Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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WO 99/20768 PCT/IB98/01615
METHODS FOR PRODUCING AMYLASE ENZYMES
The present invention relates to methods for producing new amylase enzymes
using
random mutation of DNA encoding for an amylase enryme followed by isolation of
amylase
enzyme by evaluating the ability to hydrolyze starches in the presence of
select cleaning
composition ingredients. The present invention also relates to detergent
compositions comprising
a new amylase enzyme produced by the above method and cleaning composition
ingredients.
For a number of years amylase enzymes have been used for a variety of
different purposes,
the most important of which are starch liquefaction, textile desizing, starch
modification in the
paper and pulp industry, and for brewing and baking. A further use of
amylases, which is
becoming increasingly important is the removal of starch-containing soils and
stains during the
washing of fabrics, hard surfaces and dishes.
Indeed, amylase enzymes have long been recognized in dishwashing, hard surface
cleaning and laundry compositions to provide the removal of starchy food
residues or starchy films
from dishware, flatware, glasses and hard surfaces or to provide cleaning
performance on starchy
soils as well as other soils typically encountered in laundry applications.
WO 94/02597, Novo Nordisk A/S published February 3, 1994, describes cleaning
compositions which incorporate mutant amylases. See also WO 94/18314,
Genencor, published
August 18, 1994 and WO 95/10603, Novo Nordisk A/S, published April 20, 1995.
Other amylases known for use in cleaning compositions include both a- and ~i-
amylases.
a-Amylases are known in the art and include those disclosed in U.S. Patent No.
5,003.257; EP
252,666; WO 91/00353; FR 2,676,456; EP 285,123; EP 525,610; EP 368,341; and
British Patent
Specification No. 1,296,839 (Novo).
Examples of commercial a-amylases products are Termanyl~, Ban~, Fungamyl~, and
Duramyl~, all available from Novo Nordisk A/S, Denmark.
CONF1RNIAT10N COPY
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2
Recently, new amylases have been identified and are described in WO 95/26397,
Novo
Nordisk A/S, published October 5, 1995, disclosing an a-amylase having a
specific activity at least
25% higher than the specific activity of Termamyl~ at a temperature range of
25 to SS°C and at a
pH value in the range of 8 to 10.
Thus, specific amylase enzymes have already found utility in laundry and
cleaning
compositions. However, these enzymes still have there limitations when used in
certain cleaning
composition matrixes. Most product applications create a harsh environment for
these enzymes,
and common detergent ingredients such as builders and bleaches are likely to
inactivate these
enzymes. Thus, there continues to be a need to identify and isolate new
amylase enzyme variants
for use in cleaning compositions.
The above-noted genetically engineered amylases are typically the product of
rational
design wherein specific sites within known amylase enzymes are targeted for
selected
modification of the amino acid sequence, either by substituting a different
amino acid for the
existing one and/or by eliminating one amino acid or a portion of the enzyme
chain. Clearly, this
approach can produce improved amylase enzymes, as evidenced by the above-noted
publications
and patents, but progress is slow and screening-intensive. Further, as noted
above, these amylase
enzymes may provide some benefits in cleaning product applications, but their
activity is much
reduced relative to their activity in their natural environment.
Several methods for modifying DNA strands are known, including variant
generation,
mutagenesis, and DNA shuffling. The gene to be improved is mutagenised,
recombined, and
expressed variants of the gene are selected and/or screened to generate
"positive" leads by using an
algorithm that is termed a "directed evolution" approach (as described by
Moore & Arnold,
Biotechnoloev x:458-467; 1996). This algorithm is applicable to any enzyme or
protein to be
changed, provided a suitable screen can be developed, and is described in
detail below. Random
mutagenesis, the concept of using single-mutant libraries, and the technique
of DNA shuffling
(recombination) have been described previously, most recently in WO 97/09446,
published March
13, 1997 by Novo Nordisk (random mutagenesis which uses a "catcher molecule"
that binds
specifically with - and then is used to separate - the enzyme variants that
exhibit only the desired
property).
In terms of variant generation, it is desirable to examine as many different
variants as
possible, to be assured of getting as many positive "hits" as possible.
However, it turns out that the
number of amino acid mutations introduced per enzyme is limited by the
screenable number of
mutants; i.e. screening is limited by the numbers of possible variants. For
example, for an average
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3
size enzyme of 300 amino acids, the number of possible sequences containing a
given number of
amino acid changes increases exponentially with the increasing number of
changes. For example,
the number of possible variants containing all possible single amino acid
changes is 5.7 x 103, for
all possible double changes is ~1.6 x 107, and for all possible triple changes
is ~3.1 x 1010.
Clearly, one is limited to examining single changes (or possibly double
changes if a robotics
system is used) if one is to examine all possible changes and be assured the
greatest probability of
success. This approach of screening libraries (populations) of single-mutant
variants has been
proposed previously by Moore & Arnold (Nature Biotechnology 14:458-467; 1996)
and Arnold
CChem. Eng. Sci. in press; 1996).
Thus, the science of genetic engineering has designed a variety of general
methods for
modifying DNA coding. However, for purpose of providing amylase enzymes of use
in
cleaning composition, it is necessary that the method used produce a
manageable number of
variants and that an efficient and effective screening method (which correctly
limits the still vast
array of amylase variants produced) be used to produce a reasonable number of
amylase
enzymes capable of more defined product use and testing.
It is therefore an object to provide a method for producing amylase enzymes
useful for
cleaning compositions produced by a random DNA mutation using a method
involving
mutagenesis combined with a selected screening process involving measuring for
increased starch
hydrolytic activity in the presence of cleaning composition components. The
present invention
also relates to detergent compositions comprising novel amylases having
increased hydrolytic
activity and detergent composition ingredients. These and other objects of the
present invention
will become apparent from the detailed description hereinafter.
A process for generating random mutatiori3 using "error prone" polymerase
chain reaction
("error-prone PCR") is described in Leung et al., Technique 1:11-15; 1989, and
Cadwell & Joyce,
PCR Methods Applic. 2:28-33; 1992. See also: WO 95/22625, published August 24,
1995 by
Affymax Technologies N.V.
The "directed evolution" approach to gene mutation is described in Moore &
Arnold,
Nature Biotechnoloev ]4:458-467; 1996. Screening libraries (populations) of
single-mutant
variants has been described in Moore & Arnold, Nature Biotechnology 14:458-
467; 1996, and
Arnold, Chem. Eng. Sci. in press; 1996.
WO 94/02597, Novo Nordisk A/S published February 3, 1994, describes cleaning
compositions which incorporate mutant amylases. See also WO 94/18314,
Genencor, published
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August 18, 1994 and WO 95/10603, Novo Nordisk A/S, published April 20, 1995.
Other amylases
known for use in cleaning compositions include those disclosed in U.S. Patent
No. 5,003,257; EP
252,666; WO 91/00353; FR 2,676,456; EP 285,123; EP 525,610; EP 368,341; and
British Patent
Specification No. 1,296,839 (Novo). Recently, new amylases have been
identified and are
described in WO 95/26397, Novo Nordisk A/S, published October 5, 1995,
disclosing an a-
amylase having a specific activity at least 25% higher than the specific
activity of Termamyl~ at a
temperature range of 25 to 55°C and at a pH value in the range of 8 to
10. WO 97/09446, Novo
Nordisk A/S published March 13, 1997, describes using random mutagenesis and a
"catcher
molecule" for identifying enzymes suitable for use in detergents.
SUMMARY OF THE INV .NT1C)m
The present invention relates to a method for producing DNA for new amylase
enzymes
useful for cleaning applications. This method comprises:
(a) randomly mutating, by chemical and/or enzymatic mean, single or double
site
mutations in one or more DNA encoding for an amylase enzyme;
(b) incorporating the mutated DNA from step (a) into a microorganism which is
capable of
expressing amylase enzyme;
(c) separating into individual isolates organisms from step (b) such that a
minimum
number (preferably on average about one) of the organisms containing mutated
DNA is present in
each isolate;
(d) producing amylase enzyme from one or more of the isolates;
(e) collecting the mutated DNA from one or more isolates that produce amylase
which
hydrolyzes starch at a rate of at least about 25% faster than the parent
amylase in the presence of
cleaning composition ingredients comprising at least surfactants, builders
and/or bleaches,
preferably at a level of hydrolytic activity of at Itast about 25% faster than
that of the amylase
enzyme having SEQ. ID. 1;
(f) optionally, DNA shuffling during a separate step or concurrently with any
of steps (a)
to (e); and
(g) optionally repeating, one or more times, steps (a) to (f) and then
collecting the mutated
DNA.
The present invention also relates to a method for producing organisms which
produce
amylase enzymes useful for cleaning applications. This method comprises:
(a) randomly mutating, by chemical and/or enzymatic mean, single or double
site
mutations in one or more DNA encoding for an amylase enzyme;
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(b) incorporating the mutated DNA from step (a) into a microorganism which is
capable of
expressing amylase enzyme;
(c) separating into individual isolates organisms from step (b) such that a
minimum
number (preferably on average about one) of the organisms containing mutated
DNA is present in
each isolate;
(d) producing amylase enzyme from one or more of the isolates;
(e) collecting the organisms containing mutated DNA from one or more isolates
that
produce amylase which hydrolyzes starch at a rate of at least about 25% faster
than the parent
amylase in the presence of cleaning composition ingredients comprising at
least surfactants,
builders and/or bleaches, preferably at a level of hydrolytic activity of at
least about 25% faster
than that of the amylase enzyme having SEQ.1D. 1;
(f) optionally, DNA shuffling during a separate step or concurrently with any
of steps (a)
to (e); and
(g) optionally repeating, one or more times, steps (a) to (f) and then
collecting the
organisms containing mutated DNA that produce the amylase.
The present invention also relates to detergent compositions comprising:
(a) from about 0.0001 % to about 2% by weight of an amylase enzyme, having a
level of
hydrolytic activity of at least about 25% faster than that of the amylase
enzyme having SEQ. ID. 1,
produced by an organism containing mutated DNA prepared by a method according
to the present
invention; and
(b) from about 98% to about 99.9999% of cleaning composition ingredients
comprising
one or more of surfactants, builders, and bleaching agents.
All parts, percentages and ratios used herein are expressed as percent weight
unless
otherwise specified. All documents cited are, in retevant part, incorporated
herein by reference.
The present invention method comprises the following steps, which are
described in detail
hereinafter.
(A) Random Mutation Sten:
The process of generating random mutations in genes for the present invention
methods is
accomplished by any method which randomly produces only one or two changes (on
average) per
DNA strand. A preferred process of generating such random mutations is using
"error prone"
polymerase chain reaction ("error-prone PCR") as described in detail by Leung
et al. (Technique
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1:11-15; 1989) and Cadwell & Joyce (PCR Methods Applic. 2:28-33; 1992). The
gene or gene
fragment of interest is excised from a plasmid vector that has been engineered
to contain the
appropriate restriction enzyme site. The fragment is mutagenized by error-
prone PCR, and the
population of mutant DNA molecules restricted with the appropriate enzymes and
ligated back
into the original plasmid vector to generate a mutant "library", for example
as shown in the Leung
et al. article
The overall error rate of the "error-prone" PCR process must be adjusted so
that the
average number of mutations in the population at the amino acid (protein)
level is one. Because of
the redundancy of the genetic code, this translates to an average of
approximately two mutations at
the DNA level in the variant gene library. The error rate may be adjusted, for
example, by altering
the manganese concentration in the PCR reaction (see, for example, the
reaction conditions
described in the Cadwell & Joyce article noted above). This must be done for
each gene to be
mutated at the beginning of the process. Additional details regarding the use
of enror-prone PCR
are found in WO 95/22625, published August 24, 1995 by Affymax Technologies
N.V.,
incorporated by reference herein in its entirety.
