U.S. patent application number 10/778469 was filed with the patent office on 2005-03-17 for detergent compositions containing amylase variants.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Bisgard-Frantzen, Henrik, Borchert, Torben Vedel, Svendsen, Allan.
Application Number | 20050059131 10/778469 |
Document ID | / |
Family ID | 8155525 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059131 |
Kind Code |
A1 |
Bisgard-Frantzen, Henrik ;
et al. |
March 17, 2005 |
Detergent compositions containing amylase variants
Abstract
The present invention relates to variants of a parent
.alpha.-amylase, which parent .alpha.-amylase (i) has an amino acid
sequence selected from the amino acid sequences shown in SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 7, respectively; or
(ii) displays at least 80% homology with one or more of these amino
acid sequences; and/or displays immunological cross-reactivity with
an antibody raised against an .alpha.-amylase having one of these
amino acid sequences; and/or is encoded by a DNA sequence which
hybridizes with the same probe as a DNA sequence encoding an
.alpha.-amylase having one of these amino acid sequences; in which
variant: (a) at least one amino acid residue of the parent
.alpha.-amylase has been deleted; and/or (b) at least one amino
acid residue of the parent .alpha.-amylase has been replaced by a
different amino acid residue; and/or (c) at least one amino acid
residue has been inserted relative to the parent .alpha.-amylase;
the variant having .alpha.-amylase activity and exhibiting at least
one of the following properties relative to the parent
.alpha.-amylase: increased thermostability; increased stability
towards oxidation; and reduced Ca.sup.2+ dependency; with the
proviso that the amino acid sequence of the variant is not
identical to any of the amino acid sequences shown in SEQ ID No. 1,
SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively.
Inventors: |
Bisgard-Frantzen, Henrik;
(Lyngby, DK) ; Svendsen, Allan; (Birkeroed,
DK) ; Borchert, Torben Vedel; (Copenhagen,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
8155525 |
Appl. No.: |
10/778469 |
Filed: |
February 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10778469 |
Feb 12, 2004 |
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10025648 |
Dec 19, 2001 |
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10025648 |
Dec 19, 2001 |
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09902188 |
Jul 10, 2001 |
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09902188 |
Jul 10, 2001 |
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09354191 |
Jul 15, 1999 |
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6297038 |
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09354191 |
Jul 15, 1999 |
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08600656 |
Feb 13, 1996 |
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6093562 |
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08600656 |
Feb 13, 1996 |
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PCT/DK96/00056 |
Feb 5, 1996 |
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Current U.S.
Class: |
435/204 ;
510/320 |
Current CPC
Class: |
C11D 3/38681 20130101;
D06L 1/14 20130101; C11D 3/38618 20130101; C12Y 302/01001 20130101;
C12N 9/2417 20130101; C11D 3/38609 20130101 |
Class at
Publication: |
435/204 ;
510/320 |
International
Class: |
C12N 009/32; C11D
003/386 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 3, 1995 |
DK |
0126/95 |
Mar 29, 1995 |
DK |
0336/95 |
Sep 29, 1995 |
DK |
1097/95 |
Oct 6, 1995 |
DK |
1121/95 |
Claims
1-76. (Canceled.)
77. A detergent comprising a surfactant and an alpha-amylase,
wherein the alpha-amylase is selected from the group consisting of:
an alpha-amylase comprising an amino acid sequence having at least
80% homology to SEQ ID NO:1 and which alpha-amylase Is modified by
having an amino acid deletion of two amino adds selected from the
group of amino adds equivalent to positions 180, 181, 182, 183, 184
and 185 in SEQ ID NO:1; an alpha-amylase comprising an amino acid
sequence having at least 80% homology to SEQ ID NO:2 and which
alpha-amylase is modified by having an amino acid deletion of two
amino acids selected from the group of amino acids equivalent to
positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:2; an
alpha-amylase comprising an amino acid sequence having at least 80%
homology to SEQ ID NO:3 and which alpha-amylase is modified by
having an amino acid deletion of two amino acids selected from the
group of amino acids equivalent to positions 178, 179, 180, 181,
182 and 183 in SEQ ID NO:3; and an alpha-amylase comprising an
amino acid sequence having at least 80% homology to SEQ ID NO:7 and
which alpha-amylase is modified by having an amino acid deletion of
two amino acids selected from the group of amino acids equivalent
to positions 180, 181, 182, 183, 184 and 185 in SEQ ID NO:7.
78. A detergent comprising a surfactant and an alpha-amylase,
wherein the alpha-amylase has an amino add sequence having at least
80% homology to SEQ ID NO:1 and which alpha-amylase Is modified by
having an amino acid deletion of two amino acids selected from the
group of amino acids equivalent to positions 180, 181, 182, 183,
184 and 185 In SEQ ID NO:1.
79. The detergent of claim 78, wherein the alpha amylase comprises
a deletion of three amino acids selected from the group of amino
acids equivalent to positions 180, 181, 182, 183, 184 and 185 in
SEQ ID NO:1.
80. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 182 in SEQ
ID NO. 1.
81. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 183 and 184 in SEQ
ID NO. 1.
82. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 183 in SEQ
ID NO. 1.
83. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 182 and 183 in SEQ
ID NO. 1.
84. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 182 and 184 in SEQ
ID NO. 1.
85. The detergent of claim 78, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 184 in SEQ
ID NO. 1.
86. The detergent of claim 78, wherein the alpha-amylase comprises
an amino acid sequence having at least 85% homology to SEQ ID
NO:1.
87. The detergent of claim 78, wherein the alpha-amylase comprises
an amino acid sequence having at least 90% homology to SEQ ID
NO:1.
88. A detergent comprising a surfactant and an alpha-amylase,
wherein the alpha-amylase has an amino acid sequence having at
least 80% homology to SEQ ID NO:2 and which alpha-amylase is
modified by having an amino acid deletion of two amino acids
selected from the group of amino acids equivalent to positions 180,
181, 182, 183, 184 and 185 in SEQ ID NO:2.
89. The detergent of claim 88, wherein the alpha amylase comprises
a deletion of three amino acids selected from the group of amino
acids equivalent to positions 180, 181, 182, 183.184 or 185 in SEQ
ID NO:2.
90. The detergent of claim 88, wherein the alpha amylase comprises
at least three amino acid deletions of amino acids selected from
the group of amino adds equivalent to positions 180, 181, 182, 183,
184 and 185 in SEQ ID NO:2.
91. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 182 in SEQ
ID NO. 2.
92. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 183 and 184 in SEQ
ID NO. 2.
93. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 183 in SEQ
ID NO. 2.
94. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 182 and 183 in SEQ
ID. NO. 2.
95. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 182 and 184 in SEQ
ID. NO. 2.
96. The detergent of claim 88, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 184 in SEQ
ID NO. 2.
97. The detergent of claim 88, wherein the alpha-amylase comprising
an amino acid sequence having at least 85% homology to SEQ ID
NO:2.
98. The detergent of claim 88, wherein the alpha-amylase comprising
an amino acid sequence having at least 90% homology to SEQ ID
NO:2.
99. A detergent comprising a surfactant and an alpha-amylase,
wherein the alpha-amylase has an amino acid sequence having at
least 80% homology to SEQ ID NO:3 and which alpha-amylase is
modified by having an amino acid deletion of two amino acids
selected from the group of amino adds equivalent to positions 178,
179, 180, 181, 182 and 183 in SEQ ID NO:3.
100. The detergent of claim 99, wherein the alpha amylase comprises
at least three amino acid deletions of amino acids selected from
the group of amino acids equivalent to positions 178, 179, 180,
181, 182 and 183 in SEQ ID NO:3.
101. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 179 and 180 in SEQ
ID NO. 3.
102. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 181 and 182 in SEQ
ID NO. 3.
103. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 179 and 181 in SEQ
ID NO. 3.
104. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 180 and 181 in SEQ
ID NO. 3.
105. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 180 and 182 in SEQ
ID NO. 3.
106. The detergent of claim 99, wherein the alpha-amylase comprises
a deletion at positions equivalent to positions 179 and 182 in SEQ
ID NO. 3.
107. The detergent of claim 99, wherein the alpha-amylase comprises
an amino acid sequence having at least 85% homology to SEQ ID
NO:3.
108. The detergent of claim 99, wherein the alpha-amylase comprises
an amino acid sequence having at least 90% homology to SEQ ID
NO:3.
109. A detergent comprising a surfactant and an alpha-amylase,
wherein the alpha-amylase has an amino acid sequence having at
least 80% homology to SEQ ID NO:7 and which alpha-amylase is
modified by having an amino acid deletion of two amino acids
selected from the group of amino acids equivalent to positions 180,
181, 182, 183, 184 and 185 in SEQ ID NO:7.
110. The detergent of claim 109, wherein the alpha amylase
comprises at least three amino add deletions of amino acids
selected from the group of amino acids equivalent to positions 180,
181, 182, 183, 184 and 185 in SEQ ID NO:7.
111. The detergent of claim 109, wherein the alpha amylase
comprises at least three amino acid deletions of amino adds
selected from the group of amino acids equivalent to positions 180,
181, 182.183, 184 and 185 in SEQ ID NO:7.
112. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 181 and
182 in SEQ ID NO. 7.
113. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 183 and
184 in SEQ ID NO. 7.
114. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 181 and
183 in SEQ ID NO. 7.
115. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 182 and
183 in SEQ ID NO. 7.
116. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 182 and
184 in SEQ ID NO. 7.
117. The detergent of claim 109, wherein the alpha-amylase
comprises a deletion at positions equivalent to positions 181 and
184 in SEQ ID NO. 7.
118. The detergent of claim 109, wherein the alpha-amylase
comprises an amino acid sequence having at least 85% homology to
SEQ ID NO:7.
119. The detergent of claim 109, wherein the alpha-amylase
comprises an amino add sequence having at least 90% homology to SEQ
ID NO:7.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
10/025,648, filed Dec. 19, 2001, which is a division of U.S. patent
application Ser. No. 09/902,188, filed on Jul. 10, 2001, which is a
continuation of U.S. patent application Ser. No. 09/354,191, now
U.S. Pat. No. 6,297,038, filed on Jul. 15, 1999, which is a
continuation of U.S. patent application Ser. No. 08/600,656, now
U.S. Pat. No. 6,093,562, filed on Feb. 13, 1996, which is a
continuation of application Ser. No. PCT/DK96/00056, filed on Feb.
5, 1996, which claims priority under 35 U.S.C. 119 of Danish
application serial nos. 0126/95, filed on Feb. 3, 1995, 0336/95,
filed on Mar. 29, 1995, 1097/95, filed on Sep. 29, 1995, and
1121/95, filed on Oct. 6, 1995, the contents of which are fully
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to .alpha.-amylase variants
having improved properties relative to the parent enzyme (e.g.
improved thermal and/or oxidation stability and/or reduced calcium
ion dependency), and thereby improved washing and/or dishwashing
(and/or textile desizing) performance. The invention also relates
to DNA constructs encoding the variants, and to vectors and cells
harboring the DNA constructs. The invention further relates to
methods of producing the amylase variants, and to detergent
additives and detergent compositions comprising the amylase
variants. Furthermore, the invention relates to the use of the
amylase variants for textile desizing.
BACKGROUND OF THE INVENTION
[0003] .alpha.-Amylase enzymes have been used industrially for a
number of years and for a variety of different purposes, the most
important of which are starch liquefaction, textile desizing,
starch modification in the paper and pulp industry, and for brewing
and baking. A further use of .alpha.-amylases which is becoming
increasingly important is the removal of starchy stains during
washing or dishwashing.
[0004] In recent years attempts have been made to construct
.alpha.-amylase variants having improved properties with respect to
specific uses such as starch liquefaction and textile desizing.
[0005] For instance, U.S. Pat. No. 5,093,257 discloses chimeric
.alpha.-amylases comprising an N-terminal part of a B.
stearothermophilus .alpha.-amylase and a C-terminal part of a B.
licheniformis .alpha.-amylase. The chimeric .alpha.-amylases are
stated to have unique properties, such as a different
thermostability, as compared to their parent .alpha.-amylase.
However, all of the specifically described chimeric
.alpha.-amylases were shown to have a decreased enzymatic activity
as compared to their parent .alpha.-amylases.
[0006] EP 252 666 describes hybrid amylases of the general formula
Q-R-L, in which Q is a N-terminal polypeptide residue of from 55 to
60 amino acid residues which is at least 75% homologous to the 57
N-terminal amino acid residues of a specified .alpha.-amylase from
B. amyloliquefaciens, R is a specified polypeptide, and L is a
C-terminal polypeptide comprising from 390 to 400 amino acid
residues which is at least 75% homologous to the 395 C-terminal
amino acid residues of a specified B. licheniformis
.alpha.-amylase.
[0007] Suzuki et al. (1989) disclose chimeric .alpha.-amylases, in
which specified regions of a B. amyloliquefaciens .alpha.-amylase
have been substituted for the corresponding regions of a B.
licheniformis .alpha.-amylase. The chimeric .alpha.-amylases were
constructed with the purpose of identifying regions responsible for
thermostability. Such regions were found to include amino acid
residues 177-186 and amino acid residues 255-270 of the B.
amyloliquefaciens .alpha.-amylase. The alterations of amino acid
residues in the chimeric .alpha.-amylases did not seem to affect
properties of the enzymes other than their thermostability.
[0008] WO 91/00353 discloses .alpha.-amylase mutants which differ
from their parent .alpha.-amylase in at least one amino acid
residue. The .alpha.-amylase mutants disclosed in said patent
application are stated to exhibit improved properties for
application in the degradation of starch and/or textile desizing
due to their amino acid substitutions. Some of the mutants exhibit
improved stability, but no improvements in enzymatic activity were
reported or indicated. The only mutants exemplified are prepared
from a parent B. licheniformis .alpha.-amylase and carry one of the
following mutations: H133Y or H133Y+T1491. Another suggested
mutation is A111T.
