U.S. patent application number 15/883113 was filed with the patent office on 2018-08-02 for cleaning compositions comprising amylase variants.
The applicant listed for this patent is The Procter & Gamble Company. Invention is credited to Carsten ANDERSEN, Iben DAMAGER, Chakshusmathi GHADIYARAM, Padma Venkatachalam IYER, Rajendra Kulothungan SAINATHAN.
Application Number | 20180216039 15/883113 |
Document ID | / |
Family ID | 61094358 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180216039 |
Kind Code |
A1 |
ANDERSEN; Carsten ; et
al. |
August 2, 2018 |
CLEANING COMPOSITIONS COMPRISING AMYLASE VARIANTS
Abstract
The present invention relates to cleaning compositions including
variants of an alpha-amylase and methods of treating surfaces such
as textiles with aqueous liquor including such compositions,
especially at low temperatures.
Inventors: |
ANDERSEN; Carsten;
(Bagsvaerd, DK) ; GHADIYARAM; Chakshusmathi;
(Bangalore, IN) ; IYER; Padma Venkatachalam;
(Bangalore, IN) ; SAINATHAN; Rajendra Kulothungan;
(Bangalore, IN) ; DAMAGER; Iben; (Bagsvaerd,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Procter & Gamble Company |
Cincinnati |
OH |
US |
|
|
Family ID: |
61094358 |
Appl. No.: |
15/883113 |
Filed: |
January 30, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 3/38618 20130101;
C11D 3/386 20130101; C11D 3/38681 20130101 |
International
Class: |
C11D 3/386 20060101
C11D003/386 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2017 |
IN |
201711003745 |
Claims
1. A cleaning composition comprising: a) a variant of a parent
alpha-amylase, wherein the variant (i) has at least about 80%, but
less than 100% sequence identity with the amino acid sequence set
forth in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, (ii)
comprises a modification at one or more positions corresponding to
109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence
set forth in SEQ ID NO: 1, and (iii) has alpha-amylase activity;
and b) from about 0.01 to about 99.9 wt % of a cleaning
adjunct.
2. A cleaning composition according to claim 1 wherein said variant
of a parent alpha-amylase comprises a modification at one or more
positions corresponding to positions 140, 181, 182, 183, 184, 195,
206, 243, 260, 304, and 476 of the amino acid sequence as set forth
in SEQ ID NO: 1.
3. A cleaning composition according to claim 1 wherein said variant
of a parent alpha-amylase has at least about 80% but less than 100%
sequence identity with the amino acid sequence set forth in SEQ ID
NO: 1.
4. A composition according to claim 1 wherein said variant
comprises a modification in at least two positions corresponding to
positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid
sequence as set forth in SEQ ID NO: 1.
5. A composition according to claim 1, wherein said variant
comprises a modification in at least two positions selected from
the group consisting of 1, 109 and 391.
6. A composition according to claim 1 wherein in the variant said
modification(s) is/are selected from the group consisting of: X1*,
X1A, X7A, X7K, X7E, X7N. X7Q, X7L, X7D, X109A, X109S, X140Y, X181*,
X182*, X183*, X184*, X195F, X206Y, X243F, X260G, X280S, X284H,
X284R, X284F, X304R, X320A, X320M, X320T, X320V, X320S, X323N,
X323R, X323S, X323K, X391A, X391V, and X476K.
7. A composition according to claim 1 wherein said variant has an
improved wash performance.
8. A composition according to claim 1 wherein said variant has at
least about 90%, but less than 100%, sequence identity to the amino
acid sequence of the parent alpha-amylase.
9. A composition according to claim 1 wherein in the variant the
number of modifications is 1 to 30.
10. A composition according to claim 1 wherein the variant
comprises modifications in the positions selected from the group of
positions consisting of: X1+X7; X1+X109; X1+X280; X1+X284; X1+X320;
X1+X323; X1+X391; X109+X280; X109+X284; X109+X320; X109+X323;
X109+X391; X7+X109; X7+X280; X7+X284; X7+X320; X7+X323; X7+X391;
X280+X284; X280+X320; X280+X323; X280+X391; X284+X320; X284+X323;
X284+X391; X320+X323; X320+X391; and X323+X391, wherein numbering
is according to SEQ ID NO: 1.
11. A composition according to claim 1 wherein the variant
comprises modifications in the positions corresponding to the
positions of the amino acid sequence set forth in SEQ ID NO: 2,
selected from the group consisting of: H1*+G109A+N280S+E391A;
H1*+G7K+G109A+N280S+E391A; H1*+G7E+G109A+N280S+E391A;
H1*+G7N+G109A+N280S+E391A; H1*+G7Q+G109A+N280S+E391A;
H1*+G7L+G109A+N280S+E391A; H1*+G7D+G109A+N280S+E391A;
H1*+G109A+N280S+K320A+E391A; H1*+G109A+N280S+K320M+E391A;
H1*+G109A+N280S+K320T+E391A; H1*+G109A+N280S+K320V+E391A;
H1*+G109A+N280S+M323R+E391A; H1*+G109A+N280S+K320S+E391A;
H1*+G109A+N280S+E391V; H1*+G109A+W284R+E391A;
H1*+G109A+W284F+E391A; H1*+G109A+N280S+K320A+M323S+E391A;
H1*+G109A+N280S+W284F+E391A; H1*+G109A+N280S+M323N+E391A;
H1*+G109A+N280S+M323K+E391A; H1*+G109S+N280S+E391A;
H1*+G109A+W284H+E391A; H1*+G109A+N280S+K320A+M323N+E391A;
H1*+G7A+G109A+N280S+E391A;
H1*+G7A+G109A+N280S+W284H+K320A+M323N+E391A; G7A+W284H+K320A+M323N;
G7A+K320A+M323N; K320A; G7A+K320A; H1*+G7A+G109A+N280S+E391A;
H1*+G109A+N280S+W284H+E391A; H1*+G109A+N280S+M323S+E391A;
H1*+G7A+G109A+N280S+K320A+E391A; H1*+G7A+G109A+N280S+M323S+E391A;
H1*+G7A+G109A+N280S+M323N+E391A; H1*+G7A+G109A+N280S+W284F+E391A;
H1*+G7A+G109A+N280S+W284R+E391A;
H1*+G7A+G109A+N280S+K320A+M323S+E391A; H1*+G7A+G109A+W284R+E391A;
and H1*+G7A+G109A+N280S+K320A+M323N+E391A.
12. A composition according to claim 1 wherein the parent
alpha-amylase for the variant is selected from the group consisting
of alpha-amylases having at least about 90%, identity to any of the
amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,
and 8.
13. A composition according to claim 12 wherein the parent
alpha-amylase for the variant is selected from the group consisting
of alpha-amylases having at least about 90% identity to any of the
amino acid sequences set forth in SEQ ID NOs: 1 or 2.
14. A composition according to claim 1, wherein the composition is
a liquid laundry detergent composition or unit dose detergent
composition.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to cleaning compositions
comprising variants of an alpha-amylase having improved cleaning
performance relative to its parent amylase in cold water surface
treatment processes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.
3.2.1.1) constitute a group of enzymes, which catalyse hydrolysis
of starch and other linear and branched 1,4-glucosidic oligo- and
polysaccharides.
[0004] Among the first bacterial alpha-amylases to be used were an
alpha-amylase from B. licheniformis, also known as Termamyl which
has been extensively characterized and the crystal structure has
been determined for this enzyme. Alkaline amylases, such as AA560
form a particular group of alpha-amylases that have found use in
detergents. Many of these known bacterial amylases have been
modified in order to improve their functionality in a particular
application. Bacillus amylases, such as Termamyl, AA560 (WO
2000/060060) and SP707 (described by Tsukamoto et al., 1988,
Biochem. Biophys. Res. Comm 151: 25-31) form a particular group of
alpha-amylases that have found use in detergents. These amylases
have been modified to improve the stability in detergents. WO
96/23873 e.g. disclose to delete the amino acids 181+182 or the
amino acids 183+184 of SP707 (SEQ ID NO: 7 of WO 96/23873) to
improve the stability of this amylase. WO 96/23873 further
discloses to modify the SP707 amylase by substituting M202 with
e.g. a leucine to stabilize the molecule towards oxidation. Thus,
it is known to modify amylases to improve certain properties.
[0005] For environmental reasons, it has been increasingly
important to lower the temperature in washing, dishwashing and/or
cleaning processes. However, most enzymes including amylases have a
temperature optimum which is above the temperature usually used in
low temperature washing. Alpha-amylase is a key enzyme for use in
detergent compositions and its use has become increasingly
important for removal of starchy stains during laundry washing or
dishwashing. Therefore, it is important to find alpha-amylase
variants, which retain their wash performance, stain removal effect
and/or activity when the temperature is lowered. However, despite
the efficiency of current detergent enzyme compositions, there are
many stains that are difficult to completely remove. These problems
are compounded by the increased use of low (e.g., cold water) wash
temperatures and shorter washing cycles. Thus, it is desirable to
have amylolytic enzymes that can function under low temperature and
at the same time preserve or increase other desirable properties
such as specific activity (amylolytic activity), stability and/or
wash performance to enable good cleaning in shorter washing
cycles.
[0006] Thus, it is an object of the present invention to provide
cleaning compositions comprising alpha-amylases variants which can
be used in washing, dishwashing and/or cleaning processes at low
temperature. It is a further object of the present invention to
provide a cleaning composition comprising alpha-amylase variants
which have improved wash performance at low temperature compared to
the parent alpha-amylase or compared to cleaning compositions
comprising the alpha-amylase of any of SEQ ID NOs: 1, 2, 3, 4, 5,
6, 7 or 8.
SUMMARY OF THE INVENTION
[0007] The present invention provides a cleaning composition
comprising: [0008] (a) a variant of a parent alpha-amylase, wherein
the variant comprises (i) a modification at one or more positions
corresponding to positions selected from the group consisting of
109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence
set forth in SEQ ID NO: 1, and optionally in one or more positions
corresponding to positions selected from the group consisting of
140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the
amino acid sequence as set forth in SEQ ID NO. 1, (ii) the variant
has at least 80, such as at least 90%, such as at least 95%, such
as at least 97%, but less than 100% sequence identity with the
amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,
or 8, and (iii) the variant has alpha-amylase activity; [0009] (b)
and a cleaning adjunct, preferably in an amount from 0.01 to 99.9
wt %.
[0010] The invention also provides a method of treating a surface,
preferably a textile, comprising [0011] (i) forming an aqueous wash
liquor comprising water and such a cleaning composition, [0012]
(ii) treating the surface with the aqueous wash liquor preferably
at a temperature of 5 or 10 to 40.degree. C., or preferably
35.degree. C. or less, more preferably at a temperature of
30.degree. C. or less, or at a temperature of 20.degree. C. or
less; and [0013] (iii) rinsing the surface.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides a cleaning composition
comprising: [0015] (a) a variant of a parent alpha-amylase, wherein
the variant comprises (i) a modification at one or more positions
corresponding to 109, 1, 7, 280, 284, 320, 323 and 391 of the amino
acid sequence set forth in SEQ ID NO: 1, and optionally in one or
more positions corresponding to positions 140, 181, 182, 183, 184,
195, 206, 243, 260, 304, and 476 of the amino acid sequence as set
forth in SEQ ID NO. 1, (ii) the variant has at least 80, such as at
least 90%, such as at least 95%, such as at least 97%, but less
than 100% sequence identity with the amino acid sequence set forth
in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and (iii) the variant has
alpha-amylase activity; and [0016] (b) a cleaning adjunct,
preferably in an amount from 0.01 to 99.9 wt %.
Definitions
[0017] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0018] Alpha-amylase: The term "alpha-amylase"
(alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1), constitutes a
group of enzymes, which catalyze hydrolysis of starch and other
linear and branched 1,4-glucosidic oligo- and polysaccharides. For
purposes of the present invention, alpha-amylase activity is
determined according to the procedure described in Example section.
In one aspect, the variants of the present invention have at least
20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or at least 100% of the
alpha-amylase activity of the mature polypeptide of SEQ ID NO:
1.
[0019] Amino Acid: The term "amino acid" as used herein includes
the standard twenty genetically-encoded amino acids and their
corresponding stereoisomers in the `d` form (as compared to the
natural `1` form), omega-amino acids other naturally-occurring
amino acids, unconventional amino acids (e.g.
.alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids,
etc.) and chemically derivatised amino acids. Chemical derivatives
of one or more amino acids may be achieved by reaction with a
functional side group. Such derivatised molecules include, for
example, those molecules in which free amino groups have been
derivatised to form amine hydrochlorides, p-toluene sulphonyl
groups, carboxybenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatised to form salts, methyl and ethyl esters or other types
of esters and hydrazides. Free hydroxyl groups may be derivatised
to form O-acyl or O-alkyl derivatives. Also included as chemical
derivatives are those peptides which contain naturally occurring
amino acid derivatives of the twenty standard amino acids. For
example: 4-hydroxyproline may be substituted for proline;
5-hydroxylysine may be substituted for lysine; 3-methylhistidine
may be substituted for histidine; homoserine may be substituted for
serine and ornithine for lysine. Derivatives also include peptides
containing one or more additions or deletions as long as the
requisite activity is maintained. Other included modifications are
amidation, amino terminal acylation (e.g. acetylation or
thioglycolic acid amidation), terminal carboxylamidation (e.g. with
ammonia or methylamine), and the like terminal modifications.
[0020] When an amino acid is being specifically enumerated, such as
`alanine` or `Ala` or `A`, the term refers to both 1-alanine and
d-alanine unless explicitly stated otherwise. Other unconventional
amino acids may also be suitable components for polypeptides of the
present invention, as long as the desired functional property is
retained by the polypeptide. For the peptides shown, each encoded
amino acid residue, where appropriate, is represented by a single
letter designation, corresponding to the trivial name of the
conventional amino acid. In one embodiment, the polypeptides of the
invention comprise or consist of 1-amino acids.
[0021] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell. cDNA lacks intron
sequences that may be present in the corresponding genomic DNA. The
initial, primary RNA transcript is a precursor to mRNA that is
processed through a series of steps, including splicing, before
appearing as mature spliced mRNA.
[0022] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a variant. The boundaries of the coding sequence are generally
determined by an open reading frame, which usually begins with a
start codon such as ATG, GTG or TTG and ends with a stop codon such
as TAA, TAG, or TGA. The coding sequence may be a DNA, cDNA,
synthetic, or recombinant polynucleotide.
[0023] Control sequences: The term "control sequences" means
nucleic acid sequences necessary for the expression of a
polynucleotide encoding a variant of the present invention. Each
control sequence may be native (i.e., from the same gene) or
foreign (i.e., from a different gene) to the polynucleotide
encoding the variant or native or foreign to each other. Such
control sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the polynucleotide encoding a variant.
[0024] Delta Intensity: The terms "delta intensity" or "delta
intensity value" are defined herein as the result of an intensity
measurement of a test material, e.g. a swatch CS-28 (Center For
Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, the
Netherlands) or a hard surface. The swatch is measured with a
portion of the swatch, washed under identical conditions, as
background. The delta intensity is the intensity value of the test
material washed with amylase subtracting the intensity value of the
test material washed without amylase.
[0025] Enzyme Detergency Benefit: The term "enzyme detergency
benefit" used herein, refers to the advantageous effect an enzyme
may add to a detergent compared to the same detergent without the
enzyme. Important detergency benefits which can be provided by
enzymes are stain removal with no or very little visible soils
after washing and/or cleaning, prevention or reduction of
re-deposition of soils released in the washing process (an effect
that also is termed anti-redeposition), restoring fully or partly
the whiteness of textiles which originally were white but after
repeated use and wash have obtained a greyish or yellowish
appearance (an effect that also is termed whitening). Textile care
benefits, which are not directly related to catalytic stain removal
or prevention of re-deposition of soils, may also be important for
enzyme detergency benefits. Examples of such textile care benefits
are prevention or reduction of dye transfer from one fabric to
another fabric or another part of the same fabric (an effect that
is also termed dye transfer inhibition or anti-backstaining),
removal of protruding or broken fibers from a fabric surface to
decrease pilling tendencies or remove already existing pills or
fuzz (an effect that also is termed anti-pilling), improvement of
the fabric-softness, colour clarification of the fabric and removal
of particulate soils which are trapped in the fibers of the fabric
or garment. Enzymatic bleaching is a further enzyme detergency
benefit where the catalytic activity generally is used to catalyze
the formation of bleaching component such as hydrogen peroxide or
other peroxides.
[0026] Expression: The term "expression" includes any step involved
in the production of the variant including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0027] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a variant and is operably linked to additional nucleotides
that provide for its expression.
[0028] Fragment: The term "fragment" means a polypeptide having one
or more (e.g. several) amino acids absent from the amino and/or
carboxyl terminus of the polypeptide of SEQ ID NOs:1, 2, 3, 4, 5,
6, 7, or 8; wherein the fragment has alpha-amylase activity. In one
aspect, a fragment contains at least 200 contiguous amino acid
residues of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, for example at
least 300 contiguous amino acid residues, or at least 350
contiguous amino acid residues, or at least 400 contiguous amino
acid residues, or at least 450 contiguous amino acid residues of
SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.
[0029] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, and the
like with a nucleic acid construct or expression vector comprising
a polynucleotide described herein. The term "host cell" encompasses
any progeny of a parent cell that is not identical to the parent
cell due to mutations that occur during replication.
[0030] Intensity Value: The term "intensity value" as used herein,
refers to the wash performance measurement. It is measured as the
brightness expressed as the intensity of the light reflected from
the sample when illuminated with white light. When the sample is
stained the intensity of the reflected light is lower, than that of
a clean sample. Therefore, the intensity of the reflected light can
be used to measure wash performance, where a higher intensity value
correlates with higher wash performance. Color measurements are
made with a professional flatbed scanner (Kodak iQsmart, Kodak)
used to capture an image of the washed textile. To extract a value
for the light intensity from the scanned images, 24-bit pixel
values from the image are converted into values for red, green and
blue (RGB). The intensity value (Int) is calculated by adding the
RGB values together as vectors and then taking the length of the
resulting vector:
Int= {square root over (r.sup.2+g.sup.2+b.sup.2)}
[0031] Improved property: The term "improved property" means a
characteristic associated with a variant that is improved compared
to the parent. Such improved properties include, but are not
limited to, wash performance, thermal activity, thermostability,
stability under storage conditions, and chemical stability.
[0032] Improved wash performance: The terms "improved wash
performance" or "enhanced wash performance" mean the ability of the
variant enzyme to provide a cleaning effect (e.g. stain removal) in
a wash process, such as laundry or dishwashing, that is improved
compared to that of the parent amylase or relative to the activity
of an alpha-amylase having the amino acid sequence shown in SEQ ID
NO: 2 or 1, e.g. by increased stain removal. Wash performance may
be determined using methods well known in the art, such as using an
automatic mechanical stress assay (AMSA). It will be appreciated by
persons skilled in the art that the enhanced wash performance may
be achieved under only some or perhaps all wash conditions, for
example at wash temperatures of 20.degree. C. or higher (such as at
40.degree. C.). Improved wash performance may be indicated by an
Improvement Factor (IF) above 1.0, preferably above 1.05 in one or
more of the conditions listed in example 1 for example in model
detergent A at 20.degree. C. where the alpha-amylase variant
concentration is 0.2 mg/L, or in model detergent A at 40.degree. C.
where the alpha-amylase variant concentration is 0.05 mg/L, or in
model detergent J at 20.degree. C. where the alpha-amylase variant
concentration is 0.2 mg/L, or in model detergent J at 30.degree. C.
where the alpha-amylase variant concentration is 0.05 mg/L or in
Detergent K at 20.degree. C. where the alpha-amylase variant
concentration is 0.2 mg/L. The wash conditions are described in the
Example section.
[0033] Isolated: The term "isolated" means a substance in a form or
environment which does not occur in nature. Non-limiting examples
of isolated substances include (1) any non-naturally occurring
substance, (2) any substance including, but not limited to, any
enzyme, variant, nucleic acid, protein, peptide or cofactor, that
is at least partially removed from one or more or all of the
naturally occurring constituents with which it is associated in
nature; (3) any substance modified by the hand of man relative to
that substance found in nature; or (4) any substance modified by
increasing the amount of the substance relative to other components
with which it is naturally associated (e.g., multiple copies of a
gene encoding the substance; use of a stronger promoter than the
promoter naturally associated with the gene encoding the
substance). An isolated substance may be present in a fermentation
broth sample. In one aspect, the present invention relates to an
isolated alpha-amylase variant.
[0034] Isolated polynucleotide: The term "isolated polynucleotide"
means a polynucleotide that is modified by the hand of man. In one
aspect, the isolated polynucleotide is at least 1% pure, e.g., at
least 5% pure, at least 10% pure, at least 20% pure, at least 40%
pure, at least 60% pure, at least 80% pure, at least 90% pure, and
at least 95% pure, as determined by agarose electrophoresis. The
polynucleotides may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0035] Isolated variant: The term "isolated variant" means a
variant that is modified by the hand of man. In one aspect, the
variant is at least 1% pure, e.g., at least 5% pure, at least 10%
pure, at least 20% pure, at least 40% pure, at least 60% pure, at
least 80% pure, and at least 90% pure, as determined by
SDS-PAGE.
[0036] Low temperature: "Low temperature" is a temperature of
5-40.degree. C., preferably 5-35.degree. C., preferably
5-30.degree. C., more preferably 5-25.degree. C., more preferably
5-20.degree. C., most preferably 5-15.degree. C., and in particular
5-10.degree. C. In a preferred embodiment, "Low temperature" is a
temperature of 10-35.degree. C., preferably 10-30.degree. C., or
10-25.degree. C., or 10-20.degree. C., or 10-15.degree. C.
[0037] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. It is
known in the art that a host cell may produce a mixture of two of
more different mature polypeptides (i.e., with a different
C-terminal and/or N-terminal amino acid) expressed by the same
polynucleotide.
