Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 54
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
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DETERGENT COMPOSITIONS
FIELD OF THE INVENTION
The present invention relates to detergent compositions, particularly laundry
detergents,
comprising lipolytic enzymes.
BACKGROUND OF THE INVENTION
Improved removal of greasy soils is a constant aim for detergent
manufacturers,
especially in the laundry context. In spite of the use of many effective
surfactants and
combinations of surfactants, especially when used at low water temperatures,
many surfactant-
based products still do not achieve complete removal of greasy/oily soils.
Lipase enzymes have
been used in detergents since the late 1980s for removal of fatty soils by
breakdown of fatty
soils into tri-glycerides.
Until relatively recently, the main commercially available lipase enzymes,
such as
Lipolase (trade name, Novozymes) worked particularly effectively at the lower
moisture levels
of the drying phase of the wash process. These enzymes tended to produce
significant cleaning
only in the second wash step with fat breakdown significant only on soils
remaining on
laundered clothes during the drying stage, the broken down fats then being
removed in the next
washing step. However, more recently, higher efficiency lipases have been
developed that also
work effectively during the wash phase of the cleaning process, so that as
well as cleaning in the
second washing step, a significant improvement in cleaning effect due to
lipase enzyme can be
found in the first wash-cycle. Examples of such enzymes are as described in US
6,939,702B 1,
W000/60063 and Research Disclosure IP6553D. Such enzymes are referred to below
as first
wash lipases.
In addition, consumers prefer that articles, such as garments, be as clean as
possible.
Such consumers typically associate the odor of a cleaned or treated article
with the degree of
cleanliness of such article. Thus, the effectiveness of a cleaning and/or
treatment composition,
from a consumer's perspective, is typically directly linked with the odor that
such composition
imparts to an article that is cleaned or treated with such composition.
Applicants recognized that
certain materials, such as esterases and lipases, can generate objectionable
fatty acid odors,
particularly short-chain fatty acid odors such as the odor of butyric acid.
However, such
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materials can be particularly effective cleaning agents. Unfortunately,
consumers typically
associate the odors resulting from the use of such agents with a lack of
cleanliness. Examples of
reduced odour variants with a C-terminal extension are shared in W002/062973,
but these lipase
variants do not demonstrate the strong wash performance of the first wash
lipases such as those
from W000/60063 including the variant sold under the tradename Lipex .
Thus, there remains a need for a detergent compositions comprising lipolytic
enzymes
for excellent greasy/oily soils removal while not generating any objectionable
fatty acid odors.
SUMMARY OF THE INVENTION
The present invention relates to detergent compositions comprising a detergent
ingredient and a lipase variant having an average Relative performance (RPavg)
of at least 0.8
and a Benefit-Risk (BR) of at least 1.1 at the test conditions given in the
specification.
SEQUENCE LISTING
SEQ ID NO: 1 shows the DNA sequence encoding lipase from 7'hermomyces
lanoginosus.
SEQ ID NO: 2 shows the amino acid sequence of a lipase from Thermomyces
lanoginosus.
SEQ ID NO: 3 and SEQ ID NO: 4 show sequences used for alignment example.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Lipase activity: The term "lipase activity" is defined herein as a carboxylic
ester
hydrolase activity which catalyzes the hydrolysis of triacylglycerol under the
formation of
diacylglycerol and a carboxylate. For purposes of the present invention,
lipase activity is
determined according to the procedure described in "Lipase activity" in
"Materials and
Methods". One unit of lipase activity is defined as the amount of enzyme
capable of releasing
1.0 micro mole of butyric acid per minute at 30 C, pH 7.
The polypeptides of the present invention have at least 70%, such at least 75%
or 80% or
85% or 90%, more preferably at least 95%, even more preferably 96% or 97%,
most preferably
98% or 99%, and even most preferably at least 100% of the lipase activity
measured as Relative
Performance of the polypeptide consisting of the amino acid sequence shown as
the mature
polypeptide of SEQ ID NO:2, with the substitutions T231R + N233R.
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Isolated polypeptide: The term "isolated polypeptide" as used herein refers to
a
polypeptide which is at least 20% pure, preferably at least 40% pure, more
preferably at least
60% pure, even more preferably at least 80% pure, most preferably at least 90%
pure, and even
most preferably at least 95% pure, as determined by SDS-PAGE.
Substantially pure polypeptide: The term "substantially pure polypeptide"
denotes
herein a polypeptide preparation which contains at most 10%, preferably at
most 8%, more
preferably at most 6%, more preferably at most 5%, more preferably at most 4%,
at most 3%,
even more preferably at most 2%, most preferably at most 1%, and even most
preferably at most
0.5% by weight of other polypeptide material with which it is natively
associated. It is,
therefore, preferred that the substantially pure polypeptide is at least 92%
pure, preferably at
least 94% pure, more preferably at least 95% pure, more preferably at least
96% pure, more
preferably at least 96% pure, more preferably at least 97% pure, more
preferably at least 98%
pure, even more preferably at least 99%, most preferably at least 99.5% pure,
and even most
preferably 100% pure by weight of the total polypeptide material present in
the preparation.
The polypeptides of the present invention are preferably in a substantially
pure form. In
particular, it is preferred that the polypeptides are in "essentially pure
form", i.e., that the
polypeptide preparation is essentially free of other polypeptide material with
which it is natively
associated. This can be accomplished, for example, by preparing the
polypeptide by means of
well-known recombinant methods or by classical purification methods.
Herein, the term "substantially pure polypeptide" is synonymous with the terms
"isolated
polypeptide" and "polypeptide in isolated form."
Identity: The relatedness between two amino acid sequences or between two
nucleotide
sequences is described by the parameter "identity".
For purposes of the present invention, the alignment of two amino acid
sequences is
determined by using the Needle program from the EMBOSS package
(http://emboss.org)
version 2.8Ø The Needle program implements the global alignment algorithm
described in
Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The
substitution matrix
used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.
The degree of identity between an amino acid sequence of the present invention
("invention sequence"; e.g. amino acids 1 to 269 of SEQ ID NO:2) and a
different amino acid
sequence ("foreign sequence") is calculated as the number of exact matches in
an alignment of
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the two sequences, divided by the length of the "invention sequence" or the
length of the
"foreign sequence", whichever is the shortest. The result is expressed in
percent identity.
An exact match occurs when the "invention sequence" and the "foreign sequence"
have
identical amino acid residues in the same positions of the overlap (in the
alignment example
below this is represented by "I"). The length of a sequence is the number of
amino acid residues
in the sequence (e.g. the length of SEQ ID NO:2 is 269).
In the alignment example below, the overlap is the amino acid sequence "HTWGER-
NL" of Sequence A; or the amino acid sequence "HGWGEDANL" of Sequence B. In
the
example a gap is indicated by a
Alignment example
Sequence A: ACMSHTWG ER- NL
I I I 1 11
Sequence B: HGWGEDANLAMNPS
Polypeptide Fragment: The term "polypeptide- fragment" is defined herein as a
polypeptide having one or more amino acids deleted from the amino and/or
carboxyl terminus of
SEQ ID NO: 2 or a homologous sequence thereof, wherein the fragment has lipase
activity.
Subsequence: The term "subsequence" is defined herein as a nucleotide sequence
having one or more nucleotides deleted from the 5' and/or 3' end of SEQ ID NO:
1 or a
homologous sequence thereof, wherein the subsequence encodes a polypeptide
fragment having
lipase activity.
Allelic variant: The term "allelic variant" denotes herein 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.
Substantially pure polynucleotide: The term "substantially pure
polynucleotide" as
used herein refers to a polynucleotide preparation free of other extraneous or
unwanted
nucleotides and in a form suitable for use within genetically engineered
protein production
systems. Thus, a substantially pure polynucleotide contains at most 10%,
preferably at most
8%, more preferably at most 6%, more preferably at most 5%, more preferably at
most 4%,
more preferably at most 3%, even more preferably at most 2%, most preferably
at most 1%, and
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even most preferably at most 0.5% by weight of other polynucleotide material
with which it is
natively 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, preferably at
least 92% pure, more
5 preferably at least 94% pure, more preferably at least 95% pure, more
preferably at least 96%
pure, more preferably at least 97% pure, even more preferably at least 98%
pure, most
preferably at least 99%, and even most preferably at least 99.5% pure by
weight. The
polynucleotides of the present invention are preferably in a substantially
pure form. In
particular, it is preferred that the polynucleotides disclosed herein are in
"essentially pure form",
i.e., that the polynucleotide preparation is essentially free of other
polynucleotide material with
which it is natively associated. Herein, the term "substantially pure
polynucleotide" is
synonymous with the terms "isolated polynucleotide" and "polynucleotide in
isolated form."
The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic
origin, or any
combinations thereof.
cDNA: The term "cDNA" is defined herein as a DNA molecule which can be
prepared
by reverse transcription from a mature, spliced, mRNA molecule obtained from a
eukaryotic
cell. cDNA lacks intron sequences that are usually present in the
corresponding genomic DNA.
The initial, primary RNA transcript is a precursor to mRNA which is processed
through a series
of steps before appearing as mature spliced mRNA. These steps include the
removal of intron
sequences by a process called splicing. cDNA derived from mRNA lacks,
therefore, any intron
sequences.
Nucleic acid construct: The term "nucleic acid construct" as used herein
refers to a
nucleic acid molecule, either single- or double-stranded, which is isolated
from a naturally
occurring gene or which is modified to contain segments of nucleic acids in a
manner that would
not otherwise exist in nature. 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.
Control sequence: The term "control sequences" is defined herein to include
all
components, which are necessary or advantageous for the expression of a
polynucleotide
encoding a polypeptide of the present invention. Each control sequence may be
native or
foreign to the nucleotide sequence encoding the polypeptide. Such control
sequences include,
but are not limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter, signal
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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 nucleotide sequence
encoding a
polypeptide.
Operably linked: The term "operably linked" denotes herein a configuration in
which a
control sequence is placed at an appropriate position relative to the coding
sequence of the
polynucleotide sequence such that the control sequence directs the expression
of the coding
sequence of a polypeptide.
Coding sequence: When used herein the term "coding sequence" means a
nucleotide
sequence, which directly specifies the amino acid sequence of its protein
product. The
boundaries of the coding sequence are generally determined by an open reading
frame, which
usually begins with the ATG start codon or alternative start codons such as
GTG and TTG. The
coding sequence may a DNA, cDNA, or recombinant nucleotide sequence.
Expression: The term "expression" includes any step involved in the production
of the
polypeptide including, but not limited to, transcription, post-transcriptional
modification,
translation, post-translational modification, and secretion.
Expression vector: The term "expression vector" is defined herein as a linear
or circular
DNA molecule that comprises a polynucleotide encoding a polypeptide of the
invention, and
which is operably linked to additional nucleotides that provide for its
expression.
Host cell: The term "host cell", as used herein, includes any cell type which
is
susceptible to transformation, transfection, transduction, and the like with a
nucleic acid
construct comprising a polynucleotide of the present invention.
Modification: The term "modification" means herein any chemical modification
of the
polypeptide consisting of the mature polypeptide of SEQ ID NO: 2 as well as
genetic
manipulation of the DNA encoding that polypeptide. The modification(s) can be
substitution(s),
deletion(s) and/or insertions(s) of the amino acid(s) as well as
replacement(s) of amino acid side
chain(s).