[0009] FR 2,676,456 discloses mutants of the B. licheniformis
.alpha.-amylase, in which an amino acid residue in the proximity of
His 133 and/or an amino acid residue in the proximity of Ala 209
have been replaced by a more hydrophobic amino acid residue. The
resulting .alpha.-amylase mutants are stated to have an improved
thermostability and to be useful in the textile, paper, brewing and
starch liquefaction industry.
[0010] EP 285 123 discloses a method of performing random
mutagenesis of a nucleotide sequence. As an example of such
sequence a nucleotide sequence encoding a B. stearothermophilus
.alpha.-amylase is mentioned. When mutated, an .alpha.-amylase
variant having improved activity at low pH values is obtained.
[0011] In none of the above references is it mentioned or even
suggested that .alpha.-amylase mutants may be constructed which
have improved properties with respect to the detergent
industry.
[0012] EP 525 610 relates to mutant enzymes having improved
stability towards ionic tensides (surfactants). The mutant enzymes
have been produced by replacing an amino acid residue in the
surface part of the parent enzyme with another amino acid residue.
The only mutant enzyme specifically described in EP 525 610 is a
protease. Amylase is mentioned as an example of an enzyme which may
obtain an improved stability towards ionic tensides, but the type
of amylase, its origin or specific mutations are not specified.
[0013] WO 94/02597 discloses .alpha.-amylase mutants which exhibit
improved stability and activity in the presence of oxidizing
agents. In the mutant .alpha.-amylases, one or more methionine
residues have been replaced with amino acid residues different from
Cys and Met. The .alpha.-amylase mutants are stated to be useful as
detergent and/or dishwashing additives as well as for textile
desizing.
[0014] WO 94/18314 discloses oxidatively stable .alpha.-amylase
mutants, including mutations in the M197 position of B.
licheniformis .alpha.-amylase.
[0015] EP 368 341 describes the use of pullulanase and other
amylolytic enzymes optionally in combination with an
.alpha.-amylase for washing and dishwashing.
[0016] An object of the present invention is to provide
.alpha.-amylase variants which--relative to their parent
.alpha.-amylase--possess improved properties of importance, inter
alia, in relation to the washing and/or dishwashing performance of
the variants in question, e.g. increased thermal stability,
increased stability towards oxidation, reduced dependency on
Ca.sup.2+ ion and/or improved stability or activity in the pH
region of relevance in, e.g., laundry washing or dishwashing. Such
variant .alpha.-amylases have the advantage, among others, that
they may be employed in a lower dosage than their parent
.alpha.-amylase. Furthermore, the .alpha.-amylase variants may be
able to remove starchy stains which cannot, or can only with
difficulty, be removed by .alpha.-amylase detergent enzymes known
today.
BRIEF DISCLOSURE OF THE INVENTION
[0017] A goal of the work underlying the present invention was to
improve, if possible, the stability of, inter alia, particular
.alpha.-amylases which are obtainable from Bacillus strains and
which themselves had been selected on the basis of their starch
removal performance in alkaline media (such as in detergent
solutions as typically employed in laundry washing or dishwashing)
relative to many of the currently commercially available
.alpha.-amylases. In this connection, the present inventors have
surprisingly found that it is in fact possible to improve
properties of the types mentioned earlier (vide supra) of such a
parent .alpha.-amylase by judicial modification of one or more
amino acid residues in various regions of the amino acid sequence
of the parent .alpha.-amylase. The present invention is based on
this finding.
[0018] Accordingly, in a first aspect the present invention relates
to variants of a parent .alpha.-amylase, the parent .alpha.-amylase
in question being one which:
[0019] i) has one of the amino acid sequences shown in SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively,
herein; or
[0020] ii) displays at least 80% homology with one or more of the
amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID
No. 3 and SEQ ID No. 7; and/or displays immunological
cross-reactivity with an antibody raised against an .alpha.-amylase
having one of the amino acid sequences shown in SEQ ID No. 1, SEQ
ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively; and/or is
encoded by a DNA sequence which hybridizes with the same probe as a
DNA sequence encoding an .alpha.-amylase having one of the amino
acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3
and SEQ ID No. 7, respectively.
[0021] An .alpha.-amylase variant of the invention is subject to
the proviso that it is a variant which does not have an amino acid
sequence identical to the amino acid sequence shown in SEQ ID No.
1, in SEQ ID No. 2, in SEQ ID No. 3 or in SEQ ID No. 7.
[0022] DNA sequences encoding the first three of the
.alpha.-amylase amino acid sequences in question are shown in SEQ
ID No. 4 (encoding the amino acid sequence shown in SEQ ID No. 1),
SEQ ID No. 5 (encoding the amino acid sequence shown in SEQ ID No.
2) and SEQ ID No. 6 (encoding the amino acid sequence shown in SEQ
ID No. 3).
[0023] The amino acid sequences of the SEQ ID No. 1 and SEQ ID No.
2 parent .alpha.-amylases, and the corresponding DNA sequences (SEQ
ID No. 4 and SEQ ID No. 5, respectively) are also disclosed in WO
95/26397 (under the same SEQ ID Nos. as in the present
application).
[0024] The variants of the invention are variants in which: (a) at
least one amino acid residue of the parent .alpha.-amylase has been
deleted; and/or (b) at least one amino acid residue of the parent
.alpha.-amylase has been replaced (i.e. substituted) by a different
amino acid residue; and/or (c) at least one amino acid residue has
been inserted relative to the parent .alpha.-amylase. The variants
in question have themselves .alpha.-amylase activity and exhibit at
least one of the following properties relative to the parent
.alpha.-amylase:
[0025] increased thermostability, i.e. satisfactory retention of
enzymatic activity at a temperature higher than that suitable for
use with the parent enzyme;
[0026] increased oxidation stability, i.e. increased resistance to
degradation by oxidants (such as oxygen, oxidizing bleaching agents
and the like);
[0027] reduced Ca.sup.2+ dependency, i.e. the ability to function
satisfactorily in the presence of a lower Ca.sup.2+ concentration
than in the case of the parent .alpha.-amylase. .alpha.-Amylases
with such reduced Ca.sup.2+ dependency are highly desirable for use
in detergent compositions, since such compositions typically
contain relatively large amounts of substances (such as phosphates,
EDTA and the like) which bind calcium ions strongly.
[0028] Examples of other desirable improvements or modifications of
properties (relative to the parent .alpha.-amylase in question)
which may be achieved with a variant according to the invention
are:
[0029] increased stability and/or .alpha.-amylolytic activity at
neutral to relatively high pH values, e.g. at pH values in the
range of 7-10.5, such as in the range of 8.5-10.5;
[0030] increased .alpha.-amylolytic activity at relatively high
temperatures, e.g. temperatures in the range of
40-70.quadrature.C;
[0031] increase or decrease of the isoelectric point (pI) so as to
better match the pI value for the .alpha.-amylase variant in
question to the pH of the medium (e.g. a laundry washing medium,
dishwashing medium or textile-desizing medium) in which the variant
is to be employed (vide infra); and
[0032] improved binding of a particular type of substrate, improved
specificity towards a substrate, and/or improved specificity with
respect to cleavage (hydrolysis) of substrate.
[0033] An amino acid sequence is considered to be X % homologous to
the parent .alpha.-amylase if a comparison of the respective amino
acid sequences, performed via known algorithms, such as the one
described by Lipman and Pearson in Science 227 (1985) p. 1435,
reveals an identity of X %. The GAP computer program from the GCG
package, version 7.3 (June 1993), may suitably be used, employing
default values for GAP penalties [Genetic Computer Group (1991)
Programme Manual for the GCG Package, version 7, 575 Science Drive,
Madison, Wis., USA 53711].
[0034] In the context of the present invention, "improved
performance" as used in connection with washing and dishwashing is,
as already indicated above, intended to mean improved removal of
starchy stains, i.e. stains containing starch, during washing or
dishwashing, respectively. The performance may be determined in
conventional washing and dishwashing experiments and the
improvement evaluated as a comparison with the performance of the
parent .alpha.-amylase in question. An example of a small-scale
"mini dishwashing test" which can be used an indicator of
dishwashing performance is described in the Experimental section,
below.
[0035] It will be understood that a variety of different
characteristics of an .alpha.-amylase variant, including specific
activity, substrate specificity, K.sub.m (the so-called "Michaelis
constant" in the Michaelis-Menten equation), V.sub.max [the maximum
rate (plateau value) of conversion of a given substrate determined
on the basis of the Michaelis-Menten equation], pI, pH optimum,
temperature optimum, thermoactivation, stability towards oxidants
or surfactants (e.g. in detergents), etc., taken alone or in
combination, can contribute to improved performance. The skilled
person will be aware that the performance of the variant cannot,
alone, be predicted on the basis of the above characteristics, but
would have to be accompanied by washing and/or dishwashing
performance tests.
[0036] In further aspects the invention relates to a DNA construct
comprising a DNA sequence encoding an .alpha.-amylase variant of
the invention, a recombinant expression vector carrying the DNA
construct, a cell which is transformed with the DNA construct or
the vector, as well as a method of producing an .alpha.-amylase
variant by culturing such a cell under conditions conducive to the
production of the .alpha.-amylase variant, after which the
.alpha.-amylase variant is recovered from the culture.
[0037] In a further aspect the invention relates to a method of
preparing a variant of a parent .alpha.-amylase which by virtue of
its improved properties as described above exhibits improved
washing and/or dishwashing performance as compared to the parent
.alpha.-amylase. This method comprises
[0038] a) constructing a population of cells containing genes
encoding variants of said parent .alpha.-amylase,
[0039] b) screening the population of cells for .alpha.-amylase
activity under conditions simulating at least one washing and/or
dishwashing condition,
[0040] c) isolating a cell from the population containing a gene
encoding a variant of said parent .alpha.-amylase which has
improved activity as compared with the parent .alpha.-amylase under
the conditions selected in step b),
[0041] d) culturing the cell isolated in step c) under suitable
conditions in an appropriate culture medium, and
[0042] e) recovering the .alpha.-amylase variant from the culture
obtained in step d).
[0043] The invention also relates to a variant (which is a variant
according the invention) prepared by the latter method.
[0044] In the present context, the term "simulating at least one
washing and/or dishwashing condition" is intended to indicate a
simulation of, e.g., the temperature or pH prevailing during
washing or dishwashing, or of the chemical composition of a
detergent composition to be used in the washing or dishwashing
treatment. The term "chemical composition" is intended to include
one, or a combination of two or more, constituents of the detergent
composition in question. The constituents of a number of different
detergent compositions are listed further below.
[0045] The "population of cells" referred to in step a) may
suitably be constructed by cloning a DNA sequence encoding a parent
.alpha.-amylase and subjecting the DNA to site-directed or random
mutagenesis as described herein.
[0046] In the present context the term "variant" is used
interchangeably with the term "mutant". The term "variant" is
intended to include hybrid .alpha.-amylases, i.e. .alpha.-amylases
comprising parts of at least two different .alpha.-amylolytic
enzymes. Thus, such a hybrid may be constructed, e.g., from: one or
more parts each deriving from a variant as already defined above;
or one or more parts each deriving from a variant as already
defined above, and one or more parts each deriving from an
unmodified parent .alpha.-amylase. In this connection, the
invention also relates to a method of producing such a hybrid
.alpha.-amylase having improved washing and/or dishwashing
performance as compared to any of its constituent enzymes (i.e. as
compared to any of the enzymes which contribute a part to the
hybrid), which method comprises:
[0047] a) recombining in vivo or in vitro the N-terminal coding
region of an .alpha.-amylase gene or corresponding cDNA of one of
the constituent .alpha.-amylases with the C-terminal coding region
of an .alpha.-amylase gene or corresponding cDNA of another
constituent .alpha.-amylase to form recombinants,
[0048] b) selecting recombinants that produce a hybrid
.alpha.-amylase having improved washing and/or dishwashing
performance as compared to any of its constituent
.alpha.-amylases,
[0049] c) culturing recombinants selected in step b) under suitable
conditions in an appropriate culture medium, and
[0050] d) recovering the hybrid .alpha.-amylase from the culture
obtained in step c).
[0051] In further aspects the invention relates to the use of an
.alpha.-amylase variant of the invention [including any variant or
hybrid prepared by one of the above mentioned methods] as a
detergent enzyme, in particular for washing or dishwashing, to a
detergent additive and a detergent composition comprising the
.alpha.-amylase variant, and to the use of an .alpha.-amylase
variant of the invention for textile desizing.
[0052] Random mutagenesis may be used to generate variants
according to the invention, and the invention further relates to a
method of preparing a variant of a parent .alpha.-amylase, which
method comprises
[0053] (a) subjecting a DNA sequence encoding the parent
.alpha.-amylase to random mutagenesis,
[0054] (b) expressing the mutated DNA sequence obtained in step (a)
in a host cell, and
[0055] (c) screening for host cells expressing a mutated amylolytic
enzyme which has improved properties as described above (e.g.
properties such as decreased calcium dependency, increased
oxidation stability, increased thermostability, and/or improved
activity at relatively high pH) as compared to the parent
.alpha.-amylase.