[0038] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having alpha-amylase activity.
[0039] Mutant: The term "mutant" means a polynucleotide encoding a
variant.
[0040] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic, which comprises
one or more control sequences. The term nucleic acid construct is
synonymous with the term "expression cassette" when the nucleic
acid construct contains the control sequences required for
expression of a coding sequence of the present invention.
[0041] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs the
expression of the coding sequence.
[0042] Parent or Parent alpha-amylase: The term "parent" or "parent
alpha-amylase" means an alpha-amylase to which an alteration is
made to produce the enzyme variants of the present invention. The
parent may be a naturally occurring (wild-type) polypeptide or a
variant thereof. For example, the parent may be the alpha-amylase
of SEQ ID NO:1 (known as SP722). Alternatively, it may mean the
alpha-amylase of SEQ ID NO: 2 or any suitable alpha-amylase, such
as those listed herein as SEQ ID Nos.: 3, 4, 5, 6, 7, and 8.
[0043] Sequence Identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0044] For purposes of the present invention, the degree of
sequence identity between two amino acid sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
J. Mol. Biol. 48: 443-453) as implemented in the Needle program of
the EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 3.0.0 or later. The optional parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0045] Alternatively, the parameters used may be gap open penalty
of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS
version of NCBI NUC4.4) substitution matrix. The output of Needle
labeled "longest identity" (obtained using the -nobrief option) is
used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0046] Starch removing process: The expression "starch removing
process" relates to any kind of process whereby starch is removed
(or converted) such as in washing processes where starch is removed
from textile e.g. textile cleaning such as laundry. A starch
removing process could also be hard surface cleaning such as dish
wash or it could be cleaning processes in general such as
industrial or institutional cleaning. The expression also comprises
other starch removing processes or starch conversion, ethanol
production, starch liquefaction, textile desizing, paper and pulp
production, beer making and detergents in general.
[0047] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g. several) nucleotides deleted from the 5'-
and/or 3'-end of a mature polypeptide coding sequence; wherein the
subsequence encodes a fragment having alpha-amylase activity.
[0048] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" means a polynucleotide preparation free of
other extraneous or unwanted nucleotides and in a form suitable for
use within genetically engineered polypeptide production systems.
Thus, a substantially pure polynucleotide contains at most 10%, at
most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most
2%, at most 1%, and at most 0.5% by weight of other polynucleotide
material with which it is natively or recombinantly associated. A
substantially pure polynucleotide may, however, include naturally
occurring 5'- and 3'-untranslated regions, such as promoters and
terminators. It is preferred that the substantially pure
polynucleotide is at least 90% pure, e.g., at least 92% pure, at
least 94% pure, at least 95% pure, at least 96% pure, at least 97%
pure, at least 98% pure, at least 99% pure, and at least 99.5% pure
by weight. The polynucleotides of the present invention are
preferably in a substantially pure form.
[0049] Substantially pure variant: The term "substantially pure
variant" means a preparation that contains at most 10%, at most 8%,
at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most
1%, and at most 0.5% by weight of other polypeptide material with
which it is natively or recombinantly associated. Preferably, the
variant is at least 92% pure, e.g., at least 94% pure, at least 95%
pure, at least 96% pure, at least 97% pure, at least 98% pure, at
least 99%, at least 99.5% pure, and 100% pure by weight of the
total polypeptide material present in the preparation. The variants
of the present invention are preferably in a substantially pure
form. This can be accomplished, for example, by preparing the
variant by well known recombinant methods or by classical
purification methods.
[0050] Textile Care Benefits: The term "textile care benefits", as
used herein, is defined as not being directly related to catalytic
stain removal or prevention of re-deposition of soils, are also
important for enzyme detergency benefits. Examples of such textile
care benefits are prevention or reduction of dye transfer from one
textile to another textile or another part of the same textile (an
effect that is also termed dye transfer inhibition or
anti-backstaining), removal of protruding or broken fibers from a
textile surface to decrease pilling tendencies or remove already
existing pills or fuzz (an effect that also is termed
anti-pilling), improvement of the textile-softness, color
clarification of the textile and removal of particulate soils which
are trapped in the fibers of the textile. Enzymatic bleaching is a
further enzyme detergency benefit where the catalytic activity
generally is used to catalyze the formation of bleaching component
such as hydrogen peroxide or other peroxides or other bleaching
species."
[0051] Variant: The term "variant" means a polypeptide having
alpha-amylase activity comprising an alteration/mutation, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.
several) positions relative to the parent alpha-amylase. A
substitution means a replacement of an amino acid occupying a
position with a different amino acid; a deletion means removal of
an amino acid occupying a position; and an insertion means adding
1-3 amino acids adjacent to and immediately following an amino acid
occupying a position.
[0052] Wash performance In the present context the term "wash
performance" is used as an enzyme's ability to remove starch or
starch-containing stains present on the object to be cleaned during
e.g. laundry or hard surface cleaning, such as dish washing. The
wash performance may be quantified by calculating the so-called
intensity value (Int) defined in the description of AMSA or in the
beaker wash performance test in the Methods section below.
[0053] Wild-Type Enzyme: The term "wild-type" alpha-amylase means
an alpha-amylase expressed by a naturally occurring microorganism,
such as a bacterium, yeast, or filamentous fungus found in
nature.
[0054] The term "wash performance" includes cleaning in general
e.g. hard surface cleaning as in dish wash, but also wash
performance on textiles such as laundry, and also industrial and
institutional cleaning. Improved wash performance may be measured
by comparing the delta intensities as described in the definition
herein
[0055] The term "wash performance" includes cleaning in general
e.g. hard surface cleaning as in dish wash, but also wash
performance on textiles such as laundry, and also industrial and
institutional cleaning.
Conventions for Designation of Variants
[0056] The polypeptides of the invention having alpha-amylase
activity correspond to variants of an alpha-amylase derived from
Bacillus, as shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.
TABLE-US-00001 SEO ID NO: 1
HHNGTNGTMMQYFEWHLPNDGNHWNRLRDDASNLRNRGITAIWIPPAWKG
TSQNDVGYGAYDLYDLGEFNQKGTVRTKYGTRSQLESAIHALKNNGVQVY
GDVVMNHKGGADATENVLAVEVNPNNRNQEISGDYTIEAWTKFDFPGRGN
TYSDFKWRWYHFDGVDWDQSRQFQNRIYKFRGDGKAWDWEVDSENGNYDY
LMYADVDMDHPEVVNELRRWGEWYTNTLNLDGFRIDAVKHIKYSFTRDWL
THVRNATGKEMFAVAEFWKNDLGALENYLNKTNWNHSVFDVPLHYNLYNA
SNSGGNYDMAKLLNGTVVQKHPMHAVTFVDNHDSQPGESLESFVQEWFKP
LAYALILTREQGYPSVFYGDYYGIPTHSVPAMKAKIDPILEARQNFAYGT
QHDYFDHHNIIGWTREGNTTHPNSGLATIMSDGPGGEKWMYVGQNKAGQV
WHDITGNKPGTVTINADGWANFSVNGGSVSIWVKR
[0057] For the purposes of the present invention, the mature
polypeptide disclosed in SEQ ID NO: 1 is used to determine the
corresponding amino acid residue in another alpha-amylase
polypeptide. However, the skilled person would recognize that the
sequence of SEQ ID NO: 2 may also be used to determine the
corresponding amino acid residue in another alpha-amylase
polypeptide. The amino acid sequence of another alpha-amylase is
aligned with the mature polypeptide disclosed in SEQ ID NO: 1, and
based on the alignment, the amino acid position number
corresponding the any amino acid residue in the mature polypeptide
disclosed in SEQ IDN O: 1 is determined using the Needleman-Wunsch
algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453)
as implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or
later. The parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of
BLOSUM62) substitution matrix.
[0058] Identification of the corresponding amino acid residue in
another alpha-amylase can be determined by an alignment of multiple
polypeptide sequences using several computer programs including,
but not limited to, MUSCLE (multiple sequence comparison by
log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids
Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and
Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al.,
2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007,
Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in
Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics
26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;
Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using
their respective default parameters.
[0059] When the other alpha-amylase has diverged from the mature
polypeptide of SEQ ID NO: 1 such that traditional sequence-based
comparison fails to detect their relationship (Lindahl and
Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise
sequence comparison algorithms can be used. Greater sensitivity in
sequence-based searching can be attained using search programs that
utilize probabilistic representations of polypeptide families
(profiles) to search databases. For example, the PSI-BLAST program
generates profiles through an iterative database search process and
is capable of detecting remote homologs (Atschul et al., 1997,
Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be
achieved if the family or superfamily for the polypeptide has one
or more representatives in the protein structure databases.
Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:
797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881)
utilize information from a variety of sources (PSI-BLAST, secondary
structure prediction, structural alignment profiles, and solvation
potentials) as input to a neural network that predicts the
structural fold for a query sequence. Similarly, the method of
Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to
align a sequence of unknown structure with the superfamily models
present in the SCOP database. These alignments can in turn be used
to generate homology models for the polypeptide, and such models
can be assessed for accuracy using a variety of tools developed for
that purpose.
[0060] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
E.g. the SCOP superfamilies of proteins have been structurally
aligned, and those alignments are accessible and downloadable. Two
or more protein structures can be aligned using a variety of
algorithms such as the distance alignment matrix (Holm and Sander,
1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov
and Bourne, 1998, Protein Engineering 11: 739-747), and
implementation of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567).
[0061] In describing the alpha-amylase variants of the present
invention, the nomenclature described below is adapted for ease of
reference. The accepted IUPAC single letter or three letter amino
acid abbreviation is employed.
[0062] Substitutions. For an amino acid substitution, the following
nomenclature is used: Original amino acid, position, substituted
amino acid. Accordingly, the substitution of e.g. threonine at
position 226 with alanine is designated as "Thr226Ala" or "T226A".
Multiple mutations are separated by addition marks ("+"), e.g.,
"Gly205Arg+Ser411Phe" or "G205R+S411F", representing substitutions
at positions 205 and 411 of glycine (G) with arginine (R) and
serine (S) with phenylalanine (F), respectively.
[0063] Deletions. For an amino acid deletion, the following
nomenclature is used: Original amino acid, position, *.
Accordingly, the deletion of glycine at position 181 is designated
as "Ser181*" or "S181*". Multiple deletions are separated by
addition marks ("+"), e.g., "Ser181*+Thr182*" or "S181*+T182*".
[0064] Insertions. For an amino acid insertion, the following
nomenclature is used: Original amino acid, position, original amino
acid, inserted amino acid. Accordingly, the insertion of lysine
after e.g. glycine at position 195 is designated "Gly195GlyLys" or
"G195GK". An insertion of multiple amino acids is designated
[Original amino acid, position, original amino acid, inserted amino
acid #1, inserted amino acid #2; etc.]. For example, the insertion
of lysine and alanine after glycine at position 195 is indicated as
"Gly195GlyLysAla" or "G195GKA".
[0065] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example, the sequence would thus be:
TABLE-US-00002 Parent: Variant: 195 195 195a 195b G G-K-A
[0066] Multiple modifications. Variants comprising multiple
modifications are separated by addition marks ("+"), e.g.,
"Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution
of arginine and glycine at positions 170 and 195 with tyrosine and
glutamic acid, respectively.
[0067] Different modifications. Where different alterations can be
introduced at a position, the different alterations are separated
by a comma, e.g., "Arg170Tyr,Glu" represents a substitution of
arginine at position 170 with tyrosine or glutamic acid. Thus,
"Tyr167Gly,Ala+Arg170Gly,Ala" designates the following
variants:
[0068] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala",
"Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".
Parent Alpha-Amylases
[0069] The parent alpha-amylase may be a polypeptide with at least
80% sequence identity with the polypeptide set forth in SEQ ID NO:
1.
[0070] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 1 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 1.
[0071] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 1. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 1.
[0072] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 2.
[0073] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 2 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 2.
[0074] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 2. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 2.
[0075] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 3.
[0076] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 3 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 3.
[0077] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 3. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 3.
[0078] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 4.
[0079] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 4 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 4.
[0080] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 4. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 4.
[0081] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 5.
[0082] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 5 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 5.
[0083] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 5. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 5.
[0084] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 6.
[0085] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 6 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 6.
[0086] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 6. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 6.
[0087] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 7.
[0088] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 7 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 7.
[0089] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 7. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 7.
[0090] The parent alpha-amylase may also be a polypeptide with at
least 80% sequence identity with the polypeptide set forth in SEQ
ID NO: 8.
[0091] In one aspect, the parent alpha-amylase has a sequence
identity to the polypeptide of SEQ ID NO: 8 of at least 80%, such
as at least 85%, at least 90%, e.g. at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99, or 100%, which has alpha-amylase activity. In one
aspect, the amino acid sequence of the parent alpha-amylase differs
by no more than ten amino acids, e.g. by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the polypeptide of SEQ ID NO: 8.
[0092] The parent alpha-amylase preferably comprises or consists of
the amino acid sequence of SEQ ID NO: 8. In another embodiment, the
parent alpha-amylase is an allelic variant of the polypeptide of
SEQ ID NO: 8.
[0093] The amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 8, or a fragment thereof, may be used to design nucleic
acid probes to identify and clone DNA encoding a parent from
strains of different genera or species according to methods well
known in the art. In particular, such probes can be used for
hybridization with the genomic or cDNA of the genus or species of
interest, following standard Southern blotting procedures, in order
to identify and isolate the corresponding gene therein. Such probes
can be considerably shorter than the entire sequence, but should be
at least 14, e.g., at least 25, at least 35, or at least 70
nucleotides in length. Preferably, the nucleic acid probe is at
least 100 nucleotides in length, e.g., at least 200 nucleotides, at
least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides, at least 600 nucleotides, at least 700 nucleotides, at
least 800 nucleotides, or at least 900 nucleotides in length. Both
DNA and RNA probes can be used. The probes are typically labeled
for detecting the corresponding gene (for example, with .sup.32P,
.sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed
by the present invention.
[0094] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a parent. Genomic or other DNA from
such other organisms may be separated by agarose or polyacrylamide
gel electrophoresis, or other separation techniques. DNA from the
libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material,
which is used in a Southern blot.
[0095] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled
nucleotide probe corresponding to a polynucleotide encoding SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or a subsequence thereof,
under low to very high stringency conditions. Molecules to which
the probe hybridizes can be detected using, for example, X-ray film
or any other detection means known in the art.
[0096] In one aspect, the nucleic acid probe is a polynucleotide
that encodes the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, or a fragment thereof.
[0097] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally. The carrier material is finally washed three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree. C. (very
low stringency), 50.degree. C. (low stringency), 55.degree. C.
(medium stringency), 60.degree. C. (medium-high stringency),
65.degree. C. (high stringency), or 70.degree. C. (very high
stringency).
[0098] For short probes that are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization and hybridization at about 5.degree. C. to about
10.degree. C. below the calculated T.sub.m using the calculation
according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA
48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5%
NP-40, 1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM
sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per
ml following standard Southern blotting procedures for 12 to 24
hours optimally. The carrier material is finally washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0099] The parent may be obtained from microorganisms of any genus.
For purposes of the present invention, the term "obtained from" as
used herein in connection with a given source shall mean that the
parent encoded by a polynucleotide is produced by the source or by
a cell in which the polynucleotide from the source has been
inserted. In one aspect, the parent is secreted
extracellularly.
[0100] The parent may be a bacterial alpha-amylase. For example,
the parent may be a gram-positive bacterial polypeptide such as a
Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,
Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or
Streptomyces alpha-amylase, or a gram-negative bacterial
polypeptide such as a Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, or Ureaplasma alpha-amylase.
[0101] In one aspect, the parent is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis alpha-amylase.
[0102] In another aspect, the parent is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus alpha-amylase.
[0103] In another aspect, the parent is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans alpha-amylase.
[0104] In another aspect, the parent is a Bacillus sp.
alpha-amylase, e.g., the alpha-amylase of SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID
NO: 7, or SEQ ID NO: 8.
[0105] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0106] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0107] The parent may be identified and obtained from other sources
including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) or DNA samples obtained directly from
natural materials (e.g., soil, composts, water, etc,) using the
above-mentioned probes. Techniques for isolating microorganisms and
DNA directly from natural habitats are well known in the art. The
polynucleotide encoding a parent may then be derived by similarly
screening a genomic or cDNA library of another microorganism or
mixed DNA sample. Once a polynucleotide encoding a parent has been
detected with a probe(s), the polynucleotide may be isolated or
cloned by utilizing techniques that are known to those of ordinary
skill in the art (see, e.g., Sambrook et al., 1989, supra).
[0108] The parent may be a hybrid polypeptide in which a portion of
one polypeptide is fused at the N-terminus or the C-terminus of a
portion of another polypeptide.
[0109] The parent may also be a fused polypeptide or cleavable
fusion polypeptide in which one polypeptide is fused at the
N-terminus or the C-terminus of another polypeptide. A fused
polypeptide is produced by fusing a polynucleotide encoding one
polypeptide to a polynucleotide encoding another polypeptide.
Techniques for producing fusion polypeptides are known in the art,
and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fused
polypeptide is under control of the same promoter(s) and
terminator. Fusion proteins may also be constructed using intein
technology in which fusions are created post-translationally
(Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994,
Science 266: 776-779).
[0110] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Preparation of Variants
[0111] A suitable method for obtaining a variant essential to the
present invention having alpha-amylase activity, comprises (a)
introducing into a parent alpha-amylase a modification at one or
more positions corresponding to positions 109, 1, 7, 280, 284, 320,
323 and 391 of the amino acid sequence set forth in SEQ ID NO: 1,
and optionally in one or more positions corresponding to positions
140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the
amino acid sequence as set forth in SEQ ID NO: 1, wherein each
modification is independently a substitution or deletion, and said
variant has alpha-amylase activity; and (b) recovering said
variant.
[0112] In one aspect, the a method for obtaining a variant having
alpha-amylase activity, comprises (a) introducing into a parent
alpha-amylase a modification at one or more positions corresponding
to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino
acid sequence set forth in SEQ ID NO: 1, and optionally in one or
more positions corresponding to positions 140, 181, 182, 183, 184,
195, 206, 243, 260, 304, and 476 of the amino acid sequence as set
forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, of 8, wherein the numbering
is according to SEQ ID NO: 1, and wherein each modification is
independently a substitution or deletion, and said variant has
alpha-amylase activity; and (b) recovering said variant.
[0113] In one embodiment, the modification is a substitution. In
one embodiment, the modification is a deletion.
[0114] In another embodiment, the method for obtaining a variant
having alpha-amylase activity, comprises (a) introducing into a
parent alpha-amylase a substitution at one or more positions,
wherein the substitution is selected from H1A, G7A, G109A, N280S,
W284H, K320A, M323N, and E391A of the polypeptide of SEQ ID NOs: 1,
2, 3, 4, 5, 6, 7, or 8, wherein numbering is according to SEQ ID
NO: 1 and (b) recovering the variant.
[0115] The method may further comprise introducing to the parent
alpha-amylase a deletion in one or more positions, wherein the
deletion is selected from: H1*, R181*, G182*, D183*, and G184* of
the polypeptide of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, wherein
numbering is according to SEQ ID NO: 1, and recovering the
variant.
[0116] The method may further comprise introducing to the parent
alpha-amylase a substitution in one or more positions, wherein the
substitution is selected from: W140Y, N195F, V206Y, Y243F, E260G,
G304R, and G476K of the polypeptide of SEQ ID Nos.: 1, 3, 4, 5, 6,
7, or 8, and recovering the variant.
[0117] The variants may be prepared using any mutagenesis procedure
known in the art, such as site-directed mutagenesis, synthetic gene
construction, semi-synthetic gene construction, random mutagenesis,
shuffling, etc.
[0118] Site-directed mutagenesis is a technique in which one or
more (several) mutations are created at one or more defined sites
in a polynucleotide encoding the parent.
[0119] Site-directed mutagenesis can be accomplished in vitro by
PCR involving the use of oligonucleotide primers containing the
desired mutation. Site-directed mutagenesis can also be performed
in vitro by cassette mutagenesis involving the cleavage by a
restriction enzyme at a site in the plasmid comprising a
polynucleotide encoding the parent and subsequent ligation of an
oligonucleotide containing the mutation in the polynucleotide.
Usually the restriction enzyme that digests at the plasmid and the
oligonucleotide is the same, permitting sticky ends of the plasmid
and insert to ligate to one another. See, e.g., Scherer and Davis,
1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al.,
1990, Nucleic Acids Res. 18: 7349-4966.
[0120] Site-directed mutagenesis can also be accomplished in vivo
by methods known in the art. See, e.g., U.S. Patent Application
Publication No. 2004/0171154; Storici et al., 2001, Nature
Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;
and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:
15-16.
[0121] Any site-directed mutagenesis procedure can be used in the
present invention. There are many commercial kits available that
can be used to prepare variants.
[0122] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of
interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al. (2004, Nature 432: 1050-1054) and similar
technologies wherein oligonucleotides are synthesized and assembled
upon photo-programmable microfluidic chips.
[0123] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0124] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0125] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide subsequences may then be shuffled.
Variants
[0126] The variants of a parent alpha-amylase essential to the
present invention may comprise (i) a modification at one or more
positions corresponding to positions 109, 1, 7, 280, 284, 320, 323
and 391 of the amino acid sequence set forth in SEQ ID NO: 1, and
optionally in one or more positions corresponding to positions 140,
181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the amino
acid sequence as set forth in SEQ ID NO: 1, (ii) the variant has at
least 80, such as at least 90%, such as at least 95%, such as at
least 97%, but less than 100% sequence identity with the amino acid
sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and
(iii) the variant has alpha-amylase activity. Hereby, variants are
provided which have improved washing performance at low
temperature, compared to the parent alpha-amylase or compared to
the alpha-amylase of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.
