110 Thr Ala Val Glu Val Asn
Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu 115 120 125 Glu Tyr Gln Ile
Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg Gly 130 135 140 145 Asn
Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala 150 155
160 Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly
165 170 175 Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly
Asn Tyr 180 185 190 Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His
Pro Asp Val Val 195 200 205 Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr
Ala Asn Glu Leu Ser Leu 210 215 220 225 Asp Gly Phe Arg Ile Asp Ala
Ala Lys His Ile Lys Phe Ser Phe Leu 230 235 240 Arg Asp Trp Val Gln
Ala Val Arg Gln Ala Thr Gly Lys Glu Met Phe 245 250 255 Thr Val Ala
Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn Tyr 260 265 270 Leu
Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His 275 280
285 Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg
290 295 300 305 Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu
Lys Ala Val 310 315 320 Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu Ser 325 330 335 Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu Thr 340 345 350 Arg Glu Ser Gly Tyr Pro Gln
Val Phe Tyr Gly Asp Met Tyr Gly Thr 355 360 365 Lys Gly Thr Ser Pro
Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu 370 375 380 385 Pro Ile
Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp 390 395 400
Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp Ser 405
410 415 Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
Gly 420 425 430 Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly
Glu Thr Trp 435 440 445 Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val
Lys Ile Gly Ser Asp 450 455 460 465 Gly Trp Gly Glu Phe His Val Asn
Asp Gly Ser Val Ser Ile Tyr Val 470 475 480 Gln Lys
51814DNABacillus
stearothermophilusCDS(156)..(1802)sig_peptide(156)..(257)mat_peptide(258)-
..(1802) 5aaattcgata ttgaaaacga ttacaaataa aaattataat agacgtaaac
gttcgagggt 60ttgctccctt tttactcttt ttatgcaatc gtttccctta attttttgga
agccaaaccg 120tcgaatgtaa catttgatta agggggaagg gcatt gtg cta acg
ttt cac cgc 173 Val Leu Thr Phe His Arg -30 atc att cga aaa gga tgg
atg ttc ctg ctc gcg ttt ttg ctc act gtc 221Ile Ile Arg Lys Gly Trp
Met Phe Leu Leu Ala Phe Leu Leu Thr Val -25 -20 -15 tcg ctg ttc tgc
cca aca gga cag ccc gcc aag gct gcc gca ccg ttt 269Ser Leu Phe Cys
Pro Thr Gly Gln Pro Ala Lys Ala Ala Ala Pro Phe -10 -5 -1 1 aac ggc
acc atg atg cag tat ttt gaa tgg tac ttg ccg gat gat ggc 317Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu Pro Asp Asp Gly 5 10 15 20
acg tta tgg acc aaa gtg gcc aat gaa gcc aac aac tta tcc agc ctt
365Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn Leu Ser Ser Leu
25 30 35 ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa gga aca
agc cgc 413Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly Thr
Ser Arg 40 45 50 agc gac gta ggg tac gga gta tac gac ttg tat gac
ctc ggc gaa ttc 461Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
Leu Gly Glu Phe 55 60 65 aat caa aaa ggg acc gtc cgc aca aaa tac
gga aca aaa gct caa tat 509Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys Ala Gln Tyr 70 75 80 ctt caa gcc att caa gcc gcc cac
gcc gct gga atg caa gtg tac gcc 557Leu Gln Ala Ile Gln Ala Ala His
Ala Ala Gly Met Gln Val Tyr Ala 85 90 95 100 gat gtc gtg ttc gac
cat aaa ggc ggc gct gac ggc acg gaa tgg gtg 605Asp Val Val Phe Asp
His Lys Gly Gly Ala Asp Gly Thr Glu Trp Val 105 110 115 gac gcc gtc
gaa gtc aat ccg tcc gac cgc aac caa gaa atc tcg ggc 653Asp Ala Val
Glu Val Asn Pro Ser Asp Arg Asn Gln Glu Ile Ser Gly 120 125 130 acc
tat caa atc caa gca tgg acg aaa ttt gat ttt ccc ggg cgg ggc 701Thr
Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly 135 140
145 aac acc tac tcc agc ttt aag tgg cgc tgg tac cat ttt gac ggc gtt
749Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Val
150 155 160 gat tgg gac gaa agc cga aaa ttg agc cgc att tac aaa ttc
cgc ggc 797Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr Lys Phe
Arg Gly 165 170 175 180 atc ggc aaa gcg tgg gat tgg gaa gta gac acg
gaa aac gga aac tat 845Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
Glu Asn Gly Asn Tyr 185 190 195 gac tac tta atg tat gcc gac ctt gat
atg gat cat ccc gaa gtc gtg 893Asp Tyr Leu Met Tyr Ala Asp Leu Asp
Met Asp His Pro Glu Val Val 200 205 210 acc gag ctg aaa aac tgg ggg
aaa tgg tat gtc aac aca acg aac att 941Thr Glu Leu Lys Asn Trp Gly
Lys Trp Tyr Val Asn Thr Thr Asn Ile 215 220 225 gat ggg ttc cgg ctt
gat gcc gtc aag cat att aag ttc agt ttt ttt 989Asp Gly Phe Arg Leu
Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe 230 235 240 cct gat tgg
ttg tcg tat gtg cgt tct cag act ggc aag ccg cta ttt 1037Pro Asp Trp
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Pro Leu Phe 245 250 255 260
acc gtc ggg gaa tat tgg agc tat gac atc aac aag ttg cac aat tac
1085Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu His Asn Tyr
265 270 275 att acg aaa aca gac gga acg atg tct ttg ttt gat gcc ccg
tta cac 1133Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala Pro