Artificial variant: When used herein, the term "artificial variant" means a
polypeptide
having lipase activity produced by an organism expressing a modified
nucleotide sequence of
SEQ ID NO: 1. The modified nucleotide sequence is obtained through human
intervention by
modification of the nucleotide sequence disclosed in SEQ ID NO: 1.
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Relative performance (RP): The term relative performance reflects performance
of the
enzyme variant compared to a reference enzyme when measured as the brightness
of the color of
the textile samples washed with that specific enzyme variant as described in
Example 2 of the
present specification.
Risk (R): The term "risk" and risk factor are used interchangeably in the
present
specification and is the ratio between the amount of released butyric acid
from the lipase variant
washed swatch and the amount of released butyric acid from a swatch washed
with the mature
part of the lipase of SEQ ID NO: 2, after both values have been corrected for
the amount of
released butyric acid from a non-lipase washed swatch.
Benefit-Risk factor (BR): The Benefit-Risk factor describes the wash
performance
compared to risk for odor. Thus BR=RPaõg/R.
Conventions for Designation of Variants:
In describing lipase variants according to the invention, the following
nomenclature is
used for ease of reference: Original amino acid(s):position(s):substituted
amino acid(s)
According to this nomenclature, for instance the substitution of glutamic acid
for
glycine in position 195 is shown as G195E. A deletion of glycine in the same
position is shown
as G195*, and insertion of an additional amino acid residue such as lysine is
shown as G195GK.
Where a specific lipase contains a "deletion" in comparison with other lipases
and an
insertion is made in such a position this is indicated as *36D for insertion
of an aspartic acid in
position 36.
Multiple mutations are separated by pluses, i.e.: R170Y+G195E, representing
mutations in positions 170 and 195 substituting tyrosine and glutamic acid for
arginine and
glycine, respectively.
X231 indicates the amino acid in a parent polypeptide corresponding to
position 231,
when applying the -described alignment procedure. X231R indicates that the
amino acid is
replaced with R. For SEQ ID NO:2 X is T, and T231R thus indicates a
substitution of T in
position 231 with R. Where the amino acid in a position (e.g. 231) may be
substituted by another
amino acid selected from a group of amino acids, e.g. the group consisting of
R and P.and Y,
this will be indicated by X231R/P/Y.
In all cases, the accepted IUPAC single letter or triple letter amino acid
abbreviation is
employed.
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DETAILED DESCRIPTION OF THE INVENTION
Polypeptides having Lipase Activity
The isolated polypeptides having a lipase activity, of the present invention,
are selected
from the group consisting of lipases having a RP of at least 0.8 and a BR of
at least 1.1 at the
test conditions given in the specification.
In a preferred embodiment the lipase has a RP of at least 0.9, such as 1.0 or
1.1. In an
even more preferred embodiment the lipase has a RP of at least 1.2, such as
1.3 or even 1.4.
In another preferred embodiment the lipase has a BR of at least 1.2, such as
1.3 or even
1.4. In an even more preferred embodiment the lipase has a BR of at least 1.5,
such as 1.6 or
even 1.7.
In a further aspect the polypeptide of the present invention further has a
relative
LU/A280 less thanl, such as less than 0.95 at the test conditions given in the
specification. In a
preferred embodiment the relative LU/A280 is less than 0.90, such as less than
0.85 or even less
than 0.80.
In a further aspect, the isolated polypeptides of the present invention, have
an amino acid
sequence which is comprised by or comprises SEQ ID NO:2, or an allelic variant
thereof, and
which further has BR of at least 1.1 and RP of at least 0.8. In another
aspect, the isolated
polypeptides of the present invention, have an amino acid sequence which is
comprised by or
comprises the mature part of SEQ ID NO:2, or an allelic variant thereof, and
which further has
BR of at least 1.1 and RP of at Ieast 0.8
In a still further aspect, the isolated polypeptides of the present invention,
have an amino
acid sequence which has a degree of identity to the mature polypeptide of SEQ
ID NO: 2 (i.e.,
the mature polypeptide) of at least 80%, such as at least 85% or 90%, or at
least 95%, preferably
at least 97%, most preferably at least 98%, and even most preferably at least
99%, which have
lipase activity (hereinafter "homologous polypeptides"). In a preferred
aspect, the homologous
polypeptides have an amino acid sequence which differs by ten amino acids,
preferably by five
amino acids, more preferably by four amino acids, even more preferably by
three amino acids,
most preferably by two amino acids, and even most preferably.by one amino acid
from the
mature polypeptide of SEQ ID NO: 2.
In a further aspect, the isolated polypeptides having lipase activity of the
present
invention, are encoded by polynucleotides which hybridize under very low
stringency
conditions, preferably low stringency conditions, more preferably medium
stringency
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conditions, more preferably medium-high stringency conditions, even more
preferably high
stringency conditions, and most preferably very high stringency conditions
with (i) nucleotides
644 to 732 of SEQ ID NO: 1, (ii) the cDNA sequence contained in nucleotides
644 to 732of
SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or (iv) a complementary
strand of (i), (ii), or (iii)
(J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A
Laboratory Manual,
2d edition, Cold Spring Harbor, New York). A subsequence of SEQ ID NO: 1
contains at least
100 contiguous nucleotides or preferably at least 200 contiguous nucleotides.
Moreover, the
subsequence may encode a polypeptide fragment which has lipase activity.
The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, as well as
the
amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may be used to
design a nucleic
acid probe to identify and clone DNA encoding polypeptides having lipase
activity 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, preferably at least 25, more preferably at least
35, and most preferably
at least 70 nucleotides in length. It is, however, preferred that the nucleic
acid probe is at least
'100 nucleotides in length. For example, the nucleic acid probe may be at
least 200 nucleotides,
preferably at least 300 nucleotides, more preferably at least 400 nucleotides,
or most preferably
at least 500 nucleotides in length. Even longer probes may be used, e.g.,
nucleic acid probes
which are at least 600 nucleotides, at least preferably at least 700
nucleotides, or more preferably
at least 800 nucleotides in length. Both DNA and RNA probes can be used. The
probes are
typically labeled for detecting the corresponding gene (for example, with 32F,
3H, 35 S, biotin, or
avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other organisms may,
therefore,
be screened for DNA which hybridizes with the probes described above and which
encodes a
polypeptide having lipase activity. 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. In order to identify a
clone or DNA which is
homologous with SEQ ID,NO: 1 or a subsequence thereof, the carrier material is
used in a
Southern blot.
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For purposes of the present invention, hybridization indicates that the
nucleotide
sequence hybridizes to a labeled nucleic acid probe corresponding to the
nucleotide sequence
shown in SEQ ID NO: 1, its complementary strand, or a subsequence thereof,
under very low to
very high stringency conditions. Molecules to which the nucleic acid probe
hybridizes under
5 these conditions can be detected using X-ray film.
In a still further aspect, the variant of the present invention can be
artificial variants
comprising a conservative substitution, deletion, and/or insertion of one or
more amino acids of
SEQ ID NO: 2 or the mature polypeptide thereof, said variants having a BR of
at least 1.1 and a
RP of at least 0.8. Preferably, amino acid changes are of a minor nature, that
is conservative
10 amino acid substitutions or insertions that do not significantly affect the
folding and/or activity
of the protein; small deletions, typically of one to about 30 amino acids;
small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine residue; a
small linker
peptide of up to about 20-25 residues; or a small extension that facilitates
purification by
changing net charge or another function, such as a poly-histidine tract, an
antigenic epitope or a
binding domain.
Examples of conservative substitutions are within the group of basic amino
acids
(arginine, lysine and histidine), acidic amino acids (glutamic acid and
aspartic acid), polar amino
acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine
and valine),
aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino
acids (glycine,
alanine, serine, threonine and methionine). Amino acid substitutions which do
not generally
alter specific activity are known in the art and are described, for example,
by H. Neurath and
R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly
occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly,
Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
In addition to the 20 standard amino acids, non-standard amino acids (such as
4-
hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and
alpha-methyl serine)
may be substituted for amino acid residues of a wild-type polypeptide. A
limited number of
non-conservative amino acids, amino acids that are not encoded by the genetic
code, and
unnatural amino acids may be substituted for amino acid residues. "Unnatural
amino acids"
have been modified after protein synthesis, and/or have a chemical structure
in their side
chain(s) different from that of the standard amino acids. Unnatural amino
acids can be
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chemically synthesized, and preferably, are commercially available, and
include pipecolic acid,
thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-
dimethylproline.
Alternatively, the amino acid changes are of such a nature that the physico-
chemical
properties of the polypeptides are altered. For example, amino acid changes
may improve the
thermal stability of the polypeptide, alter the substrate specificity, change
the pH optimum, and
the like.
Essential amino acids in the parent polypeptide 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 biological activity (i.e_, lipase 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 enzyme 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 which are related to a polypeptide according to the invention.
Single or multiple amino acid substitutions 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,
Biochem. 30:10832-10837; U.S. Patent 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).
Mutagenesis/shuffling methods can be combined with high-throughput, automated
screening methods to detect activity of cloned, mutagenized polypeptides
expressed by host
cells. (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 of
interest, and can be applied to polypeptides of unknown structure.
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In one aspect, the total number of amino acid substitutions, deletions and/or
insertions of
amino acids 1 to 291 of SEQ ID NO: 2 is 10, preferably 9, more preferably 8,
more preferably 7,
more preferably at most 6, more preferably at most 5, more preferably 4, even
more preferably
3, most preferably 2, and even iimost preferably 1.
Identification of regions and substitutions.
The positions referred to in Region I through Region IV below are the
positions of the
amino acid residues in SEQ ID NO:2. To find the corresponding (or homologous)
positions in a
different lipase, the procedure described in "Homology and alignment" is used.
Substitutions in Re ig on I
Region I consists of amino acid residues surrounding the N-terminal residue
El. In this
region it is preferred to substitute an amino acid of the parent lipase with a
more positive amino
acid. Amino acid residues corresponding to the following positions are
comprised by Region I:
1 to 11 and 223-239. The following positions are of particular interest: 1, 2,
4, 8, 11, 223, 227,
229, 231, 233, 234 and 236. In particular the following substitutions have
been identified:
X1N/*, X4V, X227G, X231R and X233R.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2 . In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Substitutions in Re ig on II
Region Il consists of amino acid residues in contact with substrate on one
side of the
acyl chain and one side of the alcohol part. In this region it is preferred to
substitute an amino
acid of the parent lipase with a more positive amino acid or with a less
hydrophobic amino acid.