The DNA selected for this random mutation step may be any DNA which encodes
for an a
-amylase protein. However, it is preferred herein to use the DNA which encodes
for known a-
amylases which have been evaluated for use in cleaning compositions. Such
amylase enzymes
include those described in WO 95/26397.
Specific DNA encoding for amylase enzymes useful in detergent compositions
therefore
include:
(a) a-amylases characterized by having a specific activity at least 25% higher
than the specific
activity of Termamyl~ at a temperature range of 25°C to 55°C and
at a pH value in the range of 8
to 10, measured by the Phadebas~ a-amylase'activity assay, as described in
detail in WO
95/26397.
(b) a-amylases according to (a) comprising the amino sequence shown in SEQ ID
No. 1 or an a-
amylase being at least 80% homologous with the amino acid sequence shown in
SEQ ID No. 1.
A peptide is considered to be X% homologous to the parent amylase if a
comparison of the
respective amino acid sequences, performed via algorithms, such as the one
described by Lipman
and Pearson in Science 227, 1985, p. 1435, reveals an identity of X%.
(c) a-amylases according to (a or b) wherein the a-amylase is obtainable from
an alkalophilic
Bacillus species; and in particular, from any of the strains NCIB 12289, NCIB
12512, NCIB 12513
and DSM 935.
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In the context of the present invention, the term "obtainable from" is
intended not only to
indicate an amylase produced by a Bacillus strain but also an amylase encoded
by a DNA
sequence isolated from such a Bacillus strain and produced in a host organism
transformed with
said DNA sequence.
(d) Amylases showing positive immunological cross-reactivity with antibodies
raised against an a-
amylase having an amino acid sequence corresponding to SEQ ID No. l, or
Termamyl.
(e) Variants of the following parent a-amylases which (i) have the amino acid
sequence shown in
SEQ ID No. l, or (ii) displays at least 60 %, preferably 80%, homology with
this amino acid
sequence, and/or displays immunologica) cross-reactivity with an antibody
raised against an a-
amylase having this amino acid sequence, and/or is encoded by a DNA sequence
which hybridizes
with the same probe as a DNA sequence encoding an a-amylase having this amino
acid sequence;
in which variants:
1. at least one amino acid residue of said parent a-amylase has been deleted;
and/or
2. at least one amino acid residue of said parent a-amylase has been replaced
by a different amino
acid residue; and/or
3. at least one amino acid residue has been inserted relative to said parent a-
amylase;
said variant having an a-amylase activity and exhibiting at least one of the
following properties
relative to said parent a-amylase: increased thermostability, increased
stability towards oxidation,
reduced Ca ion dependency, increased stability and/or a-amylolytic activity at
neutral to relatively
high pH values, increased a-amylolytic activity at relatively high temperature
and increase or
decrease of the isoeiectric point (pI) so as to better match the pI value for
a-amylase variant to the
pH of the medium.
(B) Incorooration of Mutated DNA into Microorganism apable of Ex~ Icing
Am~la~g
Before transformation into organisms ca~a,ble of expressing amylase (preferred
being a
Bacillus species), it is highly preferred that the population of mutated DNA
molecules (as
described herein before, averaging about 1 amino acid change in the final
protein product), cloned
in an appropriate plasmid DNA vector, be introduced into a standard,
commercially available E.
coli strain (such as XL-2 Blue from Stratagene), using a standard DNA
transformation method,
followed by isolation of resulting transformants on agar plates (all standard
methods). The
transformation method is such that each isolated colony that grows on the agar
plate has resulted
from a single, transformed cell containing a single, mutated amylase gene DNA
sequence. These
colonies are pooled together and the plasmid DNAs they contain are isolated,
to give a population
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8
of plasmid DNA molecules representative of the entire orginal (from the random
mutation step
hereinbefore) population of mutated DNA molecules.
The purpose of this preferred step is to amplify the amount of the population
of the
original, mutated DNA molecules, since transformation directly into Bacillus
species is generally
difficult with small amounts of DNA (thus it is preferred to use this
intermediate E. coli step as an
amplification step; no amylase is produced at this stage since E. coli is not
capable of producing
it). Thus, preferably following this amplification process step, the mutated
DNA molecules from
the random mutation step are introduced into an organism capable of producing
amylase enzymes
(preferably for the present process a Bacillus species) using a standard DNA
transformation
method.
The cells useful in this invention may be a cell of a higher organism such as
a mammal or
an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal
(including yeast) cell.
Examples of suitable bacteria are gram positive bacteria such as Bacillus
subtilis, Bacillus
licheniformis, Bacillus lentos, Bacillus brevis, Bacillus stearotherm hiiu .
- Q~-..~ ~1111~~
Bacillus amvloliguefaciens, Bacillus coaaulans, Bacillus circulans, Bacillus
lautus. Bacillus
megaterium. Bacillus thurinQiensis. Streptom,~ lividans or Strentom, ces
murinus, or gram
negative bacteria such as E.E. coli.
The transformation of the bacteria may, for instance, be effected by
protoplast
transformation or by using competent cells in a manner known per se.
The yeast organism may favorably be selected from a species of Saccharomyces
or
Schizosaccharomyces, e.g., Saccharom,~ cerevisiae. The filamentous fixngus may
advantageously belong to a species of As e~rg,j]j~, e.g., Asizrgillus _op~zae
or ~ergillus niQer.
Fungal cells may be transformed by a process involving protoplast formation
and transformation
of the protoplasts followed by regeneration of the~ell wall in a manner known
per se. A suitable
procedure for transformation of jll~ host cells is described in EP 238,023.
Preferred a-amylases of the invention are obtainable from an alkaliphilic
Bacillus species,
particularly from one of the Bacillus strains NCIB 12289, NCIB 12512, NCIB
12513 and DSM
9375. In the context of the present invention, the term "obtainable from" is
intended not only to
indicate an a-amylase produced by a Bacillus strain but also an a-amylase
encoded by a DNA
sequence isolated from such a Bacillus strain, modified by the present
invention process, and
produced in a host organism transformed with this mutated DNA sequence.
The strain NCIB 12289 is described in detail in EP 277,216. The strain NCIB
12289 has
been deposited according to the Budapest Treaty on the International
Recognition of the Deposits
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of Microorganisms for the Purpose of Patent Procedures, on July 8, 1996 at The
National
Collection of Industrial Bacteria (NCIB) under accession No. NCIB 12289.
The strain NCIB 12512 is described in detail in EP 277,216. The strain NCIB
12512 has
been deposited according to the Budapest Treaty on the International
Recognition of the Deposits
of Microorganisms for the Purpose of Patent Procedures, on August S, 1987 at
The National
Collection of Industrial Bacteria (NCI) under accession No. NCIB 12512.
The strain NCIB 12513 is described in detail in EP 277,216. The strain NCIB
12513 has
been deposited according to the Budapest Treaty on the International
Recognition of the Deposits
of Microorganisms for the Purpose of Patent Procedures, on August 5, 1987 at
The National
Collection of Industrial Bacteria (NCIB) under accession No. NCIB 12513.
The strain DSM 9375 has been deposited according to the Budapest Treaty on the
International Recognition of the Deposits of Microorganisms for the Purpose of
Patent Procedures,
on August 16, 1994 at Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH (DSM)
under Accession No. DSM 9375.
According to the invention, an a-amylase-encoding DNA sequence produced by
methods
described above, can be expressed, in enzyme form, using an expression vector
which typically
includes control sequences encoding a promoter, operator, ribosome binding
site, translation
initiation signal, and, optionally, a repressor gene or various activator
genes.
The recombinant expression vector carrying the DNA sequence encoding an a-
amylase
produced by the present invention method may be any vector which may
conveniently be
subjected to recombinant DNA procedures, and the choice of vector will often
depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector,
i.e. a vector which exists as an extra chromosomal entity, the replication of
which is independent
of chormosomal replication, e.g., a plasmid, a bacteriophage or an extra
chromosomal element,
minichromosome or an artificial chromosome. Alternatively, the vector may be
one which, when
introduced into a host cell, is integrated into the host cell genome and
replicated together with the
chromosomes) into which it has been integrated.
In the vector, the DNA sequence should be operably connected to a suitable
promoter
sequence. The promoter may be any DNA sequence which shows transcriptional
activity in the
host cell of choice and may be derived from genes encoding proteins either
homologous or
heterologous to the host cell. Examples of suitable promoters for directing
the transcription of the
DNA sequence encoding an a-amylase, especially in a bacterial host, are the
promoter of the ]a~
operon of E.E. coli, the Strentomvces coelicolor agarase gene ~A promoters,
the promoters of the
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Bacillus licheniformis a-amylase gene (), the promoters of the Bacillus
stearothermo hn ilus
maltogenic amylase gene (), the promoters of the Bacillus Am ly olinuefaciens
a-amylase
(amy0), the promoters of the Bacillus subtilis xylA and xylB genes, etc. For
transcription in a
fungal host, examples of useful promoters are those derived from the gene
encoding ~
TAKA amylase, Bhizomur,~r miehei aspartic proteinase, A.A. niQer neutral a-
amylase, A.A. nieer acid
stable a-amylase, ~~ glucoamylase, Rhizomucor miehei lipase, alkaline
protease,
A.~triose phosphate isomerase or A. nidulans acetamidase.
The expression vector may also comprise a suitable transcription terminator
and, in
eukaryotes, polyadenylation sequences operably connected to the DNA sequence
encoding the
desired a-amylase. Termination and polyadenylation sequences may suitably be
derived from the
same sources as the promoter.
The vector may further comprise a DNA sequence enabling the vector to
replicate in the
host cell in question. Examples of such sequences are the origins of
replication of plasmids
pUC 19, pACYC 177, pUB 110, pE 194, pAMB 1 and pIJ702.
The vector may also comprise a selectable marker, e.g., a gene the product of
which
complements a defect in the host cell such as the ,~] genes from $~ubtilis or
B. iicheniformis, or
one which confers antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or
tetracyclin resistance. Furthermore, the vector may comprise ,[],~ selection
markers such
as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or
the selection may
be accomplished by co-transformation, e.g., as described in WO 91/17243.
While intracellular expression may be advantageous in some respects, e.g.,
when using
certain bacteria as host cells, it is generally preferred that the expression
is extracellular.
Procedures suitable for constructing vectors useful for the present invention
process
encoding the desired a-amylase and containing'-the promoter, terminator, and
other elements,
respectively, are well known to persons skilled in the art (cf., for instance,
Sambrook et al. in
Molecular Cloning: A Laboratory Mad, 2nd Ed., Cold Spring Harbor, 1989).
(C) ~_epa_ration of Microorganisms into Isolates nd Am3~lase Production Steo~
The resulting transformants from the proceeding steps, containing the large
amount of
DNA produced by the random mutation step herein above, and which has been
transformed (using
standard methods, for example those described in "Biology of Bacilli:
Applications to Industry",
1992, R.H. Doi and M. McGloughlin, Editors, Butterworth-Heinemann, Stoneham,
MA., pp. 357-
359) into the appropriate species (e.g., preferably Bacillus species), are
isolated on agar plates
using standard methods. At this stage, for example, the individual Bacillus
colonies that have
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derived from single, transformed cells containing single, mutated amylase gene
DNA sequences,
are able to express the amylase gene (i.e. make variant amylase proteins).
Individual colonies (10,000) are picked from the agar plates and inoculated
into
individual wells of plastic, 96-well microtiter plates that each contain a
growth medium that results
in amylase production {e.g. 100-200 microliters volume). The medium used to
cultivate the cells
may be any conventional medium suitable for growing the host cell in question
and obtaining
expression of the a-amylase of the invention. Suitable media are available
from commercial
suppliers or may be prepared according to published recipes (e.g., as
described in catalogues of the
American Type Culture Collection).