[0127] Suitable variants may have a sequence identity of at least
80%, such as at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99%, but less than
100%, to the amino acid sequence of the parent alpha-amylase.
[0128] The isolated variants of a parent alpha-amylase suitable
herein may comprise (i) a modification at one or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of
the amino acid sequence set forth in SEQ ID NO: 1, and optionally
in one or more positions corresponding to positions 140, 181, 182,
183, 184, 195, 206, 243, 260, 304, and 476 of the amino acid
sequence as set forth in SEQ ID NO: 1, (ii) the variant has at
least 80, such as at least 90%, such as at least 95%, such as at
least 97%, but less than 100% sequence identity with the amino acid
sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and
(iii) the variant has alpha-amylase activity.
[0129] Suitable variants may have at least 80%, such as at least
85%, at least 90%, at least 95%, such as at least 96%, at least
97%, at least 98%, and at least 99%, but less than 100%, sequence
identity with the mature polypeptide of SEQ ID NO: 1.
[0130] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
2.
[0131] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
3.
[0132] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
4.
[0133] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
5.
[0134] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
6.
[0135] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
7.
[0136] In another embodiment, a suitable variant has at least 80%,
such as at least 85%, at least 90%, at least 95%, such as at least
96%, at least 97%, at least 98%, and at least 99%, but less than
100%, sequence identity with the mature polypeptide of SEQ ID NO:
8.
[0137] In one embodiment, the number of modifications in a suitable
variant for use in the present invention is 1 to 20, e.g., 1 to 10
and 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
modifications.
[0138] In one embodiment, a suitable variant comprises a
modification, such as a substitution, at one or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0139] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at two or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0140] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at three or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0141] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at four or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0142] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at five or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0143] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at six or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0144] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at seven or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0145] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at eight positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and optionally a modification at one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243,
260, 304, and 476, wherein numbering is according to SEQ ID NO:
1.
[0146] In one embodiment, a suitable variant comprises a
modification, such as a substitution, at one or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0147] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at two or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0148] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at three or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0149] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at four or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0150] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at five or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0151] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at six or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0152] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at seven or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0153] In another embodiment, a suitable variant comprises a
modification, such as a substitution, at eight positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391,
and a modification at one or more positions corresponding to
positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and
476, wherein numbering is according to SEQ ID NO: 1.
[0154] In a preferred embodiment, the variant comprises a
modification in one, two, three, four, or five positions selected
from the group consisting of 1, 7, 109, 280, and 391. In one
embodiment, the variant comprises at least one deletion and at
least one substitution in two, three, four or five positions
selected from the group consisting of 1, 7, 109, 280, and 391.
[0155] In one embodiment, a suitable variant comprises a
substitution at one, two, three, or four positions selected from 7,
109, 280, and 391.
[0156] In one embodiment, a suitable variant comprises
modifications in the positions selected from the group of positions
consisting of: X1+X7; X1+X109; X1+X280; X1+X284; X1+X320; X1+X323;
X1+X391; X109+X280; X109+X284; X109+X320; X109+X323; X109+X391;
X7+X109; X7+X280; X7+X284; X7+X320; X7+X323; X7+X391; X280+X284;
X280+X320; X280+X323; X280+X391; X284+X320; X284+X323; X284+X391;
X320+X323; X320+X391; and X323+X391, wherein numbering is according
to SEQ ID NO: 1.
[0157] In one embodiment, a suitable variant comprises
modifications in the positions selected from the group of positions
consisting of: X109+X7+X1; X109+X7+X391; X109+X7+X280;
X109+X7+X284; X109+X7+X320; X109+X7+X323; X109+X1+X391;
X109+X1+X280; X109+X1+X284; X109+X1+X320; X109+X1+X323;
X109+X391+X280; X109+X391+X284; X109+X391+X320; X109+X391+X323;
X109+X280+X284; X109+X280+X320; X109+X280+X323; X109+X284+X320;
X109+X284+X323; X109+X320+X323; X7+X1+X391; X7+X1+X280; X7+X1+X284;
X7+X1+X320; X7+X1+X323; X7+X391+X280; X7+X391+X284; X7+X391+X320;
X7+X391+X323; X7+X280+X284; X7+X280+X320; X7+X280+X323;
X7+X284+X320; X7+X284+X323; X7+X320+X323; X1+X391+X280;
X1+X391+X284; X1+X391+X320; X1+X391+X323; X1+X280+X284;
X1+X280+X320; X1+X280+X323; X1+X284+X320; X1+X284+X323;
X1+X320+X323; X391+X280+X284; X391+X280+X320; X391+X280+X323;
X391+X284+X320; X391+X284+X323; X391+X320+X323; X280+X284+X320;
X280+X284+X323; X280+X320+X323; and X284+X320+X323, wherein
numbering is according to SEQ ID NO: 1.
[0158] In one embodiment, a suitable variant comprises
modifications in the positions selected from the group of positions
consisting of: X109+X7+X1+X391; X109+X7+X1+X280; X109+X7+X1+X284;
X109+X7+X1+X320; X109+X7+X1+X323; X109+X7+X391+X280;
X109+X7+X391+X284; X109+X7+X391+X320; X109+X7+X391+X323;
X109+X7+X280+X284; X109+X7+X280+X320; X109+X7+X280+X323;
X109+X7+X284+X320; X109+X7+X284+X323; X109+X7+X320+X323;
X109+X1+X391+X280; X109+X1+X391+X284; X109+X1+X391+X320;
X109+X1+X391+X323; X109+X1+X280+X284; X109+X1+X280+X320;
X109+X1+X280+X323; X109+X1+X284+X320; X109+X1+X284+X323;
X109+X1+X320+X323; X109+X391+X280+X284; X109+X391+X280+X320;
X109+X391+X280+X323; X109+X391+X284+X320; X109+X391+X284+X323;
X109+X391+X320+X323; X109+X280+X284+X320; X109+X280+X284+X323;
X109+X280+X320+X323; X109+X284+X320+X323; X7+X1+X391+X280;
X7+X1+X391+X284; X7+X1+X391+X320; X7+X1+X391+X323; X7+X1+X280+X284;
X7+X1+X280+X320; X7+X1+X280+X323; X7+X1+X284+X320; X7+X1+X284+X323;
X7+X1+X320+X323; X7+X391+X280+X284; X7+X391+X280+X320;
X7+X391+X280+X323; X7+X391+X284+X320; X7+X391+X284+X323;
X7+X391+X320+X323; X7+X280+X284+X320; X7+X280+X284+X323;
X7+X280+X320+X323; X7+X284+X320+X323; X1+X391+X280+X284;
X1+X391+X280+X320; X1+X391+X280+X323; X1+X391+X284+X320;
X1+X391+X284+X323; X1+X391+X320+X323; X1+X280+X284+X320;
X1+X280+X284+X323; X1+X280+X320+X323; X1+X284+X320+X323;
X391+X280+X284+X320; X391+X280+X284+X323; X391+X280+X320+X323;
X391+X284+X320+X323; and X280+X284+X320+X323, wherein numbering is
according to SEQ ID NO: 1.
[0159] In one embodiment, a suitable variant comprises one or more
modifications selected from the group consisting of X1*, X1A, X7A,
X7K, X7E, X7N. X7Q, X7L, X7D, X109A, X109S, X140Y, X181*, X182*,
X183*, X184*, X195F, X206Y, X243F, X260G, X280S, X284H, X284R,
X284F, X304R, X320A, X320M, X320T, X320V, X320S, X323N, X323R,
X323S, X323K, X391A, X391V, and X476K, wherein numbering is
according to SEQ ID NO: 1.
[0160] In one particular embodiment, a suitable variant comprises
the modifications selected from the group consisting of: X1*+X1A;
X1*+X7A; X1*+X109A; X1*.+-.X280S; X1*+X284H; X1*+X320A; X1*+X323N;
X1*+X391A; X1A+X7A; X1A+X109A; X1A+X280S; X1A+X284H; X1A+X320A;
X1A+X323N; X1A+X391A; X7A+X109A; X7A+X280S; X7A+X284H; X7A+X320A;
X7A+X323N; X7A+X391A; X109A+X280S; X109A+X284H; X109A+X320A;
X109A+X323N; X109A+X391A; X280S+X284H; X280S+X320A; X280S+X323N;
X280S+X391A; X284H+X320A; X284H+X323N; X284H+X391A; X320A+X323N;
X320A+X391A; and X323N+X391A, wherein numbering is according to SEQ
ID NO: 1.
[0161] In one embodiment, a suitable variant comprises the
modifications selected from the group consisting of: X1*+X7A+X109A;
X1*+X7A+X280S; X1*+X7A+X284H; X1*+X7A+X320A; X1*+X7A+X323N;
X1*+X7A+X391A; X1*+X109A+X280S; X1*+X109A+X284H; X1*+X109A+X320A;
X1*+X109A+X323N; X1*+X109A+X391A; X1*+X280S+X284H; X1*+X280S+X320A;
X1*+X280S+X323N; X1*+X280S+X391A; X1*+X284H+X320A; X1*+X284H+X323N;
X1*+X284H+X391A; X1*+X320A+X323N; X1*+X320A+X391A; X1*+X323N+X391A;
X1A+X7A+X109A; X1A+X7A+X280S; X1A+X7A+X284H; X1A+X7A+X320A;
X1A+X7A+X323N; X1A+X7A+X391A; X1A+X109A+X280S; X1A+X109A+X284H;
X1A+X109A+X320A; X1A+X109A+X323N; X1A+X109A+X391A; X1A+X280S+X284H;
X1A+X280S+X320A; X1A+X280S+X323N; X1A+X280S+X391A; X1A+X284H+X320A;
X1A+X284H+X323N; X1A+X284H+X391A; X1A+X320A+X323N; X1A+X320A+X391A;
X1A+X323N+X391A; X7A+X109A+X280S; X7A+X109A+X284H; X7A+X109A+X320A;
X7A+X109A+X323N; X7A+X109A+X391A; X7A+X280S+X284H; X7A+X280S+X320A;
X7A+X280S+X323N; X7A+X280S+X391A; X7A+X284H+X320A; X7A+X284H+X323N;
X7A+X284H+X391A; X7A+X320A+X323N; X7A+X320A+X391A; X7A+X323N+X391A;
X109A+X280S+X284H; X109A+X280S+X320A; X109A+X280S+X323N;
X109A+X280S+X391A; X109A+X284H+X320A; X109A+X284H+X323N;
X109A+X284H+X391A; X109A+X320A+X323N; X109A+X320A+X391A;
X109A+X323N+X391A; X280S+X284H+X320A; X280S+X284H+X323N;
X280S+X284H+X391A; X280S+X320A+X323N; X280S+X320A+X391A;
X280S+X323N+X391A; X284H+X320A+X323N; X284H+X320A+X391A;
X284H+X323N+X391A; and X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1.
[0162] A preferred variant comprises modifications in the positions
corresponding to the positions selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0163] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to any one of the amylases set forth in SEQ ID Nos: 1, 2,
3, 4, 5, 6, 7, or 8.
[0164] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 1, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0165] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 1.
[0166] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 2, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0167] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 2.
[0168] A preferred variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 2, selected from the group consisting
of:
H1*+G109A+N280S+E391A;
H1*+G7K+G109A+N280S+E391A;
H1*+G7E+G109A+N280S+E391A;
H1*+G7N+G109A+N280S+E391A;
H1*+G7Q+G109A+N280S+E391A;
H1*+G7L+G109A+N280S+E391A;
H1*+G7D+G109A+N280S+E391A;
H1*+G109A+N280S+K320A+E391A;
H1*+G109A+N280S+K320M+E391A;
H1*+G109A+N280S+K320T+E391A;
H1*+G109A+N280S+K320V+E391A;
H1*+G109A+N280S+M323R+E391A;
H1*+G109A+N280S+K320S+E391A;
H1*+G109A+N280S+E391V;
H1*+G109A+W284R+E391A;
H1*+G109A+W284F+E391A;
H1*+G109A+N280S+K320A+M323S+E391A;
H1*+G109A+N280S+W284F+E391A;
H1*+G109A+N280S+M323N+E391A;
H1*+G109A+N280S+M323K+E391A;
H1*+G109S+N280S+E391A;
H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A;
H1*+G7A+G109A+N280S+E391A;
H1*+G7A+G109A+N280S+W284H+K320A+M323N+E391A;
G7A+W284H+K320A+M323N;
G7A+K320A+M323N;
K320A;
G7A+K320A;
H1*+G7A+G109A+N280S+E391A;
H1*+G109A+N280S+W284H+E391A;
H1*+G109A+N280S+M323S+E391A;
H1*+G7A+G109A+N280S+K320A+E391A;
H1*+G7A+G109A+N280S+M323S+E391A;
H1*+G7A+G109A+N280S+M323N+E391A;
H1*+G7A+G109A+N280S+W284F+E391A;
H1*+G7A+G109A+N280S+W284R+E391A;
H1*+G7A+G109A+N280S+K320A+M323S+E391A;
H1*+G7A+G109A+W284R+E391A; and
H1*+G7A+G109A+N280S+K320A+M323N+E391A.
[0169] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 3, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0170] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 3.
[0171] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 4, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0172] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 4.
[0173] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 5, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0174] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 5.
[0175] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 6, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0176] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 6.
[0177] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 7, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0178] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 7.
[0179] A suitable variant may comprise modifications in the
positions corresponding to the positions of the amino acid sequence
set forth in SEQ ID NO: 8, selected from the group consisting
of:
X1*+X109A+X280S+X391A;
X1*+X7K+X109A+X280S+X391A;
X1*+X7E+X109A+X280S+X391A;
X1*+X7N+X109A+X280S+X391A;
X1*+X7Q+X109A+X280S+X391A;
X1*+X7L+X109A+X280S+X391A;
X1*+X7D+X109A+X280S+X391A;
X1*+X109A+X280S+X320A+X391A;
X1*+X109A+X280S+X320M+X391A;
X1*+X109A+X280S+X320T+X391A;
X1*+X109A+X280S+X320V+X391A;
X1*+X109A+X280S+X323R+X391A;
X1*+X109A+X280S+X320S+X391A;
X1*+X109A+X280S+X391V;
X1*+X109A+X284R+X391A;
X1*+X109A+X284F+X391A;
X1*+X109A+X280S+X320A+X323S+X391A;
X1*+X109A+X280S+X284F+X391A;
X1*+X109A+X280S+X323N+X391A;
X1*+X109A+X280S+X323K+X391A;
X1*+X109S+X280S+X391A;
X1*+X109A+X284H+X391A;
X1*+X109A+X280S+X320A+X323N+X391A;
X1*+X7A+X109A+X280S+X391A;
X1*+X7A+X109A+X280S+X284H+X320A+X323N+X391A;
X7A+X284H+X320A+X323N;
X7A+X320A+X323N;
X320A;
X7A+X320A;
X1*+X7A+X109A+X280S+X391A;
X1*+X109A+X280S+X284H+X391A;
X1*+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X320A+X391A;
X1*+X7A+X109A+X280S+X323S+X391A;
X1*+X7A+X109A+X280S+X323N+X391A;
X1*+X7A+X109A+X280S+X284F+X391A;
X1*+X7A+X109A+X280S+X284R+X391A;
X1*+X7A+X109A+X280S+X320A+X323S+X391A;
X1*+X7A+X109A+X284R+X391A; and
[0180] X1*+X7A+X109A+X280S+X320A+X323N+X391A, wherein numbering is
according to SEQ ID NO: 1 and the variant has at least 80% sequence
identity to the amylases set forth in SEQ ID NO: 8.
[0181] It is preferred that the variant comprises a modification at
one, two, three, four or five positions selected from the group of
X1*, X1A, X7A, X109A, X280S, and X391A. In a more preferred
embodiment, the modifications at one, two, three, four or five
positions are selected from X1*, X7A, X109A, X280S, and X391A.
[0182] In one aspect, a suitable variant may comprise modifications
in the positions corresponding to
X1*+X109A+X280S+X391A,
X1*+X109A+X284H+X391A,
X1*+X109A+X280S+X320A+X323N+X391A,
X1*+X7A+X109A+X280S+X391A, and
X1*+X7A+X109A+X280S+X284H+X323N+X391A,
[0183] wherein numbering is according to SEQ ID NO: 1, and wherein
the variant has at least 80% sequence identity to SEQ ID NO:1, 2,
3, 4, 5, 6, 7, or 8.
[0184] A suitable variant may comprise variants of SEQ ID NO: 1
comprising modifications in the positions corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A,
wherein numbering is according to SEQ ID NO: 1, and wherein the
variant has at least 80% sequence identity to SEQ ID NO:1.
[0185] A suitable variant may comprise a variant of SEQ ID NO: 2
comprising modifications corresponding to H1*+G109A+N280S+E391A;
H1*+G109A+W284H+E391A; H1*+G109A+N280S+K320A+M323N+E391A;
H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 2.
[0186] In one embodiment, the invention relates to variants of SEQ
ID NO: 3 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 3.
[0187] In one embodiment, the invention relates to variants of SEQ
ID NO: 4 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 4.
[0188] In one embodiment, the invention relates to variants of SEQ
ID NO: 5 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 5.
[0189] In one embodiment, the invention relates to variants of SEQ
ID NO: 6 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 6.
[0190] In one embodiment, the invention relates to variants of SEQ
ID NO: 7 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 7.
[0191] In one embodiment, the variants may comprise variants of SEQ
ID NO: 8 comprising modifications corresponding to
H1*+G109A+N280S+E391A; H1*+G109A+W284H+E391A;
H1*+G109A+N280S+K320A+M323N+E391A; H1*+G7A+G109A+N280S+E391A; and
H1*+G7A+G109A+N280S+W284H+M323N+E391A, wherein numbering is
according to SEQ ID NO: 1, and wherein the variant has at least 80%
sequence identity to SEQ ID NO: 8.
[0192] In one embodiment, the variant for the invention further
comprises a modification in one or more positions selected from the
group of 140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476.
In a particular embodiment, the variant for the invention comprises
one or more further modifications selected from the group of
W140Y/F, R181*, G182*, D183*, G184*, N195F/Y, I206Y/F, Y243F,
E260A/D/C/Q/L/M/F/P/S/W/V/G/H/I/K/N/R/T/Y, G304R/K/E/Q, and
G476E/Q/R/K. The variant for the invention may further comprise
substitutions at two, three or four positions selected from the
group consisting of G304R, W140YF, E260GHIKNPRTY and G476EQRK. In a
more preferred embodiment, the substitutions at the two, three or
four positions are selected from the group consisting of G304R,
W140Y, E260G and G476K.
[0193] The variant for the invention may comprise the modifications
corresponding to
H1*+G109A+W140Y+D183*+G184*+N195F+I206Y+Y243F+E260G+N280S+G304R+E391A+G476-
K,
H1*+G109A+W140Y+D183*+G184*+N195F+I206Y+Y243F+E260G+W284H+G304R+E391A+G476-
K,
H1*+G109A+W140Y+D183*+G184*+N195F+I206Y+Y243F+E260G+N280S+G304R+K320A+M323-
N+E391A+G476K,
H1*+G7A+G109A+W140Y+D183*+G184*+N195F+I206Y+Y243F+E260G+N280S+G304R+E391A+-
G476K, and
H1*+G7A+G109A+W140Y+D183*+G184*+N195F+I206Y+Y243F+E260G+N280S+W284H+G304R+-
M323N+E391A+G476K,
[0194] wherein numbering is according to SEQ ID NO: 1, the variant
has at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6,
7, or 8, and is a variant of SEQ ID NO: 1
[0195] Essential amino acids in a parent can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for alpha-amylase
activity to identify amino acid residues that are critical to the
activity of the molecule. See also, Hilton et al., 1996, J. Biol.
Chem. 271: 4699-4708. The active site of the alpha-amylase or other
biological interaction can also be determined by physical analysis
of structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identities of essential
amino acids can also be inferred from analysis of identities with
polypeptides that are related to the parent.
Nucleic Acid Constructs
[0196] The nucleic acid constructs may comprise a polynucleotide
encoding a variant essential to the present invention operably
linked to one or more (several) control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences. A polynucleotide
may be manipulated in a variety of ways to provide for expression
of a variant. Manipulation of the polynucleotide prior to its
insertion into a vector may be desirable or necessary depending on
the expression vector. The techniques for modifying polynucleotides
utilizing recombinant DNA methods are well known in the art.
[0197] The control sequence may be a promoter sequence, which is
recognized by a host cell for expression of the polynucleotide. The
promoter sequence contains transcriptional control sequences that
mediate the expression of the variant. The promoter may be any
nucleic acid sequence that shows transcriptional activity in the
host cell including mutant, truncated, and hybrid promoters, and
may be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0198] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a bacterial host cell are the promoters obtained from
the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus
licheniformis alpha-amylase gene (amyL), Bacillus licheniformis
penicillinase gene (penP), Bacillus stearothermophilus maltogenic
amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),
Bacillus subtilis xylA and xylB genes, E. coli lac operon,
Streptomyces coelicolor agarase gene (dagA), and prokaryotic
beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad.
Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et
al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Gilbert et al., 1980, Scientific American 242: 74-94; and in
Sambrook et al., 1989, supra.
[0199] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are the promoters
obtained from the genes for Aspergillus nidulans acetamidase,
Aspergillus niger neutral alpha-amylase, Aspergillus niger acid
stable alpha-amylase, Aspergillus niger or Aspergillus awamori
glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus
oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787),
Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium
venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO
00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic
proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a modified promoter including a gene encoding a neutral
alpha-amylase in Aspergilli in which the untranslated leader has
been replaced by an untranslated leader from a gene encoding triose
phosphate isomerase in Aspergilli; non-limiting examples include
modified promoters including the gene encoding neutral
alpha-amylase in Aspergillus niger in which the untranslated leader
has been replaced by an untranslated leader from the gene encoding
triose phosphate isomerase in Aspergillus nidulans or Aspergillus
oryzae); and mutant, truncated, and hybrid promoters thereof.