Amino acid residues corresponding to the following positions are comprised by
Region II: 202
to 211 and 249 to 269. The following, positions are of particular interest :
202, 210, 211, 253,
254, 255, 256, 259. In particular the following substitutions have been
identified: X202G,
X21OK/W/A, X255YN/A, X256KJR and X259G/M/Q/V.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2. In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
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13
Substitutions in Region III
Region III consists of amino acid residues that form a flexible structure and
thus
allowing the substrate to get into the active site. In this region it is
preferred to substitute an
amino acid of the parent lipase with a more positive amino acid or a less
hydrophobic amino
acid. Amino acid residues corresponding to the following positions are
comprised by Region
III: 82 to 102. The following positions are of particular interest: 83, 86,
87, 90, 91, 95, 96, 99. In
particular the following substitutions have been identified: X83T, X86V and
X90A/R.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2. In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Substitutions in Re ion IV
Region IV consists of amino acid residues that bind electrostatically to a
surface. In this
region it is preferred to substitute an amino acid of the parent lipase with a
more positive amino
acid. Amino acid residues corresponding to the following positions are
comprised by Region IV:
27 and 54 to 62. The following positions are of particular interest: 27, 56,
57, 58, 60. In
particular the following substitutions have been identified: X27R, X58N/AG/T/P
and
X60V/S/G/N/R/K/AIL.
In a preferred embodiment the parent lipase has at least 80%, such as 85% or
90%, such
as at least 95% or 96% or 97% or 98% or 99%, identity to SEQ ID NO:2. In a
most preferred
embodiment the parent lipase is identical to SEQ ID NO: 2.
Amino acids at other positions
The parent lipase may optionally comprise substitutions of other amino acids,
particularly less than 10 or less than 5 such substitutions. Examples are
substitutions
corresponding to one or more of the positions 24, 37, 38, 46, 74, 81, 83, 115,
127, 131, 137, 143,
147, 150, 199, 200, 203, 206, 211, 263, 264, 265, 267 and 269 of the parent
lipase. In a
particular embodiment there is a substitution in at least one of the positions
corresponding to
position 81, 143, 147, 150 and 249. In a preferred embodiment the at least one
substitution is
selected from the group consisting of X81Q/E, X143S/C/N/D/A, X147IV1/Y,
X150G/K and
X249R/I/L.
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14
The variant may comprise substitutions outside the defined Regions I to IV,
the number
of substitutions outside of the defined Regions I-to IV is preferably less
than six, or less than
five, or less than four, or less than three, or less than two, such as five,
or four, or three, or two
or one. Alternatively, the variant does not comprise any substitution outside
of the defined
Regions I to IV.
Further substitutions may, e.g., be made according to principles known in the
art, e.g.
substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079
and WO
97/07202.
Parent lipase variants
In one aspect, said variant, when compared to said parent, comprises a total
of at least
three substitutions, said substitutions being selected from one or more of the
following groups of
substitutions:
a) at least two, or at least three, or at least four, or at least five, or at
least six, such as
two, three, four, five or six, substitutions in Region I,
b) at least one, at least two, or at least three, or at least four, or at
least five, or at least
six, such as one, two, three, four, five or six, substitution in Region II,
c) at least one, at least two, or at least three, or at least four, or at
least five, or at least
six, such as one, two, three, four, five or six, substitution in Region III,
d) and/or at least one, at least two, or at least three, or at least four, or
at least five, or
at least six, such as one, two, three, four, five or six, substitution in
Region IV.
The variant may comprise substitutions, compared to the variant's parent,
corresponding
to those substitutions listed below in Table 1.
Region I Region II Region IH Region IV Outside regions
'
X4V + X227G + X210K + X83T + X58A + X60S X 150G
X231R + X233R X256K X86V
X227G + X231R X256K X86V X58N + X60S X150G
+ X233R
X231R + X233R X255Y
X231R + X233R X202G
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X227G + X23 1R X256K X86V
+ X233R
X4V + X231R + X58N + X60S
X233R
X231R + X233R X90R X58N + X60S
X231R + X233R X255V X90A
X227G + X231R X256K X86V X58N + X60S X150G
+ X233R
X231R + X233R X211L X58N + X60S X147M
X231R + X233R X150K
Table 1: Some particular variants.
In a further particular embodiment the parent lipase is identical to SEQ ID
NO:2, and
5 the variants of Table 1 will thus be:
Region I Region II Region III Region IV Outside regions
Q4V + L227G + E210K + S83T + I86V S58A + V60S A150G
T231R + N233R P256K
L227G + T231R P256K 186V S58N + V60S A1'50G
+ N233R
T231R + N233R 1255Y
T231R + N233R 1202G
L227G + T231R + P256K I86V
N233R
Q4V + T231R + S58N + V60S
N233R
T231R + N233R 190R S58N + V60S
T231R + N233R 1255V 190A
L227G + T231R + P256K 186V S58N + V60S A150G
N233R
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16
T231R + N233R F211L S58N + VbOS L147M
T231R + N233R A150K
Table 2: Some particular variants of SEQ ID NO:2
Further substitutions may, e.g., be made according to principles known in the
art, e.g.
substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079
and WO
97/07202.
Homology and alignment
For purposes of the present invention, the degree of homology may be suitably
determined by means of computer programs known in the art, such as GAP
provided in the GCG
program package (Program Manual for the Wisconsin Package, Version 8, August
1994,
Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711)
(Needleman,
S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-45),
using GAP with the
following settings for polypeptide sequence comparison: GAP creation penalty
of 3.0 and GAP
extensiori penalty of 0.1.
In the _present invention, corresponding (or homologous) positions in the
lipase
sequences of Absidia reflexa, Absidia corymbefera, Rhizmucor miehei, Rhizopus
delemar,
Aspergillus niger, Aspergillus tubigensis, Fusarium oxysporum, Fusarium
heterosporum,
Aspergillus oryzea, Penicilium camembertii, Aspergillus foetidus, Aspergillus
niger,
Thermomyces lanoginosus (synonym: Humicola lanuginose) and Landerina
penisapora are
defined by the alignment shown in Figure 1.
To find the homologous positions in lipase sequences not shown in the
alignment, the
sequence of interest is aligned to the sequences shown in Figure 1. The new
sequence is aligned
to the present alignment in Figure 1 by using the GAP alignment to the most
homologous
sequence found by the GAP program. GAP is provided in the GCG program package
(Program
Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer
Group, 575
Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch,
C.D., (1970),
Journal of Molecular Biology, 48, 443-45). The following settings are used for
polypeptide se-
quence comparison: GAP creation penalty of 3.0 and GAP extension penalty of
0.1.
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17
The parent lipase has a homology of at least 50 % with the T. lanuginosus
lipase (SEQ ID NO:
2), particularly at least 55 %, at least 60 %, at least 75 %, at least 85 %,
at least 90 %, more than
95 % or more than 98 %. In a particular embodiment the parent lipase is
identical to the T.
lanuginosus lipase (SEQ ID NO:2).
- Benefit Risk
The Benefit Risk factor describing the performance compared to the reduced
risk for odour
smell is defined as: BR = RPaõg / R. Lipase variants described herein may have
BRs greater than
1, greater than 1.1, or even greater than I to about 1000.
-Average Relative Performance
The procedure for calculating average relative performance (RPa,g) is found in
Example 5 of the
present specification. Lipase variants described herein may have (RPa,g) of at
least 0.8, at least
1.1, at least 1.5, or even at least 2 to about 1000.
-Relative LU/A280
The relative LU/A280 is determined by LU/A280 assay found in Example 4 of the
present specification. Lipase variants described herein may have a relative
LU/A280 less than
1.00, less than 0.9, less than 0.8 or even from less than 1.00 to about 0.1.
Sources of Polypeptides Having Lipase Activity
A polypeptide of the present invention 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 polypeptide encoded by a
nucleotide
sequence is produced by the source or by a strain in which the nucleotide
sequence from the
source has been inserted. In a preferred aspect, the polypeptide obtained from
a given source is
secreted extracellularly.
A polypeptide of the present invention may be a bacterial polypeptide. For
example, the
polypeptide may be a gram positive bacterial polypeptide such as a Bacillus
polypeptide, e.g., a
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus
circulans, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium,
Bacillus stearotherrnophilus, Bacillus subtilis, or Bacillus thuringiensis
polypeptide; or a
Streptornyces polypeptide, e.g., a Streptomyces lividans or Streptomyces
murinus polypeptide; or
a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.
polypeptide.
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18
A polypeptide of the present invention may also be a fungal polypeptide, and
more
preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia,
Saccharomyces,
Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous
fungal
polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus,
Filobasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallirnastix,
Neurospora,
Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus,
Thielavia,
Tolypocladiurn, or Trichoderma polypeptide.
In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis,
Saccharomyces
cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis potypeptide having lipase
activity.
In another preferred aspect, the polypeptide is an Aspergillus aculeatus,
Aspergillus
awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus
nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus turbigensis,
Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariurn
graminearum,
Fusarium graminum, Fusariurn heterosporum, Fusarium negundi, Fusarium
oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariurn
sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecioides, Fusarium venenatum, Humicola insolens, Thermomyces
lanoginosus
(synonym: Humicola lanuginose), Mucor miehei, Myceliophthora thermophila,.
Neurospora
crassa, Penicillium purpurogenurn, Trichoderma harzianum, Trichoderma
koningid,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride
polypeptide.
In another preferred aspect, the polypeptide is a Thermornyces polypeptide.
In a more preferred aspect, the polypeptide is a Thermornyces lanuginosus
polypeptide,
e.g., the polypeptide of SEQ ID NO: 2 with mutations as disclosed in the
present application.
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.
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 und Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures
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WO 2007/087319 PCT/US2007/001803
19
(CBS), and Agricultural Research Service Patent Culture Collection, Northern
Regional
Research Center (NRRL).
Furthermore, such polypeptides may be identified and obtained from other
sources
including microorganisms isolated from nature (e.g., soil, composts, water,
etc.) using the
above-mentioned probes. Techniques for isolating microorganisms from natural
habitats are
well known in the art. The polynucleotide may then be obtained by similarly
screening a
genomic or cDNA library of another microorganism. Once a polynucleotide
sequence encoding
a polypeptide has been detected with the probe(s), the polynucleotide can be
isolated or cloned
by utilizing techniques which are well known to those of ordinary skill in the
art (see, e.g.,
Sambrook et al., 1989, supra).
Polypeptides of the present invention also include fused polypeptides or
cleavable fusion
polypeptides in which another polypeptide is fused at the N-terminus or the C-
terminus of the
polypeptide or fragment thereof_ A fused polypeptide is produced by fusing a
nucleotide
sequence (or a portion thereof) encoding another polypeptide to a nucleotide
sequence (or a
portion thereof) of the present invention. 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.
Polynucleotides
The lipase of the present invention is preferably derived from a isolated
polynucleotides having
a nucleotide sequence which encode a polypeptide of the present invention,
more preferably
nucleotide sequences which encode a polypeptide being a variant of the amino
acid sequence of
SEQ ID NO: 2 or the mature polypeptide thereof, which differ from the encoding
polynucleotide
by virtue of the degeneracy of the genetic code. The polypeptide may also be
derived from
subsequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 that have
lipase
activity, said fragments having a BR of at least 1.1 and a RP of at least 0.8.
The techniques used to isolate or clone a polynucleotide encoding a
polypeptide are
known in the art and include isolation from genomic DNA, preparation from
eDNA, or a
combination thereof. The cloning of the polynucleotides of the present
invention from such
genomic DNA can be effected, e.g., by using the well known polymerase chain
reaction (PCR)
or antibody screening of expression libraries to detect cloned DNA fragments
with shared
CA 02633798 2008-06-18
WO 2007/087319 PCT/US2007/001803
structural features_ See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application,
Academic Press, New York. Other nucleic acid amplification procedures such as
ligase chain
reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-
based
amplification (NASBA) may be used. The polynucleotides may be cloned from a
strain of
5 Thermom_yces, or another or related organism and thus, for example, may be
an allelic or species
variant of the polypeptide encoding region of the nucleotide sequence.