DETAILED DISCLOSURE OF THE INVENTION
[0056] Nomenclature
[0057] In the present description and claims, the conventional
one-letter codes for nucleotides and the conventional one-letter
and three-letter codes for amino acid residues are used. For ease
of reference, .alpha.-amylase variants of the invention are
described by use of the following nomenclature:
[0058] Original amino acid(s):position(s):substituted amino
acid(s)
[0059] According to this nomenclature, and by way of example, the
substitution of alanine for asparagine in position 30 is shown
as:
[0060] Ala 30 Asn or A30N
[0061] a deletion of alanine in the same position is shown as:
[0062] Ala 30* or A30*
[0063] and insertion of an additional amino acid residue, such as
lysine, is shown as:
[0064] Ala 30 AlaLys or A30AK
[0065] A deletion of a consecutive stretch of amino acid residues,
exemplified by amino acid residues 30-33, is indicated as
(30-33)*.
[0066] Where a specific .alpha.-amylase contains a "deletion" (i.e.
lacks an amino acid residue) in comparison with other
.alpha.-amylases and an insertion is made in such a position, this
is indicated as:
[0067] *36 Asp or *36D for insertion of an aspartic acid in
position 36.
[0068] Multiple mutations are separated by plus signs, i.e.:
[0069] Ala 30 Asp+Glu 34 Ser or A30N+E34S representing mutations in
positions 30 and 34 (in which alanine and glutamic acid replace,
i.e. are substituted for, asparagine and serine, respectively).
[0070] When one or more alternative amino acid residues may be
inserted in a given position this is indicated as: A30N,E or A30N
or A30E Furthermore, when a position suitable for modification is
identified herein without any specific modification being
suggested, it is to be understood that any other amino acid residue
may be substituted for the amino acid residue present in that
position (i.e. any amino acid residue--other than that normally
present in the position in question--chosen among A, R, N, D, C, Q,
E, G, H, I, L, K, M, F, P, S, T, W, Y and V). Thus, for instance,
when a modification (replacement) of a methionine in position 202
is mentioned, but not specified, it is to be understood that any of
the other amino acids may be substituted for the methionine, i.e.
any other amino acid chosen among
A,R,N,D,C,Q,E,G,H,I,L,K,F,P,S,T,W,Y and V.
[0071] The parent .alpha.-amylase
[0072] As already indicated, an .alpha.-amylase variant of the
invention is very suitably prepared on the basis of a parent
.alpha.-amylase having one of the amino acid sequences shown in SEQ
ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively
(vide infra).
[0073] The parent .alpha.-amylases having the amino acid sequences
shown in SEQ ID No. 1 and SEQ ID No. 2, respectively, are
obtainable from alkalophilic Bacillus strains (strain NCIB 12512
and strain NCIB 12513, respectively), both of which are described
in detail in EP 0 277 216 B1. The preparation, purification and
sequencing of these two parent .alpha.-amylases is described in WO
95/26397 [see the Experimental section herein (vide infra)].
[0074] The parent .alpha.-amylase having the amino acid sequence
shown in SEQ ID No. 3 is obtainable from Bacillus
stearothermophilus and is described in, inter alia, J. Bacteriol.
166 (1986) pp. 635-643.
[0075] The parent .alpha.-amylase having the amino acid sequence
shown in SEQ ID No. 7 (which is the same sequence as that numbered
4 in FIG. 1) is obtainable from a "Bacillus sp. #707" and is
described by Tsukamoto et al. in Biochem. Biophys. Res. Commun. 151
(1988) pp. 25-31.
[0076] Apart from variants of the above-mentioned parent
.alpha.-amylases having the amino acid sequences shown in SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively,
other interesting variants according to the invention include
variants of parent .alpha.-amylases which have amino acid sequences
exhibiting a high degree of homology, such as at least 70%
homology, preferably (as already indicated) at least 80% homology,
desirably at least 85% homology, and more preferably at least 90%
homology, e.g. .quadrature.95% homology, with at least one of the
latter four amino acid sequences.
[0077] As also already indicated above, further criteria for
identifying a suitable parent .alpha.-amylase are a) that the
.alpha.-amylase displays an immunological cross-reaction with an
antibody raised against an .alpha.-amylase having one of the amino
acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3
and SEQ ID No. 7, respectively, and/or b) that the .alpha.-amylase
is encoded by a DNA sequence which hybridizes with the same probe
as a DNA sequence encoding an .alpha.-amylase having one of the
amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID
No. 3 and SEQ ID No. 7, respectively.
[0078] As already mentioned, with regard to determination of the
degree of homology of polypeptides (such as enzymes), amino acid
sequence comparisons can be performed using known algorithms, such
as the one described by Lipman and Pearson (1985).
[0079] Assays for immunological cross-reactivity may be carried out
using an antibody raised against, or reactive with, at least one
epitope of the .alpha.-amylase having the amino acid sequence shown
in SEQ ID No. 1, or of the .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 2, or of the .alpha.-amylase having
the amino acid sequence shown in SEQ ID No. 3, or of the
.alpha.-amylase having the amino acid sequence shown in SEQ ID No.
7.
[0080] 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). Examples of suitable assay techniques well
known in the art include Western Blotting and Radial
Immunodiffusion Assay, e.g. as described by Hudson et al.
(1989).
[0081] The oligonucleotide probe for use in the identification of
suitable parent .alpha.-amylases on the basis of probe
hybridization [criterion b) above] may, by way of example, suitably
be prepared on the basis of the full or partial amino acid sequence
of an .alpha.-amylase having one of the sequences shown in SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively,
or on the basis of the full or partial nucleotide sequence
corresponding thereto.
[0082] Suitable conditions for testing hybridization involve
presoaking in 5.times.SSC and prehybridizing for 1 h at
.about.40.quadrature.C in a solution of 20% formamide, 5.times.
Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 .mu.g
of denatured sonicated calf thymus DNA, followed by hybridization
in the same solution supplemented with 100 .mu.M ATP for 18 h at
.about.40.quadrature.C, or using other methods described by, e.g.,
Sambrook et al. (1989).
[0083] Influence of Mutations on Particular Properties
[0084] From the results obtained by the present inventors it
appears that changes in a particular property, e.g. thermal
stability or oxidation stability, exhibited by a variant relative
to the parent .alpha.-amylase in question can to a considerable
extent be correlated with the type of, and positioning of,
mutation(s) (amino acid substitutions, deletions or insertions) in
the variant. It is to be understood, however, that the observation
that a particular mutation or pattern of mutations leads to changes
in a given property in no way excludes the possibility that the
mutation(s) in question can also influence other properties.
[0085] Oxidation stability: With respect to increasing the
oxidation stability of an .alpha.-amylase variant relative to its
parent .alpha.-amylase, it appears to be particularly desirable
that at least one, and preferably multiple, oxidizable amino acid
residue(s) of the parent has/have been deleted or replaced (i.e.
substituted by) a different amino acid residue which is less
susceptible to oxidation than the original oxidizable amino acid
residue.
[0086] Particularly relevant oxidizable amino acid residues in this
connection are cysteine, methionine, tryptophan and tyrosine. Thus,
for example, in the case of parent .alpha.-amylases containing
cysteine it is anticipated that deletion of cysteine residues, or
substitution thereof by less oxidizable amino acid residues, will
be of importance in obtaining variants with improved oxidation
stability relative to the parent .alpha.-amylase.
[0087] In the case of the above-mentioned parent .alpha.-amylases
having the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2
and SEQ ID No. 7, respectively, all of which contain no cysteine
residues but have a significant methionine content, the deletion or
substitution of methionine residues is particularly relevant with
respect to achieving improved oxidation stability of the resulting
variants. Thus, deletion or substitution [e.g. by threonine (T), or
by one of the other amino acids listed above] of one or more of the
methionine residues in positions M9, M10, M105, M202, M208, M261,
M309, M382, M430 and M440 of the amino acid sequences shown in SEQ
ID No. 1, SEQ ID No. 2 and SEQ ID No. 7, and/or in position M323 of
the amino acid sequence shown in SEQ ID No. 2 (or deletion or
substitution of methionine residues in equivalent positions in the
sequence of another .alpha.-amylase meeting one of the other
criteria for a parent .alpha.-amylase mentioned above) appear to be
particularly effective with respect to increasing the oxidation
stability.
[0088] In the case of the parent .alpha.-amylase having the amino
acid sequence shown in SEQ ID No. 3, relevant amino acid residues
which may be deleted or substituted with a view to improving the
oxidation stability include the single cysteine residue (C363)
and--by analogy with the sequences shown in SEQ ID No. 1 and SEQ ID
No. 3--the methionine residues located in positions M8, M9, M96,
M200, M206, M284, M307, M311, M316 and M438.
[0089] In this connection, the term "equivalent position" denotes a
position which, on the basis of an alignment of the amino acid
sequence of the parent .alpha.-amylase in question with the
"reference" .alpha.-amylase amino acid sequence in question (for
example the sequence shown in SEQ ID No. 1) so as to achieve
juxtapositioning of amino acid residues/regions which are common to
both, corresponds most closely to (e.g. is occupied by the same
amino acid residue as) a particular position in the reference
sequence in question.
[0090] Particularly interesting mutations in connection with
modification (improvement) of the oxidation stability of the
.alpha.-amylases having the amino acid sequences shown in SEQ ID
No. 1, SEQ ID No. 2 and SEQ ID No. 7, respectively, are one or more
of the following methionine substitutions (or equivalents thereof
in the amino acid sequences of other .alpha.-amylases meeting the
requirements of a parent .alpha.-amylase in the context of the
invention): M202A,R,N,D,Q,E,G,H,I,L- ,K,F,P,S,T,W,Y,V.
[0091] Further relevant methionine substitutions in the amino acid
sequence shown in SEQ ID No. 2 are:
M323A,R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y- ,V.
[0092] Particularly interesting mutations in connection with
modification (improvement) of the oxidation stability of the
.alpha.-amylase having the amino acid sequence shown in SEQ ID No.
3 are one or more of the following methionine substitutions:
M200A,R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y- ,V;
M311A,R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y,V; and
M316A,R,N,D,Q,E,G,H,I,L,K- ,F,P,S,T,W,Y,V.
[0093] Thermal stability: With respect to increasing the thermal
stability of an .alpha.-amylase variant relative to its parent
.alpha.-amylase, it appears to be particularly desirable to delete
at least one, and preferably two or even three, of the following
amino acid residues in the amino acid sequence shown in SEQ ID No.
1 (or their equivalents): F 180, R181, G182, T183, G184 and K185.
The corresponding, particularly relevant (and equivalent) amino
acid residues in the amino acid sequences shown in SEQ ID No. 2,
SEQ ID No. 3 and SEQ ID No. 7, respectively, are: F180, R181, G182,
D183, G184 and K185 (SEQ ID No. 2); F178, R179, G180,1181, G182 and
K183 (SEQ ID No. 3); and F180, R181, G182, H183, G184 and K185 (SEQ
ID No. 7).
[0094] Particularly interesting pairwise deletions of this type are
as follows: R181*+G182*; and T183*+G184* (SEQ ID No. 1);
R181*+G182*; and D183*+G184* (SEQ ID No. 2); R179*+G180*; and
I181*+G182* (SEQ ID No. 3); and R181*+G182*; and H183*+G184* (SEQ
ID No. 7) (or equivalents of these pairwise deletions in another
.alpha.-amylase meeting the requirements of a parent
.alpha.-amylase in the context of the present invention).
[0095] Other mutations which appear to be of importance in
connection with thermal stability are substitutions of one or more
of the amino acid residues from P260 to I275 in the sequence shown
in SEQ ID No. 1 (or equivalents thereof in another parent
.alpha.-amylase in the context of the invention), such as
substitution of the lysine residue in position 269.
[0096] Examples of specific mutations which appear to be of
importance in connection with the thermal stability of an
.alpha.-amylase variant relative to the parent .alpha.-amylase in
question are one or more of the following substitutions in the
amino acid sequence shown in SEQ ID No. 1 (or equivalents thereof
in another parent .alpha.-amylase in the context of the invention):
K269R; P260E; R124P; M105F,I,L,V; M208F,W,Y; L217I; V206I,L,F.
[0097] For the parent .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 2, important further (equivalent)
mutations are, correspondingly, one or more of the substitutions:
M105F,I,L,V; M208F,W,Y; L217I; V206I,L,F; and K269R.
[0098] For the parent .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 3, important further (equivalent)
mutations are, correspondingly, one or both of the substitutions:
M206F,W,Y; and L215I.
[0099] For the parent .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 7, important further (equivalent)
mutations are, correspondingly, one or more of the substitutions:
M105F,I,L,V; M208F,W,Y; L217I; and K269R.
[0100] Still further examples of mutations which appear to be of
importance, inter alia, in achieving improved thermal stability of
an .alpha.-amylase variant relative to the parent .alpha.-amylase
in question are one or more of the following substitutions in the
amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID
No. 7 (or equivalents thereof in another parent .alpha.-amylase in
the context of the invention): A354C+V479C; L351 C+M430C;
N457D,E+K385R; L355D,E+M430R,K; L355D,E+I141R,K; and N457D,E.
[0101] Ca.sup.2+ dependency: With respect to achieving decreased
Ca.sup.2+ dependency of an .alpha.-amylase variant relative to its
parent .alpha.-amylase [i.e. with respect to obtaining a variant
which exhibits 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 which, for example,
therefore is less sensitive than the parent to calcium
ion-depleting conditions such as those obtaining in media
containing calcium-complexing agents (such as certain detergent
builders)], it appears to be particularly desirable to incorporate
one or more of the following substitutions in the amino acid
sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7 (or
an equivalent substitution in another parent .alpha.-amylase in the
context of the invention): Y243F, K108R, K179R, K239R, K242R,
K269R, D163N, D188N, D192N, D199N, D205N, D207N, D209N, E190Q,
E194Q and N106D.
[0102] In the case of the amino acid sequence shown in SEQ ID No.
3, particularly desirable substitutions appear, correspondingly
(equivalently), to be one or more of the following: K107R, K177R,
K237R, K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N,
E188Q and E192Q.