Leu His 280 285 290 aac aaa ttt tat acc gct tcc aaa tca ggg ggc gca
ttt gat atg cgc 1181Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
Phe Asp Met Arg 295 300 305 acg tta atg acc aat act ctc atg aaa gat
caa ccg aca ttg gcc gtc 1229Thr Leu Met Thr Asn Thr Leu Met Lys Asp
Gln Pro Thr Leu Ala Val 310 315 320 acc ttc gtt gat aat cat gac acc
gaa ccc ggc caa gcg ctg cag tca 1277Thr Phe Val Asp Asn His Asp Thr
Glu Pro Gly Gln Ala Leu Gln Ser 325 330 335 340 tgg gtc gac cca tgg
ttc aaa ccg ttg gct tac gcc ttt att cta act 1325Trp Val Asp Pro Trp
Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr 345 350 355 cgg cag gaa
gga tac ccg tgc gtc ttt tat ggt gac tat tat ggc att 1373Arg Gln Glu
Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Ile 360 365 370 cca
caa tat aac att cct tcg ctg aaa agc aaa atc gat ccg ctc ctc 1421Pro
Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp Pro Leu Leu 375 380
385 atc gcg cgc agg gat tat gct tac gga acg caa cat gat tat ctt gat
1469Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp Tyr Leu Asp
390 395 400 cac tcc gac atc atc ggg tgg aca agg gaa ggg ggc act gaa
aaa cca 1517His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr Glu
Lys Pro 405 410 415 420 gga tcc gga ctg gcc gca ctg atc acc gat ggg
ccg gga gga agc aaa 1565Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
Pro Gly Gly Ser Lys 425 430 435 tgg atg tac gtt ggc aaa caa cac gct
gga aaa gtg ttc tat gac ctt 1613Trp Met Tyr Val Gly Lys Gln His Ala
Gly Lys Val Phe Tyr Asp Leu 440 445 450 acc ggc aac cgg agt gac acc
gtc acc atc aac agt gat gga tgg ggg 1661Thr Gly Asn Arg Ser Asp Thr
Val Thr Ile Asn Ser Asp Gly Trp Gly 455 460 465 gaa ttc aaa gtc aat
ggc ggt tcg gtt tcg gtt tgg gtt cct aga aaa 1709Glu Phe Lys Val Asn
Gly Gly Ser Val Ser Val Trp Val Pro Arg Lys 470 475 480 acg acc gtt
tct acc atc gct cgg ccg atc aca acc cga ccg tgg act 1757Thr Thr Val
Ser Thr Ile Ala Arg Pro Ile Thr Thr Arg Pro Trp Thr 485 490 495 500
ggt gaa ttc gtc cgt tgg acc gaa cca cgg ttg gtg gca tgg cct 1802Gly
Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val Ala Trp Pro 505 510 515
tgatgcctgc ga 18146549PRTBacillus stearothermophilus 6Val Leu Thr
Phe His Arg Ile Ile Arg Lys Gly Trp Met Phe Leu Leu -30 -25 -20 Ala
Phe Leu Leu Thr Val Ser Leu Phe Cys Pro Thr Gly Gln Pro Ala -15 -10
-5 Lys Ala Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
-1 1 5 10 Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn
Glu Ala 15 20 25 30 Asn Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp
Leu Pro Pro Ala 35 40 45 Tyr Lys Gly Thr Ser Arg Ser Asp Val Gly
Tyr Gly Val Tyr Asp Leu 50 55 60 Tyr Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr 65 70 75 Gly Thr Lys Ala Gln Tyr
Leu Gln Ala Ile Gln Ala Ala His Ala Ala 80 85 90 Gly Met Gln Val
Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala 95 100 105 110 Asp
Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg 115 120
125 Asn Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe
130 135 140 Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp
Arg Trp 145 150 155 Tyr His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg
Lys Leu Ser Arg 160 165 170 Ile Tyr Lys Phe Arg Gly Ile Gly Lys Ala
Trp Asp Trp Glu Val Asp 175 180 185 190 Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Leu Asp Met 195 200 205 Asp His Pro Glu Val
Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr 210 215 220 Val Asn Thr
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His 225 230 235 Ile
Lys Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln 240 245
250 Thr Gly Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile
255 260 265 270 Asn Lys Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr
Met Ser Leu 275 280 285 Phe Asp Ala Pro Leu His Asn Lys Phe Tyr Thr
Ala Ser Lys Ser Gly 290 295 300 Gly Ala Phe Asp Met Arg Thr Leu Met
Thr Asn Thr Leu Met Lys Asp 305 310 315 Gln Pro Thr Leu Ala Val Thr
Phe Val Asp Asn His Asp Thr Glu Pro 320 325 330 Gly Gln Ala Leu Gln
Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala 335 340 345 350 Tyr Ala
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr 355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser 370
375 380 Lys Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly
Thr 385 390 395 Gln His Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp
Thr Arg Glu 400 405 410 Gly Gly Thr Glu Lys Pro Gly Ser Gly Leu Ala
Ala Leu Ile Thr Asp 415 420 425 430 Gly Pro Gly Gly Ser Lys Trp Met
Tyr Val Gly Lys Gln His Ala Gly 435 440 445 Lys Val Phe Tyr Asp Leu
Thr Gly Asn Arg Ser Asp Thr Val Thr Ile 450 455 460 Asn Ser Asp Gly
Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser 465 470 475 Val Trp
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile 480 485 490
Thr Thr Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg 495
500 505 510 Leu Val Ala Trp Pro 515 731DNAArtificial
sequenceSynthetic construct 7ggtcgtaggc accgtagccc caatccgctt g
31836DNAArtificial sequenceSynthetic construct 8ggtcgtaggc
accgtagccc
caatcccatt ggctcg 36928DNAArtificial sequenceSynthetic construct