The polypeptide may be derived from polynucleotides having nucleotide
sequences
which have a degree of identity to the mature polypeptide coding sequence of
SEQ ID NO: 1 of
at least 60%, preferably at least 65%, more preferably at least 70%, more
preferably at least
10 75%, more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%,
even more preferably at least 95%, and most preferably at least 97% identity,
which encode an
active polypeptide having lipase activity and BR of at least 1.1 and RP of at
least 0.8.
Modification of a nucleotide sequence encoding a polypeptide of the present
invention
may be necessary for the synthesis of polypeptides substantially similar to
the polypeptide. The
15 term "substantially similar" to the polypeptide refers to non-naturally
occurring forms of the
polypeptide. These polypeptides may differ in some engineered way from the
polypeptide
isolated from its native source, e.g., artificial variants that differ in
specific activity, thermo
stability, pH optimum, or the like. The variant sequence may be constructed on
the basis of the
nucleotide sequence presented as the polypeptide encoding region of SEQ ID NO:
1, e.g., a
20 subsequence thereof, and/or by introduction of nucleotide substitutions
which do not give rise to
another amino acid sequence of the polypeptide encoded by the nucleotide
sequence, but which
correspond to the codon usage of the host organism intended for production of
the enzyme, or by
introduction of nucleotide substitutions which may give rise to a different
amino acid sequence.
For a general description of nucleotide substitution, see, e.g., Ford et al.,
1991, Protein
Expression and Purification 2: 95-107.
It will be apparent to those skilled in the art that such substitutions can be
made outside
the regions critical to the function of the molecule and still result in an
active polypeptide.
Amino acid residues essential to the activity of the polypeptide encoded by an
isolated
polynucleotide of the invention, and therefore preferably not subject to
substitution, may be
identified according to procedures known in the art, such as site-directed
mutagenesis or
alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science
244: 1081-
1085). In the latter technique, mutations are introduced at every positively
charged residue in
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WO 2007/087319 PCT/US2007/001803
21
the molecule, and the resultant mutant molecules are tested for lipase
activity to identify amino
acid residues that are critical to the activity of the molecule. Sites of
substrate-enzyme
interaction can also be determined by analysis of the three-dimensional
structure as determined
by such techniques as nuclear magnetic resonance analysis, crystallography or
photoaffinity
labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, Journal of
Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-
64).
The polypeptide may be derived from isolated polynucleotides encoding a
polypeptide of
the present invention, which hybridize under very low stringency conditions,
preferably low
stringency conditions, more,preferably medium stringency conditions, more
preferably medium-
high stringency conditions, even more preferably high stringency conditions,
and most
preferably very high stringency conditions with (i) of SEQ ID NO: 1, (ii) the
cDNA sequence
contained in SEQ ID NO: 1, or (iii) a complementary strand of (i) or (ii); or
allelic variarits and
subsequences thereof (Sambrook et al., 1989, supra), as defined herein.
The polypeptide may be derived from isolated polynucleotides obtained by (a)
hybridizing a population of DNA under very low, low, medium, medium-high,
high, or very
high stringency conditions with (i) nucleotides SEQ ID NO: 1, (ii) the cDNA
sequence
contained in nucleotides of SEQ ID NO: 1, or (iii) a complementary strand of
(i) or (ii); and (b)
isolating the hybridizing polynucleotide, which encodes a polypeptide having
lipase activity.
Nucleic Acid Constructs
Nucleic acid constructs comprising an isolated polynucleotide of the present
invention
can be operably linked to one or more control sequences which direct the
expression of the
coding sequence in a suitable host cell under conditions compatible with the
control sequences.
An isolated polynucleotide encoding a polypeptide of the present invention may
be
manipulated in a variety of ways to provide for expression of the polypeptide.
Manipulation of
the polynucleotide's sequence prior to its insertion into a vector may be
desirable or necessary
depending on the expression vector. The techniques for modifying
polynucleotide sequences
utilizing recombinant DNA methods are well known in the art.
The control sequence may be an appropriate promoter sequence, a nucleotide
sequence
which is recognized by a host cell for expression of a polynucleotide encoding
a polypeptide of
the present invention. The promoter sequence contains transcriptional control
sequences which
mediate the expression of the polypeptide. The promoter may be any nucleotide
sequence which
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22
shows transcriptional activity in the host cell of choice 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.
Examples of suitable promoters for directing the transcription of the nucleic
acid
constructs of the present invention, especially in a bacterial host cell, are
the promoters obtained
from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA),
Bacillus subtilis
levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL),
Bacillus
stearothermophilus maltogenic amylase gene (amy1V1), Bacillus
amyloliquefaciens alpha-
amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP),
Bacillus subtilis xylA
and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al.,
1978, Proceedings
of the National Academy of Sciences USA 75: 3727-3731), as well as the tac
promoter (DeBoer
et al., 1983, Proceedings of the Nati nal.Academy of Sciences USA 80: 21-25).
Further
promoters are described in "Useful proteins from recombinant bacteria" in
Scientific American,
1980, 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic
acid
constructs of the present invention in a filamentous fungal host cell are
promoters obtained from
the genes for Aspergillus oryzae TAKA amylase, Rhiz:omucor miehei aspartic
proteinase,
Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-
amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor
miehei lipase,
Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Aspergillus
nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium
venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),
Fusarium
oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-
glucosidase,
Trichoderma reesei cellobiohydrolase I, Trichoderma reesei endoglucanase I,
Trichoderrna
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 hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase
and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and
hybrid
promoters thereof.
In a yeast host, useful promoters are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),
Saccharomyces
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23
cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase
(ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI),
S'accharomyces cerevisiae metallothionine (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.
The control sequence may also be a suitable transcription terminator sequence,
a
sequence recognized by a host cell to terminate transcription. The terminator
sequence is
operably linked to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any
terminator which is functional in the host cell of choice may be used in the
present invention.
Preferred terminators for filamentous fungal host cells are obtained from the
genes for
Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus
nidulans
anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-
like protease.
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.
The control sequence may also be a suitable leader sequence, a nontranslated
region of
an mRNA which is important for translation by the host cell. The leader
sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the polypeptide.
Any leader
sequence that is functional in the host cell of choice may be used in the
present invention.
Preferred leaders for filamentous fungal host cells are obtained from the
genes for
Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate
isomerase.
Suitable leaders for yeast host cells are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate
kinase,
Saccharornyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence
operably
linked to the 3' terminus of the nucleotide sequence and which, when
transcribed, is recognized
by the host cell as a signal to add polyadenosine residues to transcribed
rrmRNA. Any
polyadenylation sequence which is functional in the host cell of choice may be
used in the
present invention.
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24
Preferred polyadenylation sequences for filamentous fungal host cells are
obtained from
the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,
Aspergillus
nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and
Aspergillus niger
alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and
Sherman, 1995,1VIolecular Cellular Biology 15: 5983-5990.
The control sequence may also be a signal peptide coding region that codes for
an amino
acid sequence linked to the amino terminus of a polypeptide and directs the
encoded polypeptide
into the cell's secretory pathway. The 5' end of the coding sequence of the
nucleotide sequence
may inherently contain a signal peptide coding region naturally linked in
translation reading
frame with the segment of the coding region which encodes the secreted
polypeptide.
Alternatively, the 5' end of the coding sequence may contain a signal peptide
coding region
which 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 polypeptide.
However, any signal
peptide coding region which directs the expressed polypeptide into the
secretory pathway of a
host cell of choice may be used in the present invention.
Effective signal peptide coding regions for bacterial host cells are the
signal peptide
coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic
amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
licheniformis beta-
lactamase, 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.
Effective signal peptide coding regions for filamentous fungal host cells are
the signal
peptide coding regions obtained from the genes for Aspergillus oryzae TAKA
amylase,
Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic
proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
Useful signal peptides for yeast host cells are obtained from the genes for
Saccharornyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
Other useful
signal peptide coding regions are described by Romanos et al., 1992, supra.
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The control sequence may also be a propeptide coding region that codes for an
amino
acid sequence positioned at the amino terminus of a polypeptide. 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 a mature active polypeptide by
catalytic or
5 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), Saccharomyces cerevisiae alpha-factor, Rhizomucor
miehei aspartic
proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
Where both signal peptide and propeptide regions are present at the amino
terminus of a
10 polypeptide, the propeptide region is positioned next to the amino terminus
of a polypeptide and
the signal peptide region is positioned next to the amino terminus of the
propeptide region.
It may also be desirable to add regulatory sequences which allow the
regulation of the
expression of the polypeptide relative to the growth of the host cell.
Examples of regulatory
systems are those which cause the expression of the gene to be turned on or
off in response to a
15 _ 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 GALl system may be used_ In filamentous fungi, the TAKA alpha-
amylase
promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae
glucoamylase
promoter may be used as regulatory sequences. Other examples of regulatory
sequences are
20 those which allow for gene amplification. In eukaryotic systems, these
include the dihydrofolate
reductase gene which is amplified in the presence of methotrexate, and the
metallothionein
genes which are amplified with heavy metals. In these cases, the nucleotide
sequence encoding
the polypeptide would be operably linked with the regulatory sequence.
25 Expression Vectors
Recombinant expression vectors usually comprise a polynucleotide of the
present
invention, a promoter, and transcriptional and translational stop signals. The
various nucleic
acids and control sequences described above may be joined together to produce
a recombinant
expression vector which may include one or more convenient restriction sites
to allow for
insertion or substitution of the nucleotide sequence encoding the polypeptide
at such sites.
Alternatively, a nucleotide sequence of the present invention may be expressed
by inserting the
nucleotide sequence or a nucleic acid construct comprising the sequence into
an appropriate.
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26
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.
The recombinant expression vector may be any vector (e.g., a plasmid or virus)
which
can be conveniently subjected to recombinant DNA procedures and can bring
about expression
of the nucleotide sequence. 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 vectors may be
linear or closed circular plasmids.
The vector may be an autonomously replicating vector, i.e., a vector which
exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication,
e.g., a plasmid, 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 which, 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 which together contain the total
DNA to be
introduced into the genome of the host cell, or a transposon may be used.
The vectors preferably contain one or more selectable markers which permit
easy
selection of transformed 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.
A conditionally essential gene may function as a non-antibiotic selectable
marker. Non-
limiting examples of bacterial conditionally essential non-antibiotic
selectable markers are the
dal genes from Bacillus subtilis, Bacillus licheniformis, or other Bacilli,
that are only essential
when the bacterium is cultivated in the absence of D-alanine. Also the genes
encoding enzymes
involved in the turnover of UDP-galactose can function as conditionally
essential markers in a
cell when the cell is grown in the presence of galactose or grown in a medium
which gives rise
to the presence of galactose. Non-limiting examples of such genes are those
from B. subtilis or
B. licheniformis encoding UTP-dependent phosphorylase (EC 2.7.7.10), UDP-
glucose-
dependent uridylyltransferase (EC 2.7.7.12), or UDP-galactose epimerase (EC
5.1.3.2). Also a
xylose isomerase gene such as xylA, of Bacilli can be used as selectable
markers in cells grown
in minimal medium with xylose as sole carbon source. The genes necessary for
utilizing.
gluconate, gntK, and gntP can also be used as selectable markers in cells
grown in minimal
medium with gluconate as sole carbon source. Other examples of conditionally
essential genes
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27
are known in the art. Antibiotic selectable markers confer antibiotic
resistance to such antibiotics
as ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline,
neomycin, hygromycin
or methotrexate.
Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1,
and
URA3. Selectable markers for use in a filamentous fungal host cell include,
but are not limited
to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate
reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and
trpC (anthranilate
synthase), as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS
and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene
of Streptomyces
hygroscopicus.
The vectors preferably contain 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.
For integration into the host cell genome, the vector may rely on the
polynucleotide's
sequence encoding the polypeptide or any other element of the vector for
integration into the
genome by homologous or nonhomologous 'recombination. Alternatively, the
vector may
contain 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 preferably
contain a sufficient number of nucleic acids, such as 100 to 10,000 base
pairs, preferably 400 to
10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a
high degree of
identity with 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.
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
which functions in
a cell. The term "origin of replication" or "plasmid replicator" is defined
herein as a nucleotide
sequence that enables a plasmid or vector to replicate in vivo.
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28
Examples of bacterial origins of replication are the origins of replication of
plasmids
pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and
pUB 110,
pE194, pTA1060, and pAM131 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2
micron origin of
replication, ARS 1, ARS4, the combination of ARS 1 and CEN3, and the
combination of ARS4
and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are
AMA1 and
ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids
Research 15: 9163-
9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or
vectors
comprising the gene can be accomplished according to the methods disclosed in
WO 00/24883.
More than one copy of a polynucleotide of the present invention may be
inserted into the
host cell to increase production of the gene product. 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.
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).
Host Cells
Recombinant host cells usually comprise a polynucleotide of the present
invention,
which are advantageously used in the recombinant production of the
polypeptides. A vector
comprising a polynucleotide of the present invention is introduced into a host
cell so that the
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 polypeptide
and its source.
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29
The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-
unicellular
microorganism, e.g., a eukaryote.
Useful unicellular microorganisms are bacterial cells such as gram positive
bacteria
including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus,
Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii,
Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus
stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a
Streptomyces cell, e.g.,
Streptomyces lividans and Streptomyces murinus, or gram negative bacteria such
as E. coli and
Pseudomonas sp. In a preferred aspect, the bacterial host cell is a Bacillus
lentus, Bacillus
licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In
another preferred aspect,
the Bacillus cell is an alkalophilic Bacillus.
The introduction of a vector into a bacterial host cell may, for instance, be
effected by
protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General
Genetics 168:
111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology
81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular
Biology 56: 209-
221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:
742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:
5771-5278).
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or
fungal cell.
In a preferred aspect, the host cell is a fungal cell. "Fungi" as used herein
includes the
phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined
by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th
edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the Oomycota (as
cited in
Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth
et al., 1995,
supra).
In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as
used herein
includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and
yeast
belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change
in the future, for the purposes of this invention, yeast shall be defined as
described in Biology
and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R.,
eds, Soc. App.
Bacteriol. Symposium Series No. 9, 1980).
In an even more preferred aspect, the yeast host cell is a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia;cell.
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In a most preferred aspect, the yeast host cell is a Saccharomyces
carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,
Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis
cell. In another
most preferred aspect, the 'yeast host cell is a Kluyveromyces lactis cell. In
another most
5 preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred aspect, the fungal host cell is a filamentous fungal
cell.
"Filamentous fungi" include all filamentous forms of the subdivision Eumycota
and Oomycota
(as defined by Hawksworth et al., 1995, supra). The filamentous fungi are
generally
characterized by a mycelial wall composed of chitin, cellulose, glucan,
chitosan, mannan, and
10 other complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon
catabolism is obligately aerobic. In contrast, vegetative growth by yeasts
such as
Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon
catabolism may be
fermentative.
In an even more preferred aspect, the filamentous fungal host cell is an
Acremonium,
15 Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus,
Cryptococcus,
Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix,
Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces,
Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes,
or
Trichoderma cell.
20 In a most preferred aspect, the filamentous fungal host cell is an
Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans,
Aspergillus niger or Aspergillus oryzae cell. In another most preferred
aspect, the filamentous
fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium
crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium
heterosporum.,
25 Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium
roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichio ides, Fusarium
sulphureum,
Fusarium torulosum, Fusariurn trichothecioides, or Fusarium venenatum cell. In
another most
preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta,
Ceriporiopsis
aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis
gilvescens,
30 Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or
Ceriporiopsis
subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens,
Humicola lanuginosa,
Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum,
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31
Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia
terrestris,
Trametes villosa, Trarnetes versicolor, Trichoderma harzianum, Trichoderma
koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain
cell.
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 238 023 and Yelton et al., 1984, Proceedings of the National Academy of
Sciences USA
81: 1470-1474. Suitable methods for transforming Fusarium species are
described by Malardier
er 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, Journal of Bacteriology 153:
163; and Hinnen
et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
The polypeptide of the present invention can be produced by a method
comprising (a)
cultivating a cell, which in its wild-type form is capable of producing the
polypeptide, under
conditions conducive for production of the polypeptide; and (b) recovering the
polypeptide.
Preferably, the cell is of the genus Aspergillus, and more preferably
Aspergillus Oryzae.
Methods for producing a polypeptide of the present invention can also comprise
(a)
cultivating a host cell under conditions conducive for production of the
polypeptide; and (b)
recovering the polypeptide.
Methods for producing a polypeptide of the present invention can also comprise
(a)
cultivating a host cell under conditions conducive for production of the
polypeptide, wherein the
host cell comprises a mutant nucleotide sequence having at least one mutation
in the mature
polypeptide coding region of SEQ ID NO: 1, wherein the mutant nucleotide
sequence encodes a
polypeptide which is a lipase comprised by or comprising the polypeptide of
SEQ ID NO: 2, and
(b) recovering the polypeptide. In a preferred embodiment the nucleotide
sequence encodes a
polypeptide which is a lipase comprised by or comprising the mature part of
the polypeptide of
SEQ ID NO: 2, and (b) recovering the polypeptide.
In the production methods, the cells are cultivated in a nutrient medium
suitable for
production of the polypeptide using methods well known in the art. For
example, the cell may
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32
be cultivated by shake flask cultivation, and 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
polypeptide is secreted into
the nutrient medium, the polypeptide can be recovered directly from the
medium. If the
polypeptide is not secreted, it can be recovered from cell lysates.
The polypeptides may be detected using methods known in the art that are
specific for
the polypeptides. 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 polypeptide as described herein.
The resulting polypeptide may be recovered using methods known in the art. For
example, the polypeptide may be recovered from the nutrient medium by
conventional
procedures including, but not limited to, centrifugation, filtration,
extraction, spray-drying,
evaporation, or precipitation.
The polypeptides 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).
Compositions
Preferably, the compositions are enriched in the polypeptide of the present
invention..
The term "enriched" indicates that the lipase activity of the composition has
been increased, e.g.,
with an enrichment factor of 1.1.
The composition may comprise a polypeptide of the present invention as the
major
enzymatic component, e.g., a mono-component composition. Alternatively, the
composition
may comprise multiple enzymatic activities, such as an aminopeptidase,
amylase, carbohydrase,
carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin
glycosyltransferase,
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33
deoxyribonuclease, esterase, aipha-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,
preferably
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus
oryzae; Fusarium,
preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium
culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatum, Fusariurn roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum,
Fusarium
trichothecioides, or Fusarium venenatum; Humicola, preferably Humicola
insolens or Humicola
lanuginosa; or Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
The polypeptide 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 polypeptide
composition may be in the form of a granulate or a microgranulate. The
polypeptide to be
included in the composition may be stabilized in accordance with methods known
in the art.
DETERGENT INGREDIENTS
As used herein detergent compositions include articles and cleaning and
treatment compositions.
As used herein, the term "cleaning and/or treatment composition" includes,
unless otherwise
indicated, tablet, granular or powder-form all-purpose or "heavy-duty" washing
agents,
especially Iaundry detergents; liquid, gel or paste-form all-purpose washing
agents, especially
the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand
dishwashing agents or
light duty dishwashing agents, especially those of the high-foaming type;
machine dishwashing
agents, including the various tablet, granular, liquid and rinse-aid types for
household and
institutional use. The compositions 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.
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34
The detergent composition of the present invention can comprise one or more
lipase variant(s)
of the present invention. In addition to the lipase variant(s), the detergent
composition will
further comprise a detergent ingredient. The non-limiting list of detergent
ingredients illustrated
hereinafter are suitable for use in the instant compositions and may be
desirably incorporated in
certain embodiments of the invention, for example to assist or enhance
cleaning performance,
for treatment of the substrate to be cleaned, or to) modify the aesthetics of
the cleaning
composition as is the case with colorants, dyes or the like. The precise
nature of these additional
components, and levels of incorporation thereof, will depend on the physical
form of the
composition and the nature of the cleaning operation for which it is to be
used. Suitable
detergent ingredients include, but are not limited to, surfactants, builders,
chelating agents, dye
transfer inhibiting agents, dispersants, enzymes, and enzyme stabilizers,
bleach activators,
hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric
dispersing
agents, brighteners, suds suppressors, dyes, anti-corrosion agents, tarnish
inhibitors, perfumes,
fabric softeners, carriers, hydrotropes, processing aids, solvents and/or
pigments.
Typical detergents would comprise by weight any combination of the following
ingredients: 5 -
30% surfactant, preferably anionic surfactants such as linear
alkylbenzenesulfonate and alcohol
ethoxysulfate; 0.005-0.1% protease active protein, wherein the protease is
preferably selected
from CoronaseTM, FNA, FN4 or SavinaseTM, 0.001-0.1% amylase active protein,
wherein the
amylase is preferably selected from TermamylTM NatalaseTM, StainzymeTM and
PurastarTM and
0.1-3% chelants, preferably diethylene triamine pentaacetic acid. For granular
and tablet
products, such typical detergents would additionally comprise by weight: 5-20%
bleach,
preferably sodium percarbonate; 1-4% bleach activator, preferably TAED and/or
0-30%,
preferably 5-30%, more preferably less than 10% builder, such as the
aluminosilicate Zeolite A
and/or tripolyphosphate.
Bleaching Agents - The detergent compositions of the present invention may
comprise
one or more bleaching agents.
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. Examples of suitable bleaching
agents include:
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(1) sources of hydrogen peroxide, for example, inorganic perhydrate salts,
including
alkali metal salts such as sodium salts of perborate (usually mono- or tetra-
hydrate),
percarbonate, persulphate, perphosphate, persilicate salts and mixtures
thereof. In one aspect of
the invention the inorganic perhydrate salts are selected from the group
consisting of sodium
5 salts of perborate, percarbonate and mixtures thereof soaps; and
(2) bleach activators having R-(C=O)-L wherein R is an alkyl group, optionally
branched, having, when the bleach activator is hydrophobic, from 6 to 14
carbon atoms, or from
8 to 12 carbon atoms and, when the bleach activator is hydrophilic, less than
6 carbon atoms or
even less than 4 carbon atoms; and L is leaving group. Examples of suitable
leaving groups are
10 benzoic acid and derivatives thereof - especially benzene sulphonate.