[0200] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0201] The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the variant. Any
terminator that is functional in the host cell may be used.
[0202] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, Aspergillus niger
glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
[0203] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0204] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA that is important for translation
by the host cell. The leader sequence is operably linked to the
5'-terminus of the polynucleotide encoding the variant. Any leader
sequence that is functional in the host cell may be used.
[0205] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0206] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0207] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the
variant-encoding sequence and, when transcribed, is recognized by
the host cell as a signal to add polyadenosine residues to
transcribed mRNA. Any polyadenylation sequence that is functional
in the host cell may be used.
[0208] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus nidulans
anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus
niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and
Fusarium oxysporum trypsin-like protease.
[0209] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0210] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
variant and directs the variant into the cell's secretory pathway.
The 5'-end of the coding sequence of the polynucleotide may
inherently contain a signal peptide coding region naturally linked
in translation reading frame with the segment of the coding region
that encodes the variant. Alternatively, the 5'-end of the coding
sequence may contain a signal peptide coding region that is foreign
to the coding sequence. The foreign signal peptide coding region
may be required where the coding sequence does not naturally
contain a signal peptide coding region. Alternatively, the foreign
signal peptide coding region may simply replace the natural signal
peptide coding region in order to enhance secretion of the variant.
However, any signal peptide coding region that directs the
expressed variant into the secretory pathway of a host cell may be
used.
[0211] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0212] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0213] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0214] The control sequence may also be a propeptide coding region
that encodes a propeptide positioned at the N-terminus of a
variant. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propolypeptide is
generally inactive and can be converted to an active polypeptide by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding region may be obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus
subtilis neutral protease (nprT), Myceliophthora thermophila
laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and
Saccharomyces cerevisiae alpha-factor.
[0215] Where both signal peptide and propeptide regions are present
at the N-terminus of a variant, the propeptide region is positioned
next to the N-terminus of the variant and the signal peptide region
is positioned next to the N-terminus of the propeptide region.
[0216] It may also be desirable to add regulatory sequences that
allow the regulation of the expression of the variant relative to
the growth of the host cell. Examples of regulatory systems are
those that cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the Aspergillus niger glucoamylase promoter,
Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus
oryzae glucoamylase promoter may be used. Other examples of
regulatory sequences are those that allow for gene amplification.
In eukaryotic systems, these regulatory sequences include the
dihydrofolate reductase gene that is amplified in the presence of
methotrexate, and the metallothionein genes that are amplified with
heavy metals. In these cases, the polynucleotide encoding the
variant would be operably linked with the regulatory sequence.
Expression Vectors
[0217] The recombinant expression vectors may comprise a
polynucleotide essential to the present invention, a promoter, and
transcriptional and translational stop signals. The various
nucleotide and control sequences may be joined together to produce
a recombinant expression vector that may include one or more
(several) convenient restriction sites to allow for insertion or
substitution of the polynucleotide encoding the variant at such
sites. Alternatively, the polynucleotide may be expressed by
inserting the polynucleotide or a nucleic acid construct comprising
the polynucleotide into an appropriate vector for expression. In
creating the expression vector, the coding sequence is located in
the vector so that the coding sequence is operably linked with the
appropriate control sequences for expression.
[0218] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about the expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vector may be a linear or closed
circular plasmid.
[0219] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0220] The vector preferably comprises one or more (several)
selectable markers that permit easy selection of transformed,
transfected, transduced, or the like cells. A selectable marker is
a gene the product of which provides for biocide or viral
resistance, resistance to heavy metals, prototrophy to auxotrophs,
and the like.
[0221] Examples of bacterial selectable markers are the dal genes
from Bacillus licheniformis or Bacillus subtilis, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, or tetracycline resistance. Suitable markers for yeast
host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
[0222] The vector preferably comprises an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0223] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the variant or any
other element of the vector for integration into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may comprise additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should contain a sufficient number of nucleic acids, such
as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to
10,000 base pairs, which have a high degree of identity to the
corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding nucleotide sequences. On the other hand,
the vector may be integrated into the genome of the host cell by
non-homologous recombination.
[0224] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" means a nucleotide sequence
that enables a plasmid or vector to replicate in vivo.
[0225] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of a variant. An increase in the copy number of the polynucleotide
can be obtained by integrating at least one additional copy of the
sequence into the host cell genome or by including an amplifiable
selectable marker gene with the polynucleotide where cells
containing amplified copies of the selectable marker gene, and
thereby additional copies of the polynucleotide, can be selected
for by cultivating the cells in the presence of the appropriate
selectable agent.
[0226] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra) to obtain substantially pure
variants.
Host Cells
[0227] Recombinant host cells, may comprise a polynucleotide
essential to the present invention operably linked to one or more
(several) control sequences that direct the production of a variant
for the present invention. A construct or vector comprising a
polynucleotide is introduced into a host cell so that the construct
or vector is maintained as a chromosomal integrant or as a
self-replicating extra-chromosomal vector as described earlier. The
term "host cell" encompasses any progeny of a parent cell that is
not identical to the parent cell due to mutations that occur during
replication. The choice of a host cell will to a large extent
depend upon the gene encoding the variant and its source.
[0228] The host cell may be any cell useful in the recombinant
production of a variant, e.g., a prokaryote or a eukaryote.
[0229] The prokaryotic host cell may be any gram-positive or
gram-negative bacterium. Gram-positive bacteria include, but are
not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but are not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0230] The bacterial host cell may be any Bacillus cell, including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0231] The bacterial host cell may also be any Streptococcus cell,
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0232] The bacterial host cell may also be any Streptomyces cell,
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0233] The introduction of DNA into a Bacillus cell may, for
instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using
competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol.
81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol.
56: 209-221), by electroporation (see, e.g., Shigekawa and Dower,
1988, Biotechniques 6: 742-751), or by conjugation (see, e.g.,
Koehler and Thorne, 1987, J. Bacteria 169: 5271-5278). The
introduction of DNA into an E. coli cell may, for instance, be
effected by protoplast transformation (see, e.g., Hanahan, 1983, J.
Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et
al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of
DNA into a Streptomyces cell may, for instance, be effected by
protoplast transformation and electroporation (see, e.g., Gong et
al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation
(see, e.g., Mazodier et al., 1989, J. Bacteria 171: 3583-3585), or
by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad.
Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas
cell may, for instance, be effected by electroporation (see, e.g.,
Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by
conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.
Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus
cell may, for instance, be effected by natural competence (see,
e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by
protoplast transformation (see, e.g., Catt and Jollick, 1991,
Microbios 68: 189-2070, by electroporation (see, e.g., Buckley et
al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by
conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:
409-436). However, any method known in the art for introducing DNA
into a host cell can be used.
[0234] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0235] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023 and Yelton et al., 1984, Proc.
Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for
transforming Fusarium species are described by Malardier et al.,
1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed
using the procedures described by Becker and Guarente, In Abelson,
J. N. and Simon, M. I., editors, Guide to Yeast Genetics and
Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187,
Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.
153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:
1920.
Methods of Production
[0236] The method of producing a variant, may comprise: (a)
cultivating a host cell of the present invention under conditions
suitable for the expression of the variant; and (b) recovering the
variant. Accordingly, the present invention relates to methods of
producing a variant, comprising (a) cultivating a host cell
comprising an expression vector or a polynucleotide encoding
variant comprising a modification at one or more positions
corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of
the amino acid sequence set forth in SEQ ID NO: 1, and optionally
in one or more positions corresponding to positions 140, 181, 182,
183, 184, 195, 206, 243, 260, 304, and 476 of the amino acid
sequence as set forth in SEQ ID NO: 1, under conditions suitable
for the expression of the variant; and (b) recovering the
variant.
[0237] The host cells are cultivated in a nutrient medium suitable
for production of the variant using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation, or
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the variant is secreted into the nutrient medium,
the variant can be recovered directly from the medium. If the
variant is not secreted, it can be recovered from cell lysates.
[0238] The variant may be detected using methods known in the art
that are specific for the variants. These detection methods may
include use of specific antibodies, formation of an enzyme product,
or disappearance of an enzyme substrate. For example, an enzyme
assay may be used to determine the activity of the variant.
[0239] The variant may be recovered by methods known in the art.
For example, the variant may be recovered from the nutrient medium
by conventional procedures including, but not limited to,
collection, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation.
[0240] The variant may be purified by a variety of procedures known
in the art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e g, ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989) to obtain substantially pure variants.
[0241] In an alternative aspect, the variant is not recovered, but
rather a host cell of the present invention expressing a variant is
used as a source of the variant.
[0242] The compositions may be prepared in accordance with methods
known in the art and may be in the form of a liquid or a dry
composition. For instance, the composition may be in the form of a
granulate or a microgranulate. The variant may be stabilized in
accordance with methods known in the art.
Cleaning Compositions
[0243] The present invention preferably relates to products for
and/or methods relating to and/or use of the claimed compositions
that are for air care, car care, dishwashing, fabric conditioning
(including softening), laundry detergency, laundry and rinse
additive and/or care, hard surface cleaning and/or treatment, and
other cleaning for consumer or institutional use. According to the
invention, the above alpha-amylase variants may typically be a
component in a cleaning composition, such as a solid, liquid, gel
and/or unit dose detergent composition, e.g., a laundry detergent
composition or a dishwashing detergent composition. Especially
preferred is a liquid laundry detergent composition.
[0244] Such cleaning compositions comprise a cleaning/detergent
adjunct, preferably a mixture of components. Typically the cleaning
adjunct will be present in the composition in an amount from 0.001
to 99.9 wt %, more typically from 0.01 to 80 wt % cleaning adjunct.
Suitable cleaning adjuncts comprise: surfactants, builders,
bleaches, bleach catalysts, colorants, bleach boosters, chelating
agents, dye transfer agents, deposition aids, dispersants,
additional enzymes, and enzyme stabilizers, catalytic materials,
bleach activators, hydrogen peroxide, sources of hydrogen peroxide,
optical brighteners, photoactivators, fluorescers, fabric hueing
agents, fabric conditioners, preformed peracids, polymeric
dispersing agents, clay soil removal/anti-redeposition agents,
filler salts, hydrotropes, brighteners, suds suppressors, structure
elasticizing agents, fabric softeners, hydrolyzable surfactants,
preservatives, anti-oxidants, anti-shrinkage agents, germicides,
fungicides, anti-tarnish, anti-corrosion agents, alkalinity
sources, solubilizing agents, carriers, processing aids, pigments,
dyes, perfumes and pH control agents, encapsulates, polymers. For
example, these may include: bleach ingredients such as imine bleach
boosters; sources of hydrogen peroxide such as percarbonate and/or
perborate, especially percarbonate coated with material such as
carbonate and/or sulphate salt, silicate salt, borosilicate, and
any mixture thereof; pre-formed peracid, including pre-formed
peracid in encapsulated form; transition metal catalysts; suds
suppressors or suppressor systems such as silicone based suds
suppressors and/or fatty acid based suds suppressors;
fabric-softeners such as clay, silicone and/or quaternary ammonium
compounds; flocculants such as polyethylene oxide; dye transfer
inhibitors such as polyvinylpyrrolidone, poly 4-vinylpyridine
N-oxide and/or co-polymer of vinylpyrrolidone and vinylimidazole;
fabric integrity components such as oligomers produced by the
condensation of imidazole and epichlorhydrin; soil dispersants and
soil anti-redeposition aids such as alkoxylated polyamines and
ethoxylated ethyleneimine polymers; anti-redeposition components
such as polyesters; carboxylate polymers such as maleic acid
polymers or co-polymers of maleic and acrylic acid; perfumes such
as perfume microcapsules, starch encapsulated accords, perfume
spray-on; soap rings; aesthetic particles; dyes; fillers such as
sodium sulphate, although it is preferred for the composition to be
substantially free of fillers; silicate salt such as sodium
silicate, including 1.6R and 2.0R sodium silicate, or sodium
metasilicate; co-polyesters of di-carboxylic acids and diols;
cellulosic polymers such as methyl cellulose, carboxymethyl
cellulose, hydroxyethoxycellulose, or other alkyl or alkylalkoxy
cellulose; solvents such as 1,2 propanediol, monoethanolamine;
diethylene glycol, ethanol, and any mixture thereof; hydrotropes
such as sodium cumene sulphonate, sodium xylene sulphonate, sodium
toluene sulphonate, and any mixtures; organic acids such as citric
acid; and any combination thereof. The composition may be such that
the cleaning adjunct comprises one or more selected from the group
consisting of (i) perfume microcapsule; (ii) fabric hueing agent;
(iii) protease; (iv) amphiphilic cleaning polymer; (v) lipase, or
(vi) mixtures thereof.
[0245] In another preferred aspect the composition comprises one or
more surfactants, which may be non-ionic including semi-polar
and/or anionic and/or cationic and/or zwitterionic and/or
ampholytic and/or semi-polar nonionic and/or mixtures thereof. The
surfactants are typically present at a level of from 0.1% to 60% by
weight or from 0.5 to 50 wt % or 1 to 40 wt % of the
composition.
[0246] When included therein the cleaning composition will usually
contain from about 1% to about 40% of an anionic surfactant such as
linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate
(fatty alcohol sulfate), alcohol ethoxysulfate, secondary
alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or
alkenylsuccinic acid or soap.
[0247] When included therein the cleaning agent will usually
contain from about 0.2% to about 40% of a non-ionic surfactant such
as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside,
alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0248] The cleaning composition may comprise one or more other
enzymes. Therefore a preferred composition comprises (a) a variant
of a parent alpha-amylase, wherein said variant comprises (i) a
modification at one or more positions corresponding to positions
109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence
set forth in SEQ ID NO: 1, and optionally in one or more positions
corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243,
260, 304, and 476 of the amino acid sequence as set forth in SEQ ID
NO: 1, (ii) said variant has at least 80, such as at least 90%,
such as at least 95%, such as at least 97%, but less than 100%
sequence identity with the amino acid sequence set forth in SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and (iii) said variant has
alpha-amylase activity; and (b) one or more additional enzymes
preferably selected from the group consisting of aminopeptidase,
amylase, carbohydrase, carboxypeptidase, catalase, cellulase,
chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, haloperoxidase, invertase, laccase, lipase,
mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase,
peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,
ribonuclease, transglutaminase, or xylanase. The additional
enzyme(s) may be produced, for example, by a microorganism
belonging to the genus Aspergillus, e.g., Aspergillus aculeatus,
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or
Aspergillus oryzae; Fusarium, e.g., Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sulphureum, Fusarium toruloseum, Fusarium
trichothecioides, or Fusarium venenatum; Humicola, e.g., Humicola
insolens or Humicola lanuginosa; or Trichoderma, e.g., Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride.
[0249] Preferably the composition comprises a protease or mixtures
of more than one protease, a lipase or mixtures of more than one
lipase, a peroxidase or mixtures of more than one peroxidase, one
or more additional amylolytic enzymes, e.g., an additional
alpha-amylase, glucoamylase, maltogenic amylase, preferably an
additional alpha amylase, one or mixtures of more than one CGTase
and/or a cellulase or mixtures of more than one cellulase,
mannanase (such as MANNAWAY.TM. from Novozymes, Denmark) or
mixtures of more than one mannanase, pectinase, pectate lyase,
cutinase, and/or laccase or mixtures of more than one of one or
more of these.
[0250] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent, (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective amounts.
Preferably, the product of the invention comprises at least 0.01
mg, preferably from about 0.05 to about 10, more preferably from
about 0.1 to about 6, especially from about 0.2 to about 5 mg of
active further enzyme/g of composition.
[0251] Proteases: Suitable proteases include metalloproteases
and/or serine proteases, including neutral or alkaline microbial
serine proteases, such as subtilisins (EC 3.4.21.62). Suitable
proteases include those of animal, vegetable or microbial origin.
In one aspect, such suitable protease may be of microbial origin.
The suitable proteases include chemically or genetically modified
mutants of the aforementioned suitable proteases. In one aspect,
the suitable protease may be a serine protease, such as an alkaline
microbial protease or/and a trypsin-type protease. Examples of
suitable neutral or alkaline proteases include:
[0252] (a) subtilisins (EC 3.4.21.62), including those derived from
Bacillus, such as Bacillus lentus, B. alkalophilus, B. subtilis, B.
amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described
in U.S. Pat. No. 6,312,936 B1, U.S. Pat. No. 5,679,630, U.S. Pat.
No. 4,760,025, U.S. Pat. No. 7,262,042 and WO09/021867.
[0253] (b) trypsin-type or chymotrypsin-type proteases, such as
trypsin (e.g., of porcine or bovine origin), including the Fusarium
protease described in WO 89/06270 and the chymotrypsin proteases
derived from Cellumonas described in WO 05/052161 and WO
05/052146.
[0254] (c) metalloproteases, including those derived from Bacillus
amyloliquefaciens described in WO 07/044993A2.
[0255] Preferred proteases include those derived from Bacillus
gibsonii or Bacillus Lentus.
[0256] Suitable commercially available protease enzymes include
those sold under the trade names Alcalase.RTM., Savinase.RTM.,
Primase.RTM., Durazym.RTM., Polarzyme.RTM., Kannase.RTM.,
Liquanase.RTM., Liquanase Ultra.RTM., Savinase Ultra.RTM.,
Ovozyme.RTM., Neutrase.RTM., Everlase.RTM. and Esperase.RTM. by
Novozymes A/S (Denmark), those sold under the tradename
Maxatase.RTM., Maxacal.RTM., Maxapem.RTM., Properase.RTM.,
Purafect.RTM., Purafect Prime.RTM., Purafect Ox.RTM., FN3.RTM.,
FN4.RTM., Excellase.RTM. and Purafect OXP.RTM. by Genencor
International, those sold under the tradename Opticlean.RTM. and
Optimase.RTM. by Solvay Enzymes, those available from
Henkel/Kemira, namely BLAP (sequence shown in FIG. 29 of U.S. Pat.
No. 5,352,604 with the following mutations S99D+S101
R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP
with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I)
and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D)--all from
Henkel/Kemira; and KAP (Bacillus alkalophilus subtilisin with
mutations A230V+S256G+S259N) from Kao. Further suitable proteases
are described in WO2011/03623, WO2011/140316, WO2011/140364 and
WO2012/05778.
[0257] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful lipases include lipases from
Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T.
lanuginosus) or from H. insolens, a Pseudomonas lipase, e.g., from
P. alcaligenes or P. pseudoalcaligenes, P. cepacia P. stutzeri, P.
fluorescens, Pseudomonas sp. strain SD 705, P. wisconsinensis, a
Bacillus lipase, e.g., from B. subtilis (Dartois et al. (1993),
Biochemica et Biophysica Acta, 1131, 253-360), B.
stearothermophilus or B. pumilus.
[0258] The lipase may be a "first cycle lipase" such as those
described in U.S. Pat. No. 6,939,702 B1 and US PA 2009/0217464. In
one aspect, the lipase is a first-wash lipase, preferably a variant
of the wild-type lipase from Thermomyces lanuginosus comprising
T231R and N233R mutations. The wild-type sequence is the 269 amino
acids (amino acids 23 291) of the Swissprot accession number
Swiss-Prot 059952 (derived from Thermomyces lanuginosus (Humicola
lanuginosa)). Preferred lipases would include those sold under the
tradenames Lipex.RTM., Lipolex.RTM. and Lipoclean.RTM..
[0259] Cellulases: Suitable cellulases include those of bacterial
or fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g., the fungal cellulases produced from Humicola
insolens, Myceliophthora thermophila and Fusarium oxysporum.
[0260] In one aspect, preferred enzymes include microbial-derived
endoglucanases exhibiting endo-beta-1,4-glucanase activity (E.C.
3.2.1.4), preferably selected from the group comprising: [0261] (a)
a bacterial polypeptide endogenous to a member of the genus
Bacillus which has a sequence of at least 90%, 94%, 97% and even
99% identity to the amino acid sequence SEQ ID NO:2 in U.S. Pat.
No. 7,141,403B2; [0262] (b) a glycosyl hydrolase having enzymatic
activity towards both xyloglucan and amorphous cellulose
substrates, wherein the glycosyl hydrolase is selected from GH
families 5, 12, 44 or 74; [0263] (c) a glycosyl hydrolase having a
sequence of at least 90%, 94%, 97% and even 99% identity to the
amino acid sequence SEQ ID NO:3 in WO09/148983; [0264] (d) and
mixtures thereof.
[0265] Suitable endoglucanases are sold under the tradenames
Celluclean.RTM. and Whitezyme.RTM. (Novozymes A/S, Bagsvaerd,
Denmark).
[0266] Other commercially available cellulases include
CELLUZYME.RTM., and CAREZYME.RTM. (Novozymes A/S), CLAZINASE.RTM.,
and PURADAX HA.RTM. (Genencor International Inc.), and
KAC-500(B).RTM. (Kao Corporation).
[0267] Other amylases: Preferably the composition comprises a
further amylase. Suitable further amylases include alpha-amylases
including those of bacterial or fungal origin. Chemically or
genetically modified mutants (variants) are included. A preferred
alkaline alpha-amylase is derived from a strain of Bacillus, such
as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus
stearothermophilus, Bacillus subtilis, or other Bacillus sp., such
as Bacillus sp. NCBI 12289, NCBI 12512, NCBI 12513, DSM 9375 (U.S.