These plates are incubated for 16 hours at the optimal growth temperature of
the Bacillus
species being used, during which time the inoculated colonies grow and (if
able to) secrete
amylase into the growth medium. The cells are then removed (centrifugation of
the plates), and a
portion of the supernatants, each potentially containing a different amylase
variant, are assayed as
described herein after. The a-amylase secreted from the host cells may
conveniently be recovered
from the culture medium by well-known procedures, including separating the
cells from the
medium by centrifugation or filtration, and precipitating proteinaceous
components of the medium
by means of a salt such as ammonium sulphate, followed by the use of
chromatographic
procedures such as ion exchange chromatography, affinity chromatography, or
the like.
(D) Amylase Screening
The present invention process produces an a-amylase having an activity at
least 25%
higher (preferably at least 35% higher, more preferably at least 50% higher,
and most preferably at
least 75% higher) than the activity of the parent amylase from which the DNA
was obtained and
mutated by the random mutation step of this process (preferably at a
temperature in the range of
25°C to 55°C). Further preferred are amylases pibduced by the
present methods which not only
have a specific activity at least 25% greater than the parent amylase from
which the DNA was
obtained from the random mutation step, but further this amylase has a
specific activity at least
25% greater than the commercially available amylase Teramamyl~. These
increased activities are
as measured in the presence of cleaning composition ingredients selected
preferably from
surfactants, builders and/or bleaching agents, as described in detail
hereinafter.
a-Amylase activity for comparison versus the parent amylase is preferably
determined by
a method employing Phadebas~ tablets as substrate. Phadebas tablets (Phadebas~
Amylase Test,
supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-
colored starch polymer
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12
which has been mixed with bovine serum albumin and a buffer substance and
tableted. One
Phadebas tablet is added to 6 ml of detergent solution and vortexed for 15
seconds.
180 pl of the Phadebas suspension containing cleaning composition ingredients,
comprising at least surfactant and/or builder and/or bleaching agents as
described in detail herein
after, is added to each well in a 96-well plate. [It is preferred that the
suspension contain a fully
formulated detergent composition, comprising surfactant and builder, and also
preferably one or
more ingredients selected from bleaching agents, detergent polymers, and/or
chelants, as described
in greater detail hereinafter.] To each well, 20 pl of enzyme which is the
supernatant from the cell
growth described herein before is added and incubated for 15 min. 1 ml of NaOH
is added after 15
min of incubation and vortexed for 1 S sec. The reaction solution is then
filtered. The starch,
which is hydrolyzed by the alpha-amylase, gives soluble blue fragments and the
absorbance of the
resulting blue solution is measured spectrophotometrically at 620 nm. It is
important that the
measured absorbance is at 620 nm range in the 0.2-2.0 absorbance units to
maintain the linearity
between activity and absorbance. The reaction solutions must be diluted to
have absorbance in
this region. Higher absorbance readings is indicative of higher amylolytic
activity under the
specified conditions of the reaction.
Alternatively, kinetic assay can be performed instead of taking measurements
at a given
time by measuring absorbances at several time intervals. The above protocol
must be followed for
each time interval. The slope of the curve of the new libraries vs. the slope
of the parent amylase
is compared to assess increased amylolytic activity.
A specific procedure to screen the improved amylase in a Loprodyne "Silent
Monitor" 96-
well assay plates (Pall Biosupport) for development in granular detergents is
described as follows.
A wash solution is prepared in 6 gpg hardness (3:1 CaCl2:MgCl2) with 0.07 ppm
of an amylase
with Sequence No. 1 and 1 g/L of the following detergent composition.
ppm in wash solution % in composition
I. Bleach
1. Nonanoyloxybenzene 16 2.4
sulfonate
2. Perborate 20 3.1
II. Builders
1. Na Carbonate 153 23
2. Alumino silicate 287 44
3. Sodium silicate 6 0.9
III. Surfactants:
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13
1. Linear C 12 alkyl benzene sulfonate 74 11
2. C 12-C 14 sodium alkyl sulfate 13 2.0
condense with an average of 6
ethylene oxides per mol
3. NaCl4-15 alkyl sulphate 90 13.6
TOTAL 659 100
The pH of above solution is adjusted to approximately 10.5. 5 mls of this
solution is
added to 20 mls of de-ionized water. The Phadebas solution is prepared by
adding 1 Phadebas
tablet in 5 mls of de-ionized water. Phadebas tablets are from Pharmacia and
Upjohn
(manufactured in Sweden, distributed by Pharmacia Diagnostics Div. of Electro-
Nucleonics, Inc.
Fairfield, New Jersey). After mixing the Phadebas solution, 143 p,l is added
into each well of the
96 well plate. To each well, containing the Phadebas solution, 29 pl of
diluted wash solution is
added. The reaction is progressed for 20 minutes and stopped via addition of
29 pl 1M NaOH per
well. The contents of the loprodyne plate is filtered using a Pall vacuum
manifold into a clean 96-
well Dynatech plate. The concentration of liberated dye in the filtrate is
read at 620 nm.
An amylase of this invention delivers at least 25% increased Phadebas
hydrolysis versus
the amylase having SEQ. ID No. 1 dosed at 0.07 ppm protein at 25°C in
the presence of the
cleaning composition ingredients.
(E) Mutated DNA Collection:
The DNA sequence encoding an a-amylase produced by the present invention
process
may be isolated from any cell or microorganism producing the a-amylase in
question, using
various methods well known in the art. Preferably, cells from wells on the 96-
well plates that
generated supernatants having a positive result (bj~-demonstrating improved
amylolytic activity as
described hereinbefore) are then isolated and, preferably re-screened (i.e.,
inoculation into 96-well
plates; growth; re-assay) to confirm that the variant amylases they are
producing are indeed
improved. These cells from individual wells on the 96-well plated are then
grown up (separately)
in a larger scale, and the plasmid DNA isolated from them using standard
methods. These DNAs
(each DNA preparation represents a single amylase variant) can then be used in
subsequent rounds
of mutagenesis and screening (preferably, only one or two of the DNAs
representing the best
results are used as the starting points for the next cycle of mutagenesis).
Alternatively, these positive DNAs (usually only 4 or 5) can be recombined by
DNA
shuffling (as described herein after) to make all possible combinations of the
positive mutations.
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14
After DNA shuffling, the resultant combinations are introduced into E.coli,
then Bacillus, and the
resultant cells screened to potentially identify "better positives", in the
same way as described
hereinbefore. The best positive variant from the DNA shuffling step can then
either be taken as
the end result, or itself used as the starting point for the next cycle of
mutagenesis (with or without
shuffling).
(F) ~tional DNA Shuffling:
DNA shuffling is a method for recombining unique, but homologeous DNA
sequences,
whereby all possible combinations of each unique sequence can be generated. In
essence, the
sequences of interest (e.g., gene fragments, as described above) are
enzymatically fragmented, and
the fragments used in an assembly PCR reaction (where single-stranded pieces
of different variants
can prime each other) to reassemble combinations of the individual mutations.
In effect what
results is a massively parallel reaction, where ~]] possible combinations of
the individual positive
mutations are put together, and it is this pool of recombinants that, when
cloned into the
appropriate plasmid vector (same one as described above) constitutes the
"shuffled library". The
method has been described in detail in WO 95/22625. While DNA shuffling is
optional, it is a
highly preferred optional part of the present invention process, since it
allows all possible
combinations of positive variants isolated from screening of mutagenic
libraries to be constructed
rapidly, and tested.
The above steps may be repeated one or more times to produce further mutated
DNA
and/or organisms as desired which produce amylase enzyme.
leaning omposition Ingredients and Deterg n ompositions
The detergent compositions of the invention contain cleaning composition
ingedients as
described hereinafter. The precise nature of these 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.
The detergent compositions according to the invention can be liquid, paste,
gels, bars,
tablets, powder or granular forms. Granular compositions can also be in
"compact" form, the
liquid compositions can also be in a "concentrated" form.
The compositions of the invention may for example, be formulated as hand and
machine laundry detergent compositions including laundry additive compositions
and
compositions suitable for use in the soaking and/or pretreatment of stained
fabrics, rinse added
fabric softener compositions. Pre-or post treatment of fabric include gel,
spray and liquid fabric
conditioning compositions.
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WO 99/20768 PCT/IB98/01615
IS
When formulated as compositions suitable for use in a laundry machine washing
method, the compositions of the invention preferably contain both a surfactant
and a builder
compound and additionally one or more detergent components preferably selected
from organic
polymeric compounds, bleaching agents, additional enzymes, suds suppressors,
dispersants,
lime-soap dispersants, soil suspension and anti-redeposition agents and
corrosion inhibitors.
Laundry compositions can also contain softening agents, as additional
detergent components.
The compositions of the invention can also be used as detergent additive
products. Such
additive products are intended to supplement or boost the performance of
conventional detergent
compositions.
If needed the density of the laundry detergent compositions herein ranges from
400 to
1200 g/litre, preferably 600 to 950 g/litre of composition measured at
20°C.
The "compact" form of the compositions herein is best reflected by density
and, in terms
of composition, by the amount of inorganic filler salt; inorganic filler salts
are conventional
ingredients of detergent compositions in powder form; in conventional
detergent compositions,
the filler salts are present in substantial amounts, typically 17-35% by
weight of the total
composition.
In the compact compositions, the filler salt is present in amounts not
exceeding 15% of
the total composition, preferably not exceeding 10%, most preferably not
exceeding 5% by
weight of the composition.
The inorganic filler salts, such as meant in the present compositions are
selected from
the alkali and alkaline-earth-metal salts of sulphates and chlorides.
A preferred filler salt is sodium sulphate.
Liquid detergent compositions according to the present invention can also be
in a
"concentrated form", in such case, the liquid detergent compositions according
the present
invention will contain a lower amount of water, compared to conventional
liquid detergents.
Typically the water content of the concentrated liquid detergent is preferably
less than
40%, more preferably less than 30%, most preferably less than 20% by weight of
the detergent
composition.
Preferably, the detergent compositions according to the present invention
comprise a
surfactant or surfactant system wherein the surfactant can be selected from
nonionic and/or
anionic and/or cationic surfactants and/or ampholytic and/or zwitterionic
and/or semi-polar
nonionic surfactants.
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WO 99/20768 PCT/IB98/01615
16
The surfactant is typically present at a level of from about 0.1%, more
preferably about
1% by weight of the deteregent compositions to about 60%, more preferably
about 35%, most
preferably 30% about by weight of the detergent compositions.
The surfactant is preferably formulated to be compatible with enzyme
components
present in the composition. In liquid or gel compositions the surfactant is
most preferably
formulated such that it promotes, or at least does not degrade, the stability
of any enzyme in
these compositions.
Examples of suitable nonionic, anionic, cationic, ampholytic, zwitterionic and
semi-
polar nonionic surfactants are disclosed in U.S. Patent Nos. 5,707,950 and
5,576,282.
Highly preferred nonionic surfactants are polyhydroxy fatty acid amide
surfactants of
the formula:
R2-C(O)-N(Rl)-Z,
wherein R1 is H, or RI is C1_4 hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl
or a mixture
thereof, R2 is CS-31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a
linear
hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain,
or an alkoxylated
derivative thereof. Preferably, R1 is methyl, R2 is a straight C11-15 alkyl or
C16-18 alkyl or
alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from
a reducing sugar
such as glucose, fructose, maltose, lactose, in a reductive amination
reaction.