[0103] As well as the above-mentioned replacements of D residues
with N residues, or of E residues with Q residues, other relevant
substitutions in the context of reducing Ca.sup.2+ dependency are
replacement of the D and/or E residues in question with any other
amino acid residue.
[0104] Further substitutions which appear to be of importance in
the context of achieving reduced Ca.sup.2+ dependency are pairwise
substitutions of the amino acid residues present at: positions 113
and 151, and positions 351 and 430, in the amino acid sequences
shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7; and at:
positions 112 and 150, and positions 349 and 428, in the amino acid
sequence shown in SEQ ID No. 3 (or equivalent pairwise
substitutions in another parent .alpha.-amylase in the context of
the invention), i.e. pairwise substitutions of the following amino
acid residues:
[0105] G113+N151 (in relation to SEQ ID No. 1); A 113+T151 (in
relation to SEQ ID No. 2 and SEQ ID No. 7); and G112+T150 (in
relation to SEQ ID No. 3); and
[0106] L351+M430 (in relation to SEQ ID No. 1, SEQ ID No. 2 and SEQ
ID No. 7); and L349+I428 (in relation to SEQ ID No. 3).
[0107] Particularly interesting pairwise substitutions of this type
with respect to achieving decreased Ca.sup.2+ dependency are the
following:
[0108] G113T+N151I (in relation to SEQ ID No. 1); A113T+T151I (in
relation to SEQ ID No. 2 and SEQ ID No. 7); and G112T+T150I (in
relation to SEQ ID No. 3); and L351C+M430C (in relation to SEQ ID
No. 1, SEQ ID No. 2 and SEQ ID No. 7); and L349C+I428C (in relation
to SEQ ID No. 3).
[0109] In connection with substitutions of relevance for Ca.sup.2+
dependency, some other substitutions which appear to be of
importance in stabilizing the enzyme conformation, and which it is
contemplated may achieve this by, e.g., enhancing the strength of
binding or retention of calcium ion at or within a calcium binding
site within the .alpha.-amylolytic enzyme, are one or more of the
following substitutions in the amino acid sequences shown in SEQ ID
No. 1, SEQ ID No. 2 and SEQ ID No. 7 (or an equivalent substitution
in another parent .alpha.-amylase in the context of the invention):
G304W,F,Y,R,I,L,V,Q,N; G305A,S,N,D,Q,E,R,K; and H408Q,E.
[0110] Corresponding (equivalent) substitutions in the amino acid
sequence shown in SEQ ID No. 3 are: G302W,F,Y,R,I,L,V,Q,N; and
G303A,S,N,D,Q,E,R,K.
[0111] Further mutations which appear to be of importance in the
context of achieving reduced Ca.sup.2+ dependency are pairwise
deletions of amino acids (i.e. deletion of two amino acids) at
positions selected among R181, G182, T183 and G184 in the amino
acid sequence shown in SEQ ID No. 1 (or equivalent positions in the
amino acid sequence of another .alpha.-amylase meeting the
requirements of a parent .alpha.-amylase in the context of the
invention). Such pairwise deletions are thus the following:
[0112] R181*+G182*; T183*+G184*; R181*+T183*; G182*+T183*;
G182*+G184*; and R181*+G184*(SEQ ID No. 1);
[0113] R181*+G182*; D183*+G184*; R181*+D183*; G182*+D183*;
G182*+G184*; and R181*+G184*(SEQ ID No. 2);
[0114] R179*+G180*; I181*+G182*; R179*+I181*; G180*+I181*;
G180*+G182*; and R179*+G182*(SEQ ID No. 3); and
[0115] R181*+G182*; H183*+G184*; R181*+H183*; G182*+H183*;
G182*+G184*; and R181*+G184*(SEQ ID No. 7); (or equivalents of
these pairwise deletions in another .alpha.-amylase meeting the
requirements of a parent .alpha.-amylase in the context of the
present invention).
[0116] Isoelectric point (pI): Preliminary results indicate that
the washing performance, e.g. the laundry washing performance, of
an .alpha.-amylase is optimal when the pH of the washing liquor
(washing medium) is close to the pI value for the .alpha.-amylase
in question. It will thus be desirable, where appropriate, to
produce an .alpha.-amylase variant having an isoelectric point (pI
value) which is better matched to the pH of a medium (such as a
washing medium) in which the enzyme is to be employed than the
isoelectric point of the parent .alpha.-amylase in question.
[0117] With respect to decreasing the isoelectric point, preferred
mutations in the amino acid sequence shown in SEQ ID No. 1 include
one or more of the following substitutions: Q86E, R124P, S154D,
T183D, V222E, P260E, R310A, Q346E, Q391E, N437E, K444Q and R452H.
Appropriate combinations of these substitutions in the context of
decreasing the isoelectric point include: Q391E+K444Q; and
Q391E+K444Q+S154D.
[0118] Correspondingly, preferred mutations in the amino acid
sequence shown in SEQ ID No. 3 with respect to decreasing the
isoelectric point include one or more of the substitutions: L85E,
S153D, I181D, K220E, P258E, R308A, P344E, Q358E and S435E.
[0119] With respect to increasing the isoelectric point, preferred
mutations in the amino acid sequence shown in SEQ ID No. 2 include
one or more of the following substitutions: E86Q,L; D154S; D183T,I;
E222V,K; E260P; A310R; E346Q,P; E437N,S; and H452R.
[0120] In the Experimental section below, the construction of a
number of variants according to the invention is described.
[0121] .alpha.-Amylase variants of the invention will, apart from
having one or more improved properties as discussed above,
preferably be such that they have a higher starch hydrolysis
velocity at low substrate concentrations than the parent
.alpha.-amylase. Alternatively, an .alpha.-amylase variant of the
invention will preferably be one which has a higher V.sub.maxand/or
a lower K.sub.m than the parent .alpha.-amylase when tested under
the same conditions. In the case of a hybrid .alpha.-amylase, the
"parent .alpha.-amylase" to be used for the comparison should be
the one of the constituent enzymes having the best performance.
[0122] V.sub.max and K.sub.m (parameters of the Michaelis-Menten
equation) may be determined by well-known procedures.
[0123] Methods of Preparing .alpha.-amylase Variants
[0124] 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.
[0125] Cloning a DNA Sequence Encoding an .alpha.-amylase
[0126] 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.
[0127] 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.
[0128] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g. the
phosphoamidite method described by S. L. Beaucage and M. H.
Caruthers (1981) or the method described by Matthes et al. (1984).
In the phosphoamidite method, oligonucleotides are synthesized,
e.g. in an automatic DNA synthesizer, purified, annealed, ligated
and cloned in appropriate vectors.
[0129] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et
al. (1988).
[0130] Site-directed mutagenesis
[0131] 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.
[0132] 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.
[0133] Random Mutagenesis
[0134] 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.
[0135] For region-specific random mutagenesis with a view to
improving the thermal stability, the following codon positions, in
particular, may appropriately be targeted (using one-letter amino
acid abbreviations and the numbering of the amino acid residues in
the sequence in question):
1 In the amino acid sequence shown in SEQ ID No. 1: 120-140 =
VEVNRSNRNQETSGEYAIEAW 178-187 = YKFRGTGKAW 264-277 = VAEFWKNDLGAIEN
In the amino acid sequence shown in SEQ ID No. 2: 120-140 =
VEVNPNNRNQEISGDYTIEAW 178-187 = YKFRGDGKAW 264-277 = VAEFWKNDLGALEN
In the amino acid sequence shown in SEQ ID No. 3: 119-139 =
VEVNPSDRNQEISGTYQIQAW 176-185 = YKFRGIGKAW 262-275 = VGEYWSYDINKLHN
In the amino acid sequence shown in SEQ ID No. 7: 120-140 =
VEVNPNNRNQEVTGEYTIEAW 178-187 = YKFRGHGKAW 264-277 =
VAEFWKNDLGAIEN
[0136] With a view to achieving reduced Ca.sup.2+ dependency, the
following codon positions, in particular, may appropriately be
targeted:
2 In the amino acid sequence shown in SEQ ID No. 1: 178-209 =
YKFRGTGKAWDWEVDTENGNYDYLMYADVDMD 237-246 = AVKHIKYSFT In the amino
acid sequence shown in SEQ ID No. 2: 178-209 =
YKFRGDGKAWDWEVDSENGNYDYLMYADVDMD 237-246 = AVKHIKYSFT In the amino
acid sequence shown in SEQ ID No. 7: 178-209 =
YKFRGHGKAWDWEVDTENGNYDYLMYADIDMD 237-246 = AVKHIKYSFT
[0137] With a view to achieving improved binding of a substrate
(i.e. improved binding of a carbohydrate species--such as amylose
or amylopectin--which is a substrate for .alpha.-amylolytic
enzymes) by an .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. 1 (or equivalent codon positions for another parent
.alpha.-amylase in the context of the invention) may particularly
appropriately be targeted:
3 In the amino acid sequence shown in SEQ ID No. 1: 15-20 = WYLPND
52-58 = SQNDVGY 72-78 = KGTVRTK 104-111 = VMNHKGGA 165-174 =
TDWDQSRQLQ 194-204 = ENGNYDYLMYA 234-240 = RIDAVKH 332-340 =
HDSQPGEAL
[0138] 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.
[0139] 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.
[0140] The mutagenizing agent may, e.g., be one which induces
transitions, transversions, inversions, scrambling, deletions,
and/or insertions.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] When PCR-generated mutagenesis is used, either a chemically
treated or non-treated gene encoding a parent .alpha.-amylase
enzyme is subjected to PCR under conditions that increase the
misincorporation of nucleotides (Deshler 1992; Leung et al.,
Technique, Vol.1, 1989, pp. 11-15).
[0145] A mutator strain of E. coli (Fowler et al., Molec. Gen.
Genet., 133, 1974, pp. 179-191), S. cereviseae or any other
microbial organism may be used for the random mutagenesis of the
DNA encoding the amylolytic enzyme by e.g. transforming a plasmid
containing the parent enzyme into the mutator strain, growing the
mutator strain with the plasmid and isolating the mutated plasmid
from the mutator strain. The mutated plasmid may subsequently be
transformed into the expression organism.
[0146] 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 harbored in the
cell. Finally, the DNA to be mutagenized may be in isolated form.
It will be understood that the DNA sequence to be subjected to
random mutagenesis is preferably a cDNA or a genomic DNA
sequence.
[0147] 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.
[0148] 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.
[0149] The mutated DNA sequence may further comprise a DNA sequence
encoding functions permitting expression of the mutated DNA
sequence.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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:
[0154] 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.
[0155] The filter used for binding the enzymatic activity may be
any protein binding filter e.g. nylon or nitrocellulose. The top
filter carrying the colonies of the expression organism may be any
filter that has no or low affinity for binding proteins e.g.
cellulose acetate or Durapore.TM.. 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.
[0156] The enzymatic activity may be detected by a dye,
flourescence, precipitation, pH indicator, IR-absorbance or any
other known technique for detection of enzymatic activity.
[0157] 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. .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.quadrature.or
65.quadrature.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 color intensity due to
increased liberation of Cibacron Red.
[0158] 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.quadrature.C, 10.quadrature.C or
30.quadrature.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.
[0159] Methods of Preparing Hybrid .alpha.-amylases
[0160] As an alternative to site-specific mutagenesis,
.alpha.-amylase variants which are hybrids of at least two
constituent .alpha.-amylases may be prepared by combining the
relevant parts of the respective genes in question.
[0161] Naturally occurring enzymes may be genetically modified by
random or site directed mutagenesis as described above.
Alternatively, part of one enzyme may be replaced by a part of
another to obtain a chimeric enzyme. This replacement can be
achieved either by conventional in vitro gene splicing techniques
or by in vivo recombination or by combinations of both techniques.
When using conventional in vitro gene splicing techniques, a
desired portion of the .alpha.-amylase gene coding sequence may be
deleted using appropriate site-specific restriction enzymes; the
deleted portion of the coding sequence may then be replaced by the
insertion of a desired portion of a different .alpha.-amylase
coding sequence so that a chimeric nucleotide sequence encoding a
new .alpha.-amylase is produced. Alternatively, .alpha.-amylase
genes may be fused, e.g. by use of the PCR overlay extension method
described by Higuchi et al. 1988.
[0162] The in vivo recombination techniques depend on the fact that
different DNA segments with highly homologous regions (identity of
DNA sequence) may recombine, i.e. break and exchange DNA, and
establish new bonds in the homologous regions. Accordingly, when
the coding sequences for two different but homologous amylase
enzymes are used to transform a host cell, recombination of
homologous sequences in vivo will result in the production of
chimeric gene sequences. Translation of these coding sequences by
the host cell will result in production of a chimeric amylase gene
product. Specific in vivo recombination techniques are described in
U.S. Pat. No. 5,093,257 and EP 252 666.
[0163] Alternatively, the hybrid enzyme may be synthesized by
standard chemical methods known in the art. For example, see
Hunkapiller et al. (1984). Accordingly, peptides having the
appropriate amino acid sequences may be synthesized in whole or in
part and joined to form hybrid enzymes (variants) of the
invention.
[0164] Expression of .alpha.-amylase Variants
[0165] According to the invention, a mutated
.alpha.-amylase-encoding DNA sequence 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.
[0166] 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.