Pat. No. 7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO
97/00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred further
amylases may be selected from: (a) variants described in WO
94/02597, WO 94/18314, WO96/23874 and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions versus the enzyme listed as SEQ ID No. 2 in WO 96/23874:
15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202,
208, 209, 243, 264, 304, 305, 391, 408, and 444; (b) variants
described in WO 96/23873, WO00/60060, WO06/002643 and
WO2017/192657, especially the variants with one or more
substitutions in the following positions versus the AA560 enzyme
listed as SEQ ID No. 12 in WO 06/002643: 26, 30, 33, 82, 37, 106,
118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 203, 214, 231,
246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303,
304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419,
421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484,
preferably that also contain the deletions of D183* and G184*; (c)
variants exhibiting at least 90% identity with SEQ ID No. 4 in
WO06/002643, the wild-type enzyme from Bacillus SP722, especially
variants with deletions in the 183 and 184 positions and variants
described in WO 00/60060, which is incorporated herein by
reference; (d) variants exhibiting at least 95% identity with the
wild-type enzyme from Bacillus sp. 707 (SEQ ID NO:7 in U.S. Pat.
No. 6,093,562), especially those comprising one or more of the
following mutations M202, M208, S255, R172, and/or M261. Preferably
said amylase comprises one or more of M202L, M202V, M202S, M202T,
M202I, M202Q, M202W, S255N and/or R172Q. Particularly preferred are
those comprising the M202L or M202T mutations; (e) variants
described in WO 09/149130, preferably those exhibiting at least 90%
identity with SEQ ID NO: 1 or SEQ ID NO:2 in WO 09/149130, the
wild-type enzyme from Geobacillus Stearophermophilus or a truncated
version thereof; (f) variants exhibiting at least 89% identity with
SEQ ID NO:1 in WO2016091688, especially those comprising deletions
at positions H183+G184 and additionally one or more mutations at
positions 405, 421, 422 and/or 428; (g) variants exhibiting at
least 60% amino acid sequence identity with the "PcuAmyl
.alpha.-amylase" from Paenibacillus curdlanolyticus YK9 (SEQ ID
NO:3 in WO2014099523); (h) variants exhibiting at least 60% amino
acid sequence identity with the "CspAmy2 amylase" from Cytophaga
sp. (SEQ ID NO:1 in WO2014164777); (i) variants exhibiting at least
85% identity with AmyE from Bacillus subtilis (SEQ ID NO:1 in
WO2009149271); (j) variants exhibiting at least 90% identity with
the wild-type amylase from Bacillus sp. KSM-K38 with accession
number AB051102; (k) variants exhibiting at least 80% identity with
the mature amino acid sequence of AAI10 from Bacillus sp (SEQ ID
NO:7 in WO2016180748); (l) variants exhibiting at least 80%
identity with the mature amino acid sequence of Alicyclobacillus
sp. amylase (SEQ ID NO:8 in WO2016180748); or mixtures thereof.
Where present, the composition of the invention preferably
comprises from at least 0.01 mg, preferably from about 0.05 to
about 10, more preferably from about 0.1 to about 6, especially
from about 0.2 to about 5 mg of active further amylase/g of
composition.
[0268] Suitable commercially available alpha-amylases include
DURAMYL.RTM., LIQUEZYME.RTM., TERMAMYL.RTM., TERMAMYL ULTRA.RTM.,
NATALASE.RTM., SUPRAMYL.RTM., STAINZYME.RTM., STAINZYME PLUS.RTM.,
FUNGAMYL.RTM., ATLANTIC.RTM., INTENSA.RTM. and BAN.RTM. (Novozymes
A/S, Bagsvaerd, Denmark), KEMZYM.RTM. AT 9000 Biozym Biotech
Trading GmbH Wehlistrasse 27b A-1200 Wien Austria, RAPIDASE.RTM.,
PURASTAR.RTM., ENZYSIZE.RTM., OPTISIZE HT PLUS.RTM., POWERASE.RTM.,
PREFERENZ S.RTM. series (including PREFERENZ S10000 and PREFERENZ
S2000.RTM. and PURASTAR OXAM.RTM. (DuPont., Palo Alto, Calif.) and
KAM.RTM. (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku Tokyo
103-8210, Japan). In one aspect, suitable amylases include
ATLANTIC.RTM., STAINZYME.RTM., POWERASE.RTM., INTENSA.RTM. and
STAINZYME PLUS.RTM. and mixtures thereof.
[0269] Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bac-terial or fungal origin. Chemically modified or
protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g., from C.
cinereus, and variants thereof as those described in WO 93/24618,
WO 95/10602, and WO 98/15257.
[0270] Commercially available peroxidases include GUARDZYME.RTM.
(Novozymes A/S).
[0271] Other enzymes: Other preferred enzymes include pectate
lyases sold under the tradenames Pectawash.RTM., Pectaway.RTM. and
mannanases sold under the tradenames Mannaway.RTM. (all from
Novozymes A/S, Bagsvaerd, Denmark), and Purabrite.RTM. (Genencor
International Inc., Palo Alto, Calif.).
[0272] The detergent enzyme(s) 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 separate
additive or a combined additive, can be formulated, e.g.,
granulate, a liquid, a slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0273] Non-dusting granulates may be produced and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonyl-phenols 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. Film-forming coating materials may be
applied for example by fluid bed techniques. 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.
[0274] The composition may comprise a fabric hueing agent
(sometimes referred to as shading, bluing or whitening agents).
Typically the hueing agent provides a blue or violet shade to
fabric. Hueing agents can be used either alone or in combination to
create a specific shade of hueing and/or to shade different fabric
types. This may be provided for example by mixing a red and
green-blue dye to yield a blue or violet shade. Hueing agents may
be selected from any known chemical class of dye, including but not
limited to acridine, anthraquinone (including polycyclic quinones),
azine, azo (e.g., monoazo, disazo, trisazo, tetrakisazo, polyazo),
including premetallized azo, benzodifurane and benzodifuranone,
carotenoid, coumarin, cyanine, diazahemicyanine, diphenylmethane,
formazan, hemicyanine, indigoids, methane, naphthalimides,
naphthoquinone, nitro and nitroso, oxazine, phthalocyanine,
pyrazoles, stilbene, styryl, triarylmethane, triphenylmethane,
xanthenes and mixtures thereof.
[0275] Suitable fabric hueing agents include dyes, dye-clay
conjugates, and organic and inorganic pigments. Suitable dyes
include small molecule dyes and polymeric dyes. Suitable small
molecule dyes include small molecule dyes selected from the group
consisting of dyes falling into the Colour Index (C.I.)
classifications of Direct, Basic, Reactive or hydrolysed Reactive,
Solvent or Disperse dyes for example that are classified as Blue,
Violet, Red, Green or Black, and provide the desired shade either
alone or in combination. In another aspect, suitable small molecule
dyes include small molecule dyes selected from the group consisting
of Colour Index (Society of Dyers and Colourists, Bradford, UK)
numbers Direct Violet dyes such as 9, 35, 48, 51, 66, and 99,
Direct Blue dyes such as 1, 71, 80 and 279, Acid Red dyes such as
17, 73, 52, 88 and 150, Acid Violet dyes such as 15, 17, 24, 43, 49
and 50, Acid Blue dyes such as 15, 17, 25, 29, 40, 45, 75, 80, 83,
90 and 113, Acid Black dyes such as 1, Basic Violet dyes such as 1,
3, 4, 10 and 35, Basic Blue dyes such as 3, 16, 22, 47, 66, 75 and
159, Disperse or Solvent dyes such as those described in EP1794275
or EP1794276, or dyes as disclosed in U.S. Pat. No. 7,208,459
B2,and mixtures thereof. In another aspect, suitable small molecule
dyes include small molecule dyes selected from the group consisting
of Colour Index numbers Acid Violet 17, Direct Blue 71, Direct
Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29,
Acid Blue 113 or mixtures thereof.
[0276] Suitable polymeric dyes include polymeric dyes selected from
the group consisting of polymers containing covalently bound
(sometimes referred to as conjugated) chromogens, (dye-polymer
conjugates), for example polymers with chromogens co-polymerized
into the backbone of the polymer and mixtures thereof. Polymeric
dyes include those described in WO2011/98355, WO2011/47987,
US2012/090102, WO2010/145887, WO2006/055787 and WO2010/142503.
[0277] In another aspect, suitable polymeric dyes include polymeric
dyes selected from the group consisting of fabric-substantive
colorants sold under the name of Liquitint.RTM. (Milliken,
Spartanburg, S.C., USA), dye-polymer conjugates formed from at
least one reactive dye and a polymer selected from the group
consisting of polymers comprising a moiety selected from the group
consisting of a hydroxyl moiety, a primary amine moiety, a
secondary amine moiety, a thiol moiety and mixtures thereof. In
still another aspect, suitable polymeric dyes include polymeric
dyes selected from the group consisting of Liquitint.RTM. Violet
CT, carboxymethyl cellulose (CMC) covalently bound to a reactive
blue, reactive violet or reactive red dye such as CMC conjugated
with C.I. Reactive Blue 19, sold by Megazyme, Wicklow, Ireland
under the product name AZO-CM-CELLULOSE, product code S-ACMC,
alkoxylated triphenyl-methane polymeric colourants, alkoxylated
thiophene polymeric colourants, and mixtures thereof.
[0278] Preferred hueing dyes include the alkoxylated thiophene azo
whitening agents found in US2008/0177090 which may be optionally
anionic, such as those selected from Examples 1-42 in Table 5 of
WO2011/011799. Other preferred dyes are disclosed in U.S. Pat. No.
8,138,222.
[0279] Suitable dye clay conjugates include dye clay conjugates
selected from the group comprising at least one cationic/basic dye
and a smectite clay, and mixtures thereof. In another aspect,
suitable dye clay conjugates include dye clay conjugates selected
from the group consisting of one cationic/basic dye selected from
the group consisting of C.I. Basic Yellow 1 through 108, C.I. Basic
Orange 1 through 69, C.I. Basic Red 1 through 118, C.I. Basic
Violet 1 through 51, C.I. Basic Blue 1 through 164, C.I. Basic
Green 1 through 14, C.I. Basic Brown 1 through 23, CI Basic Black 1
through 11, and a clay selected from the group consisting of
Montmorillonite clay, Hectorite clay, Saponite clay and mixtures
thereof. In still another aspect, suitable dye clay conjugates
include dye clay conjugates selected from the group consisting of:
Montmorillonite Basic Blue B7 C.I. 42595 conjugate, Montmorillonite
Basic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3
C.I. 42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040
conjugate, Montmorillonite Basic Red R1 C.I. 45160 conjugate,
Montmorillonite C.I. Basic Black 2 conjugate, Hectorite Basic Blue
B7 C.I. 42595 conjugate, Hectorite Basic Blue B9 C.I. 52015
conjugate, Hectorite Basic Violet V3 C.I. 42555 conjugate,
Hectorite Basic Green G1 C.I. 42040 conjugate, Hectorite Basic Red
R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black 2 conjugate,
Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite Basic Blue B9
C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555
conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite
Basic Red R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2
conjugate and mixtures thereof.
[0280] Suitable pigments include pigments selected from the group
consisting of flavanthrone, indanthrone, chlorinated indanthrone
containing from 1 to 4 chlorine atoms, pyranthrone,
dichloropyranthrone, monobromodichloropyranthrone,
dibromodichloropyranthrone, tetrabromopyranthrone,
perylene-3,4,9,10-tetracarboxylic acid diimide, wherein the imide
groups may be unsubstituted or substituted by C1-C3-alkyl or a
phenyl or heterocyclic radical, and wherein the phenyl and
heterocyclic radicals may additionally carry substituents which do
not confer solubility in water, anthrapyrimidinecarboxylic acid
amides, violanthrone, isoviolanthrone, dioxazine pigments, copper
phthalocyanine which may contain up to 2 chlorine atoms per
molecule, polychloro-copper phthalocyanine or
polybromochloro-copper phthalocyanine containing up to 14 bromine
atoms per molecule and mixtures thereof.
[0281] In another aspect, suitable pigments include pigments
selected from the group consisting of Ultramarine Blue (C.I.
Pigment Blue 29), Ultramarine Violet (C.I. Pigment Violet 15) and
mixtures thereof. Builders--The cleaning composition may further
contain builders, such as builders based on carbonate, bicarbonate
or silicates which may be Zeolites, such as Zeolite A, Zeolite MAP
(Maximum Aluminium type P). Zeolites, useable in laundry preferably
has the formula
Na.sub.12(AlO.sub.2).sub.12(SiO.sub.2).sub.12.27H.sub.2O and the
particle size is usually between 1-10 .mu.m for zeolite A and 0.7-2
.mu.m for zeolite MAP. Other builders are Sodium metasilicate
(Na.sub.2SiO.sub.3.nH.sub.2O or Na.sub.2Si.sub.2O.sub.5.n H.sub.2O)
strong alkaline and preferably used in dish wash. In preferred
embodiments, the amount of a detergent builder may be above 5%,
above 10%, above 20%, above 30%, above 40% or above 50%, and may be
below 80%, 65%. In a dishwash detergent, the level of builder is
typically 40-65%, particularly 50-65% or even 75-90%.
[0282] Encapsulates--The composition may comprise an encapsulate.
In one aspect, an encapsulate comprising a core, a shell having an
inner and outer surface, said shell encapsulating said core.
[0283] In one aspect of said encapsulate, said core may comprise a
material selected from the group consisting of perfumes;
brighteners; dyes; insect repellants; silicones; waxes; flavors;
vitamins; fabric softening agents; skin care agents in one aspect,
paraffins; enzymes; anti-bacterial agents; bleaches; sensates; and
mixtures thereof; and said shell may comprise a material selected
from the group consisting of polyethylenes; polyamides;
polystyrenes; polyisoprenes; polycarbonates; polyesters;
polyacrylates; aminoplasts, in one aspect said aminoplast may
comprise a polyureas, polyurethane, and/or polyureaurethane, in one
aspect said polyurea may comprise polyoxymethyleneurea and/or
melamine formaldehyde; polyolefins; polysaccharides, in one aspect
said polysaccharide may comprise alginate and/or chitosan; gelatin;
shellac; epoxy resins; vinyl polymers; water insoluble inorganics;
silicone; and mixtures thereof.
[0284] In one aspect of said encapsulate, said core may comprise
perfume. Such encapsulates are perfume microcapsules.
[0285] In one aspect of said encapsulate, said shell may comprise
melamine formaldehyde and/or cross linked melamine
formaldehyde.
[0286] In a one aspect, suitable encapsulates may comprise a core
material and a shell, said shell at least partially surrounding
said core material, is disclosed. At least 75%, 85% or even 90% of
said encapsulates may have a fracture strength of from about 0.2
MPa to about 10 MPa, from about 0.4 MPa to about 5 MPa, from about
0.6 MPa to about 3.5 MPa, or even from about 0.7 MPa to about 3
MPa; and a benefit agent leakage of from 0% to about 30%, from 0%
to about 20%, or even from 0% to about 5%.
[0287] In one aspect, at least 75%, 85% or even 90% of said
encapsulates may have a particle size of from about 1 microns to
about 80 microns, about 5 microns to 60 microns, from about 10
microns to about 50 microns, or even from about 15 microns to about
40 microns.
[0288] In one aspect, at least 75%, 85% or even 90% of said
encapsulates may have a particle wall thickness of from about 30 nm
to about 250 nm, from about 80 nm to about 180 nm, or even from
about 100 nm to about 160 nm.
[0289] In one aspect, said encapsulates' core material may comprise
a material selected from the group consisting of a perfume raw
material and/or optionally a material selected from the group
consisting of vegetable oil, including neat and/or blended
vegetable oils including caster oil, coconut oil, cottonseed oil,
grape oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil,
safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil,
castor oil, lemon oil and mixtures thereof; esters of vegetable
oils, esters, including dibutyl adipate, dibutyl phthalate, butyl
benzyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl
phosphate and mixtures thereof; straight or branched chain
hydrocarbons, including those straight or branched chain
hydrocarbons having a boiling point of greater than about
80.degree. C.; partially hydrogenated terphenyls, dialkyl
phthalates, alkyl biphenyls, including monoisopropylbiphenyl,
alkylated naphthalene, including dipropylnaphthalene, petroleum
spirits, including kerosene, mineral oil and mixtures thereof;
aromatic solvents, including benzene, toluene and mixtures thereof;
silicone oils; and mixtures thereof.
[0290] In one aspect, said encapsulates' wall material may comprise
a suitable resin including the reaction product of an aldehyde and
an amine, suitable aldehydes include, formaldehyde. Suitable amines
include melamine, urea, benzoguanamine, glycoluril, and mixtures
thereof. Suitable melamines include, methylol melamine, methylated
methylol melamine, imino melamine and mixtures thereof. Suitable
ureas include, dimethylol urea, methylated dimethylol urea,
urea-resorcinol, and mixtures thereof.
[0291] In one aspect, suitable formaldehyde scavengers may be
employed with the encapsulates, for example, in a capsule slurry
and/or added to a consumer product before, during or after the
encapsulates are added to such consumer product.
[0292] Suitable capsules can be purchased from Appleton Papers Inc.
of Appleton, Wis. USA.
[0293] In addition, the materials for making the aforementioned
encapsulates can be obtained from Solutia Inc. (St Louis, Mo.
U.S.A.), Cytec Industries (West Paterson, New Jersey U.S.A.),
sigma-Aldrich (St. Louis, Mo. U.S.A.), CP Kelco Corp. of San Diego,
Calif., USA; BASF AG of Ludwigshafen, Germany; Rhodia Corp. of
Cranbury, N.J., USA; Hercules Corp. of Wilmington, Del., USA;
Agrium Inc. of Calgary, Alberta, Canada, ISP of New Jersey U.S.A.,
Akzo Nobel of Chicago, Ill., USA; Stroever Shellac Bremen of
Bremen, Germany; Dow Chemical Company of Midland, Mich., USA; Bayer
AG of Leverkusen, Germany; Sigma-Aldrich Corp., St. Louis, Mo.,
USA.
[0294] In one aspect, the composition may comprise an enzyme
stabilizer selected from the group consisting of (a) inorganic
salts selected from the group consisting of calcium salts,
magnesium salts and mixtures thereof; (b) carbohydrates selected
from the group consisting of oligosaccharides, polysaccharides and
mixtures thereof; (c) mass efficient reversible protease inhibitors
selected from the group consisting of phenyl boronic acid and
derivatives thereof; and (d) mixtures thereof.
[0295] In another embodiment, the composition comprises: (1)
reversible protease inhibitors such as a boron containing compound;
(2) 1-2 propane diol; (3) calcium formate and/or sodium formate;
and (4) any combination thereof.
[0296] In one aspect, the composition may comprise a structurant
selected from the group consisting of diglycerides and
triglycerides, ethylene glycol distearate microcrystalline
cellulose, cellulose-based materials, microfiber cellulose,
biopolymers, xanthan gum, gellan gum, and mixtures thereof.
Polymers
[0297] The consumer product may comprise one or more polymers.
Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly
(ethylene glycol), poly(vinyl alcohol),
poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates
such as polyacrylates, maleic/acrylic acid copolymers and lauryl
methacrylate/acrylic acid co-polymers and amphiphilic polymers.
Amphiphilic Cleaning Polymers
[0298] Preferably, the amphiphilic cleaning polymer is a compound
having the following general structure:
bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n)(CH.sub.3)--N.sup.+--C.sub.xH.sub-
.2x--N.sup.+--(CH.sub.3)-bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n),
wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or
sulphonated variants thereof.
[0299] Amphiphilic alkoxylated grease cleaning polymers of the
present invention refer to any alkoxylated polymer having balanced
hydrophilic and hydrophobic properties such that they remove grease
particles from fabrics and surfaces. Specific embodiments of the
amphiphilic alkoxylated grease cleaning polymers of the present
invention comprise a core structure and a plurality of alkoxylate
groups attached to that core structure. These may comprise
alkoxylated polyalkylenimines, preferably having an inner
polyethylene oxide block and an outer polypropylene oxide
block.
[0300] The core structure may comprise a polyalkylenimine structure
comprising, in condensed form, repeating units of formulae (I),
(II), (III) and (IV):
##STR00001##
wherein # in each case denotes one-half of a bond between a
nitrogen atom and the free binding position of a group A.sup.1 of
two adjacent repeating units of formulae (I), (II), (III) or (IV);
* in each case denotes one-half of a bond to one of the alkoxylate
groups; and A.sup.1 is independently selected from linear or
branched C.sub.2-C.sub.6-alkylene; wherein the polyalkylenimine
structure consists of 1 repeating unit of formula (I), x repeating
units of formula (II), y repeating units of formula (III) and y+1
repeating units of formula (IV), wherein x and y in each case have
a value in the range of from 0 to about 150; where the average
weight average molecular weight, Mw, of the polyalkylenimine core
structure is a value in the range of from about 60 to about 10,000
g/mol.
[0301] The core structure may alternatively comprise a
polyalkanolamine structure of the condensation products of at least
one compound selected from N-(hydroxyalkyl)amines of formulae (I.a)
and/or (I.b),
##STR00002##
wherein A are independently selected from C.sub.1-C.sub.6-alkylene;
R.sup.1, R.sup.1*, R.sup.2, R.sup.2*, R.sup.3, R.sup.3*, R.sup.4,
R.sup.4*, R.sup.5 and R.sup.5* are independently selected from
hydrogen, alkyl, cycloalkyl or aryl, wherein the last three
mentioned radicals may be optionally substituted; and R.sup.6 is
selected from hydrogen, alkyl, cycloalkyl or aryl, wherein the last
three mentioned radicals may be optionally substituted.
[0302] The plurality of alkylenoxy groups attached to the core
structure are independently selected from alkylenoxy units of the
formula (V)
* A.sup.2-O .sub.m CH.sub.2--CH.sub.2--O .sub.n A.sup.3-O .sub.p--R
(V)
wherein * in each case denotes one-half of a bond to the nitrogen
atom of the repeating unit of formula (I), (II) or (IV); A.sup.2 is
in each case independently selected from 1,2-propylene,
1,2-butylene and 1,2-isobutylene; A.sup.3 is 1,2-propylene; R is in
each case independently selected from hydrogen and
C.sub.1-C.sub.4-alkyl; m has an average value in the range of from
0 to about 2; n has an average value in the range of from about 20
to about 50; and p has an average value in the range of from about
10 to about 50.