Highly preferred anionic surfactants include alkyl alkoxylated sulfate
surfactants hereof
are water soluble salts or acids of the formula RO(A)mS03M wherein R is an
unsubstituted
C I0-C24 alkyl or hydroxyalkyl group having a C 10-C24 alkyl component,
preferably a C 12-C20
alkyl or hydroxyalkyl, more preferably C 12-C 1 g alkyl or hydroxyalkyl, A is
an ethoxy or
propoxy unit, m is greater than zero, typically between about 0.5 and about 6,
more preferably
between about 0.5 and about 3, and M is H or a canon which can be, for
example, a metal cation
(e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or
substituted-
ammonium canon. Alkyl ethoxylated sulfates as well as alkyl propoxylated
sulfates are
contemplated herein.
. When included therein, the laundry detergent compositions of the present
invention
typically comprise from about 1%, more preferably about 3% by weight of such
such anionic
surfactants to about 40%, more preferably about 20% by weight of such anionic
surfactants.
Highly preferred cationic surfactants are the water-soluble quaternary
ammonium
compounds useful in the present composition having the formula
R1R2R3R4N+X-
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WO 99/20768 PCT/IB98/01615
17
wherein R1 is Cg-C16 alkyl, each of R2, R3 and R4 is independently Cl-C4
alkyl, C1-C4
hydroxy alkyl, benryl, and -(C2H40)xH where x has a value from 2 to 5, and X
is an anion. Not
more than one of R2, R3 or R4 should be benzyl.
When included therein, the detergent compositions of the present invention
typically
comprise from about 0.2%, more preferably about 1 % by weight of such cationic
surfactants to
about 25%, more preferably about 8% by weight of such cationic surfactants.
When included therein, the detergent compositions of the present invention
typically
comprise from about 0.2%, more preferably about 1 % by weight of such
ampholytic surfactants
to about 15%, more preferably about 10% by weight of such ampholytic
surfactants.
When included therein, the detergent compositions of the present invention
typically
comprise from about 0.2%, more preferably about 1 % by weight of such
zwitterionic surfactants
to about 15%, more preferably about 10% by weight of such zwitterionic
surfactants.
When included therein, the detergent compositions of the present invention
typically
comprise from about 0.2%, more preferably 1 % by weight of such semi-polar
nonionic
surfactants to about 15%, more preferably about 10% by weight of such semi-
polar nonionic
surfactants.
The detergent compositions of the present invention can also comprise from
about
0.001 % to about 100% of one or more (preferably a mixture of two or more) mid-
chain branched
surfactants, preferably mid-chain branched alkyl alkoxy alcohols having the
formula:
R R' R2
I I I
CH3CH2(CH~"CH(CH2),~CH(CH~yCH(CH~~(EO/PO)mOH
mid-chain branched alkyl sulfates having the formula:
R R' R2
I I I
CH3CH~(CH~CH(CH~,~CHf CH2~,CH(CH2~OS03M
and mid-chain branched alkyl alkoxy sulfates having the formula:
R R' R2
I
CH3CH~(CHI,rCH(CHI,~CH(CH2~,CH(CH~~EO/PO~"OSO~IvI
wherein the total number of carbon atoms in the branched primary alkyl moiety
of these
formulae (including the R, Rl, and R2 branching, but not including the carbon
atoms which
comprise any EO/PO alkoxy moiety) is from 14 to 20, and wherein further for
this surfactant
mixture the average total number of carbon atoms in the branched primary alkyl
moieties having
the above formula is within the range of greater than 14.5 to about 17.5
(preferably from about
15 to about 17); R, R1, and R2 are each independently selected from hydrogen,
C1-C3 alkyl, and
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WO 99/20768 PCT/IB98/01615
18
mixtures thereof, preferably methyl; provided R, R1, and R2 are not all
hydrogen and, when z is
1, at least R or R1 is not hydrogen. M is a water soluble cation and may
comprises more than
one type of cation, for example, a mixture of sodium and potassium. The index
w is an integer
from 0 to 13; x is an integer from 0 to 13; y is an integer from 0 to 13; z is
an integer of at least
1; provided w + x + y + z is from 8 to 14. EO and PO represent ethyleneoxy
units and
propyleneoxy units having the formula:
CH3 CH3
-CHCH20- or -CH2CH0
respectively, however, other alkoxy units inter alia 1,3-propyleneoxy, butoxy,
and mixtures
thereof are suitable as alkoxy units appended to the mid-chain branched alkyl
moieties.
The mid-chain branched surfactants are preferably mixtures which comprise a
surfactant
system. Therefore, when the surfactant system comprises an alkoxylated
surfactant, the index m
indicates the average degree of alkoxylation within the mixture of
surfactants. As such, the
index m is at least about 0.01, preferably within the range of from about 0.1,
more preferably
from about 0.5, most preferably from about 1 to about 30, preferably to about
10, more
preferably to about 5. When considering a mid-chain branched surfactant system
which
comprises only alkoxylated surfactants, the value of the index m represents a
distribution of the
average degree of alkoxylation corresponding to m, or it may be a single
specific chain with
alkoxylation (e.g., ethoxylation and/or propoxylation) of exactly the number
of units
corresponding to m.
The preferred mid-chain branched surfactants of the present invention which
are suitable
for use in the surfactant systems of the present invention have the formula:
CH3
CHI(CH~eCH(CH2~CH~(EO/PO~"OSO~IvI
or the formula:
CH3 CH3
CH3(CH~dCH(CH~CHCH2(EO/PO~"OSO~ivI
wherein a, b, d, and a are integers such that a + b is from 10 to 16 and d + a
is from 8 to 14; M is
selected from sodium, potassium, magnesium, ammonium and substituted ammonium,
and
mixtures thereof.
The surfactant systems of the present invention which comprise mid-chain
branched
surfactants are preferably formulated in two embodiments. A first preferred
embodiment
comprises mid-chain branched surfactants which are formed from afeedstock
which comprises
25% or less of mid-chain branched alkyl units. Therefore, prior to admixture
with any other
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WO 99/20768 PCT/IB98/01615
19
conventional surfactants, the mid-chain branched surfactant component will
comprise 25% or
less of surfactant molecules which are non-linear surfactants.
A second preferred embodiment comprises mid-chain branched surfactants which
are
formed from a feedstock which comprises from about 25% to about 70% of mid-
chain branched
alkyl units. Therefore, prior to admixture with any other conventional
surfactants, the mid-chain
branched surfactant component will comprise from about 25% to about 70%
surfactant
molecules which are non-linear surfactants.
The surfactant systems of the laundry detergent compositions of the present
invention
can also comprise from about 0.001 %, preferably from about 1 %, more
preferably from about
5%, most preferably from about 10% to about 100%, preferably to about 60%,
more preferably
to about 30% by weight, of the surfactant system, of one or more (preferably a
mixture of two or
more) mid-chain branched alkyl arylsulfonate surfactants, preferably
surfactants wherein the aryl
unit is a benzene ring having the formula:
R1R2L R3
CM q+~
b
S03
wherein L is an acyclic hydrocarbyl moiety comprising from 6 to 18 carbon
atoms; R~, R', and
R3 are each independently hydrogen or C~-C3 alkyl, provided R' and R2 are not
attached at the
terminus of the L unit; M is a water soluble cation having charge q wherein a
and b are taken
together to satisfy charge neutrality.
Bleaching System
The compositions of the present inventiori~referably comprise a bleaching
system.
Bleaching systems typically comprise a "bleaching agent" (source of hydrogen
peroxide) and an
"initiator" or "catalyst". When present, bleaching agents will typically be at
levels of from
about I %, preferably from about 5% to about 30%, preferably to about 20% by
weight of the
composition. If present, the amount of bleach activator will typically be from
about 0.1 %,
preferably from about 0.5% to about 60%, preferably to about 40% by weight, of
the bleaching
composition comprising the bleaching agent-plus-bleach activator.
Bleaching Agents - Hydrogen peroxide sources are described in detail in the
herein
incorporated Kirk Othmer's Encyclopedia of Chemical Technology, 4th Ed ( 1992,
John Wiley &
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Sons), Vol. 4, pp. 271-300 "Bleaching Agents (Survey)", and include the
various foams of
sodium perborate and sodium percarbonate, including various coated and
modified forms.
The preferred source of hydrogen peroxide used herein can be any convenient
source,
including hydrogen peroxide itself. For example, perborate, e.g., sodium
perborate {any hydrate
but preferably the mono- or tetra-hydrate), sodium carbonate peroxyhydrate or
equivalent
percarbonate salts, sodium pyrophosphate peroxyhydrate, urea peroxyhydrate, or
sodium
peroxide can be used herein. Also useful are sources of available oxygen such
as persulfate
bleach (e.g., OXONE, manufactured by DuPont). Sodium perborate monohydrate and
sodium
percarbonate are particularly preferred. Mixtures of any convenient hydrogen
peroxide sources
can also be used.
A preferred percarbonate bleach comprises dry particles having an average
particle size
in the range from about 500 micrometers to about 1,000 micrometers, not more
than about 10%
by weight of said particles being smaller than about 200 micrometers and not
more than about
10% by weight of said particles being larger than about 1,250 micrometers.
Optionally, the
percarbonate can be coated with a silicate, borate or water-soluble
surfactants. Percarbonate is
available from various commercial sources such as FMC, Solvay and Tokai Denka.
Compositions of the present invention may also comprise as the bleaching agent
a
chlorine-type bleaching material. Such agents are well known in the art, and
include for
example sodium dichloroisocyanurate ("NaDCC"). However, chlorine-type bleaches
are less
preferred for compositions which comprise enzymes.
~,8) Bleach Activators - Preferably, the peroxygen bleach component in the
composition
is formulated with an activator (peracid precursor). The activator is present
at levels of from
about 0.01%, preferably from about 0.5%, more preferably from about 1% to
about 15%,
preferably to about 10%, more preferably to about 8%, by weight of the
composition. Preferred
activators are selected from the group consisting of tetraacetyl ethylene
diamine (TAED),
benzoylcaprolactam (BzCL), 4-nitrobenzoylcaprolactam, 3-
chlorobenzoyl~aprolactam,
benzoyloxybenzenesulphonate (BOBS), nonanoyloxybenzenesulphonate (HOBS),
phenyl
benzoate (PhBz), decanoyloxybenzenesulphonate (C10-OBS), benzoylvalerolactam
(BZVL),
octanoyloxybenzenesulphonate (Cg-OBS), perhydrolyzable esters and mixtures
thereof, most
preferably benzoylcaprolactam and benzoylvalerolactam. Particularly preferred
bleach
activators in the pH range from about 8 to about 9.5 are those selected having
an OBS or VL
leaving group.
Preferred hydrophobic bleach activators include, but are not limited to,
nonanoyloxybenzenesulphonate (HOBS), 4-[N-(nonaoyl) amino hexanoyloxy]-benzene
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WO 99/20768 PCT/IB98/01615
21
sulfonate sodium salt (NACA-OBS) an example of which is described in U.S.
Patent No.
5,523,434, lauryloxybenzenesulphonate (LOBS or C12-OBS), 10-
undecenoyloxybenzenesulfonate (UDOBS or CI 1-OBS with unsaturation in the 10
position),
and decanoyloxybenzoic acid (DOBA).
Preferred bleach activators are those described in U.S. 5,698,504 Christie et
al., issued
December 16, 1997; U.S. 5,695,679 Christie et al. issued December 9, 1997;
U.S. 5,686,401
Willey et al., issued November 11, 1997; U.S. 5,686,014 Hartshorn et al.,
issued November 11,
1997; U.S. 5,405,412 Willey et al., issued April 11, 1995; U.S. 5,405,413
Willey et al., issued
April 11, 1995; U.S. S,I30,045 Mitchel et al., issued July 14, 1992; and U.S.
4,412,934 Chung et
al., issued November 1, 1983, and copending patent applications U. S. Serial
Nos. 08/709,072,
08/064,564, all of which are incorporated herein by reference.