[0167] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA sequence
encoding an .alpha.-amylase variant of the invention, especially in
a bacterial host, are the promoter of the lac operon of E. coli,
the Streptomyces coelicolor agarase gene dagA promoters, the
promoters of the Bacillus licheniformis .alpha.-amylase gene
(amyL), the promoters of the Bacillus stearothermophilus maltogenic
amylase gene (amyM), the promoters of the Bacillus
Amyloliquefaciens .alpha.-amylase (amyQ), the promoters of the
Bacillus subtilis xylA and xylB genes etc. For transcription in a
fungal host, examples of useful promoters are those derived from
the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei
aspartic proteinase, A. niger neutral .alpha.-amylase, A. niger
acid stable .alpha.-amylase, A. niger glucoamylase, Rhizomucor
miehei lipase, A. oryzae alkaline protease, A. oryzae triose
phosphate isomerase or A. nidulans acetamidase.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] Procedures suitable for constructing vectors of the
invention encoding an .alpha.-amylase variant, and containing the
promoter, terminator and other elements, respectively, are well
known to persons skilled in the art [cf., for instance, Sambrook et
al. (1989)].
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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).
[0179] 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
[0180] Owing to their activity at alkaline pH values,
.alpha.-amylase variants of the invention are well suited for use
in a variety of industrial processes. In particular, they find
potential applications as a component in washing, dishwashing and
hard surface cleaning detergent compositions (vide infra), but may
also be useful in the production of sweeteners and ethanol from
starch. Conditions for conventional starch-converting processes and
liquefaction and/or saccharification processes are described in,
for instance, U.S. Pat. No. 3,912,590, EP 252,730 and EP
63,909.
[0181] Some areas of application of .alpha.-amylase variants of the
invention are outlined below.
[0182] Paper-related applications: .alpha.-Amylase variants of the
invention possess properties of value in the production of
lignocellulosic materials, such as pulp, paper and cardboard, from
starch-reinforced waste paper and waste cardboard, especially where
repulping occurs at a pH above 7, and where amylases can facilitate
the disintegration of the waste material through degradation of the
reinforcing starch.
[0183] .alpha.-Amylase variants of the invention are well suited
for use in the deinking/recycling processes of making paper out of
starch-coated or starch-containing waste printed paper. It is
usually desirable to remove the printing ink in order to produce
new paper of high brightness; examples of how the variants of the
invention may be used in this way are described in
PCT/DK94/00437.
[0184] .alpha.-Amylase variants of the invention may also be very
useful in modifying starch where enzymatically modified starch is
used in papermaking together with alkaline fillers such as calcium
carbonate, kaolin and clays. With alkaline .alpha.-amylase variants
of the invention it is feasible to modify the starch in the
presence of the filler, thus allowing for a simpler, integrated
process.
[0185] Textile desizing: .alpha.-Amylase variants of the invention
are also well suited for use in textile desizing. In the textile
processing industry, .alpha.-amylases are traditionally used as
auxiliaries in the desizing process to facilitate the removal of
starch-containing size which has served as a protective coating on
weft yarns during weaving.
[0186] Complete removal of the size coating after weaving is
important to ensure optimum results in subsequent processes in
which the fabric is scoured, bleached and dyed. Enzymatic starch
degradation is preferred because it does not harm the fibers of the
textile or fabric.
[0187] In order to reduce processing costs and increase mill
throughput, the desizing processing is sometimes combined with the
scouring and bleaching steps. In such cases, non-enzymatic
auxiliaries such as alkali or oxidation agents are typically used
to break down the starch, because traditional .alpha.-amylases are
not very compatible with high pH levels and bleaching agents. The
non-enzymatic breakdown of the starch size does lead to some fibre
damage because of the rather aggressive chemicals used.
[0188] .alpha.-Amylase variants of the invention exhibiting
improved starch-degrading performance at relatively high pH levels
and in the presence of oxidizing (bleaching) agents are thus well
suited for use in desizing processes as described above, in
particular for replacement of non-enzymatic desizing agents
currently used. The .alpha.-amylase variant may be used alone, or
in combination with a cellulase when desizing cellulose-containing
fabric or textile.
[0189] Beer production: .alpha.-Amylase variants of the invention
are also believed to be very useful in beer-making processes; in
such processes the variants will typically be added during the
mashing process.
[0190] Applications in detergent additives and detergent
compositions for washing or dishwashing: Owing to the improved
washing and/or dishwashing performance which will often be a
consequence of improvements in properties as discussed above,
numerous .alpha.-amylase variants (including hybrids) of the
invention are particularly well suited for incorporation into
detergent compositions, e.g. detergent compositions intended for
performance in the pH range of 7-13, particularly the pH range of
8-11. According to the invention, the .alpha.-amylase variant may
be added as a component of a detergent composition. As such, it may
be included in the detergent composition in the form of a detergent
additive.
[0191] Thus, a further aspect of the invention relates to a
detergent additive comprising an .alpha.-amylase variant according
to the invention. The enzymes may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e. a separated
additive or a combined additive, can be formulated, e.g., as a
granulate, liquid, slurry, etc. Preferred enzyme formulations for
detergent additives are granulates (in particular non-dusting
granulates), liquids (in particular stabilized liquids), slurries
or protected enzymes (vide infra).
[0192] The detergent composition as well as the detergent additive
may additionally comprise one or more other enzymes conventionally
used in detergents, such as proteases, lipases, amylolytic enzymes,
oxidases (including peroxidases), or cellulases.
[0193] It has been found that substantial improvements in washing
and/or dishwashing performance may be obtained when .alpha.-amylase
is combined with another amylolytic enzyme, such as a pullulanase,
an iso-amylase, a beta-amylase, an amyloglucosidase or a CGTase.
Examples of commercially available amylolytic enzymes suitable for
the given purpose are AMG.quadrature., Novamyl.quadrature. and
Promozyme.quadrature., all of which available from Novo Nordisk
A/S, Bagsvaerd, Denmark. Accordingly, a particular embodiment of
the invention relates to a detergent additive comprising an
.alpha.-amylase variant of the invention in combination with at
least one other amylolytic enzyme (e.g. chosen amongst those
mentioned above).
[0194] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452, and may
optionally be coated by methods known in the art; further details
concerning coatings are given below. When a combination of
different detergent enzymes is to be employed, the enzymes may be
mixed before or after granulation.
[0195] 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.
[0196] As already indicated, a still further aspect of the
invention relates to a detergent composition, e.g. for laundry
washing, for dishwashing or for hard-surface cleaning, comprising
an .alpha.-amylase variant (including hybrid) of the invention, and
a surfactant.
[0197] The detergent composition of the invention may be in any
convenient form, e.g. as powder, granules or liquid. A liquid
detergent may be aqueous, typically containing up to 90% of water
and 0-20% of organic solvent, or non-aqueous, e.g. as described in
EP Patent 120,659.
[0198] Detergent Compositions
[0199] When an .alpha.-amylase variant of the invention is employed
as a component of a detergent composition (e.g. a laundry washing
detergent composition, or a dishwashing detergent composition), it
may, for example, be included in the detergent composition in the
form of a non-dusting granulate, a stabilized liquid, or a
protected enzyme. As mentioned above, 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
molecular 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 GB 1483591.
[0200] Enzymes added in the form of liquid enzyme preparations may,
as indicated above, be stabilized by, e.g., the addition of 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.
[0201] Protected enzymes for inclusion in a detergent composition
of the invention may be prepared, as mentioned above, according to
the method disclosed in EP 238,216.
[0202] 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%
water and 0-30% organic solvent, or nonaqueous.
[0203] The detergent composition comprises one or more surfactants,
each of which may be anionic, nonionic, cationic, or amphoteric
(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),
alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol
ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide,
ethoxylated fatty acid monoethanolamide, fatty acid
monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as
described in WO 92/06154).
[0204] The detergent composition may additionally comprise one or
more other enzymes, such as pullulanase, esterase, lipase,
cutinase, protease, cellulase, peroxidase, or oxidase, e.g.,
laccase.
[0205] Normally the detergent contains 1-65% of a detergent builder
(although some dishwashing detergents may contain even up to 90% 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).
[0206] The detergent builders may be subdivided into
phosphorus-containing and non-phosphorous-containing types.
Examples of phosphorus-containing inorganic alkaline detergent
builders include the water-soluble salts, especially alkali metal
pyrophosphates, orthophosphates, polyphosphates and phosphonates.
Examples of non-phosphorus-containing inorganic builders include
water-soluble alkali metal carbonates, borates and silicates, as
well as layered disilicates and the various types of
water-insoluble crystalline or amorphous alumino silicates of which
zeolites are the best known representatives.
[0207] Examples of suitable organic builders include alkali metal,
ammonium or substituted ammonium salts of succinates, malonates,
fatty acid malonates, fatty acid sulphonates, carboxymethoxy
succinates, polyacetates, carboxylates, polycarboxylates,
aminopolycarboxylates and polyacetyl carboxylates.
[0208] The detergent may also be unbuilt, i.e. essentially free of
detergent builder.
[0209] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose (CMC; typically in the form of the
sodium salt), poly(vinylpyrrolidone) (PVP), polyethyleneglycol
(PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as
polyacrylates, polymaleates, maleic/acrylic acid copolymers and
lauryl methacrylate/acrylic acid copolymers.
[0210] The detergent composition may contain bleaching agents of
the chlorine/bromine-type or the oxygen-type. The bleaching agents
may be coated or encapsulated. Examples of inorganic
chlorine/bromine-type bleaches are lithium, sodium or calcium
hypochlorite or hypobromite as well as chlorinated trisodium
phosphate. The bleaching system may also 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).
[0211] 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. The bleaching system may also comprise
peroxyacids of, e.g., the amide, imide, or sulfone type.
[0212] In dishwashing detergents 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 or NOBS.
[0213] 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 such
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. The
enzymes of the invention may also be stabilized by adding
reversible enzyme inhibitors, e.g., of the protein type (as
described in EP 0 544 777 B1) or the boronic acid type.
[0214] The detergent may also contain other conventional detergent
ingredients such as, e.g., fabric conditioners including clays,
deflocculant material, foam boosters/foam depressors (in
dishwashing detergents foam depressors), suds suppressors,
anti-corrosion agents, soil-suspending agents,
anti-soil-redeposition agents, dyes, dehydrating agents,
bactericides, optical brighteners, or perfume.
[0215] The pH (measured in aqueous solution at use concentration)
will usually be neutral or alkaline, e.g. in the range of 7-11.
[0216] Particular forms of laundry detergent compositions within
the scope of the invention include:
[0217] 1) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
4 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.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)
[0218] 2) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
5 Linear alkylbenzenesulfonate (calculated as acid) 6-11% Alcohol
ethoxysulfate (e.g. C.sub.12-18 alcohol, 1-2 EO or 1-3% 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
NaAlSiO.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, PVP, 1-6% PEG) Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g. suds suppressors,
perfume) 0-5%
[0219] 3) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
6 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.2SO4) 0-4% Sodium perborate (as NaBO.sub.3.H.sub.2O) 8-16%
TAED 2-8% Phosphonate (e.g. EDTMPA) 0-1% 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)
[0220] 4) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
7 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, PVP, 1-3% PEG)
Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g. suds suppressors, perfume) 0-5%
[0221] 5) An aqueous liquid detergent composition comprising
8 Linear alkylbenzenesulfonate (calculated as acid) 15-21% Alcohol
ethoxylate (e.g. C.sub.12-15 alcohol, 7 EO or C.sub.12-15 12-18%
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.sup.2-) 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 0-5% suppressors, perfume,
optical brightener)
[0222] 6) An aqueous structured liquid detergent composition
comprising
9 Linear alkylbenzenesulfonate (calculated as acid) 15-21% Alcohol
ethoxylate (e.g. C.sub.12-15 alcohol, 7 EO, or 3-9% C.sub.12-15
alcohol, 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.sup.2-) 0-2% Carboxybethylcellulose 0-2% Polymers
(e.g. PEG, PVP) 0-3% Anchoring polymers such as, e.g., lauryl 0-3%
methacrylate/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 0-5%
suppressors, perfume, optical brighteners)
[0223] 7) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
10 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.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)
[0224] 8) A detergent composition formulated as a granulate
comprising
11 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%
[0225] 9) A detergent composition formulated as a granulate
comprising
12 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.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%
[0226] 10) An aqueous liquid detergent composition comprising
13 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.12-15
alcohol, 5 EO) Soap as fatty acid (e.g. lauric acid) 0-3%
Aminoethanol 1-5% Sodium citrate 5-10% Hydrotrope (e.g. sodium
toluene sulfonate) 2-6% Borate (as B.sub.4O.sub.7.sup.2-) 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)
[0227] 11) An aqueous liquid detergent composition comprising
14 Linear alkylbenzenesulfonate (calculated as acid) 20-32% Alcohol
ethoxylate (e.g. C.sub.12-15 alcohol, 7 EO, or C.sub.12-15 6-12%
alcohol, 5 EO) Aminoethanol 2-6% Citric acid 8-14% Borate (as
B.sub.4O.sub.7.sup.2-) 1-3% Polymer (e.g. maleic/acrylic acid
copolymer, 0-3% anchoring polymer such as, e.g., lauryl
methacrylate/acrylic acid copolymer) Glycerol 63-8% Enzymes
(calculated as pure enzyme protein) 0.0001-0.1% Minor ingredients
(e.g. hydrotropes, dispersants, 60-5% perfume, optical
brighteners)
[0228] 12) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
15 Anionic surfactant (linear alkylbenzenesulfonate, 25-40% alkyl
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.3.4H.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%
[0229] 13) Detergent formulations as described in 1)-12) wherein
all or part of the linear alkylbenzenesulfonate is replaced by
(Cl.sub.2--Cl.sub.8)alkyl sulfate.
[0230] 14) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
16 (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)
[0231] 15) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/l comprising
17 (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)
[0232] 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.
[0233] 17) Detergent compositions as described in 1), 3), 7), 9)
and 12) wherein perborate is replaced by percarbonate.
[0234] 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, 1994, pp. 637-639.