[0303] Specific embodiments of the amphiphilic alkoxylated grease
cleaning polymers may be selected from alkoxylated
polyalkylenimines having an inner polyethylene oxide block and an
outer polypropylene oxide block, the degree of ethoxylation and the
degree of propoxylation not going above or below specific limiting
values. Specific embodiments of the alkoxylated polyalkylenimines
according to the present invention have a minimum ratio of
polyethylene blocks to polypropylene blocks (n/p) of about 0.6 and
a maximum of about 1.5(x+2y+1).sup.1/2. Alkoxykated
polyalkyenimines having an n/p ratio of from about 0.8 to about
1.2(x+2y+1).sup.1/2 have been found to have especially beneficial
properties.
[0304] The alkoxylated polyalkylenimines according to the present
invention have a backbone which consists of primary, secondary and
tertiary amine nitrogen atoms which are attached to one another by
alkylene radicals A and are randomly arranged. Primary amino
moieties which start or terminate the main chain and the side
chains of the polyalkylenimine backbone and whose remaining
hydrogen atoms are subsequently replaced by alkylenoxy units are
referred to as repeating units of formulae (I) or (IV),
respectively. Secondary amino moieties whose remaining hydrogen
atom is subsequently replaced by alkylenoxy units are referred to
as repeating units of formula (II). Tertiary amino moieties which
branch the main chain and the side chains are referred to as
repeating units of formula (III).
[0305] Since cyclization can occur in the formation of the
polyalkylenimine backbone, it is also possible for cyclic amino
moieties to be present to a small extent in the backbone. Such
polyalkylenimines containing cyclic amino moieties are of course
alkoxylated in the same way as those consisting of the noncyclic
primary and secondary amino moieties.
[0306] The polyalkylenimine backbone consisting of the nitrogen
atoms and the groups A', has an average molecular weight Mw of from
about 60 to about 10,000 g/mole, preferably from about 100 to about
8,000 g/mole and more preferably from about 500 to about 6,000
g/mole.
[0307] The sum (x+2y+1) corresponds to the total number of
alkylenimine units present in one individual polyalkylenimine
backbone and thus is directly related to the molecular weight of
the polyalkylenimine backbone. The values given in the
specification however relate to the number average of all
polyalkylenimines present in the mixture. The sum (x+2y+2)
corresponds to the total number amino groups present in one
individual polyalkylenimine backbone.
[0308] The radicals A.sup.1 connecting the amino nitrogen atoms may
be identical or different, linear or branched
C.sub.2-C.sub.6-alkylene radicals, such as 1,2-ethylene,
1,2-propylene, 1,2-butylene, 1,2-isobutylene, 1,2-pentanediyl,
1,2-hexanediyl or hexamethylen. A preferred branched alkylene is
1,2-propylene. Preferred linear alkylene are ethylene and
hexamethylene. A more preferred alkylene is 1,2-ethylene.
[0309] The hydrogen atoms of the primary and secondary amino groups
of the polyalkylenimine backbone are replaced by alkylenoxy units
of the formula (V).
* A.sup.2-O .sub.m CH.sub.2--CH.sub.2--O .sub.n A.sup.3-O .sub.p--R
(V)
[0310] In this formula, the variables preferably have one of the
meanings given below:
[0311] A.sup.2 in each case is selected from 1,2-propylene,
1,2-butylene and 1,2-isobutylene; preferably A.sup.2 is
1,2-propylene. A.sup.3 is 1,2-propylene; R in each case is selected
from hydrogen and C.sub.1-C.sub.4-alkyl, such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl; preferably
R is hydrogen. The index m in each case has a value of 0 to about
2; preferably m is 0 or approximately 1; more preferably m is 0.
The index n has an average value in the range of from about 20 to
about 50, preferably in the range of from about 22 to about 40, and
more preferably in the range of from about 24 to about 30. The
index p has an average value in the range of from about 10 to about
50, preferably in the range of from about 11 to about 40, and more
preferably in the range of from about 12 to about 30.
[0312] Preferably the alkylenoxy unit of formula (V) is a
non-random sequence of alkoxylate blocks. By non-random sequence it
is meant that the [-A.sup.2-O--].sub.m is added first (i.e.,
closest to the bond to the nitrogen atom of the repeating unit of
formula (I), (II), or (III)), the [--CH.sub.2--CH.sub.2--O--].sub.n
is added second, and the [-A.sup.3-O--].sub.p is added third. This
orientation provides the alkoxylated polyalkylenimine with an inner
polyethylene oxide block and an outer polypropylene oxide
block.
[0313] The substantial part of these alkylenoxy units of formula
(V) is formed by the ethylenoxy units
--[CH.sub.2--CH.sub.2--O)].sub.n-- and the propylenoxy units
--[CH.sub.2--CH.sub.2(CH.sub.3)--O].sub.p--. The alkylenoxy units
may additionally also have a small proportion of propylenoxy or
butylenoxy units -[A.sup.2-O].sub.m--, i.e. the polyalkylenimine
backbone saturated with hydrogen atoms may be reacted initially
with small amounts of up to about 2 mol, especially from about 0.5
to about 1.5 mol, in particular from about 0.8 to about 1.2 mol, of
propylene oxide or butylene oxide per mole of NH-- moieties
present, i.e. incipiently alkoxylated.
[0314] This initial modification of the polyalkylenimine backbone
allows, if necessary, the viscosity of the reaction mixture in the
alkoxylation to be lowered. However, the modification generally
does not influence the performance properties of the alkoxylated
polyalkylenimine and therefore does not constitute a preferred
measure.
[0315] The amphiphilic alkoxylated grease cleaning polymers are
present in the fabric and home care products, including but not
limited to detergents, of the present invention at levels ranging
from about 0.05% to 10% by weight of the fabric and home care
product. Embodiments of the fabric and home care products may
comprise from about 0.1% to about 5% by weight. More specifically,
the embodiments may comprise from about 0.25 to about 2.5% of the
grease cleaning polymer.
[0316] Carboxylate polymer--The consumer products of the present
invention may also include one or more carboxylate polymers such as
a maleate/acrylate random copolymer or polyacrylate homopolymer. In
one aspect, the carboxylate polymer is a polyacrylate homopolymer
having a molecular weight of from 4,000 Da to 9,000 Da, or from
6,000 Da to 9,000 Da.
[0317] Soil release polymer--The consumer products of the present
invention may also include one or more soil release polymers having
a structure as defined by one of the following structures (I), (II)
or (III):
--[(OCHR.sup.1--CHR.sup.2).sub.a--O--OC--Ar--CO--].sub.d (I)
--[(OCHR.sup.3--CHR.sup.4).sub.b--O--OC-sAr--CO--].sub.e (II)
--[(OCHR.sup.5--CHR.sup.6).sub.c--OR.sup.7].sub.f (III)
[0318] wherein:
[0319] a, b and c are from 1 to 200;
[0320] d, e and f are from 1 to 50;
[0321] Ar is a 1,4-substituted phenylene;
[0322] sAr is 1,3-substituted phenylene substituted in position 5
with SO.sub.3Me;
[0323] Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-,
or tetraalkylammonium wherein the alkyl groups are C.sub.1-C.sub.18
alkyl or C.sub.2-C.sub.10 hydroxyalkyl, or mixtures thereof;
[0324] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are
independently selected from H or C.sub.1-C.sub.18 n- or iso-alkyl;
and
[0325] R.sup.7 is a linear or branched C.sub.1-C.sub.18 alkyl, or a
linear or branched C.sub.2-C.sub.30 alkenyl, or a cycloalkyl group
with 5 to 9 carbon atoms, or a C.sub.8-C.sub.30 aryl group, or a
C.sub.6-C.sub.30 arylalkyl group.
[0326] Suitable soil release polymers are polyester soil release
polymers such as Repel-o-tex polymers, including Repel-o-tex SF,
SF-2 and SRP6 supplied by Rhodia. Other suitable soil release
polymers include Texcare polymers, including Texcare SRA100,
SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325 supplied by
Clariant. Other suitable soil release polymers are Marloquest
polymers, such as Marloquest SL supplied by Sasol.
[0327] Cellulosic polymer--The consumer products of the present
invention may also include one or more cellulosic polymers
including those selected from alkyl cellulose, alkyl alkoxyalkyl
cellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose. In
one aspect, the cellulosic polymers are selected from the group
comprising carboxymethyl cellulose, methyl cellulose, methyl
hydroxyethyl cellulose, methyl carboxymethyl cellulose, and
mixtures thereof. In one aspect, the carboxymethyl cellulose has a
degree of carboxymethyl substitution from 0.5 to 0.9 and a
molecular weight from 100,000 Da to 300,000 Da.
[0328] The detergent may contain a bleaching system, which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of,
e.g., the amide, imide, or sulfone type. In general, when a
bleaching agent is used, the compositions of the present invention
may comprise from about 0.1% to about 50% or even from about 0.1%
to about 25% bleaching agent by weight of the subject cleaning
composition.
[0329] Chelating Agents--The consumer products herein may contain a
chelating agent. Suitable chelating agents include copper, iron
and/or manganese chelating agents and mixtures thereof. When a
chelating agent is used, the subject consumer product may comprise
from about 0.005% to about 15% or even from about 3.0% to about 10%
chelating agent by weight of the subject consumer product. Suitable
chelants include DTPA (Diethylene triamine pentaacetic acid), HEDP
(Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine
penta(methylene phosphonic acid)),
1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,
ethylenediamine, diethylene triamine, ethylenediaminedisuccinic
acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA),
triethylenetetraaminehexaacetic acid (TTHA),
N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine
(DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives
thereof.
[0330] The enzyme variants of the invention may be stabilized using
conventional stabilizing agents, and/or protease inhibitors e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, salts such as sodium chloride and potassium chloride,
lactic acid, formic acid, boric acid, or a boric acid derivative,
e.g., an aromatic borate ester, or a phenyl boronic acid derivative
such as 4-formylphenyl boronic acid, or a peptide aldehyde such as
di-, tri- or tetrapeptide aldehydes or aldehyde analogues (either
of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X
(X=halogen), B0 is a single amino acid residue (preferably with an
optionally substituted aliphatic or aromatic side chain); and B1
consists of one or more amino acid residues (preferably one, two or
three), optionally comprising an N-terminal protection group, or as
described in WO09118375, WO98/13459) or a protease inhibitor of the
protein type such as RASI, BASI, WASI (bifunctional
alpha-amylase/subtilisin inhibitors of rice, barley and wheat) or
CI2 or SSI. In some embodiments, the enzymes employed herein are
stabilized by the presence of water-soluble sources of zinc (II),
calcium (II) and/or magnesium (II) ions in the finished
compositions that provide such ions to the enzymes, as well as
other metal ions (e.g., barium (II), scandium (II), iron (II),
manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II),
Nickel (II), and oxovanadium (IV)).
[0331] The composition may also contain other conventional
detergent ingredients such as e.g. fabric conditioners including
clays, foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil re-deposition agents, dyes,
bactericides, optical brighteners, hydrotropes, tarnish inhibitors,
organic solvents such as ethanol or perfumes. Furthermore, the
detergent could contain a pre-spotter or a booster, which is added
to the wash to increase the general cleaning level, some of these
additives may also be used as a pre-treatment agent applied to the
textile before the washing step.
[0332] It is at present contemplated that in the detergent
compositions any enzyme, in particular the enzyme essential to the
present invention, may be added in an amount corresponding to
0.001-100 mg of enzyme protein per liter of wash liquor, preferably
0.005-5 mg of enzyme protein per liter of wash liquor, more
preferably 0.01-1 mg of enzyme protein per liter of wash liquor and
in particular 0.1-1 mg of enzyme protein per liter of wash liquor.
However, the compositions of the present invention comprise at
least 0.0001 to about 0.1% weight percent of pure enzyme protein,
such as from about 0.0001% to about 0.01%, from about 0.001% to
about 0.01% or from about 0.001% to about 0.01%. However, when
using a formulated enzyme the detergent composition comprises from
about 0.02% to about 20% weight percent, such as or from about
0.05% to about 15% weight, or from about 0.05 to about 20%, or from
about 0.05% to about 5%, or from about 0.05% to about 3%.
[0333] The alpha-amylase variants useful in the present invention
may additionally be incorporated in the detergent formulations
disclosed in WO 97/07202, which is hereby incorporated by
reference.
[0334] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste, a gel or a liquid. The composition may be a powder-form
all-purpose "heavy-duty" washing agent, a paste-form all-purpose, a
heavy-duty liquid type, a liquid fine-fabric, a hand dishwashing
agent, a light duty dishwashing agent, a high-foaming type. a
machine dishwashing agent, a various tablet, a dishwash granular, a
dish wash liquid, a rinse-aid type. The composition can also be in
unit dose packages, including those known in the art and those that
are water soluble, water insoluble and/or water permeable. A liquid
detergent may be aqueous, typically containing up to 70% water and
0-30% organic solvent, or non-aqueous or a solution containing more
than 0.5 g/L of the detergent composition.
[0335] The composition of the invention may for example be
formulated as a hand or machine laundry detergent composition
including a laundry additive composition suitable for pre-treatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
hand or machine dishwashing operations. The detergent may be a
powder, or granulated form, or it may be in the form of a liquid,
gel or paste or in the form of a unit dose product such as a tablet
or pouch, including multi-compartment pouches, or the detergent can
be in the form of a sheet.
EXAMPLES
[0336] pNP-G7 Assay for Determination of Alpha-Amylase Activity
[0337] The alpha-amylase activity may be determined by a method
employing the G7-pNP substrate. G7-pNP which is an abbreviation for
4,6-ethylidene(G.sub.7)-p-nitrophenyl(G.sub.1)-.alpha.,D-maltoheptaoside,
a blocked oligosaccharide which can be cleaved by an endo-amylase,
such as an alpha-amylase. Following the cleavage, the
alpha-Glucosidase included in the kit digest the hydrolysed
substrate further to liberate a free PNP molecule which has a
yellow color and thus can be measured by visible spectophometry at
.lamda.=405 nm (400-420 nm.). Kits containing G7-pNP substrate and
alpha-Glucosidase is manufactured by Roche/Hitachi (cat. No.
11876473).
Reagents:
[0338] The G7-pNP substrate from this kit contains 22 mM
4,6-ethylidene-G7-pNP and 52.4 mM HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH
7.0).
The alpha-Glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl,
12.6 mM MgCl.sub.2, 0.075 mM CaCl.sub.2, .gtoreq.4 kU/L
alpha-glucosidase).
[0339] The substrate working solution is made by mixing 1 mL of the
alpha-Glucosidase reagent with 0.2 mL of the G7-pNP substrate. This
substrate working solution is made immediately before use.
[0340] Dilution buffer: 50 mM MOPS, 0.05% (w/v) Triton X100
(polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether
(C.sub.14H.sub.22O(C.sub.2H.sub.4O).sub.n (n=9-10))), 1 mM CaCl2,
pH8.0.
Procedure:
[0341] The amylase sample to be analyzed was diluted in dilution
buffer to ensure the pH in the diluted sample is 7. The assay was
performed by transferring 20 .mu.l diluted enzyme samples to 96
well microtiter plate and adding 80 .mu.l substrate working
solution. The solution was mixed and pre-incubated 1 minute at room
temperature and absorption is measured every 20 sec. over 5 minutes
at OD 405 nm.
[0342] The slope (absorbance per minute) of the time dependent
absorption-curve is directly proportional to the specific activity
(activity per mg enzyme) of the alpha-amylase in question under the
given set of conditions. The amylase sample should be diluted to a
level where the slope is below 0.4 absorbance units per minute.
Automatic Mechanical Stress Assay (AMSA) for Laundry
[0343] In order to assess the wash performance in laundry washing
experiments are performed, using the Automatic Mechanical Stress
Assay (AMSA). With the AMSA, the wash performance of a large
quantity of small volume enzyme-detergent solutions can be
examined. The AMSA plate has a number of slots for test solutions
and a lid firmly squeezing the laundry sample, the textile to be
washed against all the slot openings. During the washing time, the
plate, test solutions, textile and lid are vigorously shaken to
bring the test solution in contact with the textile and apply
mechanical stress in a regular, periodic oscillating manner For
further description see WO02/42740 especially the paragraph
"Special method embodiments" at page 23-24.
General Wash Performance Description
[0344] A test solution comprising water (10.degree. dH), detergent,
e.g. 5.1 g/L European liquid detergent as described below and the
enzyme of the invention, e.g. at concentration of 0, 0.8 and/or 1.2
mg enzyme protein/L, is prepared. Fabrics stained with starch (e.g.
CS-28 from Center For Testmaterials BV, P.O. Box 120, 3133 KT,
Vlaardingen, The Netherlands) is added and washed for 20 minutes at
20.degree. C. After thorough rinse under running tap water and
drying in the dark, the light intensity or reflectance values of
the stained fabrics are subsequently measured as a measure for wash
performance. The test with 0 mg enzyme protein/L is used as a blank
to obtain a delta remission value. Preferably mechanical action is
applied during the wash step, e.g. in the form of shaking, rotating
or stirring the wash solution with the fabrics.
The AMSA wash performance experiments were conducted under the
experimental conditions specified below:
TABLE-US-00003 TABLE 1 AMSA experimental conditions Laundry liquid
detergent dosage 5.7 g/L European (EU) liquid detergent (cf.
Example 1A), or 0.8 g/L Northern America (US) liquid detergent (cf.
Example 1B) Test solution volume 160 micro L pH as is Wash time 20
minutes Temperature 20.degree. C. Water hardness 10.degree.dH,
Ca.sup.2+:Mg.sup.2+:HCO.sub.3.sup.- = 3:1:6 Enzyme concentration in
test 0.8 and 1.2 Mg/L solution Test material CS-28 (Rice starch on
cotton)
[0345] Amylase dilution buffer: Amylase was diluted in ultrapure
water (MilliQ water) with a small concentration of calcium (0.1 mM)
to stabilize the amylase during storage and 0.01% Triton X-100 to
reduce risk of adsorption of enzyme protein to containers and
pipettes.
[0346] Water hardness was adjusted to 10.degree. dH by addition of
CaCl.sub.2, MgCl2, and NaHCO.sub.3
(Ca.sup.2+:Mg.sup.2+:HCO.sub.3.sup.-=3:1:4.5) to the test system.
After washing the textiles were flushed in tap water and dried.
[0347] The wash performance is measured as the brightness of the
color of the textile washed. Brightness can also be expressed as
the intensity of the light reflected from the sample when
illuminated with white light. When the sample is stained the
intensity of the reflected light is lower, than that of a clean
sample. Therefore, the intensity of the reflected light can be used
to measure wash performance.
[0348] Color measurements are made with a professional flatbed
scanner (Kodak iQsmart, Kodak, Midtager 29, DK-2605 Brondby,
Denmark), which is used to capture an image of the washed
textile.
[0349] To extract a value for the light intensity from the scanned
images, 24-bit pixel values from the image are converted into
values for red, green and blue (RGB). The intensity value (Int) is
calculated by adding the RGB values together as vectors and then
taking the length of the resulting vector:
Int= {square root over (r.sup.2+g.sup.2+b.sup.2)}.
[0350] Results of the AMSA laundry test of different variants are
shown in Table 1 and 2. In the result the index is 100. The
performance result of the parent alpha-amylase is assigned the
value of 100 and the results of the variants are compared to this
value.
TOM Wash Performance
[0351] Water hardness was adjusted to the strength described below
by addition of CaCl.sub.2, MgCl.sub.2 and NAHCO.sub.3. Wash
solutions were prepared with desired amount of detergent,
temperature and water hardness in a bucket as described below.
Detergent was dissolved during magnet stirring for 10 minutes (wash
solution was used within 30 to 60 min after preparation).
[0352] Temperature and rotation (rpm) in the water bath in the
Terg-O-toMeter were set according to the settings below in Table 2.
When temperature was adjusted according to settings (tolerance is
+/-0.5.degree. C.) wash solution was added to TOM beaker according
to the amount described below.
[0353] Agitation in the beaker was at 200 rpm. 2 handmade rice
starch swatches (HM CS-28), 2 handmade tapioca starch swatches (HM
CS-29) and ballast were added to each of the beakers and wash
carried out according to time stated below. Swatches were rinsed in
cold tap water for 5 minutes and placed in a washing bag and rinsed
in washing machine (AEG OKO LAVAMAT 86820) on "STIVN" program. The
swatches were sorted and let to dry between filter paper in a
drying cupboard without heat overnight.
[0354] Textile sample HM CS-28 (rice starch on cotton, 5.times.5
cm, starch applied in 2.5 cm in diameter circle) and HM CS-29
(tapioca starch on cotton, 5.times.5 cm, starch applied in 2.5 cm
in diameter circle) and HM CS-26 (corn starch on cotton, 5.times.5
cm, starch applied in 2.5 cm in diameter circle) were obtained from
Center for Test Materials BV, P.O. Box 120, 3133 KT Vlaardingen,
the Netherlands.
[0355] White knitted cotton was used as ballast and was obtained
from Warwick Equest Ltd, Unit 55, Consett Business Park, Consett,
County Durham, DH8 6BN UK.
TABLE-US-00004 TABLE 2 Experimental conditions European conditions
European conditions using WE SUD model using WE HDL detergent model
detergent Detergent dosage 1.87 g/L 5.30 g/L Enzyme concentration
0.065 mg enzyme 0.2 mg enzyme protein/L in wash solution protein/L
Water hardness 20.6.degree.dH (Ca2+:Mg2+:HCO3- = 4:1:7.5) Test
solution volume 1000 ml Wash time 5 minutes Rotation 200 rpm pH as
is Temperature 15.degree. C.