The mole ratio of peroxygen bleaching compound (as Av0) to bleach activator in
the
present invention generally ranges from at least 1:1, preferably from about
20:1, more preferably
from about 10:1 to about 1:1, preferably to about 3:1.
Quaternary substituted bleach activators may also be included. The present
detergent
compositions preferably comprise a quaternary substituted bleach activator
(QSBA) or a
quaternary substituted peracid (QSP); more preferably, the former. Preferred
QSBA structures
are further described in U.S. 5,686,01 S Willey et al., issued November 11,
1997; U.S. 5,654,421
Taylor et al., issued August S, 1997; U.S. 5,460,747 Gosselink et al., issued
October 24, 1995;
U.S. 5,584,888 Miracle et al., issued December I7, 1996; and U.S. 5,578,136
Taylor et al.,
issued November 26, 1996; all of which are incorporated herein by reference.
Highly preferred bleach activators useful herein are amide-substituted as
described in
U.S. 5,698,504, U.S. 5,695,679, and U.S. 5,686,014 each of which are cited
herein above.
Preferred examples of such bleach activators include: (6-octanamidocaproyl)
oxybenzenesulfonate, (6-nonanamidocaproyl~xybenzenesulfonate,
(6-decanamidocaproyl~xybenzenesulfonate and mixtures thereof.
Other useful activators, disclosed in U.S. 5,698,504, U.S. 5,695,679, U.S.
5,686,014
each of which is cited herein above and U.S. 4,966,723Hodge et al., issued
October 30, 1990,
include benzoxazin-type activators, such as a C5H4 ring to which is fused in
the 1,2-positions a
moiety --C(OpC(R 1 ~N-.
Depending on the activator and precise application, good bleaching results can
be
obtained from bleaching systems having with in-use pH of from about 6 to about
13,
preferably from about 9.0 to about 10.5. Typically, for example, activators
with electron-
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22
withdrawing moieties are used for near-neutral or sub-neutral pH ranges.
Alkalis and buffering
agents can be used to secure such pH.
Acyl lactam activators, as described in U.S. 5,698,504, U.S. 5,695,679 and
U.S.
5,686,014, each of which is cited herein above, are very useful herein,
especially the acyl
caprolactams (see for example WO 94-28102 A) and acyl valerolactams (see U.S.
5,503,639
Willey et al., issued April 2, 1996 incorporated herein by reference).
lb) Organic Peroxides, especially is yl Peroxides - These are extensively
illustrated in
Kirk Othmer, Encyclopedia of Chemical Technology, Vol. 17, John Wiley and
Sons, 1982 at
pages 27-90 and especially at pages 63-72, all incorporated herein by
reference. If a diacyl
peroxide is used, it will preferably be one which exerts minimal adverse
impact on
spotting/filming.
(c) Metal-containing Bleach Catalysts - The present invention compositions and
methods utilize metal-containing bleach catalysts that are effective for use
in cleaning
compositions. Preferred are manganese and cobalt-containing bleach catalysts.
One type of metal-containing bleach catalyst is a catalyst system comprising a
transition
metal cation of defined bleach catalytic activity, such as copper, iron,
titanium, ruthenium
tungsten, molybdenum, or manganese cations, an auxiliary metal cation having
little or no
bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate
having defined
stability constants for the catalytic and auxiliary metal cations,
particularly
ethylenediaminetetraacetic acid, ethylenediarninetetra (methylenephosphonic
acid) and water-
soluble salts thereof. Such catalysts are disclosed in U.S. 4,430,243 Bragg,
issued February 2,
1982.
M~ganese Metal Compjexes
If desired, the compositions herein can be catalyzed by means of a manganese
compound. Such compounds and levels of use are well known in the art and
include, for
example, the manganese-based catalysts disclosed in U.S. 5,576,282 Miracle et
al., issued
November 19, 1996; U.S. 5,246,621 Favre et al., issued September 21, 1993;
U.S. 5,244,594
Favre et al., issued September 14, 1993; U.S. 5,194,416 Jureller et al.,
issued March 16, 1993;
U.S. 5,114,606 van Vliet et al., issued May 19, 1992; and European Pat. App.
Pub. Nos. 549,271
A 1, 549,272 A 1, 544,440 A2, and 544,490 A 1; Preferred examples of these
catalysts include
MnIV2(u-O~(1,4,7-trimethyl-1,4,7-triazacyclononaneh(PF6)2, MnII~(u-O)1(u-
OAc)2(1,4,7-
trimethyl-1,4,7-triazacyclononaneh(C104)2, MnIV4(u-O)6(1,4,7-
triazacyclononane)4(C104)4,
MnIIIMnIV4(u-O)1(u-OAc)2-(1,4,7-trimethyl-1,4,7-triazacyclononane}2(C104)3,
MnIV(1,4,7-
trimethyl-1,4,7-triazacyclononane)- (OCH3)3(PF6), and mixtures thereof. Other
metal-based
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WO 99/20768 PCT/IB98/01615
23
bleach catalysts include those disclosed in U.S. 4,430,243 included by
reference herein above
and U.S. 5,114,611 van Kralingen, issued May 19, 1992. The use of manganese
with various
complex ligands to enhance bleaching is also reported in the following: U.S.
4,728,455 Rerek,
issued March 1, 1988; U.S. 5,284,944 Madison, issued Februaary 8, 1994; U.S.
5,246,612 van
Dijk et al., issued September 21, 1993; U.S. 5,256,779 Kerschner et al.,
issued October 26, 2993;
U.S. 5,280,117 Kerschner et al., issued 3anuary 18, 1994; U.S. 5,274,147
Kerschner et al., issued
December 28, 1993; U.S. 5,153,161 Kerschner et al., issued October 6, 1992;
and U.S.
5,227,084 Martens et al., issued July 13, 1993.
Cobalt bleach catalysts useful herein are known, and are described, for
example, in U.S.
5,597,936 Perkins et al., issued January 28, 1997; U.S. 5,595,967 Miracle et
al., January 21,
1997; U.S. 5,703,030 Perkins et al., issued December 30, 1997; and M. L. Tobe,
"Base
Hydrolysis of Transition-Metal Complexes", Adv. Inorg. Bioinorg. Mech..
(1983), 2, pages 1-94.
The most preferred cobalt catalyst useful herein are cobalt pentaamine acetate
salts having the
formula [Co(NH3)SOAc] Ty, wherein "OAc" represents an acetate moiety and "Ty"
is an anion,
and especially cobalt pentaamine acetate chloride, [Co(NH3)SOAc]CI2; as well
as
[Co(NH3)SOAc](OAc)2; [Co(NH3)SOAc](PF6)2; [Co(NH3)SOAc](S04); [Co-
(NH3)SOAc](BF4)2; and [Co(NH3)SOAc](N03)2 (herein "PAC").
These cobalt catalysts are readily prepared by known procedures, such as
taught for
example in U.S. 5,597,936, U.S. 5,595,967, U.S. 5,703,030, cited herein above,
the Tobe article
and the references cited therein, and in U.S. Patent 4,810,410, to Diakun et
al, issued March
7,1989, J. Chem. Ed. ( 1989), ~ø ( 12), 1043-45; The Synthesis and
Characterization of Inorganic
Compounds, W.L. Jolly (Prentice-Hall; 1970), pp. 461-3; Ino~g. Chem.. l$, 1497-
1502 ( 1979);
Ino~g. Chem.. ~, 2881-2885 ( 1982); Inorg. Chem,~.]_$, 2023-2025 ( 1979);
Inorg. Synthesis, 173-
176 (1960); and Journal ofPhysical Chemistry, 5~, 22-25 (1952).
Transition Metal Complexes of Macr~oly~~r li Rigid igandc - Compositions
herein
may also suitably include as bleach catalyst a transition metal complex of a
macropolycyclic
rigid ligand. The phrase "macropolycyclic rigid ligand" is sometimes
abbreviated as "MRL" in
discussion below. The amount used is a catalytically effective amount,
suitably about 1 ppb or
more, for example up to about 99.9%, more typically about 0.001 ppm or more,
preferably from
about 0.05 ppm to about 500 ppm (wherein "ppb" denotes parts per billion by
weight and "ppm"
denotes parts per million by weight).
Suitable transition metals e.g., Mn are illustrated hereinafter.
"Macropolycyclic" means
a MRL is both a macrocycle and is polycyclic. "Polycyclic" means at least
bicyclic. The term
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
24
"rigid" as used herein herein includes "having a superstructure" and "cross-
bridged". "Rigid"
has been defined as the constrained converse of flexibility: see D.H. Busch.,
Chemical Reviews
(1993), 2~, 847-860, incorporated by reference. More particularly, "rigid" as
used herein means
that the MRL must be determinably more rigid than a macrocycle ("parent
macrocycle") which
is otherwise identical (having the same ring size and type and number of atoms
in the main ring)
but lacking a superstructure (especially linking moieties or, preferably cross-
bridging moieties)
found in the MRL's. In determining the comparative rigidity of macrocycles
with and without
superstructures, the practitioner will use the free form (not the metal-bound
form) of the
macrocycles. Rigidity is well-known to be useful in comparing macrocycles;
suitable tools for
determining, measuring or comparing rigidity include computational methods
(see, for example,
Zimmer, Chem~a) Reviews. (1995), 95(38), 2629-2648 or Hancock et al.,
Inorganica Chimica
,~, (1989), 164, 73-84.
Preferred MRL's herein are a special type of ultra-rigid ligand which is cross-
bridged. A
"cross-bridge" is nonlimitingly illustrated in 1.11 hereinbelow. In 1.1 I, the
cross-bridge is a-
CH2CH2- moiety. It bridges N1 and N8 in the illustrative structure. By
comparison, a "same-
side" bridge, for example if one were to be introduced across N 1 and N 12 in
1.11, would not be
sufficient to constitute a "cross-bridge" and accordingly would not be
preferred.
Suitable metals in the rigid ligand complexes include Mn(II), Mn(III), Mn(IV),
Mn(V),
Fe(II), Fe(III), Fe(IV), Co(I), Co(II), Co(III), Ni(I), Ni(II), Ni(III),
Cu(I), Cu(II), Cu(III),Cr(II),
Cr(III), Cr(IV), Cr(V), Cr(VI), V(III), V(IV), V(V), Mo(IV), Mo(V), Mo(VI),
W(IV), W(V),
W(VI), Pd(II), Ru(II), Ru(III), and Ru(IV). Preferred transition-metals in the
instant transition-
metal bleach catalyst include manganese, iron and chromium.
More generally, the MRL's (and the corresponding transition-metal catalysts)
herein
suitably comprise:
(a) at least one macrocycle main ring comprising four or more heteroatoms; and
(b) a covalently connected non-metal superstructure capable of increasing the
rigidity of the
macrocycle, preferably selected from
(i) a bridging superstructure, such as a linking moiety;
(ii) a cross-bridging superstructure, such as a cross-bridging linking moiety;
and
(iii) combinations thereof.
The term "superstructure" is used herein as defined in the literature by Busch
et al., see,
for example, articles by Busch in "Chemical Reviews".
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
Preferred superstructures herein not only enhance the rigidity of the parent
macrocycle,
but also favor folding of the macrocycle so that it co-ordinates to a metal in
a cleft. Suitable
superstructures can be remarkably simple, for example a linking moiety such as
any of those
illustrated in Fig. 1 and Fig. 2 below, can be used.
\(c j
2
Fig. 1
wherein n is an integer, for example from 2 to 8, preferably less than 6,
typically 2 to 4, or
T
(CH2) ~(CHz)n
Z
Fig. 2
wherein m and n are integers from about 1 to 8, more preferably from 1 to 3; Z
is N or CH; and
T is a compatible substituent, for example H, alkyl, trialkylammonium,
halogen, nitro, sulfonate,
or the like. The aromatic ring in 1.10 can be replaced by a saturated ring, in
which the atom in Z
connecting into the ring can contain N, O, S or C.