Suitable bleach activators
include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate,
decanoyl
oxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzene
sulphonate, tetraacetyl
ethylene diamine (TAED) and nonanoyloxybenzene sulphonate (NOBS)_ Suitable
bleach
aotivators are also disclosed in WO 98/17767. While any suitable bleach
activator may be
15 employed, in one aspect of the invention the subject cleaning composition
may comprise NOBS,
TAED or mixtures thereof.
(3) Pre-formed peracids.
When present, the peracid and/or bleach activator is generally present in the
composition
in an amount of from about 0.1 to about 60 wt%, from about 0.5 to about 40 wt
% or even from
20 about 0.6 to about 10 wt% based on the composition. One or more hydrophobic
precursors
thereof may be used in combination with one or more hydrophilic peracid or
precursor thereof.
The amounts of hydrogen peroxide source and peracid or bleach activator may be
selected such that the molar ratio of available oxygen (from the peroxide
source) to peracid is
from 1: i to 35:1, or even 2:1 to 10:1.
Surfactants - The detergent compositions according to the present invention
may
comprise a surfactant or surfactant system wherein the surfactant can be
selected from nonionic
surfactants, anionic surfactants, cationic surfactants, ampholytic
surfactants, zwitterionic
surfactants, semi-polar nonionic surfactants and mixtures thereof. When
present, surfactant is
typically present at a level of from about 0.1% to about 60%, from about 0.1%
to about 40%,
from about 0.1% to about 12%, from about 1% to about 50% or even from about 5%
to about
40% by weight of the subject composition.
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When included therein the detergent 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.
The detergent may optionally contain from about 0.2% to about 40% of a non-
ionic
surfactant such as alcohol ethoxylate, nonylphenol ethoxylate,
alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid
monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl
derivatives of
glucosamine ("glucamides").
Builders - The detergent compositions of the present invention may comprise
one or
more detergent builders or builder systems. When a builder is used, the
subject composition will
typically comprise at least about 1%, from about 5% to about 60% or even from
about 10% to
about 40% builder by weight of the subject composition. Builders include, but
are not limited
to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates,
alkali metal
silicates or layered silicates, alkaline earth and alkali metal carbonates,
aluminosilicate builders
and the various alkali metal, ammonium and substituted ammonium salts of
polyacetic acids
such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as
polycarboxylates
such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid,
polymaleic acid, benzene
1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts
thereof.
Chelating Agents - The detergent compositions 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 composition 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 composition.
Brighteners - The detergent compositions of the present invention can also
contain
additional components that may alter appearance of articles being cleaned,
such as fluorescent
brighteners. These brighteners absorb in the UV-range and emit in the visible.
Suitable
fluorescent brightener levels include lower levels of from about 0.01, from
about 0.05, from
about 0.1 or even frorri about 0.2 wt % to upper levels of 0.5 or even 0.75 wt
%.
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37
Dispersants - The compositions of the present invention can also contain
dispersants.
Suitable water-soluble organic materials include the homo- or co-polymeric
acids or their salts,
in which the polycarboxylic acid comprises at least two carboxyl radicals
separated from each
other by not more than two carbon atoms.
Enzymes - In addition to the lipase variant(s) of the present invention the
detergent
composition can comprise one or more further enzymes which provide cleaning
performance
and/or fabric care benefits such as a protease, another lipase, a cutinase, an
amylase, a
carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a
galactanase, a xylanase, an
oxidase, e.g., a laccase, and/or a peroxidase.
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.
Suitable proteases include those of animal, vegetable or microbial origin.
Microbial
origin is preferred. Chemically modified or protein engineered mutants are
included. The
protease may be a serine protease or a metallo protease, preferably an
alkaline microbial
protease or a trypsin-like protease. Examples of alkaline proteases are
subtilisins, especially
those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,
subtilisin 309, subtilisin
147 and subtilisin 168 (described in WO 89/06279), SEQ ID no 4 and SEQ ID no 7
in WO
05/103244. Other suitable serin proteases include those from Micrococcineae
spp especially
Cellulonas spp and variants thereof as disclosured in W02005052146. Examples
of trypsin-like
proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium
protease described in
WO 89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO
98/20115,
WO 98/20116, and WO 98/34946, especially the variants with substitutions in
one or more of
the following positions: 27, 36, 57, 68, 76, 87, 97, 101, 104, 106, 120, 123,
167, 170, 194, 206,
218, 222, 224, 235, 245, 252 and 274, and amongst other variants with the
following mutations:
(K27R, V 104Y, N 123 S, T124A), (N76D, S 103A, V1041), or (S 101 G, S 103A,
V1041, G159D,
A232V, Q236H, Q245R, N248D, N252K). Other examples of useful proteases are the
variants
described in WO 05/052146 especially the variants with substitutions in one or
more of the
following positions: 14, 16, 35, 65, 75, 76, 79, 123, 127, 159 and 179
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38
Preferred commercially available protease enzymes include AlcalaseTM,
SavinaseTM,
PrimaseTM, DuralaseTM, EsperaseTM, CoronaseTM, PolarzymeTM and KannaseTM
(Novozymes
A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect
PrimeTM, Purafect
OxPTM, FNA, FN2, FN3 and FN4 (Genencor International Inc.).
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 (synonymous T. lanuginosus) as
described in
EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a
Pseudomonas
lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376),
P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO
95/06720 and
WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis
(Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,- 253-360), B.
stearothermophilus
(JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO
94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96100292, WO 95130744, WO
94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
Other commercially available lipase enzymes include LipolaseT"', Lipolase
U1traT"' and
LipexTM (Novozymes A/S).
Suitable amylases (a and/or (3) include those of bacterial or fungal origin.
Chemically
modified or protein engineered mutants are included. Amylases include, for
example, a-
amylases obtained from Bacillus, e.g. a special strain of B. lieheniformis,
described in more
detail in GB 1,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO
94/18314,
WO 96/23873, and WO 97/43424, especially the variants with substitutions in
one or more of
the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208,
209, 243, 264, 304, 305, 391, 408, and 444.
Commercially available amylases are DuramylTM, TermamylTM, StainzymeTM
Stainzyme UltraTM, FungamylTM and BANT"1 (Novozymes A./S), RapidaseTm and
PurastarTM
(from Genencor International Inc.).
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
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39
produced from Humicola insolens, Myceliophthora thermophila and Fusarium
oxysporum
disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO
89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having
colour care
benefits. Examples of such cellulases are cellulases described in EP 0 495
257, EP 0 531 372,
WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants
such as
those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US
5,763,254,
WO 95/2447 1, WO 98112307 and PCT/DK98/00299.
Commercially available cellulases include Renozymem, CellueleanTM, EndolaseTM,
CelluzymeTM, and CarezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM
(Genencor
International Inc.), and KAC-500(B)TM (Kao Corporation).
Peroxidases/Oxidases:
Suitable peroxidases/oxidases include those of plant, bacterial 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.
Commercially available peroxidases include GuardzymeTM (Novozymes A/S).
When present in a cleaning composition, the aforementioned enzymes may be
present at levels
from about 0.00001% to about 2%, from about 0.0001% to about 1% or even from
about
0.001% to about 0.5% enzyme protein by weight of the composition.
Enzyme Stabilizers - Enzymes for use in detergents can be stabilized by
various
techniques. The enzymes employed herein can be stabilized by the presence of
water-soluble
sourc~s of calcium and/or magnesium ions in the finished compositions that
provide such ions to
the enzymes. Further conventional stabilizing agents, e.g., a polyol such as
propylene glycol or
glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid
derivative, e.g., an
aromatic borate ester, or a phenyl boronic acid derivative such as 4-
formylphenyl boronic acid,
may also be used and the composition may be formulated as described in e.g. WO
92/19709 and
WO 92/19708.
Solvents - Suitable solvents include water and other solvents such as
lipophilic fluids.
Examples of suitable lipophilic fluids include siloxanes, other silicones,
hydrocarbons, glycol
ethers, glycerine derivatives such as glycerine ethers, perfluorinated amines,
perfluorinated and
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hydrofluoroether solvents, low-volatility nonfluorinated organic solvents,
diol solvents, other
environmentally-friendly solvents and mixtures thereof.
WASHING METHOD
5 The present invention includes a method for cleaning and /or treating a
situs inter alia a surface
or fabric. Such method includes the steps of contacting an embodiment of
Applicants' cleaning
composition, in neat form or diluted in a wash liquor, with at least a portion
of a surface or
fabric then optionally rinsing such surface or fabric. The surface or fabric
may be subjected to a
washing step prior to the aforementioned rinsing step. For purposes of the
present invention,
10 washing includes but is not limited to, scrubbing, and mechanical
agitation. 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
15 Applicants' cleaning composition, cleaning additive or mixture thereof. The
fabric may
comprise most any fabric capable of being laundered in normal consumer 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 100 ppm, preferably 500ppm to about 15,000 ppm
in solution.
The water temperatures typically range from about 5 C to about 90 C. The
invention may be
20 particularly beneficial at low water temperatures such as below 30 C or
below 25 or 20 C. The
water to fabric ratio is typically from about 1:1 to about 30:1.
LIPASE VARIANTS EXAMPLES
25 .
Chemicals used as buffers and substrates are commercial products of at least
reagent grade.
- Media and Solutions: LAS (Surfac PSTM) and Zeolite A (Wessalith PTM). Other
ingredients
used are standard laboratory reagents.
- Materials: EMPA221 from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St.
Gallen,
30 Switzerland
Example 1: Production of enzyme
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A plasmid containing the gene encoding the lipase is constructed and
transformed into a
suitable host cell using standard methods of the art.
Fermentation is carried out as a fed-batch fermentation using a constant
medium
temperature of 34 C and a start volume of 1.2 liter. The initial pH of the
medium is set to 6.5.
Once the pH has increased to 7.0 this value is maintained through addition of
10% H3P04. The
level of dissolved oxygen in the medium is controlled by varying the agitation
rate and using a
fixed aeration rate of 1.0 liter air per liter medium per minute. The feed
addition rate is
maintained at a constant level during the entire fed-batch phase.
The batch medium contains maltose syrup as carbon source, urea and yeast
extract as nitrogen
source and a mixture of trace metals and salts. The feed added continuously
during the fed-batch
phase contains maltose syrup as carbon source whereas yeast extract and urea
is added in order
to assure a sufficient supply of nitrogen.
Purification of the lipase may be done by use of standard methods known in the
art, e.g.
by filtering the fermentation supernatant and subsequent hydrophobic
chromatography and
anion exchange, e.g. as described in EP 0 851 913 EP, Example 3.
Example 2: AMSA - Automated Mechanical Stress Assay - for calculation of RP.
The enzyme variants of the present application are tested using the 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 meehanical
stress. For further description see WO 02/42740 especially the paragraph
"Special method
embodiments" at page 23-24. The containers, which contain the detergent test
solution, consist
of cylindrical holes (6 mm diam, 10 mm depth) in a metal plate. The stained
fabric (test
material) lies on the top of the metal plate and is used as a lid and seal on
the containers.