Detergents and Test Materials were as Follows:
TABLE-US-00005 Laundry Detergent K liquid deter- gent Test HM CS-28
(Rice starch on cotton, 5 .times. 5 cm swatch with starch material
applied in 2.5 cm in diameter circle), HM CS-29 (tapioca starch on
cotton, 5 .times. 5 cm swatch with starch applied in 2.5 cm in
diameter circle). Ballast White knitted cotton in size 5 .times. 5
cm added to a total weight of 40 g (40 g including all swatches
i.e. ballast and test material).
[0356] The wash performance was measured as the brightness of the
color of the textile washed expressed in remission values (REM).
Remission measurements were made using a Macbeth 7000 Color Eye
spectrophotometer. Each of the dry swatches was measured. As there
is a risk of interference from the back-ground, the swatches were
placed on top of 2 layers of fabric during the measurement of the
remission. The remission was measured at 460 nm. The UV filter was
not included. An average result for remission for the swatches was
calculated.
The wash performance of different variants is shown in Table 5 as
Improvement Factor (IF) and is calculated as shown below:
IF = REM Variant - REM Blank REM Reference enzyme - REM Blank
##EQU00001##
Example 1
Wash Performance of Alpha-Amylases Using Automatic Mechanical
Stress Assay
[0357] In order to assess the wash performance of the
alpha-amylases in a detergent base composition, washing experiments
may be performed using Automatic Mechanical Stress Assay (AMSA).
With the AMSA test the wash performance of a large quantity of
small volume enzyme-detergent solutions can be examined. The AMSA
plate has a number of slots for test solutions and a lid firmly
squeezing the textile swatch to be washed against all the slot
openings. During the washing time, the plate, test solutions,
textile and lid are vigorously shaken to bring the test solution in
contact with the textile and apply mechanical stress in a regular,
periodic oscillating manner For further description see WO
02/42740, especially the paragraph "Special method embodiments" at
page 23-24.
General Wash Performance Description
[0358] A test solution comprising water (6.degree. dH or 15.degree.
dH), 0.79 g/L detergent, e.g., model detergent J as described
below, and the enzyme of the invention at concentration of 0 or 0.2
mg enzyme protein/L, is prepared. Fabrics stained with starch
(CS-28 from Center For Test materials BV, P.O. Box 120, 3133 KT,
Vlaardingen, The Netherlands) is added and washed for 10 minutes at
20.degree. C. and 40.degree. C., or alternatively 10 minutes at
20.degree. C. and 30.degree. C. as specified in the examples. After
thorough rinse under running tap water and drying in the dark, the
light intensity values of the stained fabrics are subsequently
measured as a measure for wash performance. The test with 0 mg
enzyme protein/L is used as a blank and corresponds to the
contribution from the detergent. Preferably mechanical action is
applied during the wash step, e.g. in the form of shaking, rotating
or stirring the wash solution with the fabrics. The AMSA wash
performance experiments may be conducted under the experimental
conditions specified below:
TABLE-US-00006 TABLE A Experimental condition Detergent Liquid
Model detergent J (see Table B) Detergent dosage 0.79 g/L Test
solution volume 160 micro L pH As is Wash time 10 minutes
Temperature 20.degree. C. or 30.degree. C. Water hardness
6.degree.dH Enzyme concentration in test 0.2 mg enzyme protein/L
and 0.05 mg enzyme protein/L Test material CS-28 (Rice starch
cotton)
TABLE-US-00007 TABLE B Model detergent J Content of % active
Compound compound (% w/w) component (% w/w) LAS 5.15 5.00 AS 5.00
4.50 AEOS 14.18 10.00 Coco fatty acid 1.00 1.00 AEO 5.00 5.00 MEA
0.30 0.30 MPG 3.00 3.00 Ethanol 1.50 1.35 DTPA (as Na5 salt) 0.25
0.10 Sodium citrate 4.00 4.00 Sodium formate 1.00 1.00 Sodium
hydroxide 0.66 0.66 H.sub.2O, ion exchanged 58.95 58.95
Water hardness was adjusted to 6.degree. dH by addition of
CaCl.sub.2, MgCl.sub.2, and NaHCO.sub.3
(Ca.sup.2+:Mg.sup.2+:HCO.sup.3-=2:1:4.5) to the test system. After
washing the textiles were flushed in tap water and dried.
TABLE-US-00008 TABLE C Experimental condition Detergent Liquid
Model detergent A (see Table D) Detergent dosage 3.33 g/L Test
solution volume 160 micro L pH As is Wash time 10 minutes
Temperature 20.degree. C. or 40.degree. C. Water hardness
15.degree.dH Enzyme concentration in test 0.2 mg enzyme protein/L,
0.05 mg enzyme protein/L Test material CS-28 (Rice starch
cotton)
TABLE-US-00009 TABLE D Model detergent A Content of % active
Compound compound (% w/w) component (% w/w) LAS 12.00 11.60 AEOS,
SLES 17.63 4.90 Soy fatty acid 2.75 2.48 Coco fatty acid 2.75 2.80
AEO 11.00 11.00 Sodium hydroxide 1.75 1.80 Ethanol/Propan-2-ol 3.00
2.70/0.30 MPG 6.00 6.00 Glycerol 1.71 1.70 TEA 3.33 3.30 Sodium
formate 1.00 1.00 Sodium citrate 2.00 2.00 DTMPA 0.48 0.20 PCA 0.46
0.18 Phenoxy ethanol 0.50 0.50 H.sub.2O, ion exchanged 33.64
33.64
Water hardness was adjusted to 15.degree. dH by addition of
CaCl.sub.2, MgCl.sub.2, and NaHCO.sub.3
(Ca.sup.2+:Mg.sup.2+:HCO.sup.3-=4:1:7.5) to the test system. After
washing the textiles were flushed in tap water and dried.
TABLE-US-00010 TABLE E Experimental condition Detergent Detergent
Composition K Detergent dosage 5.3 g/L Test solution volume 160
micro L pH As is Wash time 10 minutes Temperature 20.degree. C. or
40.degree. C. Water hardness 15.degree.dH Enzyme concentration in
test 0.2 mg enzyme protein/L, 0.05 mg enzyme protein/L Test
material CS-28 (Rice starch cotton)
TABLE-US-00011 TABLE F Detergent K Content of compound (wt %
Compound active) Sodium alkylbenzene 8.7 sulfonate Sodium alkyl
ethoxy 3 1.0 sulfate C12-18 alkyl 1.5-7- 5.3 ethoxylate Citric Acid
3.1 Optical Brightener 0.05 Polypropylene Glycol 1.1 Phosphonated
chelant 0.5 Minors (dyes perfumes, to 100% enzymes, enzyme
stabilisers, solvents, structurants, polymers) and water
Water hardness was adjusted to 15.degree. dH by addition of
CaCl.sub.2, MgCl.sub.2, and NaHCO.sub.3
(Ca.sup.2+:Mg.sup.2+:HCO.sup.3-=4:1:7.5) to the test system. After
washing the textiles were flushed in tap water and dried.
[0359] The wash performance is measured as the brightness expressed
as the intensity of the light reflected from the sample when
illuminated with white light. When the sample is stained the
intensity of the reflected light is lower, than that of a clean
sample. Therefore, the intensity of the reflected light can be used
to measure wash performance.
[0360] Color measurements are made with a professional flatbed
scanner (EPSON Expression 10000XL, EPSON) used to capture an image
of the washed textile.
[0361] To extract a value for the light intensity from the scanned
images, 48.fwdarw.24 Bit Color pixel values from the image are
converted into values for red, green and blue (RGB). The intensity
value (Int) is calculated by adding the RGB values together as
vectors and then taking the length of the resulting vector:
Int= {square root over (r.sup.2+g.sup.2+b.sup.2)}.
[0362] The wash performance of the variants according to the
invention are shown in the tables below. Table 3 shows the results
obtained from the experiment accessing the wash performance in
model detergents A (Table D) and J (Table B) in different
concentrations (0.05 mg enzyme/L detergent and 0.2 mg enzyme/L
detergent), and at different temperatures (20.degree. C. and
40.degree. C.). Table 4 shows the results obtained from the
experiment accessing the wash performance in detergent K (Table F)
in different concentrations (0.05 mg enzyme/L detergent and 0.2 mg
enzyme/L detergent) and at different temperatures (20.degree. C.
and 40.degree. C.).
TABLE-US-00012 TABLE 3 Results of wash performance in Model
detergents 0.05 mg/ 0.2 mg/ 0.05 mg/ 0.2 mg/ 0.05 mg/ 0.2 mg/ 0.05
mg/ 0.2 mg/ L-A- L-A- L-A- L-A- L-J- L-J- L-J- L-J- Mutations 20 C.
20 C. 40 C. 40 C. 20 C. 20 C. 30 C. 30 C. Reference--SEQ ID NO: 2
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 SEQ ID NO: 2 + H1* + G7A + G109A +
N280S + 4.7 1.5 2.3 1.1 2.7 1.1 3.5 1.5 W284H + K320A + M323N +
E391A SEQ ID NO: 2 + G7A + W284H + K320A + M323N 4.7 1.4 1.8 1.0
3.0 1.1 2.0 1.2 SEQ ID NO: 2 + G7A + K320A + M323N 4.3 1.6 2.0 1.0
2.0 1.1 2.0 1.2 SEQ ID NO: 2 + K320A 4.7 1.3 2.3 1.0 3.7 1.0 4.5
1.5 SEQ ID NO: 2 + G7A + K320A 4.3 1.4 2.3 1.1 3.3 1.1 2.5 1.3 SEQ
ID NO: 2 + H1* + G7A + G109A + N280S + 1.7 1.2 1.3 1.3 1.5 1.3 1.1
1.4 E391A SEQ ID NO: 2 + H1* + G109A + N280S + 2.3 1.7 2.8 1.4 5.0
2.0 5.5 1.8 W284H + E391A SEQ ID NO: 2 + H1* + G109A + N280S +
E391A 0.8 1.0 1.0 1.1 1.0 1.1 1.3 1.1 SEQ ID NO: 2 + H1* + G109A +
N280S + 1.3 1.9 1.5 1.3 1.5 1.5 1.6 1.6 M323S + E391A SEQ ID NO: 2
+ H1* + G7A + G109A + N280S + 1.5 1.2 1.3 1.2 1.2 1.1 1.7 1.1 K320A
+ E391A SEQ ID NO: 2 + H1* + G7A + G109A + N280S + 1.3 1.3 1.5 1.1
1.0 1.0 1.2 1.1 M323S + E391A SEQ ID NO: 2 + H1* + G7A + G109A +
N280S + 1.4 1.3 1.3 1.2 1.0 1.2 1.6 1.1 M323N + E391A SEQ ID NO: 2
+ H1* + G7A + G109A + N280S + 1.3 1.4 1.3 1.2 0.9 1.2 1.6 1.0 W284F
+ E391A SEQ ID NO: 2 + H1* + G7A + G109A + N280S + 1.1 1.3 1.0 1.0
0.7 1.2 1.4 0.9 W284R + E391A SEQ ID NO: 2 + H1* + G7A + G109A +
N280S + 1.1 1.3 0.9 1.1 0.5 1.2 0.8 1.0 K320A + M323S + E391A SEQ
ID NO: 2 + H1* + G7A + G109A + W284R + 1.0 1.2 0.9 1.1 0.6 1.1 1.1
1.0 E391A SEQ ID NO: 2 + H1* + G7A + G109A + N280S + 1.0 1.2 0.8
1.0 0.8 1.2 0.9 1.0 K320A + M323N + E391A
TABLE-US-00013 TABLE 4 Results of wash performance in detergent K
Mutations 0.05 mg/L-K -20 C. 0.2 mg/L-K -20 C. 0.05 mg/L-K -40 C.
0.2 mg/L-K -40 C. Reference - SEQ ID NO: 2 1.0 1.0 1.0 1.0 SEQ ID
NO: 2 + H1* + G109A + N280S + E391A 1.1 1.0 2.0 1.1 SEQ ID NO: 2 +
H1* + G7K + G109A + N280S + E391A 1.2 1.6 0.9 1.1 SEQ ID NO: 2 +
H1* + G7E + G109A + N280S + E391A 1.2 1.6 0.6 1.0 SEQ ID NO: 2 +
H1* + G7N + G109A + N280S + E391A 0.9 1.6 1.0 1.1 SEQ ID NO: 2 +
H1* + G7Q + G109A + N280S + E391A 1.6 1.6 1.1 1.1 SEQ ID NO: 2 +
H1* + G7L + G109A + N280S + E391A 1.6 1.6 1.4 1.3 SEQ ID NO: 2 +
H1* + G7D + G109A + N280S + E391A 1.9 1.6 1.5 1.3 SEQ ID NO: 2 +
H1* + G109A + N280S + K320A + E391A 0.4 0.9 1.0 1.3 SEQ ID NO: 2 +
H1* + G109A + N280S + K320M + E391A 1.5 1.1 1.8 1.3 SEQ ID NO: 2 +
H1* + G109A + N280S + K320T + E391A 1.3 1.1 1.5 1.1 SEQ ID NO: 2 +
H1* + G109A + N280S + K320V + E391A 1.0 0.9 1.7 1.1 SEQ ID NO: 2 +
H1* + G109A + N280S + M323R + E391A 0.7 0.9 1.3 1.0 SEQ ID NO: 2 +
H1* + G109A + N280S + K320S + E391A 1.8 1.3 1.8 1.3 SEQ ID NO: 2 +
H1* + G109A + N280S + E391V 1.3 1.1 2.0 1.3 SEQ ID NO: 2 + H1* +
G109A + W284R + E391A 1.2 1.3 1.2 1.2 SEQ ID NO: 2 + H1* + G109A +
W284F + E391A 1.7 1.3 1.8 1.2 SEQ ID NO: 2 + H1* + G109A + N280S +
K320A + M323S + 2.0 1.4 1.4 1.2 E391A SEQ ID NO: 2 + H1* + G109A +
N280S + W284F + E391A 0.9 0.9 1.3 1.0 SEQ ID NO: 2 + H1* + G109A +
N280S + M323N + E391A 2.0 1.6 2.0 1.3 SEQ ID NO: 2 + H1* + G109A +
N280S + M323K + E391A 2.3 1.6 2.0 1.6 SEQ ID NO: 2 + H1* + G109S +
N280S + E391A 2.5 1.3 2.3 1.1 SEQ ID NO: 2 + H1* + G109A + W284H +
E391A 1.5 1.2 1.3 1.1 SEQ ID NO: 2 + H1* + G109A + N280S + K320A +
M323N + 1.6 1.1 1.2 1.1 E391A SEQ ID NO: 2 + H1* + G7A + G109A +
N280S + E391A 1.5 1.4 1.2 1.2
[0363] As can be seen from Table 1 and Table 2, all the tested
variants have an improved wash performance compared to the
reference (SEQ ID NO: 2) in at least one of the tested
conditions.
Example 2--Wash Performance of Alpha-Amylases in Liquid Detergent
K
[0364] The wash performance of the tested variant and corresponding
parent alpha-amylase (SEQ ID NO: 2) were tested as described above.
The results are given as (performance of variant minus performance
of blank) divided by (performance of parent minus performance of
blank).
TABLE-US-00014 TABLE 5 Wash performance in TOM scale WE HDL Model
Detergent IF IF HM CS-28 HM CS-29 Reference SEQ ID NO: 2 1.00 1.00
SEQ ID NO: 2 + H1* + G109A + 1.28 1.58 W284H + E391A SEQ ID NO: 2 +
H1* + G109A + N280S + 1.13 1.61 K320A + M323N + E391A SEQ ID NO: 2
+ H1* + G7A + G109A + 1.22 2.02 N280S + E391A
TABLE-US-00015 TABLE 6 Wash performance in TOM scale WE SUD Model
Detergent 1 L, 5 min wash IF IF 0.13 mg enzyme protein/L HM CS-29
HM CS-26 Reference SEQ ID NO: 2 1.00 1.00 SEQ ID NO: 2 + H1* +
G109A + 1.00 1.00 W284H + E391A SEQ ID NO: 2 + H1* + G109A + 1.08
1.11 N280S + K320A + M323N + E391A SEQ ID NO: 2 + H1* + G7A + G109A
+ 1.10 1.13 N280S + E391A
TABLE-US-00016 TABLE 7 Wash Performance in Full Scale Washing
Machine Test NA HDL Model Detergent Full scale, IF IF 0.072 mg
enzyme protein/L HM CS-29 HM CS-26 Reference SEQ ID NO: 2 1.00 1.00
SEQ ID NO: 2 + H1* + G109A + 1.07 1.02 W284H + E391A SEQ ID NO: 2 +
H1* + G109A + N280S + 1.22 1.32 K320A + M323N + E391A SEQ ID NO: 2
+ H1* + G7A + G109A + 1.32 1.41 N280S + E391A
The invention described and claimed herein is not to be limited in
scope by the specific aspects herein disclosed, since these aspects
are intended as illustrations of several aspects of the invention.
Any equivalent aspects are intended to be within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims. In the case of conflict, the present disclosure
including definitions will control.
Method of Use
[0365] The present invention includes a method for cleaning and/or
treating a situs inter alia a surface or fabric. In one aspect,
such method comprises the steps of optionally washing and/or
rinsing said surface or fabric, contacting said surface or fabric
with any consumer product disclosed in this specification then
optionally washing and/or rinsing said surface or fabric is
disclosed.
[0366] As used herein, washing includes but is not limited to,
scrubbing, and mechanical agitation. Drying of such surfaces or
fabrics may be accomplished by any one of the common means employed
either in domestic or industrial settings. Such means include but
are not limited to forced air or still air drying at ambient or
elevated temperatures at pressures between 5 and 0.01 atmospheres
in the presence or absence of electromagnetic radiation, including
sunlight, infrared, ultraviolet and microwave irradiation. In one
aspect, said drying may be accomplished at temperatures above
ambient by employing an iron wherein, for example, said fabric may
be in direct contact with said iron for relatively short or even
extended periods of time and wherein pressure may be exerted beyond
that otherwise normally present due to gravitational force. In
another aspect, said drying may be accomplished at temperatures
above ambient by employing a dryer. Apparatus for drying fabric is
well known and it is frequently referred to as a clothes dryer. In
addition to clothes such appliances are used to dry many other
items including towels, sheets, pillowcases, diapers and so forth
and such equipment has been accepted as a standard convenience in
many nations of the world substantially replacing the use of
clothes lines for drying of fabric. Most dryers in use today use
heated air which is passed over and or through the fabric as it is
tumbled within the dryer. The air may be heated, for example,
either electronically, via gas flame, or even with microwave
radiation. Such air may be heated from about 15.degree. C. to about
400.degree. C., from about 25.degree. C. to about 200.degree. C.,
from about 35.degree. C. to about 100.degree. C., or even from
about 40.degree. C. to about 85.degree. C. and used in the dryer to
dry a surface and/or a fabric. As will be appreciated by one
skilled in the art, the cleaning compositions of the present
invention are ideally suited for use in laundry applications.
Accordingly, the present invention includes a method for laundering
a fabric. The method comprises the steps of contacting a fabric to
be laundered with a said cleaning laundry solution comprising at
least one embodiment of Applicants' cleaning composition, cleaning
additive or mixture thereof. The fabric may comprise most any
fabric capable of being laundered in normal consumer or
institutional use conditions. The solution preferably has a pH of
from about 8 to about 10.5. The compositions may be employed at
concentrations of from about 500 ppm to about 15,000 ppm in
solution. The water temperatures typically range from about
5.degree. C. to about 90.degree. C. The water to fabric ratio is
typically from about 1:1 to about 30:1.
DETERGENT EXAMPLES
Examples 1-6
[0367] Granular laundry detergent compositions designed for hand
washing or top-loading washing machines.
TABLE-US-00017 1 2 3 4 5 6 (wt %) (wt %) (wt %) (wt %) (wt %) (wt
%) Linear alkylbenzenesulfonate 20 22 20 15 20 20 C.sub.12-14
Dimethylhydroxyethyl 0.7 0.2 1 0.6 0.0 0 ammonium chloride AE3S 0.9
1 0.9 0.0 0.5 0.9 AE7 0.0 0.0 0.0 1 0.0 3 Sodium tripolyphosphate 5
0.0 4 9 2 0.0 Zeolite A 0.0 1 0.0 1 4 1 1.6R Silicate
(SiO.sub.2:Na.sub.2O at 7 5 2 3 3 5 ratio 1.6:1) Sodium carbonate
25 20 25 17 18 19 Polyacrylate MW 4500 1 0.6 1 1 1.5 1 Random graft
copolymer.sup.1 0.1 0.2 0.0 0.0 0.0 0.0 Carboxymethyl cellulose 1
0.3 1 1 1 1 Protease (Savinase .RTM., 0.1 0.1 0.1 0.1 0.1 32.89 mg
active/g) Lipase--Lipex .RTM. 0.03 0.07 0.3 0.1 0.07 0.4 (18 mg
active/g) *Amylase of the present 0.63 1.0 2.0 0.44 0.88 0.3
invention (mg active) Fluorescent Brightener 1 0.06 0.0 0.06 0.18
0.06 0.06 Fluorescent Brightener 2 0.1 0.06 0.1 0.0 0.1 0.1 DTPA
0.6 0.8 0.6 0.25 0.6 0.6 MgSO.sub.4 1 1 1 0.5 1 1 Sodium
Percarbonate 0.0 5.2 0.1 0.0 0.0 0.0 Sodium Perborate 4.4 0.0 3.85
2.09 0.78 3.63 Monohydrate NOBS 1.9 0.0 1.66 0.0 0.33 0.75 TAED
0.58 1.2 0.51 0.0 0.015 0.28 Sulphonated zinc 0.0030 0.0 0.0012
0.0030 0.0021 0.0 phthalocyanine S-ACMC 0.1 0.0 0.0 0.0 0.06 0.0
Direct Violet 9 0.0 0.0 0.0003 0.0005 0.0003 0.0 Acid Blue 29 0.0
0.0 0.0 0.0 0.0 0.0003 Sulfate/Moisture Balance *Amylase of the
present invention is shown as mgs of active enzyme per 100 g of
detergent.