Suitable MRL's are further nonlimitingly illustrated by the following
compound:
3
2 4
n5,
14 N a N 6
13 12 b 8 7
/N .,N
11~ 9
Fig. 3
This is a MRL in accordance with the invention which is a highly preferred,
cross-
bridged, methyl-substituted (all nitrogen atoms tertiary) derivative ofcyclam.
Formally, this
iigand is named 5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2~lexadecane using
the extended
von Baeyer system. See "A Guide to IUPAC Nomenclature of Organic Compounds:
Recommendations 1993", R. Panico, W.H. Powell and J-C Richer (F.ds.),
Blackwell Scientific
Publications, Boston, 1993; see especially section R-2.4.2.1.
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
26
Transition-metal bleach catalysts of Macrocyclic Rigid Ligands which are
suitable for
use in the invention compositions can in general include known compounds where
they conform
with the definition herein, as well as, more preferably, any of a large number
of novel
compounds expressly designed for the present laundry or cleaning uses, and non-
limitingly
illustrated by any of the following:
Dichloro-5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2)hexadecane Manganese(II)
Diaquo-5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane Manganese(II)
Hexafluorophosphate
Aquo-hydroxy-5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane
Manganese(III)
Hexafluorophosphate
Diaquo-5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2)hexadecane Manganese(II)
Tetrafluoroborate
Dichloro-5,12-dimethyl-1,5,8,12-tetraazabicyclo[6.6.2)hexadecane
Manganese(III)
Hexafluorophosphate
Dichloro-5,12-di-n-butyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane
Manganese(II)
Dichloro-5,12-dibenzyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane Manganese(II)
Dichloro-5-n-butyl-12-methyl-1,5,8,12-tetraaza-bicyclo[6.6.2]hexadecane
Manganese(II)
Dichloro-5-n-octyl-12-methyl-1,5,8,12-tetraaza-bicyclo[6.6.2]hexadecane
Manganese(II)
Dichloro-5-n-butyl-12-methyl-1,5,8,12-tetraaza-bicyclo[6.6.2]hexadecane
Manganese(II).
As a practical matter, and not by way of limitation, the compositions and
cleaning
processes herein can be adjusted to provide on the order of at least one part
per hundred million
of the active bleach catalyst species in the aqueous washing medium, and will
preferably provide
from about 0.01 ppm to about 25 ppm, more preferably from about 0.05 ppm to
about 10 ppm,
and most preferably from about 0.1 ppm to about 5 ppm, of the bleach catalyst
species in the
wash liquor. In order to obtain such levels in the vKash liquor of an
automatic washing process,
typical compositions herein will comprise from about 0.0005% to about 0.2%,
more preferably
from about 0.004% to about 0.08%, of bleach catalyst, especially manganese or
cobalt catalysts,
by weight of the cleaning compositions.
The following are non-limiting examples of adjunct ingredients useful in the
laundry
compositions of the present invention, said adjunct ingredients include
builders, optical
brighteners, soil release polymers, dye transfer agents, dispersents, enzymes,
suds suppressers,
dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators,
fluorescers, fabric
conditioners, hydrolyzable surfactants, preservatives, anti-oxidants,
chelants, stabilizers, anti-
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
27
shrinkage agents, anti-wrinkle agents, germicides, fungicides, anti corrosion
agents, and
mixtures thereof.
Builders - The laundry detergent compositions of the present invention
preferably
comprise one or more detergent builders or builder systems. When present, the
compositions
will typically comprise at least about 1 % builder, preferably from about 5%,
more preferably
from about 10% to about 80%, preferably to about 50%, more preferably to about
30% by
weight, of detergent builder.
The level of builder can vary widely depending upon the end use of the
composition and
its desired physical form. When present, the compositions will typically
comprise at least about
I% builder. Formulations typically comprise from about 5% to about 50%, more
typically
about 5% to about 30%, by weight, of detergent builder. Granular formulations
typically
comprise from about 10% to about 80%, more typically from about I S% to about
50% by
weight, of the detergent builder. Lower or higher levels of builder, however,
are not meant to be
excluded.
Inorganic or P-containing detergent builders include, but are not limited to,
the alkali
metal, ammonium and alkanolammonium salts of polyphosphates (exemplified by
the
tripolyphosphates, pyrophosphates, and glassy polymeric meta-phosphates),
phosphonates,
phytic acid, silicates, carbonates (including bicarbonates and
sesquicarbonates), sulphates, and
aluminosilicates. However, non-phosphate builders are required in some
locales. Importantly,
the compositions herein function surprisingly well even in the presence of the
so-called "weak"
builders (as compared with phosphates) such as citrate, or in the so-called
"underbuilt" situation
that may occur with zeolite or layered silicate builders.
Examples of silicate builders are the alkali metal silicates, particularly
those having a
Si02:Na20 ratio in the range 1.6:1 to 3.2:1 and layered silicates, such as the
layered sodium
silicates described in U.S. 4,664,839 Rieck, issued May I2, 1987. NaSKS-6 is
the trademark for
a crystalline layered silicate marketed by Hoechst (commonly abbreviated
herein as "SKS-6").
Unlike zeolite builders, the Na SKS-6 silicate builder does not contain
aluminum. NaSKS-6 has
the delta-Na2Si05 morphology form of layered silicate. It can be prepared by
methods such as
those described in German DE-A-3,417,649 and DE-A-3,742,043. SKS-6 is a highly
preferred
layered silicate for use herein, but other such layered silicates, such as
those having the general
formula NaMSix02x+1 ~yH20 wherein M is sodium or hydrogen, x is a number from
1.9 to 4,
preferably 2, and y is a number from 0 to 20, preferably 0 can be used herein.
Various other
layered silicates from Hoechst include NaSKS-5, NaSKS-7 and NaSKS-11, as the
alpha, beta
and gamma forms. As noted above, the delta~Ia2Si05 (NaSKS-6 form) is most
preferred for
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
28
use herein. Other silicates may also be useful such as for example magnesium
silicate, which
can serve as a crispening agent in granular formulations, as a stabilizing
agent for oxygen
bleaches, and as a component of suds control systems.
Examples of carbonate builders are the alkaline earth and alkali metal
carbonates as
disclosed in German Patent Application No. 2,321,001 published on November 1
S, 1973.
Aluminosilicate builders are useful in the present invention. Aluminosilicate
builders
are of great importance in most currently marketed heavy duty granular
detergent compositions,
and can also be a significant builder ingredient in liquid detergent
formulations.
Aluminosilicate builders include those having the empirical formula:
~Mz(~lO2h,)'xH2O
wherein z and y are integers of at least 6, the molar ratio of z to y is in
the range from 1.0 to
about 0.5, and x is an integer from about 15 to about 264.
Useful aluminosilicate ion exchange materials are commercially available.
These
aluminosilicates can be crystalline or amorphous in structure and can be
naturally-occurring
aluminosilicates or synthetically derived. A method for producing
aluminosilicate ion exchange
materials is disclosed in U.S. 3,985,669, Krummel et al, issued October 12,
1976. Preferred
synthetic crystalline aluminosilicate ion exchange materials useful herein are
available under the
designations Zeolite A, Zeolite P (B), Zeolite MAP and Zeolite X. In an
especially preferred
embodiment, the crystalline aluminosilicate ion exchange material has the
formula:
Nal2~(A102)12(Si02)12)'~20
wherein x is from about 20 to about 30, especially about 27. This material is
known as Zeolite
A. Dehydrated zeolites (x = 0 - 10) may also be used herein. Preferably, the
aluminosilicate has
a particle size of about 0.1-10 microns in diameter.
Organic detergent builders suitable for the,~urposes of the present invention
include,
but are not restricted to, a wide variety of polycarboxylate compounds. As
used herein, "poly-
carboxylate" refers to compounds having a plurality of carboxylate groups,
preferably at least 3
carboxylates. Polycarboxylate builder can generally be added to the
composition in acid form,
but can also be added in the form of a neutralized salt. When utilized in salt
form, alkali metals,
such as sodium, potassium, and lithium, or alkanolammonium salts are
preferred.
Included among the polycarboxylate builders are a variety of categories of
useful mato-
rials. One important category of polycarboxylate builders encompasses the
ether polycarboxy-
lates, including oxydisuccinate, as disclosed in U.S. 3,128,287 Berg, issued
April 7, 1964, U.S.
3,635,830 Lamberti et al., issued January 18, 1972, and U.S. 3,936,448
Lamberti, issued
February 3, 1976. See also "TMS/TDS" builders of U.S. 4,663,071 Bush et al.,
issued May 5,
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
29
1987. Suitable ether polycarboxylates also include cyclic compounds,
particularly alicyclic
compounds, such as those described in U.S. 3,923,679 Rapko, issued December 2,
1975; U.S.
4,158,635 Crutchfield et al., issued June 19, 1979; U.S. 4,120,874 Crutchfield
et al., issued
October 17, 1978; and U.S. 4,102,903 Crutchfield et al., issued July 25, 1978.
Other useful detergency builders include the ether hydroxypolycarboxylates,
copoly-
mers of malefic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-
trihydroxy benzene-2, 4, 6-
trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali
metal, ammonium and
substituted ammonium salts ofpolyacetic acids such as ethylenediamine
tetraacetic acid and
nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid,
succinic acid, oxy-
disuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid,
carboxymethyloxysuccinic
acid, and soluble salts thereof.
Citrate builders, e.g., citric acid and soluble salts thereof (particularly
sodium salt), are
polycarboxylate builders of particular importance for heavy duty liquid
detergent formulations
due to their availability from renewable resources and their biodegradability.
Citrates can also
be used in granular compositions, especially in combination with zeolite
and/or layered silicate
builders. Oxydisuccinates are also especially useful in such compositions and
combinations.
Also suitable in the detergent compositions of the present invention are the
3,3-dicar-
boxy-4-oxa-1,6-hexanedioates and the related compounds disclosed in U.S.
4,566,984, Bush,
issued January 28, 1986. Useful succinic acid builders include the CS-C20
alkyl and alkenyl
succinic acids and salts thereof. A particularly preferred compound of this
type is do-
decenylsuccinic acid. Specific examples of succinate builders include:
laurylsuccinate,
myristylsuccinate, palmitylsuccinate, 2-dodecenylsuccinate (preferred), 2-
pentadecenylsucci-
nate, and the like. Laurylsuccinates are the preferred builders of this group,
and are described in
European Patent Application 86200690.5/0,200,263, published November 5, 1986.
Other suitable polycarboxylates are disclosed in U.S. 4,144,226, Crutchfield
et al.,
issued March 13, 1979 and in U.S. 3,308,067, Diehl, issued March 7, 1967. See
also Diehl U.S.
Patent 3,723,322.
Fatty acids, e.g., C12-Clg monocarboxylic acids, can also be incorporated into
the
compositions alone, or in combination with the aforesaid builders, especially
citrate and/or the
succinate builders, to provide additional builder activity. Such use of fatty
acids will generally
result in a diminution of sudsing, which should be taken into account by the
formulator.
In situations where phosphorus-based builders can be used, and especially in
the for-
mulation of bars used for hand-laundering operations, the various alkali metal
phosphates such
as the well-known sodium tripolyphosphates, sodium pyrophosphate and sodium
orthophosphate
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
- 30
can be used. Phosphonate builders such as ethane-1-hydroxy-1,1-diphosphonate
and other
known phosphonates (see, for example, U.S. Patents 3,159,581; 3,213,030;
3,422,021;
3,400,148 and 3,422,137) can also be used.