Another metal plate lies on the top of the stained fabric to avoid any
spillage from each
container. The two metal plates together with the stained fabric are vibrated
up -and down at a
frequency of 30 Hz with an amplitude of 2 mm.
The assay is conducted under the experimental conditions specified below:
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42
0.5 g/l LAS
Test solution 0.52 g/1 Na2CO3
1.07 gIl Zeolite A
0.52 g/l Trisodium citrate
Test solution volume 160 micro 1
pH As is (=9.9)
Wash time 20 minutes
Temperature 30 C
15 dH
Water hardness
Ratio of Ca2+/Mg2/NaHCO3 : 4:1:7.5
Enzyme concentration in test solution 0.125, 0.25, 0.50, 1.0 mg enzyme
protein/liter (mg ep / 1)
Wash performance: After washing the
textile pieces are immediately flushed in tap
Drying water and air-dried at 85C in 5 min
Odour: After washing the textile pieces are
immediately flushed in tap water and dried
at room temperature (20 C) for 2 hours
Cream turmeric swatch as described below
Test material
(EMPA221 used as cotton textile)
Table 3
Cream-turmeric swatches are prepared by mixing 5 g of turmeric (Santa Maria,
Denmark) with 100 g cream (38% fat, Aria, Denmark) at 50 C, the mixture is
left at this
temperature for about 20 minutes and filtered (50 C) to remove any un-
dissolved particles. The
mixture is cooled to 20 C and woven cotton swatches, EMPA221, are immersed in
the cream-
turmeric mixture and afterwards allowed to dry at room temperature over night
and frozen until
use. The preparation of cream-tumeric swatches is disclosed in the patent
application PA 2005
00775, filed 27 May 2005.
The performance of the enzyme variant is measured as the brightness of the
colour of
the textile samples washed with that specific enzyme variant. Brightness can
also be expressed
as the intensity of the light reflected from the textile sample when luminated
with white light.
When the textile is stained the intensity of the reflected light is lower,
than that of a clean textile.
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43
Therefore the intensity of the reflected light can be used to measure wash
performance of an
enzyme variant.
Color measurements are made with a professional flatbed scanner (PFU
DL2400pro),
which is used to capture an image of the washed textile samples. The scans are
made with a
resolution of 200 dpi and with an output color depth of 24 bits. In order to
get accurate results,
the scanner is frequently calibrated with a Kodak reflective IT8 target.
To extract a value for the light intensity from the scanned images, a special
designed
software application is used (Novozymes Color Vector Analyzer). The program
retrieves the 24
bit pixel values from the image and converts them 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 -rZ+g2+b2
The wash performance (P) of the variants is calculated in accordance with the
below
formula: P = Int(v) - Int(r) where
Int(v) is the light intensity value of textile surface washed with the tested
enzyme and
Int(r) is the light intensity value of textile surface washed without the
tested enzyme_
A relative performance score is given as the result of the AMSA wash in
accordance with the definition: Relative Performance scores (RP) are summing
up the
performanees (P) of the tested enzyme variants against the reference enzyme:
RP = P(test enzyme) / P(reference enzyme)
RPavg indicates the average relative performance compared to the reference
enzyme at all four
enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep/1)
RPavg = avg(RP(0.125), RP(0.25) RP(0.5), RP(1.0))
A variant is considered to exhibit improved wash performance, if it performs
better than
the reference.
In the context of the present invention the reference enzyme is the lipase of
SEQ ID NO:2 with
the substitutions T231R + N233R.
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Example 3: GC - Gas Chromatograph - for calculation of risk factor.
The butyric acid release from the lipase washed swatches are measured by Solid
Phase
Micro Extraction Gas Chromatography (SPME-GC) using the following method. Four
textile
pieces (5 mm in diameter), washed in the specified solution in Table 1
containing I mg/ L
lipase, are transferred to a Gas Chromatograph (GC) vial. The samples are
analysed on a Varian
3800 GC equipped with a Stabilwax- DA w/Integra-Guard column (30m, 0.32 mm ID
and 0.25
micro-m df) and a Carboxen PDMS SPME fibre (75 micro-m). Each sample is
preincubated for
min at 40 C followed by 20 min sampling with the SPME fibre in the head-space
over the
textile pieces. The sample is subsequently injected onto the column (injector
temperature =
10 250 C). Column flow = 2 ml Helium/min. Column oven temperature gradient: 0
min = 40 C, 2
min = 40 C, 22 min = 240 C, 32 min = 240 C. The butyric acid is detected by
FID detection and
the amount of butyric acid is calculated based on a butyric acid standard
curve.
The Risk Performance Odour, R, of a lipase variant is the ratio between the
amount of
released butyric acid from the lipase variant washed swatch and the amount of
released butyric
acid from a swatch washed with the mature part of the lipase of SEQ ID NO: 2,
after both values
have been corrected for the amount of released butyric acid from a non-lipase
washed swatch.
The risk (R) of the variants is calculated in accordance with the below
formula:
Odour =measured in g butyric acid developed at 1 mg enzyme protein / 1
corrected
for blank
(xtest enzyme = Odour test enzyme - Blank
areference enzyme = Odour reference enzyme - Blank
R = atest enzyme / areferencc enzyme
A variant is considered to exhibit reduced odor compared to the reference, if
the R factor is
lower than 1.
i
Example 4: Activity (LU) relative to absorbance at 280nm
The activity of a lipase relative to the absorbance at 280 nm is determined by
the
following assay LU/A280:
A substrate for lipase is prepared by emulsifying tributyrin (glycerin
tributyrate) using
gum Arabic as emulsifier. The hydrolysis of tributyrin at 30 C at pH 7 or 9
is followed in a pH-
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stat titration experiment. One unit of lipase activity (1 LU) equals the
amount of enzyme capable
of releasing I micro mol butyric acid/min at pH 7.
The absorbance of the purified lipase at 280 nm is measured (A280) and the
ratio
LU/A280 is calculated. The relative LU/A280 is calculated as the LU/A280 of
the variant
5 divided by the LU/A280 of a reference enzyme. In the context of the present
invention the
reference enzyme is the mature part of SEQ ID NO:2 with the mutations T231R
and N233R.
Example 5: BR - Benefit Risk
The Benefit Risk factor describing the performance compared to the reduced
risk for odour
10 smell is thus defined as: BR = RPa,g / R
A variant is considered to exhibit improved wash performance and reduced odor,
if the BR
factor is higher than 1.
Applying the above methods the following results are obtained:
Variant Mutations in mature part of polypeptide RP BR LU/A280
of SEQ ID NO: 2
1 I202G + T231R +N233R 0.84 1.41 not
determined
2 I86V + L227G + T23 IR + N233R + 1.08 1.52 1700
P256K
3 Q4V + S58N + V60S + T231R + N233R 0.87 1.73 1950
4 S58N + V60S + I90R + T231R,N233R 1.06 1.27 2250
5 I255Y + T231R + N233R 1.19 1.17 3600
6 190A + T231R + N233R + 1255V 1.13 1.14 2700
Referenc T231R + N233R 1.00 1.00 3650
e
7 G91A + E99K + T231R+N233R + 0.43 not 850
Q249R + 270H + 271T + 272P + 273S + determined
274S + 275G + 276R + 277G + 278G +
279H + 280R
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46
8 G91A + E99K + T231R, N233R + 0.13 not 500
Q249R + 270H + 27IT + 272P + 273S + determined
274S + 275G + 276R + 277G + 278G
Table 4
The reference lipase and variants 7 and 8 in Table 4 are described in WO
2000/060063.
Example 6
BR - BeneCt Risk
The Benefit Risk was measured for the variants listed in Table 5. The Benefit
Risk
factor was measured in the same way as described in Example 5 and it was found
to be above 1
for all the listed variants.
Variant Mutations in SEQ ID NO: 2
Reference T231R + N233R
9 L97V+ T231R+N233R
A150G+T231R+N233R
11 I90R+T231R+N233R
12 1202V+T231R+N233R
13 L227G+ T231R+ N233R+ P256K
14 I90A+ T231R+ N233R
T231R+N233R+ 1255P
16 I90V+1255V+T231R+N233R
17 F211L+ L227G+ T231R+ N233R+ I255L+ P256K
18 S58N+ V60S+ T231R+ N233R+ Q249L
19 S58N+ V60S+ T231R+ N233R+ Q249I
A150G+ L227G+ T231R+ N233R+ P256K
21 K46L+ S58N+ V60S+ T231R+ N233R+ Q249L+ D2541
22 Q4L+ E43T+ K46I+ S58N+ V60S+ T231R+ N233R+ Q249L+ D2541
23 Q4L+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
24 K461+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254L
K46L+ S58N+ V60S+ K2231+ T231R+ N233R+ D254I
26 E43T+ K46I+ S58N+ V60S+ T231R+ N233R+ Q249L+ D254I
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27 S58N+ V60S+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
28 K24R+ K46R+ K74R+ 186V+ K98R+ K127R+ D137K+ A150G+ K223R+
T231R+ N233R
29 S58A+V60A+ I86V+T231R+N233R
30 K24R+ K46R+ S58N+ V60S+ K74R+ I86V+ K98R+ K127R+ D137K+ K223R+
T231R+ N233R
31 S58A+ V60A+ I86V--- A150G+ T231R+ N233R
32 S58N+ V60V+ D62G+ T23IR+ N233R
33 Q4V+ S58N+ V60S+ I86V+ T231R+ N233R+ Q249L
34 Q4V+ S58N+ V60S+ I86V+ A150G+ T231 R+ N233R+ 1255V
35 Q4V+ S58N+ V60S+ I90A+ A150G+ T231R+ N233R+ 1255V
36 Y53A+ S58N+ V60S+ T231R+ N233R+ P256L
37 I202L+ T231R+ N233R+ I255A
38 S58A+ V60S+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
39 D27R+ S58N+ V60S+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
40 V60K+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
41 Q4V+ S58A+ V60S+ S83T+ I86V+ A150G+ E210K+ L227G+ T231R+ N233R+
P256K.