9ctgtgactgg tgagtactca accaagtc 2810478PRTAspergillus oryzae 10Ala
Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10
15 Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr
20 25 30 Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile
Asp Lys 35 40 45 Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile
Trp Ile Thr Pro 50 55 60 Val Thr Ala Gln Leu Pro Gln Thr Thr Ala
Tyr Gly Asp Ala Tyr His 65 70 75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr
Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95 Ala Asp Asp Leu Lys Ala
Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110 Tyr Leu Met Val
Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125 Gly Ser
Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140
Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln 145
150 155 160 Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro
Asp Leu 165 170 175 Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr
Asp Trp Val Gly 180 185 190 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly
Leu Arg Ile Asp Thr Val 195 200 205 Lys His Val Gln Lys Asp Phe Trp
Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220 Val Tyr Cys Ile Gly Glu
Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys 225 230 235 240 Pro Tyr Gln
Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro
Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265
270 Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu
275 280 285 Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala
Ser Tyr 290 295 300 Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala
Phe Ile Ile Leu 305 310 315 320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala
Gly Gln Glu Gln His Tyr Ala 325 330 335 Gly Gly Asn Asp Pro Ala Asn
Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350 Pro Thr Asp Ser Glu
Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365 Arg Asn Tyr
Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380 Trp
Pro Ile Tyr Lys Asp Asp Ile Thr Ile Ala Met Arg Lys Gly Thr 385 390
395 400 Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser
Gly 405 410 415 Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr
Ala Gly Gln 420 425 430 Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val
Thr Val Gly Ser Asp 435 440 445 Gly Asn Val Pro Val Pro Met Ala Gly
Gly Leu Pro Arg Val Leu Tyr 450 455 460 Pro Thr Glu Lys Leu Ala Gly
Ser Lys Ile Cys Ser Ser Ser 465 470 475 111458DNAUnknownBacillus
species 11cat cat aat gga aca aat ggt act atg atg caa tat ttc gaa
tgg tat 48His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp Tyr 1 5 10 15 ttg cca aat gac ggg aat cat tgg aac agg ttg agg
gat gac gca gct 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg
Asp Asp Ala Ala 20 25 30 aac tta aag agt aaa ggg ata aca gct gta
tgg atc cca cct gca tgg 144Asn Leu Lys Ser Lys Gly Ile Thr Ala Val
Trp Ile Pro Pro Ala Trp 35 40 45 aag ggg act tcc cag aat gat gta
ggt tat gga gcc tat gat tta tat 192Lys Gly Thr Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat ctt gga gag ttt aac
cag aag ggg acg gtt cgt aca aaa tat gga 240Asp Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 aca cgc aac cag
cta cag gct gcg gtg acc tct tta aaa aat aac ggc 288Thr Arg Asn Gln
Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 att cag
gta tat ggt gat gtc gtc atg aat cat aaa ggt gga gca gat 336Ile Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110
ggt acg gaa att gta aat gcg gta gaa gtg aat cgg agc aac cga aac
384Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125 cag gaa acc tca gga gag tat gca ata gaa gcg tgg aca aag
ttt gat 432Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys
Phe Asp 130 135 140 ttt cct gga aga gga aat aac cat tcc agc ttt aag
tgg cgc tgg tat 480Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys
Trp Arg Trp Tyr 145 150 155 160 cat ttt gat ggg aca gat tgg gat cag
tca cgc cag ctt caa aac aaa 528His Phe Asp Gly Thr Asp Trp Asp Gln
Ser Arg Gln Leu Gln Asn Lys 165 170 175 ata tat aaa ttc agg gga aca
ggc aag gcc tgg gac tgg gaa gtc gat 576Ile Tyr Lys Phe Arg Gly Thr
Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 aca gag aat ggc aac
tat gac tat ctt atg tat gca gac gtg gat atg 624Thr Glu Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 gat cac cca
gaa gta ata cat gaa ctt aga aac tgg gga gtg tgg tat 672Asp His Pro
Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 acg
aat aca ctg aac ctt gat gga ttt aga ata gat gca gtg aaa cat 720Thr
Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230
235 240 ata aaa tat agc ttt acg aga gat tgg ctt aca cat gtg cgt aac
acc 768Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn
Thr 245 250 255 aca ggt aaa cca atg ttt gca gtg gct gag ttt tgg aaa
aat gac ctt 816Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu 260 265 270 ggt gca att gaa aac tat ttg aat aaa aca agt
tgg aat cac tcg gtg 864Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser
Trp Asn His Ser Val 275 280 285 ttt gat gtt cct ctc cac tat aat ttg
tac aat gca tct aat agc ggt 912Phe Asp Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Asn Ser Gly 290 295 300 ggt tat tat gat atg aga aat