The detergent compositions herein may also optionally contain one or more iron
and/or
manganese chelating agents. Such chelating agents can be selected from the
group consisting of
amino carboxylates, amino phosphonates, polyfunctionally-substituted aromatic
chelating agents
and mixtures therein, all as hereinafter defined. Without intending to be
bound by theory, it is
believed that the benefit of these materials is due in part to their
exceptional ability to remove
iron and manganese ions from washing solutions by formation of soluble
chelates.
Examples of suitable chelating agents and levels of use are described in U.S.
Pat. Nos.
5,576,282 and 5,728,671.
A preferred biodegradable chelator for use herein is ethylenediamine
disuccinate
("EDDS"), especially the [S,S] isomer as described in U.S. Patent 4,704,233,
November 3, 1987,
to Hartman and Perkins.
The compositions herein may also contain water-soluble methyl glycine diacetic
acid
(MGDA) salts (or acid form) as a chelant or co-builder useful with, for
example, insoluble
builders such as zeolites, layered silicates and the like.
If utilized, these chelating agents will generally comprise from about 0.1 %
by weight of
the detergent compositions herein to about 15%, more preferably 3.0% by weight
of the
detergent compositions herein.
The detergent compositions of the present invention may also include one or
more
compounds, dye transfer inhibiting agents, for inhibiting dye transfer from
one fabric to another
of solubilized and suspended dyes encountered during fabric laundering and
conditioning
operations involving colored fabrics.
Suitable polymeric dye transfer inhibiting agents include, but are not limited
to,
polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-
vinylpyrrolidone
and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or
mixtures thereof.
Examples of such dye transfer inhibiting agents are disclosed in U.S. Pat.
Nos. 5,707,950 and
5,707,951.
Additional suitable dye transfer inhibiting agents include, but are not
limited to, cross-
linked polymers. Cross-linked polymers are polymers whose backbone are
interconnected to a
certain degree; these links can be of chemical or physical nature, possibly
with active groups on
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WO 99/20768 PCT/IB98/01615
- 31
the backbone or on branches. Cross-linked polymers have been described in the
Journal of
Polymer Science, volume 22, pages 1035-1039.
In one embodiment, the cross-linked polymers are made in such a way that they
form a
three-dimensional rigid structure, which can entrap dyes in the pores formed
by the three-
dimensional structure.
In another embodiment, the cross-linked polymers entrap dyes by swelling.
Suitable cross-linked polymers are described in the co-pending European patent
application 94870213.9.
Addition of such polymers also enhances the performance of the enzymes within
the
detergent compositions herein.
The dye transfer inhibiting agents have the ability to complex or adsorb
fugitive dyes
wash out of dyed fabrics before the dyes have the opportunity to become
attached to other
articles in the wash.
When present in the detergent compositions herein, the dye transfer inhibiting
agents are
present at levels from about 0.0001%, more preferably about 0.01%, most
preferably about
0.05% by weight of the detergent compositions to about 10%, more preferably
about 2%, most
preferably about 1 % by weight of the detergent compositions.
Dispersants
The detergent composition of the present invention can also contain
dispersants.
Suitable water-soluble organic salts are 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.
Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts
are
polyacrylates of MW 2000-5000 and their copolyc~ers with malefic anhydride,
such copolymers
having a molecular weight of from 1,000 to 100,000.
Especially, copolymer of acrylate and methylacrylate such as the 480N having a
molecular weight of 4000, at a level from 0.5-20% by weight of composition can
be added in the
detergentcompositions of the present invention.
The compositions of the invention may contain a lime soap peptiser compound,
which
has a lime soap dispersing power (LSDP), as defined hereinafter of no more
than 8, preferably
no more than 7, most preferably no more than 6. The lime soap peptiser
compound is preferably
present at a level from 0% to 20% by weight.
A numerical measure of the effectiveness of a lime soap peptiser is given by
the lime
soap dispersant power (LSDP) which is determined using the lime soap
dispersant test as
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WO 99/20768 PCT/IB98/01615
- 32
described in an article by H.C. Borghetty and C.A. Bergman, J. Am. Oil. Chem.
Soc., volume
27, pages 88-90, (1950). This lime soap dispersion test method is widely used
by practitioners in
this art field being referred to, for example, in the following review
articles; W.N. Linfield,
Surfactant science Series, Volume 7, page 3; W.N. Linfield, Tenside surf.
det., volume 27, pages
159-163, (1990); and M.K. Nagarajan, W.F. Masler, Cosmetics and Toiletries,
volume 104,
pages 71-73, (1989). The LSDP is the % weight ratio of dispersing agent to
sodium oleate
required to disperse the lime soap deposits formed by 0.025g of sodium oleate
in 30m1 of water
of 333ppm CaCo3 (Ca:Mg=3:2) equivalent hardness.
Surfactants having good lime soap peptiser capability will include certain
amine oxides,
betaines, sulfobetaines, alkyl ethoxysulfates and ethoxylated alcohols.
Exemplary surfactants having a LSDP of no more than 8 for use in accord with
the
present invention include C 16-C 1 g dimethyl amine oxide, C 12-C 1 g alkyl
ethoxysulfates with an
average degree of ethoxylation of from 1-5, particularly C 12-C 15 alkyl
ethoxysulfate surfactant
with a degree of ethoxylation of amount 3 (LSDP=4), and the C 14-C 15
ethoxylated alcohols
with an average degree of ethoxylation of either 12 (LSDP=6) or 30, sold under
the tradenames
Lutensol A012 and Lutensol A030 respectively, by BASF GmbH.
Polymeric lime soap peptisers suitable for use herein are described in the
article by
M.K. Nagarajan, W.F. Masler, to be found in Cosmetics and Toiletries, volume
104, pages 71-
73, (1989).
Hydrophobic bleaches such as 4-[N-octanoyl-6-aminohexanoylJbenzene sulfonate,
4-
[N-nonanoyl-6-aminohexanoylJbenzene sulfonate, 4-[N-decanoyl-6-
aminohexanoyl]benzene
sulfonate and mixtures thereof; and nonanoyloxy benzene sulfonate together
with hydrophilic /
hydrophobic bleach formulations can also be used as lime soap peptisers
compounds.
Conventional detergent i,~,~
The detergent compositions can comprise in addition to the amylase of the
present
invention one or more detergent enzymes which provide cleaning performance
and/or fabric
care benefits. Such enzymes can include proteases, amylases, cellulases and
lipases. Such
materials are known in the art and are commercially available under such
trademarks as . They
may be incorporated into the non-aqueous liquid detergent compositions herein
in the form of
suspensions, "marumes" or "prills". Another suitable type of enzyme comprises
those in the
form of slurries of enzymes in nonionic surfactants, e.g., the enzymes
marketed by Novo Nordisk
under the tradename "SL" or the microencapsulated enzymes marketed by Novo
Nordisk under
CA 02307324 2000-04-17
.. WO 99/20768 PCT/IB98/01615
- 33
the tradename "LDP." Suitable enzymes and levels of use are described in U.S.
Pat. No.
5,576,282.
Enzymes added to the compositions herein in the form of conventional enzyme
prills are
especially preferred for use herein. Such prills will generally range in size
from about 100 to
1,000 microns, more preferably from about 200 to 800 microns and will be
suspended
throughout the non-aqueous liquid phase of the composition. Prills in the
compositions of the
present invention have been found, in comparison with other enzyme forms, to
exhibit especially
desirable enzyme stability in terms of retention of enzymatic activity over
time. Thus,
compositions which utilize enzyme prills need not contain conventional enzyme
stabilizing such
as must frequently be used when enzymes are incorporated into aqueous liquid
detergents.
Examples of suitable enzymes include, but are not limited to, hemicellulases,
peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,
esterases, cutinases,
pectinases, keratanases, reductases, oxidases, phenoloxidases, lipoxygenases,
ligninases,
pullulanases, tannases, pentosanases, malanases, 13-glucanases,
arabinosidases, hyaluronidase,
chondroitinase, lactase, and known amylases, or mixtures thereof. A preferred
combination is a
detergent composition having a cocktail of conventional applicable enzymes
tike protease,
lipase, cutinase and/or cellulase in conjunction with the amylase of the
present invention.
Examples of such suitable enzymes are disclosed in U.S. Patent Nos. 5,576,282,
5,728,671 and 5,707,950
Suitable proteases are the subtilisins which are obtained from particular
strains of B.
subtilis and B. licheniformis (subtilisin BPN and BPN'). One suitable protease
is obtained from
a strain of Bacillus, having maximum activity throughout the pH range of 8-12,
developed and
sold as ESPERASE~ by Novo Industries A/S of Denmark, hereinafter "Novo". The
preparation
of this enzyme and analogous enzymes is described in GB 1,243,784 to Novo.
Other suitable
proteases include ALCALASE~, DURAZYM~ and SAVINASE~ from Novo and
MAXATASE~~ MAXACAL~, PROPERASE~ and MAXAPEM~ (protein engineered
Maxacal) from Gist-Brocades. Proteolytic enzymes also encompass modified
bacterial serine
proteases, such as those described in European Patent Application Serial
Number 87 303761.8,
filed April 28, 1987 (particularly pages 17, 24 and 98), and which is called
herein "Protease B",
and in European Patent Application 199,404, Venegas, published October 29,
1986, which refers
to a modified bacterial serine protealytic enzyme which is called "Protease A"
herein. More
preferred is what is called herein "Protease C", which is a variant of an
alkaline serine protease
from Bacillus in which lysine replaced arginine at position 27, tyrosine
replaced valine at
position 104, serine replaced asparagine at position 123, and alanine replaced
threonine at
CA 02307324 2000-04-17
WO 99/20768 PCT/IB98/01615
34
position 274. Protease C is described in EP 90915958:4, corresponding to WO
91/06637,
Published May 16, 1991. Genetically modified variants, particularly of
Protease C, are also
included herein. See also a high pH protease from Bacillus sp. NCIMB 40338
described in WO
93/18140 A to Novo. Enzymatic detergents comprising protease, one or more
other enzymes,
and a reversible protease inhibitor are described in WO 92/03529 A to Novo.
When desired, a
protease having decreased adsorption and increased hydrolysis is available as
described in WO
95/07791 to Procter & Gamble. A recombinant trypsin-like protease for
detergents suitable
herein is described in WO 94/25583 to Novo.
In more detail, the protease referred to as "Protease D" is a carbonyl
hydrolase variant
having an amino acid sequence not found in nature, which is derived from a
precursor carbonyl
hydrolase by substituting a different amino acid for a plurality of amino acid
residues at a
position in said carbonyl hydrolase equivalent to position +76, preferably
also in combination
with one or more amino acid residue positions equivalent to those selected
from the group
consisting of +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128,
+135, +156,
+166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265, and/or
+274
according to the numbering of Bacillus amyloliguefaciens subtilisin, as
described in WO
95/10615 published April 20, 1995 by Genencor International. Also suitable for
the present
invention are proteases described in patent applications EP 251 446 and
W091/06637 and
protease BLAP~ described in W091/02792. The proteolytic enzymes are
incorporated in the
detergent compositions of the present invention a level of from 0.0001 % to
2%, preferably from
0.001% to 0.2%, more preferably from 0.005% to 0.1% pure enzyme by weight of
the
composition.
Useful proteases are also described in PCT publications: WO 95/30010 published
November 9, 1995 by The Procter & Gamble Company; WO 95/30011 published
November 9,
1995 by 'The Procter & Gamble Company; WO 95/29979 published November 9, 1995
by The
Procter & Gamble Company.
The cellulases usable in the present invention include both bacterial or
fungal cellulase.
Preferably, they will have a pH optimum of between 5 and 9.5. Suitable
cellulases are disclosed
in U.S. Patent 4,435,307, Barbesgoard et al, which discloses fungal cellulase
produced from
Humicola insolens. Suitable cellulases are also disclosed in GB-A-2.075.028;
GB-A-2.095.275
and DE-OS-2.247.832.