[0235] 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.
[0236] Particular forms of dishwashing detergent compositions
within the scope of the invention include:
[0237] 1) POWDER AUTOMATIC DISHWASHING COMPOSITION
18 Nonionic surfactant 0.4-2.5% Sodium metasilicate 0-20% Sodium
disilicate 3-20% Sodium triphosphate 20-40% Sodium carbonate 0-20%
Sodium perborate 2-9% Tetraacetylethylenediamine (TAED) 1-4% Sodium
sulphate 5-33% Enzymes 0.0001-0.1%
[0238] 2) POWDER AUTOMATIC DISHWASHING COMPOSITION
19 Nonionic surfactant (e.g. alcohol ethoxylate) 1-2% Sodium
disilicate 2-30% Sodium carbonate 10-50% Sodium phosphonate 0-5%
Trisodium citrate dihydrate 9-30% Nitrilotrisodium acetate (NTA)
0-20% Sodium perborate monohydrate 5-10% Tetraacetylethylenediamine
(TAED) 1-2% Polyacrylate polymer (e.g. maleic acid/acrylic acid
6-25% copolymer) Enzymes 0.0001-0.1% Perfume 0.1-0.5% Water
5-10
[0239] 3) POWDER AUTOMATIC DISHWASHING COMPOSITION
20 Nonionic surfactant 0.5-2.0% Sodium disilicate 25-40% Sodium
citrate 30-55% Sodium carbonate 0-29% Sodium bicarbonate 0-20%
Sodium perborate monohydrate 0-15% Tetraacetylethylenediamine
(TAED) 0-6% Maleic acid/acrylic acid copolymer 0-5% Clay 1-3%
Poly(amino acids) 0-20% Sodium polyacrylate 0-8% Enzymes
0.0001-0.1%
[0240] 4) POWDER AUTOMATIC DISHWASHING COMPOSITION
21 Nonionic surfactant 1-2% Zeolite MAP 15-42% Sodium disilicate
30-34% Sodium citrate 0-12% Sodium carbonate 0-20% Sodium perborate
monohydrate 7-15% Tetraacetylethylenediamine (TAED) 0-3% Polymer
0-4% Maleic acid/acrylic acid copolymer 0-5% Organic phosphonate
0-4% Clay 1-2% Enzymes 0.0001-0.1% Sodium sulphate Balance
[0241] 5) POWDER AUTOMATIC DISHWASHING COMPOSITION
22 Nonionic surfactant 1-7% Sodium disilicate 18-30% Trisodium
citrate 10-24% Sodium carbonate 12-20% Monopersulphate (2
KHSO.sub.5.KHSO.sub.4.K.sub.2SO.sub.4) 15-21% Bleach stabilizer
0.1-2% Maleic acid/acrylic acid copolymer 0-6%
Diethylenetriaminepentaacetate, pentasodium salt 0-2.5% Enzymes
0.0001-0.1% Sodium sulphate, water Balance
[0242] 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING
SURFACTANT SYSTEM
23 Nonionic surfactant 0-1.5% Octadecyl dimethylamine N-oxide
dihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyl dimethylamine
0-4% N-oxide dihydrate and hexadecyldimethyl amine N- oxide
dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5%
(hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis
(hydroxyethyl)amine N-oxide anhydrous C.sub.13-C.sub.15 alkyl
ethoxysulfate with an average degree 0-10% of ethoxylation of 3
C.sub.12-C.sub.15 alkyl ethoxysulfate with an average degree 0-5%
of ethoxylation of 3 C.sub.13-C.sub.15 ethoxylated alcohol with an
average degree 0-5% of ethoxylation of 12 A blend of
C.sub.12-C.sub.15 ethoxylated alcohols with an 0-6.5% average
degree of ethoxylation of 9 A blend of C.sub.13-C.sub.15
ethoxylated alcohols with an 0-4% average degree of ethoxylation of
30 Sodium disilicate 0-33% Sodium tripolyphosphate 0-46% Sodium
citrate 0-28% Citric acid 0-29% Sodium carbonate 0-20% Sodium
perborate monohydrate 0-11.5% Tetraacetylethylenediamine (TAED)
0-4% Maleic acid/acrylic acid copolymer 0-7.5% Sodium sulphate
0-12.5% Enzymes 0.0001-0.1%
[0243] 7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION
24 Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0%
Alkali metal silicate 3.0-15.0% Alkali metal phosphate 20.0-40.0%
Liquid carrier selected from higher glycols, 25.0-45.0%
polyglycols, polyoxides, glycolethers Stabilizer (e.g. a partial
ester of phosphoric acid and 0.5-7.0% a C.sub.16-C.sub.18 alkanol)
Foam suppressor (e.g. silicone) 0-1.5% Enzymes 0.0001-0.1%
[0244] 8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION
25 Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0%
Sodium silicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium
citrate 0.0-1.5% Stabilizing system (e.g. mixtures of finely
divided 0.5-7.0% silicone and low molecular weight dialkyl
polyglycol ethers) Low molecule weight polyacrylate polymer
5.0-15.0% Clay gel thickener (e.g. bentonite) 0.0-10.0%
Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes 0.0001-0.1% Liquid
carrier selected from higher lycols, Balance polyglycols,
polyoxides and glycol ethers
[0245] 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION
26 C.sub.12-C.sub.14 fatty acid 0-0.5% Block co-polymer surfactant
1.5-15.0% Sodium citrate 0-12% Sodium tripolyphosphate 0-15% Sodium
carbonate 0-8% Aluminum tristearate 0-0.1% Sodium cumene sulphonate
0-1.7% Polyacrylate thickener 1.32-2.5% Sodium polyacrylate
2.4-6.0% Boric acid 0-4.0% Sodium formate 0-0.45% Calcium formate
0-0.2% Sodium n-decydiphenyl oxide disulphonate 0-4.0% Monoethanol
amine (MEA) 0-1.86% Sodium hydroxide (50%) 1.9-9.3% 1,2-Propanediol
0-9.4% Enzymes 0.0001-0.1% Suds suppressor, dye, perfumes, water
Balance
[0246] 10) LIQUID AUTOMATIC DISHWASHING COMPOSITION
27 Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30%
Sodium dodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid
0-10% Sodium disilicate monohydrate 18-33% Sodium citrate dihydrate
18-33% Sodium stearate 0-2.5% Sodium perborate monohydrate 0-13%
Tetraacetylethylenediamine (TAED) 0-8% Maleic acid/acrylic acid
copolymer 4-8% Enzymes 0.0001-0.1%
[0247] 11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING
PROTECTED BLEACH PARTICLES
28 Sodium silicate 5-10% Tetrapotassium pyrophosphate 15-25% Sodium
triphosphate 0-2% Potassium carbonate 4-8% Protected bleach
particles, e.g. chlorine 5-10% Polymeric thickener 0.7-1.5%
Potassium hydroxide 0-2% Enzymes 0.0001-0.1% Water Balance
[0248] 11) Automatic dishwashing compositions as described in 1),
2), 3), 4), 6) and 10), wherein perborate is replaced by
percarbonate.
[0249] 12) Automatic dishwashing compositions as described in 1)-6)
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
369,1994, pp. 637-639.
[0250] An .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 variant may be
added in an amount corresponding to 0.00001-1 mg (calculated as
pure enzyme protein) of .alpha.-amylase per liter of wash/dishwash
liquor.
[0251] The present invention is further described with reference to
the appended drawing, in which:
[0252] FIG. 1 is an alignment of the amino acid sequences of four
parent .alpha.-amylases in the context of the invention. The
numbers on the extreme left designate the respective amino acid
sequences as follows:
[0253] 1: the amino acid sequence shown in SEQ ID No. 1;
[0254] 2: the amino acid sequence shown in SEQ ID No. 2;
[0255] 3: the amino acid sequence shown in SEQ ID No. 3; and
[0256] 4: the amino acid sequence shown in SEQ ID No. 7.
[0257] The numbers on the extreme right of the figure give the
running total number of amino acids for each of the sequences in
question. It should be noted that for the sequence numbered 3
(corresponding to the amino acid sequence shown in SEQ ID No. 3),
the alignment results in "gaps" at the positions corresponding to
amino acid No. 1 and amino acid No. 175, respectively, in the
sequences numbered 1 (SEQ ID No. 1), 2 (SEQ ID No. 2) and 4 (SEQ ID
No. 7).
[0258] FIG. 2 is a restriction map of plasmid pTVB106.
[0259] FIG. 3 is a restriction map of plasmid pPM103.
[0260] FIG. 4 is a restriction map of plasmid pTVB112.
[0261] FIG. 5 is a restriction map of plasmid pTVB114.
EXPERIMENTAL SECTION
[0262] The preparation, purification and sequencing of the parent
.alpha.-amylases having the amino acid sequences shown in SEQ ID
No. 1 and SEQ ID No. 2 (from Bacillus strains NCIB 12512 and NCIB
12513, respectively) is described in WO 95/26397. The pI values and
molecular weights of these two parent .alpha.-amylases (given in WO
95/26397) are as follows:
[0263] SEQ ID No. 1: pI about 8.8-9.0 (determined by isoelectric
focusing on LKB Ampholine.quadrature. PAG plates); molecular weight
approximately 55 kD (determined by SDS-PAGE).
[0264] SEQ ID No. 2: pI about 5.8 (determined by isoelectric
focusing on LKB Ampholine.quadrature. PAG plates); molecular weight
approximately 55 kD (determined by SDS-PAGE).
[0265] Purification of .alpha.-amylase Variants of the
Invention
[0266] The construction and expression of variants according to the
invention is described in Example 2, below. The purification of
variants of the invention is illustrated here with reference to
variants of the amino acid sequences shown in SEQ ID No. 1 and SEQ
ID No. 2, respectively:
[0267] Purification of SEQ ID No. 1 variants (PI approx. 9.0): The
fermentation liquid containing the expressed .alpha.-amylase
variant is filtered, and ammonium sulfate is added to a
concentration of 15% of saturation. The liquid is then applied onto
a hydrophobic column (Toyopearl butyl/TOSOH). The column is washed
with 20 mM dimethyl-glutaric acid buffer, pH 7.0. The
.alpha.-amylase is bound very tightly, and is eluted with 25% w/w
2-propanol in 20 mM dimethylglutaric acid buffer, pH 7.0. After
elution, the 2-propanol is removed by evaporation and the
concentrate is applied onto a cation exchanger
(S-Sepharose.quadrature. FF, Pharmacia, Sweden) equilibrated with
20 mM dimethylglutaric acid buffer, pH 6.0.
[0268] The amylase is eluted using a linear gradient of 0-250 mM
NaCl in the same buffer. After dialysis against 10 mM borate/KCl
buffer, pH 8.0, the sample is adjusted to pH 9.6 and applied to an
anion exchanger (Q-Sepharose.quadrature. FF, Pharmacia)
equilibrated with 10 mM borate/KCl buffer, pH 9.6. The amylase is
eluted using a linear gradient of 0-250 mM NaCl. The pH is adjusted
to 7.5. The .alpha.-amylase is pure as judged by rSDS-PAGE. All
buffers contain 2 mM CaCl.sub.2 in order to stabilize the
amylase.
[0269] Purification of SEQ ID No. 2 variants (pl approx. 5.8): The
fermentation liquid containing the expressed .alpha.-amylase
variant is filtered, and ammonium sulfate is added to a
concentration of 15% of saturation. The liquid is then applied onto
a hydrophobic column (Toyopearl butyl/TOSOH). The bound amylase is
eluted with a linear gradient of 15%-0% w/w ammonium sulfate in 10
mM Tris buffer, pH 8.0. After dialysis of the eluate against 10 mM
borate/KCl buffer, pH 8.0, the liquid is adjusted to pH 9.6 and
applied onto an anion exchanger (Q-Sepharose.quadrature. FF,
Pharmacia) equilibrated with the same buffer. The amylase is
step-eluted using 150 mM NaCl.
[0270] After elution the amylase sample is dialyzed against the
same buffer, pH 8.0, in order to remove the NaCl. After dialysis,
the pH is adjusted to 9.6 and the amylase is bound once more onto
the anion exchanger. The amylase is eluted using a linear gradient
of 0-250 mM NaCl. The pH is adjusted to 7.5. The amylase is pure as
judged by rSDS-PAGE. All buffers contain 2 mM CaCl.sub.2 in order
to stabilize the amylase.
[0271] Determination of .alpha.-amylase Activity
[0272] .alpha.-Amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-colored starch polymer which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0273] For the determination of every single measurement one tablet
is suspended in a tube containing 5 ml 50 mM Britton-Robinson
buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid,
0.1 mM CaCl.sub.2, pH adjusted to the value of interest with NaOH).
The test is performed in a water bath at the temperature of
interest. The .alpha.-amylase to be tested is diluted in x ml of 50
mM Britton-Robinson buffer. 1 ml of this .alpha.-amylase solution
is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is
hydrolyzed by the .alpha.-amylase giving soluble blue fragments.
The absorbance of the resulting blue solution, measured
spectrophotometrically at 620 nm, is a function of the
.alpha.-amylase activity.
[0274] It is important that the measured 620 nm absorbance after 15
minutes of incubation (testing time) is in the range of 0.2 to 2.0
absorbance units at 620 nm. In this absorbance range there is
linearity between activity and absorbance (Lambert-Beer law). The
dilution of the enzyme must therefore be adjusted to fit this
criterion.