42 Q4V+ V60K+ S83T+ 186V+ A150G+ L227G+ T231R+- N233R+ P256K
43 D27R+ V60K+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
44 Q4N+ L6S+ S58N+ V60S+ 186V+ A150G+ L227G+ T231R+ N233R+ P256K
45 E1N+ V60K+ I86V+ A150G+ L227G+ T231R+ N233R+ P256K
46 V60K+ I86V+ A150G+ K223N+ G225S+ T231R+ N233R+ P256K
47 E210V+ T231R+ N233R+ Q249R
48 S58N+ V60S+ E210V+ T231R+ N233R+ Q249R
49 Q4V+ V60K+ I90R+ T231R+ N233R+ I255V
50 Q4V+ V60K+ A150G+ T231R+ N233R
51 V60K+ S83T+ T231R+ N233R
52 V60K+ A150G+ T231R+ N233R+ 1255V
53 T231R+ N233G-s- D234G
54 S58N+ V60S+ I86V+ A150G+ E210K+ L227G+ T231R+ N233R+ Q249R+ -
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P256K
55 S58N+ V60S+ I86V+ A150G+ E210K+ L2270+ T231R+ N233R+ I255A+ P256K
56 S58N+ V60S+ I86V+ A150G+ G156R+ E210K+ L227G+ T231R+ N233R+
I255A+ P256K
57 S58T+ V60K+ I86V+ N94K+ A150G+ E210V+ L227G+ T231R+ N233R+ P256K
58 S58T+ V60K+ I86V+ D102A+ A150G+ L227G+ T23 1R+ N233R+ P256K
59 S58T+ V60K+ I86V+ D102A+ A150G+ E210V+ L227G+ T231R+ N233R+
P256K
60 S58T+ V60K+ S83T+ 186V+ N94K+ A150G+ E210V+ L227G+ T231R+ N233R+
P256K
61 S58A+ V60S+ I86V+ T143S+ A150G+ L227G+ T231R+ N233R+ P256K
62 G91S+ D96V+ D254R
63 V60L+ G91M+ T231W+ Q249L
64 T37A+ D96A+ T231R+ N233R+ Q249G
65 E56G+E87D+T231R+N233R+D254A
66 E210K+T231R+N233R
67 D27H+E87Q+D96N+T231R+N233R-;-D254V
68 F181L+E210V+T231R+N233R
69 D27N+ D96G+ T231R+ N233R
70 D96N+ T231R+ N233R
71 T231 R+ N2331+ D234G
72 S58K+ V60L+ E210V+ Q249R
73 S58H+ V60L+ E210V+ Q249R
74 Q4V+ F55V+ 186V+ T231R+ N233R+ I255V
75 Q4V+ S58T+ V60K+ T199L+ N200A+ E210K+ T231R+ N233R+ 1255A+ P256K
76 Q4V+ D27N+ V60K+ T231R+ N233R
77 I90F+ I202P+ T231R+ N233R+ I255L
78 S58N+ V60S+ D158N+ T231R+ N233R
79 S58N+ V60S+ S115K+ T231R+ N233R
80 S58N+ V60S+ L147M+ A150G+ F211L+ T231R+ N233R
81 V60K+ A150G+ T231R+ N233R
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82 190V+L2270+T231R+N233R+ P256K
83 T231R+N233R+ 1255S
84 186G+ T231R+ N233R
85 V60K+ 1202V+ E210K+ T231R+ N233R+ 1255A+ P256K
86 1900+ 1202L+ T231R+ N233R+ 1255S
87 S58G+ V60G+ T231R+ N233R
Tab[e 5
The reference lipase is described in WO 2000/060063.
DETERGENT EXAMPLES
Abbreviated component identifications for the examples are as follows:
LAS Sodium linear Cl I_13 alkyl benzene sulphonate.
CxyAS S odium C 1 x- C 1 y alkyl sulfate.
CxyEzS Clx - Cly sodium alkyl sulfate condensed with an average of z
moles of ethylene oxide.
CxyEy ClX - Cly alcohol with an average of ethoxylation of z
QAS R2.N+(CH3)2(CZH4OH) with R2 = Clo-Ci2
Silicate Amorphous Sodium Silicate (SiO2:Na2O ratio = 1.6-3.2:1).
Zeolite A Hydrated Sodium Aluminosilicate of formula Na)2(AIO2SiO2)12.
27Hz0 having a primary particle size in the range from 0.1 to 10
micrometers (Weight expressed on an anhydrous basis).
(Na-)SKS-6 Crystalline layered silicate of formula 6-Na2Si2O5.
Citrate Tri-sodium citrate dihydrate.
Citric Anhydrous citric acid.
Carbonate Anhydrous sodium carbonate.
Sulphate Anhydrous sodium sulphate.
MA/AA Random copolymer of 4:1 acrylate/maleate, average molecular
weight about 70,000-80,000.
AA polymer Sodium polyacrylate polymer of average molecular weight 4,500.
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PB 1/ PB4 Anhydrous sodium perborate monohydrate / tetrahydrate.
PC3 Anhydrous sodium percarbonate [ 2.74 Na2CO3.3HZOa ]
TAED Tetraacetyl ethylene diamine.
NOBS Nonanoyloxybenzene sulfonate in the form of the sodium salt.
DTPA Diethylene triamine pentaacetic acid.
HEDP Hydroxyethane di phosphonate
EDDS Na salt of Ethylenediamine-N,N'-disuccinic acid, (S,S) isomer
STPF Sodium tripolyphosphate
Protease Proteolytic enzyme sold under the tradename Savinase
Alcalase , Everlase , Coronase , Polarzyme , by Novozymes
A/S, Properase , Purafect , Purafect MA and Purafect Ox
sold by Genencor and proteases described in patents WO 91/06637
and/or WO 95/10591 and/or EP 0 251 446 such as FNA, FN3
and/or FN4.
Amylase Amylolytic enzyme sold under the tradename Purastar , Purafect
Oxam sold by Genencor; Termamyl , Fungamyl(D Duramyl ,
Stainzyme and NatalaseO sold by Novozymes A/S .
Lipase Any lipase variant 1 to 5 described in example 5 table 2, and
combinations thereof.
Mannanase Mannaway sold by Novozymes
CMC or HEC Carboxymethyl or Hydroxyethyl or ester modified cellulose.
or EMC
SS Agglom. Suds Suppressor agglomerate: 12% Silicone/silica, 18% stearyl
alcohol,70% starch in granular form.
TEPAE Tetreaethylenepentaamine ethoxylate.
pH Measured as a 1% solution in distilled water at 20 C.
Example A
Bleaching detergent compositions having the form of granular laundry
detergents are
exemplified by the following formulations.
B C I) E F
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LAS 20 22 20 15 20 20
QAS 0.7 1 1 0.6 0.0 0.7
C25E3S 0.9 0.0 0.9 0.0 0.0 0.9
C25E7 0.0 0.5 0.0 1 3 1
STPP 23 30 23 17 12 23
Zeolite A 0.0 0.0 0.0 0.0 10 0.0
Silicate 7 7 7 7 7 7
Carbonate 15 14 15 18 15 15
AA Polymer 1 0.0 1 1 1.5 1
CMC 1 1 1 1 1 1
Protease 32.89mg/g 0.1 0.07 0.1 0.1 0.1 0.1
Amylase 8.65mg/g 0.1 0.1 0.1 0.0 0.1 0.1
Lipase 18mg/g 0.03 0.07 0.3 0.1 0.07 0.1
Brightener -Tinopal AMS (Ciba) 0.06 0.0 0.06 0.18 0.06 0.06
Brightener -Tinopal CBS-X (Cib 0.1 0.06 0.1 0.0 0.1 0.1
DTPA 0.6 0.3 0.6 0.25 0.6 0.6
MgSO4 1 1 1 0.5 1 1
PC3 0.0 5.2 0.1 0.0 0.0 0.0
pB 1 4.4 0.0 3.85 2.09 0.78 3.63
NOBS 1.9 0.0 1.66 1.77 0.33 0.75
TAED 0.58 1.2 0.51 0.0 0.015 0.28
Balance Balance to Balance Balance Balance Balance
Sulphate/Moisture to 100% 100% to 100% to 100% to 100% to 100%
Any of the compositions in Example A is used to launder fabrics at a
concentration of 600 -
10000 ppm in water, with typical median conditions of 2500ppm, 250C, and a
25:1 water:cloth
ratio. The typical pH is about 10 but can be can be adjusted by altering the
proportion of acid to
Na- salt form of alkylbenzenesulfonate.
Examgle B
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Bleaching detergent compositions having the form of granular laundry
detergents are
exemplified by. the following formulations.
A B C D
LAS 8 7.1 7 6.5
C25E3S 0 4.8 0 5.2
C68S 1 0 1 0
C25E7 2.2 0 3.2 0
QAS 0.75 0.94 0.98 0.98
(Na-)SKS-6 4.1 0 4.8 0
Zeolite A 20 0 17 0
Citric 3 5 3 4
Carbonate 15 20 14 20
Silicate 0.08 0 0.11 0
Soil release agent 0.75 0.72 0.71 0.72
MA/AA 1.1 3.7 1.0 3.7
CMC 0.15 1.4 0.2 1.4
Protease (56.00mg active/g) 0.37 0.4 0.4 0.4
Termamyl (21.55mg active/g) 0.3 0.3 0.3 0.3
Lipase (18.O0mg active/g) 0.05 0.15 0.1 0.5
Amylase (8.65mg active/g) 0.1 0.14 0.14 0.3
TAED 3.6 4.0 3.6 4.0
PC3 13 13.2 13 13.2
EDDS 0.2 0.2 0.2 0.2
HEDP 0.2 0.2 0.2 0.2
MgSOa 0.42 0.42 0.42 0.42
Perfume 0.5 0.6 0.5 0.6
SS Agglom. 0.05 0.1 0.05 0.1
Soap 0.45 0.45 0.45 0.45
Sulphate 22 33 24 30
Water & Miscellaneous Balance to 100%
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Any of the above compositions in Example B is used to launder fabrics at a
concentration
of 10,000 ppm in water, 20-90 OC, and a 5:1 water:cloth ratio. The typical pH
is about 10 but
can be can be adjusted by altering the proportion of acid to Na-salt form of
alkylbenzenesulfonate.
.
Example C
A B C D E F
(wt%) (wt%) (wt%) (wt%) (wt%) (wt%)
C25E1.8S 11 10 4 6.32 6.0 8.2
LAS 4 5.1 8 3.3 4.0 3.0
Sodium formate 1.6 0.09 1.2 0.04 1.6 1.2
Sodium hydroxide 2.3 3.8 1.7 1.9 2.3 . 1.7
Monoethanolamine 1.4 1.490 1.0 0.7 1.35 1.0
Diethylene glycol 5.5 0.0 4.1 0.0 5.500 4.1
C23E9 0.4 0.6 0.3 0.3 2 0.3
DTPA 0.15 0.15 0.11 0.07 0.15 0.11
Citric Acid 2.5 3.96 1.88 1.98 2.5 1.88
C12_14 dimethyl
Amine Oxide 0.3 0.73 0.23 0.37 0.3 0.225
C12_18 Fatty Acid 0.8 1.9 0.6 0.99 0.8 0.6
Borax 1.43 1.5 1.1 0.75 1.43 1.07
Ethanol 1.54 1.77 1.15 0.89 1.54 1.15
TEPAE' 0.3 0.33 0.23 0.17 0.0 0.0
ethoxylated
hexamethylene
diamine2 0.8 0.81 0.6 0.4 0.0 0.0
1,2-Propanediol 0.0 6.6 0.0 3.3 0.0 0.0
Protease* 36.4 36.4 27.3 18.2 36.4 27.3
Mannanase * 1.1 1.1 0.8 0.6 1.1 0.8
Amylase* 7.3 7.3 5.5 3.7 7.3 5.5
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Lipase* 10 3.2 0.5 3.2 2.4 3.2
Water, perfume,
dyes & others Balance Balance Balance Balance Balance Balance
* Numbers quoted in mg enzyme/ lOOg
~ as described in US 4,597,898.
2 available under the tradename LUTENSIT from BASF and such as those
described in WO
01/05874
All documents cited in the Detailed Description of the Invention are in
relevant part
incorporated herein by reference: the citation of any document is not to be
construed as an
admission that it is prior art with respect to the present invention. 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.
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 ctaims all such changes and
modifications that are
within the scope of this invention.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 54
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