att tta aat ggt tct gtg gtg caa aaa 960Gly Tyr Tyr Asp Met Arg Asn
Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 cat cca aca cat
gcc gtt act ttt gtt gat aac cat gat tct cag ccc 1008His Pro Thr His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 ggg gaa
gca ttg gaa tcc ttt gtt caa caa tgg ttt aaa cca ctt gca 1056Gly Glu
Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350
tat gca ttg gtt ctg aca agg gaa caa ggt tat cct tcc gta ttt tat
1104Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365 ggg gat tac tac ggt atc cca acc cat ggt gtt ccg gct atg
aaa tct 1152Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met
Lys Ser 370 375 380 aaa ata gac cct ctt ctg cag gca cgt caa act ttt
gcc tat ggt acg 1200Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe
Ala Tyr Gly Thr 385 390 395 400 cag cat gat tac ttt gat cat cat gat
att atc ggt tgg aca aga gag 1248Gln His Asp Tyr Phe Asp His His Asp
Ile Ile Gly Trp Thr Arg Glu 405 410 415 gga aat agc tcc cat cca aat
tca ggc ctt gcc acc att atg tca gat 1296Gly Asn Ser Ser His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggt cca ggt ggt aac
aaa tgg atg tat gtg ggg aaa aat aaa gcg gga 1344Gly Pro Gly Gly Asn
Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 caa gtt tgg
aga gat att acc gga aat agg aca ggc acc gtc aca att 1392Gln Val Trp
Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 aat
gca gac gga tgg ggt aat ttc tct gtt aat gga ggg tcc gtt tcg 1440Asn
Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470
475 480 gtt tgg gtg aag caa taa 1458Val Trp Val Lys Gln 485
12485PRTUnknownSynthetic Construct 12His His Asn Gly Thr Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly
Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys
Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys
Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55
60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn
Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys
Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val
Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala
Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn
Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp
Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185
190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val
Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp
Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp
Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala
Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn
Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val
Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly
Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310
315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln
Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys
Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr
Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His
Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln
Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr
Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn
Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430
Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435
440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr
Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly
Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485
13483PRTArtificial sequenceSynthetic construct 13Val Asn Gly Thr
Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln
His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35
40 45 Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly
Glu 50 55 60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
Lys Ser Glu 65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg
Asn Val Gln Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala
Gly Ala Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn
Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile
Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr
Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165
170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly
Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His
Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr
Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala
Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln
Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala
Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu
Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290
295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys
Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln
Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu
Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp
Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410
415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly
Glu Thr 435 440 445 Trp His Asp Ile Thr Gly
Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly
Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val
Gln Arg
* * * * *