[0275] Under a specified set of conditions (temp., pH, reaction
time, buffer conditions) 1 mg of a given .alpha.-amylase will
hydrolyze a certain amount of substrate and a blue color will be
produced. The color intensity is measured at 620 nm. The measured
absorbance is directly proportional to the specific activity
(activity/mg of pure .alpha.-amylase protein) of the
.alpha.-amylase in question under the given set of conditions. Thus
testing different .alpha.-amylases of interest (including a
reference .alpha.-amylase, in this case the parent .alpha.-amylase
in question) under identical conditions, the specific activity of
each of the .alpha.-amylases at a given temperature and at a given
pH can be compared directly, and the ratio of the specific activity
of each of the .alpha.-amylases of interest relative to the
specific activity of the reference .alpha.-amylase can be
determined.
[0276] Mini Dishwashing Assay
[0277] The following mini dishwashing assay was used: A suspension
of starchy material was boiled and cooled to 20.quadrature.C. The
cooled starch suspension was applied on small, individually
identified glass plates (approx. 2.times.2 cm) and dried at a
temperature of ca. 140.quadrature.C in a drying cabinet. The
individual plates were then weighed. For assay purposes, a solution
of standard European-type automatic dishwashing detergent (5 g/l)
having a temperature of 55.quadrature.C was prepared. The detergent
was allowed a dissolution time of 1 minute, after which the
.alpha.-amylase in question was added to the detergent solution
(contained in a beaker equipped with magnetic stirring) so as to
give an enzyme concentration of 0.5 mg/l. At the same time, the
weighed glass plates, held in small supporting clamps, were
immersed in a substantially vertical position in the
.alpha.-amylase/detergent solution, which was then stirred for 15
minutes at 55.quadrature.C. The glass plates were then removed from
the .alpha.-amylase/detergent solution, rinsed with distilled
water, dried at 60.quadrature.C in a drying cabinet and re-weighed.
The performance of the .alpha.-amylase in question [expressed as an
index relative to a chosen reference .alpha.-amylase (index
100)--in the example below (Example 1) the parent .alpha.-amylase
having the amino acid sequence shown in SEQ ID No. 1] was then
determined from the difference in weight of the glass plates before
and after treatment, as follows: 1 Index =
weightlossforplatetreatedwith-amylase
weightlossforplatetreatedwithre- ference .cndot. 100
[0278] The following examples further illustrate the present
invention. They are not intended to be in any way limiting to the
scope of the invention as claimed.
EXAMPLE 1
Mini Dishwashing Test of Variants of Parent .alpha.-amylase Having
the Amino Acid Sequence Shown in SEQ ID No. 1
[0279] The above-described mini dishwashing test was performed at
pH 10.5 with the parent .alpha.-amylase having the amino acid
sequence shown in SEQ ID No. 1 and the following variants thereof
(the construction and purification of which is described below):
T183*+G184*; Y243F; and K269R. The test gave the following
results:
29 Parent (SEQ ID No. 1) Index: 100 T183* + G184* Index: 120 Y243F
Index: 120 K269R Index: 131
[0280] It is apparent that the each of the tested variants
T183*+G184*(which exhibits, inter alia, higher thermal stability
than the parent .alpha.-amylase), Y243F (which exhibits lower
calcium ion dependency than the parent .alpha.-amylase) and K269R
(which exhibits lower calcium ion dependency and higher stability
at high pH than the parent .alpha.-amylase) exhibits significantly
improved dishwashing performance relative to the parent
.alpha.-amylase.
EXAMPLE 2
Construction of Variants of the Parent .alpha.-amylases Having the
Amino Acid Sequences Shown in SEQ ID No. 1 and SEQ ID No. 2,
Respectively
[0281] Primers: DNA primers employed in the construction of
variants as described below include the following [all DNA primers
are written in the direction from 5' to 3' (left to right); P
denotes a 5' phosphate]:
30 #7113: GCT GCG GTG ACC TCT TTA AAA AAT AAC GGC Y296: CC ACC GCT
ATT AGA TGC ATT GTA C #6779: CTT ACG TAT GCA GAC GTC GAT ATG GAT
CAC CC #6778: G ATC CAT ATC GAC GTC TGC ATA CGT AAG ATA GTC #3811:
TT A(C/G)G GGC AAG GCC TGG GAC TGG #7449: C CCA GGC CTT GCC C(C/G)T
AAA TTT ATA TAT TTT GTT TTG #3810: G GTT TCG GTT CGA AGG ATT CAC
TTC TAC CGC #7450: GCG GTA GAA GTG AAT CCT TCG AAC CGA AAC CAG B1:
GGT ACT ATC GTA ACA ATG GCC GAT TGC TGA CGC TGT TAT TTG C #6616: P
CTG TGA CTG GTG AGT ACT CAA CCA AGT C #8573: CTA CTT CCC AAT CCC
AAG CTT TAC CTC GGA ATT TG #8569: CAA ATT CCG AGG TAA AGC TTG GGA
TTG GGA AGT AG #8570: TTG AAC AAC CGT TCC ATT AAG AAG
[0282] A: Construction of Variants of the Parent .alpha.-amylase
Having the Amino Acid Sequence Shown in SEQ ID No. 1
[0283] Description of plasmid pTVB106: The parent .alpha.-amylase
having the amino acid sequence shown in SEQ ID No. 1 and variants
thereof are expressed from a plasmid-borne gene, SF16, shown in
FIG. 2. The plasmid, pTVBl06, contains an origin of replication
obtained from plasmid pUB 110 (Gryczan et al., 1978) and the cat
gene conferring resistance towards chloramphenicol. Secretion of
the amylase is aided by the Termamyl[ ] signal sequence that is
fused precisely, i.e. codon No. 1 of the mature protein, to the
gene encoding the parent .alpha.-amylase having the nucleotide and
amino acid sequence (mature protein) shown in SEQ ID No. 4 and SEQ
ID No. 1, respectively. The Termamyl promoter initiates
transcription of the gene.
[0284] Plasmid pTVB106 is similar to pDN1528 (see laid-open Danish
patent application No. 1155/94). Some unique restriction sites are
indicated on the plasmid map in FIG. 2, including BstBI, BamHI,
BstEII, EcoNI, DrdI, AflIII, DraIII, Xmal, SalI and BgilII.
[0285] Construction of variant M202T: The PCR overlap extension
mutagenesis method is used to construct this variant (Higuchi et
al., 1988). An approximately 350 bp DNA fragment of pTVB106 is
amplified in a PCR reaction A using primers #7113 and mutagenic
primer#6778. In a similar PCR reaction B, an approximately 300 bp
DNA fragment is amplified using primers Y296 and #6779. The
complete DNA fragment spanning the mutation site (M202) from primer
#7113 to primer Y296 is amplified in PCR C using these primers and
purified DNA fragments from reactions A and B.
[0286] PCR C DNA is digested with restriction endonucleases BstEII
and AflIII, and the 480 bp fragment is ligated with plasmid pTVB106
digested with the same enzymes and transformed into a low-protease
and low-amylase Bacillus subtilis strain (e.g. strain SHA273
mentioned in WO 92/11357).
[0287] Other M202 variants are constructed in a similar manner.
[0288] Construction of variants T183*+G184*and R181*+G182*: The PCR
overlap extension mutagenesis method is used to construct these
variants (Higuchi et al., 1988). The mutagenic oligonucleotides are
synthesized using a mixture (equal parts) of C and G in one
position; two different mutations can therefore be constructed by
this procedure. An approximately 300 bp DNA fragment of pTVB106 is
amplified in a PCR reaction A using primers #7113 and mutagenic
primer #7449. In a similar PCR reaction B, an approximately 400 bp
DNA fragment is amplified using primers Y296 and #3811. The
complete DNA fragment spanning the mutation site (amino acids
181-184) from primer #7113 to primer Y296 is amplified in PCR C
using these primers and purified DNA fragments from reactions A and
B.
[0289] PCR C DNA is digested with restriction endonucleases BstEII
and AflIII and the 480 bp fragment is ligated with plasmid pTVB106
digested with the same enzymes and transformed into a low-protease
and low-amylase B. subtilis strain (e.g. strain SHA273 mentioned in
WO 92/11357). Sequencing of plasmid DNA from these transformants
identifies the two correct mutations: i.e. R181*+G182*and
T183*+G184*.
[0290] Construction of variant R124P: The PCR overlap extension
mutagenesis method is used to construct this variant in a manner
similar to the construction of variant M202T (vide supra). PCR
reaction A (with primers #3810 and B1) generates an approximately
500 bp fragment, and PCR reaction B (primers 7450 and Y296)
generates an approximately 550 bp fragment. PCR reaction C based on
the product of PCR reaction A and B and primers B1 and Y296 is
digested with restriction endonucleases BstEII and AflIII, and the
resulting 480 bp fragment spanning amino acid position 124 is
subcloned into pTVB106 digested with the same enzymes and
transformed into B. subtilis as previously described.
[0291] Construction of variant R124P+T183*+G184*: For the
construction of the variant combining the R124P and the
T183*+G184*mutations, two EcoNI restriction sites (one located at
position 1.774 kb, i.e. between the R124P mutation and the
T183*+G184*mutation, and one located at position 0.146 kb) were
utilized. The approximately 1630 bp EcoNI fragment of the
pTVB106-like plasmid containing the T183*+G184*mutation was
subcloned into the vector part (approximately 3810 bp DNA fragment
containing the origin of replication) of another pTVB106-like
plasmid containing the R124P mutation digested with the same
enzyme. Transformation into Bacillus subtilis was carried out as
previously described.
[0292] Construction of variants G182*+G184*; R181*+T183*; Y243F;
K269R; and L351C+M430C: These variants were constructed as
follows:
[0293] A specific mutagenesis vector containing a major part of the
coding region for the amino acid sequence shown in SEQ ID No. 1 was
prepared. The important features of this vector (which is denoted
pPM103) include an origin of replication derived from the pUC
plasmid, the cat gene conferring resistance towards chloramphenicol
and a frameshift-mutation-containing version of the bla gene, the
wild-type version of which normally confers resistance towards
ampicillin (amp.sup.R phenotype). This mutated version of the bla
gene results in an amp.sup.S phenotype. The plasmid pPM103 is shown
in FIG. 3, and the E. coli origin of replication, the 5'-truncated
version of the SF16 amylase gene, and ori, bla, cat and selected
restriction sites are indicated on the plasmid.
[0294] Mutations are introduced in the gene of interest as
described by Deng and Nickoloff [Anal. Biochem. 200 (1992), pp.
81-88], except that plasmids with the "selection primer" (#6616)
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 using the selection by restriction-enzyme digestion
outlined by Deng and Nickoloff. Chemicals and enzymes used for the
mutagenesis were obtained from the Chameleon.quadrature.
mutagenesis kit from Stratagene (catalogue number 200509).
[0295] After verification of the DNA sequence in variant plasmids,
the truncated gene containing the desired alteration is subcloned
from the pPM103-like plasmid into pTVB 106 as an approximately 1440
bp BstBI-SalI fragment and transformed into Bacillus subtilis for
expression of the variant enzyme.
[0296] For the construction of the pairwise deletion variant
G182*+G184*, the following mutagenesis primer was used:
31 P CTC TGT ATC GAC TTC CCA GTC CCA AGC TTT TGT CCT GAA TTT ATA
TAT TTT GTT TTG AAG
[0297] For the construction of the pairwise deletion variant
R181*+T183*, the following mutagenesis primer was used:
32 P CTC TGT ATC GAG TTC CCA GTC CCA AGC TTT GCC TCC GAA TTT ATA
TAT TTT GTT TTG AAG
[0298] For the construction of the substitution variant Y243F, the
following mutagenesis primer was used:
33 P ATG TGT AAG CCA ATC GCG AGT AAA GCT AAA TTT TAT ATG TTT CAC
TGC ATC
[0299] For the construction of the substitution variant K269R, the
following mutagenesis primer was used:
34 P GC ACC AAG GTC ATT TCG CCA GAA TTC AGC CAC TG
[0300] For the construction of the pairwise substitution variant
L351C+M430C, the following mutagenesis primers were used
simultaneously:
35 1) P TGT CAG AAC CAA CGC GTA TGC ACA TGG TTT AAA CCA TTG 2) P
ACC ACC TGG ACC ATC GCT GCA GAT GGT GGC AAG GCC TGA ATT
[0301] Construction of variant L351C+M430C+T183*+G184*: This
variant was constructed by combining the L351 C+M430C pairwise
substitution mutation and the T183*+G184*pairwise deletion mutation
by subcloning an approximately 1430 bp HindIII-AflII fragment
containing L351C+M430C into a pTVB106-like plasmid (with the
T183*+G184*mutations) digested with the same enzymes.
[0302] Construction of variant Y243F+T183*+G184*: This variant was
constructed by combining the Y243F mutation and the
T183*+G184*mutation by subcloning an approximately 1148 bp DrdI
fragment containing T183*+G184*into a pTVB106-like plasmid (with
the Y243 mutation) digested with the same enzyme.
[0303] Bacillus subtilis transformants were screened for
.alpha.-amylase activity on starch-containing agar plates and the
presence of the correct mutations was checked by DNA
sequencing.
[0304] Construction of variant Y243F+T183*+G184*+L351C+M430C: The
L351C+M430C pairwise substitution mutation was subcloned as an
approximately 470 bp XmaI-SalI fragment into a pTVB106-like vector
(containing Y243F+T183*+G184*) digested with the same enzymes.