Examples 7-12
[0368] Granular laundry detergent compositions designed for
front-loading automatic washing machines.
TABLE-US-00018 7 8 9 10 11 12 (wt %) (wt %) (wt %) (wt %) (wt %)
(wt %) Linear alkylbenzenesulfonate 8 7.1 7 6.5 7.5 7.5 AE3S 0 4.8
0 5.2 4 4 C12-14 Alkylsulfate 1 0 1 0 0 0 AE7 2.2 0 3.2 0 0 0
C.sub.10-12 Dimethyl 0.75 0.94 0.98 0.98 0 0 hydroxyethylammonium
chloride Crystalline layered silicate 4.1 0 4.8 0 0 0
(.delta.-Na.sub.2Si.sub.2O.sub.5) Zeolite A 5 0 5 0 2 2 Citric Acid
3 5 3 4 2.5 3 Sodium Carbonate 15 20 14 20 23 23 Silicate 2R
(SiO.sub.2:Na.sub.2O at 0.08 0 0.11 0 0 0 ratio 2:1) Soil release
agent 0.75 0.72 0.71 0.72 0 0 Acrylic Acid/Maleic Acid 1.1 3.7 1.0
3.7 2.6 3.8 Copolymer Carboxymethylcellulose 0.15 1.4 0.2 1.4 1 0.5
Protease--Purafect .RTM. 0.2 0.2 0.3 0.15 0.12 0.13 (84 mg
active/g) Lipase--Lipex .RTM. 0.05 0.15 0.1 0 0 0 (18.00 mg
active/g) Cellulase--Celluclean .TM. 0 0 0 0 0.1 0.1 (15.6 mg
active/g) *Amylase of the present 4.0 2.0 1.0 0.7 6.0 3.0 invention
(mg active) Amylase.sup.4 0.15 0.04 0.03 -- 0.01 0.16 TAED 3.6 4.0
3.6 4.0 2.2 1.4 Percarbonate 13 13.2 13 13.2 16 14 Na salt of
Ethylenediamine- 0.2 0.2 0.2 0.2 0.2 0.2 N,N'-disuccinic acid,
(S,S) isomer (EDDS) Hydroxyethane di 0.2 0.2 0.2 0.2 0.2 0.2
phosphonate (HEDP) MgSO.sub.4 0.42 0.42 0.42 0.42 0.4 0.4 Perfume
0.5 0.6 0.5 0.6 0.6 0.6 Suds suppressor agglomerate 0.05 0.1 0.05
0.1 0.06 0.05 Soap 0.45 0.45 0.45 0.45 0 0 Sulphonated zinc 0.0007
0.0012 0.0007 0 0 0 phthalocyanine (active) S-ACMC 0.01 0.01 0 0.01
0 0 Direct Violet 9 (active) 0 0 0.0001 0.0001 0 0 Sulfate/Water
& Balance Miscellaneous *Amylase of the present invention is
shown as mgs of active enzyme per 100 g of detergent.
Examples 13-18 Heavy Duty Liquid Laundry Detergent Compositions
TABLE-US-00019 [0369] 13 14 15 16 17 18 (wt %) (wt %) (wt %) (wt %)
(wt %) (wt %) C.sub.12-15 Alkylethoxy(1.8)sulfate 14.7 11.6 16.31
17.29 C.sub.11.8 Alkylbenzene sulfonate 4.3 11.6 8.3 7.73 11.7 7.73
C.sub.16-17 Branched alkyl sulfate 1.7 1.29 3.09 3.3 C.sub.12-14
Alkyl-9-ethoxylate 0.9 1.07 1.31 1.31 C.sub.12 dimethylamine oxide
0.6 0.64 1.03 1.03 Citric acid 3.5 0.65 3 0.66 2.27 0.67
C.sub.12-18 fatty acid 1.5 2.32 3.6 1.52 0.82 1.52 Sodium Borate
(Borax) 2.5 2.46 1.2 2.53 2.53 Sodium C.sub.12-14 alkyl ethoxy 3
sulfate 2.9 3.9 C.sub.14-15 alkyl 7-ethoxylate 4.2 1.9 C.sub.12-14
Alkyl-7-ethoxylate 1.7 0.5 Ca chloride dihydrate 0.045 Ca formate
0.09 0.09 0.09 0.09 A compound: 1.2 0.66
bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n)(CH.sub.3)--N.sup.+--
C.sub.xH.sub.2x--N.sup.+--(CH.sub.3)--
bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n); n is 20 to 30; x is 3 to
8, optionally sulphated or sulphonated Random graft
co-polymer.sup.1 1.46 0.5 0.83 Ethoxylated Polyethylenimine.sup.2
1.5 1.29 1.44 1.44 Diethylene triamine pentaacetic acid 0.34 0.64
0.34 0.34 Diethylene triamine penta 0.3 0.3 (methylene phosphonic
acid) 1-hydroxyethyidene-1,1-diphosphonic 0.18 acid
Dihydroxybenzene-3,5-disulfonic acid 0.19 disodium salt hydrate
Tinopal AMS-GX 0.06 0.29 Tinopal CBS-X 0.2 0.17 0.29 Tinopal TAS-X
B36 0.091 Amphiphilic alkoxylated grease 1.28 1 0.4 1.93 1.93
cleaning polymer.sup.3 CHEC 0.2 Ethanol 2 1.58 1.6 5.4 1.2 3.57
Propylene Glycol 3.9 3.59 1.3 4.3 3.8 Diethylene glycol 1.05 1.54
1.15 1.15 Polyethylene glycol 0.06 0.04 0.1 0.1 *Amylase of the
present invention 15.0 10.0 5.0 8.0 4.25 11.7 (mg active)
Amylase.sup.4 0.01 0.1 0.15 0.12 -- 0.05 Monoethanolamine 3.05 2.41
0.4 1.26 0.31 1.13 NaOH 2.44 1.8 3.01 3.84 0.24 Sodium Cumene
Sulphonate 1 0.95 Sodium Formate 0.11 0.09 0.2 0.12 Water,
Aesthetics (Dyes, perfumes) balance and Minors (Enzymes including
lipase, protease, additional amylase each at 0.2% active protein,
solvents, structurants) .sup.1Random graft copolymer is a polyvinyl
acetate grafted polyethylene oxide copolymer having a polyethylene
oxide backbone and multiple polyvinyl acetate side chains. The
molecular weight of the polyethylene oxide backbone is about 6000
and the weight ratio of the polyethylene oxide to polyvinyl acetate
is about 40 to 60 and no more than 1 grafting point per 50 ethylene
oxide units. .sup.2Polyethylenimine (MW = 600) with 20 ethoxylate
groups per --NH. .sup.3Amphiphilic alkoxylated grease cleaning
polymer is a polyethylenimine (MW = 600) with 24 ethoxylate groups
per --NH and 16 propoxylate groups per --NH *Amylase of the present
invention is shown as mgs of active enzyme per 100 g of
detergent.
Examples 19-21 Heavy Duty Liquid Laundry Detergent Composition
TABLE-US-00020 [0370] 19 20 21 (wt %) (wt %) (wt %) Sodium
Alkylbenzene sulfonate 21.0 10.2 3.53 C.sub.124-18 Alkyl
1.5-9-ethoxylate 18.0 6.32 0.88 Branched Alkyl Sulfate 2.44 Sodium
Alkyl ethoxy 1-3 sulfate 1.17 14.81 Citric Acid 3.14 2.05 C.sub.12
Dimethylamine oxide 0.56 C.sub.12-18 Fatty acid 15.0 2.59 1.48
Protease (Purafect Prime .RTM., 40.6 mg active/g) 1.5 0.52 1.64
Mannanase (Mannaway .RTM., 11 mg active/g) 0.1 0.06 Xyloglucanase
(Whitezyme .RTM., 20 mg active/g) 0.2 0.06 Lipase (lLipex) 0.1 0.2
0.05 *Amylase of the present invention (mg active) 5.9 2.3 12.8
bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n)(CH.sub.3)--N.sup.+--C.sub.xH.sub.-
2x--N.sup.+--(CH.sub.3)-- 2.0 0.63
bis((C.sub.2H.sub.5O)(C.sub.2H.sub.4O)n), wherein n = from 20 to
30, and x = from 3 to 8, optionally sulphated or sulphonated Random
graft co-polymer.sup.1 1.07 Ethoxylated Polyethylenimine.sup.2 0.8
1.51 Amphiphilic alkoxylated polymer.sup.3 Amylase.sup.4
Phosphonated chelant 0.8 0.41 0.53 Hydrotrope 0.93 Brightener 0.2
0.09 0.19 Ethoxylated thiophene Hueing Dye 0.004 Minors: dyes,
perfume, perfume micro capsules, Balance Balance Balance enzymes,
enzyme stabilizers, solvents, structurants, pH modifying agents
*Amylase of the present invention is shown as mgs of active enzyme
per 100 g of detergent. **Based on total cleaning and/or treatment
composition weight, a total of no more than 7% water.
Raw Materials and Notes For Composition Examples 1-21
[0371] Linear alkylbenzenesulfonate having an average aliphatic
carbon chain length C.sub.11-C.sub.18 C.sub.12-18
Dimethylhydroxyethyl ammonium chloride AE3S is C.sub.12-15 alkyl
ethoxy (3) sulfate AE7 is C.sub.12-15 alcohol ethoxylate, with an
average degree of ethoxylation of 7 AE9 is C.sub.12-16 alcohol
ethoxylate, with an average degree of ethoxylation of 9 HSAS is a
mid-branched primary alkyl sulfate with carbon chain length of
about 16-17 as disclosed in U.S. Pat. No. 6,020,303 and U.S. Pat.
No. 6,060,443 Polyacrylate MW 4500 is supplied by BASF
Carboxymethyl cellulose is Finnfix.RTM. V supplied by CP Kelco,
Arnhem, Netherlands CHEC is a cationically modified hydroxyethyl
cellulose polymer. Phosphonate chelants are, for example,
diethylenetetraamine pentaacetic acid (DTPA) Hydroxyethane di
phosphonate (HEDP) Savinase.RTM., Natalase.RTM., Stainzyme.RTM.,
Lipex.RTM., Celluclean.TM., Mannaway.RTM. and Whitezyme.RTM. are
all products of Novozymes, Bagsvaerd, Denmark. Purafect.RTM.,
Purafect Prime.RTM. are products of Genencor International, Palo
Alto, Calif., USA Fluorescent Brightener 1 is Tinopal.RTM. AMS,
Fluorescent Brightener 2 is Tinopal.RTM. CBS-X, Direct Violet 9 is
Pergasol.RTM. Violet BN-Z NOBS is sodium
nonanoyloxybenzenesulfonate TAED is tetraacetylethylenediamine
S-ACMC is carboxymethylcellulose conjugated with C.I. Reactive Blue
19 product name AZO-CM-CELLULOSE Soil release agent is
Repel-o-tex.RTM. PF Acrylic Acid/Maleic Acid Copolymer is molecular
weight 70,000 and acrylate:maleate ratio 70:30 EDDS is a sodium
salt of ethylenediamine-N,N'-disuccinic acid, (S,S) isomer Suds
suppressor agglomerate is supplied by Dow Corning, Midland, Mich.,
USA HSAS is mid-branched alkyl sulfate Liquitint.RTM. Violet CT is
supplied by Milliken, Spartanburg, S.C., USA
Examples 22-26 Unit Dose Laundry Detergent Compositions
[0372] Such unit dose formulations can comprise one or multiple
compartments.
TABLE-US-00021 22 23 24 25 26 (wt %) (wt %) (wt %) (wt %) (wt %)
Alkylbenzene sulfonic acid 14.5 14.5 14.5 14.5 14.5 C.sub.12-18
alkyl ethoxy 3 sulfate 7.5 7.5 7.5 7.5 7.5 C.sub.12-18 alkyl
7-ethoxylate 13.0 13.0 13.0 13.0 13.0 Citric Acid 0.6 0.6 0.6 0.6
0.6 Fatty Acid 14.8 14.8 14.8 14.8 14.8 *Amylase of this invention
(mg active) 6 12 8 2 10 Ethoxylated Polyethylenimine.sup.1 4.0 4.0
4.0 4.0 4.0 Protease (Purafect Prime .RTM., 40.6 mg 1.4 2.0 0.9 1.2
0 active/g) Cellulase (Celluclean, active protein) 0.1 0.2 0.1
Amylase.sup.4 (active protein) a) to k) herein 0.1 0.05 0.1 0.2 0.1
Hydroxyethane diphosphonic acid 1.2 1.2 1.2 1.2 1.2 Brightener 0.3
0.3 0.3 0.3 0.3 P-diol 15.8 13.8 13.8 13.8 13.8 Glycerol 6.1 6.1
6.1 6.1 6.1 MEA 8.0 8.0 8.0 8.0 8.0 TIPA -- -- 2.0 -- -- TEA -- 2.0
-- -- -- Cumene sulphonate -- -- -- -- 2.0 cyclohexyl dimethanol --
-- -- 2.0 -- Water 10 10 10 10 10 Structurant 0.14 0.14 0.14 0.14
0.14 Perfume 1.9 1.9 1.9 1.9 1.9 Buffers (monoethanolamine) To pH
8.0 Solvents (1,2 propanediol, ethanol) To 100% *Amylase of the
present invention is shown as mgs of active enzyme per 100 g of
detergent. .sup.1Polyethylenimine (MW = 600) with 20 ethoxylate
groups per --NH.
Example 27 Multiple Compartment Unit Dose Composition
[0373] Multiple compartment unit dose laundry detergent
formulations of the present invention are provided below. In these
examples, the unit dose has three compartments, but similar
compositions can be made with two, four or five compartments. The
film used to encapsulate the compartments is polyvinyl alcohol.
TABLE-US-00022 27 Base composition 1 (wt %) Glycerol (min 99) 5.3
1,2-propanediol 10.0 Citric Acid 0.5 Monoethanolamine 10.0 Caustic
soda -- Dequest 2010 1.1 Potassium sulfite 0.2 *Amylase of this
invention (mg active) 8.0 Nonionic Marlipal C24EO7 20.1 HLAS 24.6
Optical brightener FWA49 0.2 C12-15 Fatty acid 16.4 Polymer
Lutensit Z96 2.9 Polyethyleneimine ethoxylate PEI600 E20 1.1 MgCl2
0.2 Solvents (1,2 propanediol, ethanol) To 100%
Multi-Compartment Formulations
TABLE-US-00023 [0374] Composition 1 2 Compartment A B C A B C
Volume of each 40 ml 5 ml 5 ml 40 ml 5 ml 5 ml compartment Active
material in Wt. % Perfume 1.6 1.6 1.6 1.6 1.6 1.6 Dyes <0.01
<0.01 <0.01 <0.01 <0.01 <0.01 TiO2 0.1 -- -- -- 0.1
-- Sodium Sulfite 0.4 0.4 0.4 0.3 0.3 0.3 Acusol 305 1.2 2 -- --
Hydrogenated castor oil 0.14 0.14 0.14 0.14 0.14 0.14 Base
Composition 1 Add to Add to Add to Add to Add to Add to 100% 100%
100% 100% 100% 100% *Amylase of the present invention is shown as
mgs of active enzyme per 100 g of detergent.
[0375] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "40 mm" is intended to mean "about 40 mm".
[0376] Every document cited herein, including any cross referenced
or related patent or application, is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or claimed
herein or that it alone, or in any combination with any other
reference or references, teaches, suggests or discloses any such
invention. Further, to the extent that any meaning or definition of
a term in this document conflicts with any meaning or definition of
the same term in a document incorporated by reference, the meaning
or definition assigned to that term in this document shall
govern.
[0377] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the
invention. It is therefore intended to cover in the appended claims
all such changes and modifications that are within the scope of
this invention.
Sequence CWU 1
1
81485PRTBacillus halmapalus 1His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His
Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg
Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr
Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70
75 80 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn
Gly 85 90 95 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly
Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn
Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile
Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr
Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly
Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr
Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195
200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp
Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala
Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu
Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val
Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr
Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro
Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn
Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305 310 315
320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro
Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser
Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala
Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe
Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr
Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly
Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440
445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile
450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser
Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485 2483PRTBacillus
halmapalus 2His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg
Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile
Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln
Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115
120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Tyr Thr Lys Phe
Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp
Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser
Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Ala
Trp Asp Trp Glu Val Asp Ser Glu 180 185 190 Phe Gly Asn Tyr Asp Tyr
Leu Met Tyr Ala Asp Tyr Asp Met Asp His 195 200 205 Pro Glu Val Val
Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn 210 215 220 Thr Leu
Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235
240 Phe Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly
245 250 255 Lys Gly Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
Gly Ala 260 265 270 Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His
Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala
Ser Asn Ser Arg Gly Asn 290 295 300 Tyr Asp Met Ala Lys Leu Leu Asn
Gly Thr Val Val Gln Lys His Pro 305 310 315 320 Met His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu 325 330 335 Ser Leu Glu
Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu
Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360
365 Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile
370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr
Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp
Thr Arg Glu Gly Asn 405 410 415 Thr Thr His Pro Asn Ser Gly Leu Ala
Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Glu Lys Trp Met Tyr
Val Gly Gln Asn Lys Ala Gly Gln Val 435 440 445 Trp His Asp Ile Thr
Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp
Ala Asn Phe Ser Val Asn Lys Gly Ser Val Ser Ile Trp 465 470 475 480
Val Lys Arg 3485PRTBacillus sp. 3His His Asn Gly Thr Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn
His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp
Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly
Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65
70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser
Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys
Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val
Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr Thr
Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn
Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp
Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile
Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185
190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val
Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp
Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp
Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala
Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn
Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val
Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly
Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310
315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln
Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys
Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr
Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His
Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu
Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln Asn Asp Tyr
Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn
Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430
Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435
440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr
Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly
Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 4485PRTBacillus
sp. 4His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser
Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp
Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu
Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val
Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala
Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120
125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp
130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg
Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg
Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly His Gly Lys
Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val
Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr
Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245
250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp
Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn
His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn
Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe
Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Ser His Ala Val
Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu
Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala
Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser 370
375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly
Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly
Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Ser Lys Trp Met
Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Ser Asp Ile
Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly
Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile
Trp Val Asn Lys 485 5485PRTBacillus sp. 5His His Asn Gly Thr Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp
Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu
Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45
Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50
55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys
Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His
Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Trp Val Arg Ala Val Glu
Val Asn Pro Ser Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Asp Tyr
Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly
Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe
Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg 165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180
185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp
Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly
Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile
Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp
Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Asn Met Phe
Ala Val Ala Glu Phe Trp Lys Asn Asp Ile 260 265 270 Gly Ala Ile Glu
Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp
Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly 290 295 300
Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305
310 315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser
Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe
Lys Pro Leu Ala 340 345 350 Cys Ala Leu Thr Leu Thr Arg Asp
Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile
Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro
Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln
Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu 405 410
415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys
Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly
Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val
Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Asn 485
6483PRTBacillus sp. 6His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn
Arg Leu Asn Ser Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile
Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln
Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly
Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr
Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90
95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn
Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp
Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser
Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp
Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg
Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His 195 200 205 Pro
Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215
220 Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys
225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser
Ala Thr Gly 245 250 255 Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu Gly Ala 260 265 270 Ile Glu Asn Tyr Leu Gln Lys Thr Asn
Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Lys Ser Gly Gly Asn 290 295 300 Tyr Asp Met Arg Asn
Ile Phe Asn Gly Thr Val Val Gln Arg His Pro 305 310 315 320 Ser His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu 325 330 335
Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340
345 350 Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly
Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg
Ser Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala
Tyr Gly Pro Gln His 385 390 395 400 Asp Tyr Leu Asp His Pro Asp Val
Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser His Pro Lys Ser
Gly Leu Ala Thr Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys
Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr
Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455 460
Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr 465
470 475 480 Val Gln Lys 7483PRTBacillus licheniformis 7Ala Asn Leu
Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn
Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25
30 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu
His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn
His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val
Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu
His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg
Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155
160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn
Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp
His Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp
Tyr Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala
Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val
Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val
Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr
Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280
285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu
Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro
Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro
Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser
Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile
Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405
410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala
Gly Glu Thr 435 440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro
Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn
Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Arg
8481PRTArtificial SequenceFusion protein 8Val Asn Gly Thr Leu Met
Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln His Trp
Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly
Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser 35 40 45
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu 50
55 60 Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly
Glu 65 70 75 80 Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile
Asn Val Tyr 85 90 95 Gly Asp Val Val Ile Asn His Lys Gly Gly Ala
Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asp Pro Ala
Asp Arg Asn Arg Val Ile Ser 115 120 125 Gly Glu His Leu Ile Lys Ala
Trp Thr His Phe His Phe Pro Gly Arg 130 135 140 Gly Ser Thr Tyr Ser
Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160 Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln 165 170 175
Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp 180
185 190 Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala
Ala 195 200 205 Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu
Gln Leu Asp 210 215 220 Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
Phe Ser Phe Leu Arg 225 230 235 240 Asp Trp Val Asn His Val Arg Glu
Lys Thr Gly Lys Glu Met Phe Thr 245 250 255 Val Ala Glu Tyr Trp Gln
Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu 260 265 270 Asn Lys Thr Asn
Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr 275 280 285 Gln Phe
His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys 290 295 300
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr 305
310 315 320 Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
Ser Thr 325 330 335 Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu Thr Arg 340 345 350 Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly Thr Lys 355 360 365 Gly Asp Ser Gln Arg Glu Ile Pro
Ala Leu Lys His Lys Ile Glu Pro 370 375 380 Ile Leu Lys Ala Arg Lys
Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr 385 390 395 400 Phe Asp His
His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser 405 410 415 Val
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly 420 425
430 Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His
435 440 445 Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
Glu Gly 450 455 460 Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser
Ile Tyr Val Gln 465 470 475 480 Arg
* * * * *