[0305] Construction of variant Y243F+T183*+G184*+L351C+M430C+Q391
E+K444Q: A pPM103-like vector containing the mutations
Y243F+T183*+G184*+L351C+M43- 0C was constructed by substituting the
truncated version of SF16 in pPM103 with the approximately 1440 bp
BstB1-SalI fragment of the pTVB106-like vector containing the five
mutations in question. The Q391 E and K444Q mutations were
introduced simultaneously into the pPM103-like vector (containing
Y243F+T183*+G184*+L351C+M430C) by the use of the following two
mutagenesis primers in a manner similar to the previously described
mutagenesis on pPM103:
36 P GGC AAA AGT TTG ACG TGC CTC GAG AAG AGG GTC TAT P TTG TCC CGC
TTT ATT CTG GCC AAC ATA CAT CCA TTT
[0306] B: Construction of Variants of the Parent .alpha.-amylase
Having the Amino Acid Sequence Shown in SEQ ID No. 2
[0307] Description of plasmid pTVB112: A vector, denoted pTVB112,
to be used for the expression in B. subtilis of the .alpha.-amylase
having the amino acid sequence shown in SEQ ID No. 2 was
constructed. This vector is very similar to pTVB106 except that the
gene encoding the mature .alpha.-amylase of SEQ ID No. 2 is
inserted between the PstI and the HindIII sites in pTVB106. Thus,
the expression of this .alpha.-amylase (SEQ ID No. 2) is also
directed by the amyL promoter and signal sequence. The plasmid
pTVB112 is shown in FIG. 4.
[0308] Construction of variant D183*+G184*: The construction of
this variant was achieved using the PCR overlap extension
mutagenesis method referred to earlier (vide supra). Primers #8573
and B1 were used in PCR reaction A, and primers #8569 and #8570
were used in PCR reaction B. The purified fragments from reaction A
and reaction B and primers 1 B and #8570 were used in PCR reaction
C, resulting in an approximately 1020 bp DNA fragment. This
fragment was digested with restriction endonucleases PstI and MluI,
and subcloned into the expression vector and transformed into B.
subtilis.
[0309] Construction of further variants: By analogy with the
construction (vide supra) of the plasmid pPM103 used in the
production of mutants of the amino acid sequence of SEQ ID No. 1, a
plasmid (denoted pTVB114; shown in FIG. 5) was constructed for the
continued mutagenesis on variant D183*+G184*(SEQ.ID No. 2).
Mutations were introduced in pTVB114 (SEQ ID No. 2; D183*+G184*) in
a manner similar to that for pPM103 (SEQ ID No. 1).
[0310] For the construction of the pairwise deletion variants
R181*+D183*and R181*+G182*, it was chosen to alter the flanking
amino acids in the variant D183*+G184*instead of deleting the
specified amino acids in the wild type gene for SEQ ID No. 2. The
following mutagenesis primer was used for the mutagenesis with
pTVB114 as template:
37 PCC CAA TCC CAA GCT TTA CCA (T/C)CG AAC TTG TAG ATA CG
[0311] The presence of a mixture of two bases (T/C) at one position
allows for the presence of two different deletion flanking amino
acid based on one mutagenesis primer. DNA sequencing of the
resulting plasmids verifies the presence of either the one or the
other mutation. The mutated gene of interest is subcloned as a
PstI-DraIII fragment into pTVB112 digested with the same enzymes
and transformed into B. subtilis.
[0312] For the construction of G182*+G184*and R181*+G184*, the
following mutagenesis primer was used with pTVB114 as template:
38 PCC CAA TCC CAA GCT TTA TCT C(C/G)G AAC TTG TAG ATA CG
[0313] As before, the presence of a mixture of two bases (C/G) at
one position allows for the presence of two different deletion
flanking amino acid based on one mutagenesis primer. DNA sequencing
of the resulting plasmids verifies the presence of either the one
or the other mutation. The mutated gene of interest is subcloned as
a PstI-DraIII fragment into pTVB112 digested with the same enzymes
and transformed into B. subtilis.
[0314] For the construction of D183*+G 184*+M202L the following
mutagenesis primer was used:
39 PGA TCC ATA TCG ACG TCT GCA TAC AGT AAA TAA TC
[0315] For the construction of D183*+G 184*+M202I the following
mutagenesis primer was used:
40 PGA TCC ATA TCG ACG TCT GCA TAA ATT AAA TAA TC
EXAMPLE 3
DETERMINATION OF OXIDATION STABILITY OF M202 SUBSTITUTION VARIANTS
OF THE PARENT .alpha.-amylases HAVING THE AMINO ACID SEQUENCES
SHOWN IN SEQ ID No. 1 AND SEQ ID No. 2
[0316] A: Oxidation Stability of Variants of the Sequence in SEQ ID
No. 1
[0317] The measurements were made using solutions of the respective
variants in 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM
phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2, pH adjusted
to the value of interest with NaOH), pH 9.0, to which hydrogen
peroxide was added (at time t=0) to give a final concentration of
200 mM H.sub.2O.sub.2. The solutions were then incubated at
40.quadrature.C in a water bath.
[0318] After incubation for 5, 10, 15 and 20 minutes after addition
of hydrogen peroxide, the residual .alpha.-amylase activity was
measured using the Phadebas assay described above. The residual
activity in the samples was measured using 50 mM Britton-Robinson
buffer, pH 7.3, at 37.quadrature.C (see Novo analytical publication
AF207-1/1, available on request from Novo Nordisk A/S). The decline
in activity was measured relative to a corresponding reference
solution of the same enzyme at 0 minutes which was not incubated
with hydrogen peroxide (100% activity).
[0319] The percentage of initial activity as a function of time is
shown in the table below for the parent enzyme (SEQ ID No. 1) and
for the variants in question.
41 % Activity after incubation for (minutes) Variant 0 5 10 15 20
M202L 100 90 72 58 27 M202F 100 100 87 71 43 M202A 100 99 82 64 30
M202I 100 91 75 59 28 M202T 100 87 65 49 20 M202V 100 100 87 74 43
M202S 100 100 85 68 34 Parent 100 51 26 13 2
[0320] All the M202 substitution variants tested clearly exhibit
significantly improved stability towards oxidation relative to the
parent .alpha.-amylase (SEQ ID No. 1).
[0321] B: Oxidation Stability of Variants of the Sequence in SEQ ID
No. 2
[0322] Measurements were made as described above using the parent
.alpha.-amylase in question (SEQ ID No. 2), the variant
M202L+D183*+G184*(designated L in the table below) and the variant
M202I+D183*+G184*(designated L in the table below), respectively.
In this case, incubation times (after addition of hydrogen
peroxide) of 5, 10, 15 and 30 minutes were employed. As in the
table above, the percentage of initial activity as a function of
time is shown in the table below for the parent enzyme and for the
variants in question.
42 % Activity after incubation for (minutes) Variant 0 5 10 15 30 L
100 91 85 71 43 I 100 81 61 44 18 Parent 100 56 26 14 4
[0323] The two "substitution+pairwise deletion" variants tested
(which both comprise an M202 substitution) clearly exhibit
significantly improved stability towards oxidation relative to the
parent .alpha.-amylase (SEQ ID No. 2).
EXAMPLE 4
Determination of Thermal Stability of Variants of the Parent
.alpha.-amylases Having the Amino Acid Sequences Shown in SEQ ID
No. 1 and SEQ ID No. 2
[0324] A: Thermal Stability of Dairwise Deletion Variants of the
Sequence in SEQ ID No. 1
[0325] Measurements were made using solutions of the respective
variants in 50 mM Britton-Robinson buffer (vide supra), pH 9.0. The
solutions were incubated at 65.degree. C. in a water bath, and
samples were withdrawn after incubation for the indicated periods
of time. The residual .alpha.-amylase activity of each withdrawn
sample was measured using the Phadebas assay, as described above.
The decline in activity was measured relative to a corresponding
reference solution of the same enzyme at 0 minutes which was not
incubated (100% activity).
[0326] The percentage of initial activity as a function of time is
shown in the table below for the parent enzyme (SEQ ID No. 1) and
for the following pairwise deletion variants in question:
[0327] Variant 1: R181*+G182*
[0328] Variant 2: R181*+T183*
[0329] Variant 3: G182*+G184*
[0330] Variant 4: T183*+G184*
[0331] Variant 5: T183*+G184*+R124P
43 % Activity after incubation for (minutes) Variant 0 5 10 15 30
45 60 1 100 81 66 49 24 14 8 2 100 80 53 39 17 8 3 3 100 64 40 28
10 4 2 4 100 64 43 34 20 8 5 5 100 78 73 66 57 47 38 Parent 100 13
2 0 0 0 0
[0332] It is apparent that all of the pairwise deletion variants
tested exhibit significantly improved thermal stability relative to
the parent .alpha.-amylase (SEQ ID No. 1), and that the thermal
stability of Variant 5, which in addition to the pairwise deletion
mutation of Variant 4 comprises the substitution R124P, is markedly
higher than that of the other variants. Since calorimetric results
for the substitution variant R124P (comprising only the
substitution R124P) reveal an approximately 7.quadrature.C
thermostabilization thereof relative to the parent .alpha.-amylase,
it appears that the thermostabilizing effects of the mutation R124P
and the pairwise deletion, respectively, reinforce each other.
[0333] B: Thermal Stability of Pairwise Deletion Variants of the
Sequence in SEQ ID No. 2
[0334] Corresponding measurements were made for the parent enzyme
(SEQ ID No. 2) and for the following pairwise deletion
variants:
[0335] Variant A: D183*+G184*
[0336] Variant B: R181*+G182*
[0337] Variant C: G182*+G184*
44 % Activity after incubation for (minutes) Variant 0 5 10 15 30 A
100 87 71 63 30 B 100 113 85 76 58 C 100 99 76 62 34 Parent 100 72
55 44 18
[0338] Again, it is apparent that the pairwise deletion variants in
question exhibit significantly improved thermal stability relative
to the parent .alpha.-amylase (SEQ ID No. 2).
[0339] C: Thermal Stability of a Multi-Combination Variant of the
Sequence in SEQ ID No. 1
[0340] Corresponding comparative measurements were also made for
the following variants of the amino acid sequence shown in SEQ ID
No. 1:
[0341] Variant 4: T183*+G184*
[0342] Variant 6: L351 C+M430C
[0343] Variant 7: Y243F
[0344] Variant 8: Q391 E+K444Q
[0345] Variant 9: T183*+G184*+L351C+M430C+Y243F+Q391 E+K444Q
45 % Activity after incubation for (minutes) Variant 0 5 10 15 30 4
100 66 41 22 7 6 100 87 73 65 43 7 100 14 2 1 0 8 100 69 46 31 14 9
100 92 93 89 82
[0346] Again, it appears that the thermostabilizing effect of
multiple mutations, each of which has a thermostabilizing effect,
is--at least qualitatively--cumulative.
EXAMPLE 5
CALCIUM-BINDING AFFINITY OF .alpha.-amylase VARIANTS OF THE
INVENTION
[0347] 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 a series 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 .mu.g/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.quadrature.C).
[0348] 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.Nlog([Ca]))+K.sub.diss/(-
K.sub.diss+[Ca])(.alpha..sub..orgate.-.beta..sub..orgate.log([Ca]))
[0349] 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 au on
the logarithm of the calcium concentration. K.sub.diss is the
apparent calcium-binding constant for an equilibrium process as
follows: 2 N -Ca .cndot. K diss U + Ca ( N = nativeenzyme ; U =
unfoldedenzyme )
[0350] 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.quadrature.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.
[0351] The following values for K.sub.diss were obtained for the
parent .alpha.-amylases having the amino acid sequences shown in
SEQ ID No. 1 and SEQ ID No. 2, and for the indicated
.alpha.-amylase variants according to the invention (the parent
.alpha.-amylase being indicated in parentheses):
46 Variant K.sub.diss (mol/l) D183* + G184* (SEQ ID No. 2) 1.2
(.+-.0.5) .times. 10.sup.-4 L351C + M430C + T183* + G184* 1.7
(.+-.0.5) .times. 10.sup.-3 (SEQ ID No. 1) T183* + G184* (SEQ ID
No. 1) 4.3 (.+-.0.7) .times. 10.sup.-3 SEQ ID No. 2 (parent) 4.2
(.+-.1.2) .times. 10.sup.-2 SEQ ID No. 1 (parent) 3.5 (.+-.1.1)
.times. 10.sup.-1
[0352] It is apparent from the above that the calcium-binding
affinity of the latter .alpha.-amylolytic enzymes decreases in a
downward direction through the above table, i.e. that the pairwise
deletion variant D183*+G184*(SEQ ID No. 2) binds calcium most
strongly (i.e. has the lowest calcium dependency) whilst the parent
.alpha.-amylase of SEQ ID No. 1 binds calcium least strongly (i.e.
has the highest calcium dependency).
REFERENCES CITED IN THE SPECIFICATION
[0353] Suzuki et al., the Journal of Biological Chemistry, Vol.
264, No. 32, Issue of November 15, pp. 18933-18938 (1989).
[0354] Hudson et al., Practical Immunology, Third edition (1989),
Blackwell Scientific Publications.
[0355] Lipman and Pearson (1985) Science 227, 1435.
[0356] Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor, 1989.
[0357] S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22,
1981, pp. 1859-1869.
[0358] Matthes et al., The EMBO J. 3, 1984, pp. 801-805.
[0359] R. K. Saiki et al., Science 239, 1988, pp. 487-491.
[0360] Morinaga et al., 1984, Biotechnology 2, pp. 646-639.
[0361] Nelson and Long, Analytical Biochemistry 180, 1989,
pp.147-151.
[0362] Hunkapiller et al., 1984, Nature 310, pp. 105-111.
[0363] R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general
method of in vitro preparation and specific mutagenesis of DNA
fragments: study of protein and DNA interactions. Nucl. Acids Res.
16, pp. 7351-7367.
[0364] Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.
[0365] Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.
[0366] S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp.
1680-1682.
[0367] Boel et al., 1990, Biochemistry 29, pp. 6244-6249.
[0368] Deng and Nickoloff, 1992, Anal. Biochem. 200, pp. 81-88.
Sequence CWU 1
1
* * * * *