Examples of such cellulases are cellulases produced by a strain of Humicola
insolens
(Humicola grisea var. thermoidea), particularly the Humicola strain DSM 1800.
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WO 99/Z0768 PCT/IB98/01615
Other suitable cellulases are cellulases originated from Humicola insolens
having a
molecular weight of about SOICDa, an isoelectric point of 5.5 and containing
415 amino acids;
and a '43kD endogiucanase derived from Humicola insolens, DSM 1800, exhibiting
cellulase
activity; a preferred endoglucanase component has the amino acid sequence
disclosed in PCT
Patent Application No. WO 91/17243. Also suitable cellulases are the EGIII
cellulases from
Trichoderma longibrachiatum described in W094/21801, Genencor, published
September 29,
1994. Especially suitable cellulases are the cellulases having color care
benefits. Examples of
such cellulases are ce11u1ases described in European patent application No.
91202879.2, filed
November 6, 1991 (Novo). Carezyme and Celluzyme (Novo Nordisk A/S) are
especially useful.
See also W091/17243.
Peroxidase enzymes are known in the art, and include, for example, horseradish
peroxidase, ligninase and haloperoxidase such as chloro- and bromo-peroxidase.
Peroxidase-
containing detergent compositions are disclosed, for example, in U.S. Patent
Nos. 5,576,282,
5,728,671 and 5,707,950, PCT International Applications WO 89/099813,
W089/09813 and in
European Patent application EP No. 91202882.6, filed on November 6, 1991 and
EP No.
96870013.8, filed February 20, 1996. Also suitable is the laccase enzyme.
Preferred enhancers are substitued phenthiazine and phenoxasine 10-
Phenothiazinepropionicacid (PPT), 10-ethylphenothiazine-4-carboxylic acid
(EPC), 10-
phenoxazinepropionic acid (POP) and 10-methylphenoxazine (described in WO
94/12621) and
substitued syringates (C3-CS substitued alkyl syringates) and phenols. Sodium
percarbonate or
perborate are preferred sources of hydrogen peroxide.
Said peroxidases are normally incorporated in the detergent composition at
levels from
0.0001 % to 2% of active enzyme by weight of the detergent composition.
Other preferred enzymes that can be in~Iuded in the detergent compositions of
the
present invention include lipases. Suitable lipase enzymes for detergent usage
include those
produced by microorganisms of the Pseudomonas group, such as Pseudomonas
stutzeri ATCC
19.154, as disclosed in British Patent 1,372,034. Suitable lipases include
those which show a
positive immunological cross-reaction with the antibody of the lipase,
produced by the
microorganism Pseudomonas fluorescent IAM 1057. This lipase is available from
Amano
Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P "Amano,"
hereinafter
referred to as "Amano-P". Other suitable commercial lipases include Amano-CES,
lipases ex
Chromobacter viscosum, e.g. Chromobacter viscosum var. lipolyticum NRRLB 3673
from Toyo
Jozo Co., Tagata, Japan; Chromobacter viscosum lipases from U.S. Biochemical
Corp., U.S.A.
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36
and Disoynth Co., The Netherlands, and lipases ex Pseudomonas gladioli.
Especially suitable
lipases are lipases such as M 1 LIPASE~ and LIPOMAX~ (Gist-Brocades) and
LIPOLASE~
and LIPOLASE ULTRA~(Novo) which have found to be very effective when used in
combination with the compositions of the present invention.
Also suitable are cutinases [EC 3.1.1.50] which can be considered as a special
kind of
lipase, namely lipases which do not require interfacial activation. Addition
of cutinases to
detergent compositions have been described in e.g. WO 88/09367 (Genencor).
The lipases and/or cutinases are normally incorporated in the detergent
composition at
levels from 0.0001% to 2% of active enzyme by weight of the detergent
composition.
Known amylases (a and/or (3) can be included for removal of carbohydrate-based
stains.
WO 94/02597, Novo Nordisk A/S published February 03, 1994, describes cleaning
compositions
which incorporate mutant amylases. See also W094/18314, Genencor, published
August 18,
1994 and W095/I0603, Novo Nordisk A/S, published April 20, 1995. Other
amylases known
for use in detergent compositions include both a- and (3-amylases. a-Amylases
are known in the
art and include those disclosed in US Pat. 5,003,257; EP 252,666; WO 91/00353;
FR 2,676,456;
EP 285,123; EP 525,610; EP 368,341; and British Patent Specification No.
1,296,839 (Novo).
Other suitable amylase are stability-enhanced amylases including PURAFACT OX
AM~
described in WO 94/18314, published August 18, 1994 and W096/05295, Genencor;
published
Februaury 22, 1996 and amylase variants from Novo Nordisk A/S, disclosed in WO
95/10603,
published April 95.
Examples of commercial a-amylases products are TERMAMYL~, BAN~,
FUNGAMYL~ and DURAMYL~, all available from Novo Nordisk A/S Denmark.
W095/26397 describes other suitable amylases : a-amylases characterized by
having a specific
activity at least 25% higher than the specii-ic activity of TERMAMYL~ at a
temperature range
of 25°C to 55°C and at a pH value in the range of 8 to 10,
measured by the Phadebas~ a-
amylase activity assay. Other amylolytic enzymes with improved properties with
respect to the
activity level and the combination of thermostability and a higher activity
level are described in
W095/35382.
The above-mentioned enzymes may be of any suitable origin, such as vegetable,
animal,
bacterial, fungal and yeast origin. Purified or non-purified forms of these
enzymes may be used.
Also included by definition, are mutants of native enzymes. Mutants can be
obtained e.g. by
protein and/or genetic engineering, chemical and/or physical modifications of
native enzymes.
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WO 99/20768 PCT/IB98/01615
' 37
Common practice as well is the expression of the enzyme via host organisms in
which the
genetic material responsible for the production of the enzyme has been cloned.
Said enzymes are normally incorporated in the detergent composition at levels
from
0.0001% to 2% of active enzyme by weight of the detergent composition. The
enzymes can be
added as separate single ingredients (prills, granulates, stabilized liquids,
etc. containing one
enzyme ) or as mixtures of two or more enzymes ( e.g. cogranulates).
Other suitable detergent ingredients that can be added are enzyme oxidation
scavengers.
Examples of such enzyme oxidation scavengers are ethoxylated tetraethylene
polyamines.
A range of enzyme materials and means for their incorporation into synthetic
detergent
compositions is also disclosed in WO 93/07263 and WO 93/07260 to Genencor
International,
WO 89/08694 to Novo, and U.S. 3,553,139, January 5, 1971 to McCarty et al.
Enzymes are
further disclosed in U.S. 4,101,457, Place et al, July 18, 1978, and in U.S.
4,507,219, Hughes,
March 26, 1985. Enzyme materials useful for liquid detergent formulations, and
their
incorporation into such formulations, are disclosed in U.S. 4,261,868, Hora et
al, April 14, 1981.
F,~zvme Stabilizers
Enzymes for use in detergents can be stabilized by various techniques. Enzyme
stabilization techniques are disclosed and exemplified in U.S. 3,600,319,
August 17, 1971,
Gedge et al, EP 199,405 and EP 200,586, October 29, 1986, Venegas. Enzyme
stabilization
systems are also described, for example, in U.S. 3,519,570. A useful Bacillus,
sp. ACI3 giving
proteases, xylanases and cellulases, is described in WO 9401532 to Novo. The
enrymes
employed herein can be stabilized by the presence of water-soluble sources of
calcium and/or
magnesium ions in the finished compositions which provide such ions to the
enzymes. Suitable
enzyme stabilizers and levels of use are described in U.S. Pat. No. 5,576,282.
Other Detergent Ingredients
The detergent compositions herein may also optionally contain one or more
of the following: polymeric dispersing agents, clay soil removal/anti-
redeposition
agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing
agents,
fabric softeners, carriers, hydrotropes, processing aids and/or pigments.
Suitable
examples of such other detergent ingredients and levels of use are found in
U.S.
Patent No. 5,576,282.
Method of washing
The compositions of the invention may be used in essentially any washing or
cleaning
methods, including soaking methods, pretreatment methods and methods with
rinsing steps for
which a separate rinse aid composition may be added.
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The process described herein comprises contacting fabrics with a laundering
solution in
the usual manner and exemplified hereunder.
The process of the invention is conveniently carried out in the course of the
cleaning
process. The method of cleaning is preferably carried out at 5°C to
95°C, especialiy between
10°C and 60°C. The pH of the treatment solution is preferably
from 7 to 11.
The following examples are meant to exemplify compositions of the present
invention,
but are not necessarily meant to limit or otherwise define the scope of the
invention. In the
detergent compositions, the enzyme levels are expressed by pure enzyme by
weight of the total
composition and unless otherwise specified, the detergent ingredients are
expressed by weight of
the total compositions. The abbreviated component identifications herein have
the following
meanings:
LAS . Sodium linear C12 alkyl benzene sulphonate
TAS . Sodium tallow alkyl sulphate
CXYAS . Sodium C1X - Cly alkyl sulfate
25EY . A C12_C15 predominantly linear primary alcohol condensed with an
average of Y moles of ethylene oxide
CXYEZ . A C I X - C 1 y predominantly linear primary alcohol condensed with
an average of Z moles of ethylene oxide
XYEZS . C 1 X - C 1 y sodium, alkyl sulfate condensed with an average of Z
moles of ethylene oxide per mole
QAS . R2.N+(CH3)2(C2H40H) with R2 = C12-C14
Soap . Sodium linear alkyl carboxylate derived from a 80/20 mixture of
tallow and coconut oils.
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WO 99/20768 PCT/IB98/01615
39
Nonionic ~ C13-C1S mixed ethoxylated/propoxylated fatty alcohol with an
average degree of ethoxylation of 3.8 and an average degree of
propoxylation of 4.S sold under the tradename P(urafac LF404 by
BASF Gmbh.
CFAA ~ C12-C14 alkyl N-methyl glucamide
TFAA . C 16-C1 g alkyl N-methyl glucamide.
TPKFA . C 12-C 14 topped whole cut fatty acids.
DEQA . Di-(tallow-oxy-ethyl) dimethyl ammonium
chloride.
Neodol 4S-13 . C 14-C 1 S linear primary alcohol ethoxylate,
sold by Shell
Chemical CO.
Silicate . Amorphous Sodium Silicate (Si02:Na20
ratio = 2.0)
NaSKS-6 . Crystalline layered silicate of formula
8-Na2Si20S,
Carbonate . Anhydrous sodium carbonate with a particle
size between
200 Pm and 900p,m.
Bicarbonate . Anhydrous sodium bicarbonate with a particle size between
400 Pm and 1200Fm.
STPP . Anhydrous sodium tripolyphosphate
MA/AA . Copolymer of 1:4 maleic/acrylic acid, average molecular
weight about 70,000-80,000
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WO 99/Z0768 PCT/IB98/01615
Zeolite A . Hydrated Sodium Aluminosilicate of formulaNal2(A102Si02)12
. 27H20 having a primary particle size in the range from
0.1 to 10 micrometers
Citrate . Tri-sodium citrate dihydrate of activity 86,4% with a
particle size distribution between 425 pm and 850 Vim.
Citric . Anhydrous citric acid
PB1 . Anhydrous sodium perborate monohydrate bleach, empirical
formula NaB02.H202
PB4 . Anhydrous sodium perborate tetrahydrate
Percarbonate . Anhydrous sodium percarbonate bleach of empirical formula
2Na2C03.3H202
TAED . Tetraacetyl ethylene diamine.
NOBS . Nonanoyloxybenzene sulfonate in the form of the sodium salt.
Photoactivated Bleach . Sulfonated zinc phtalocyanine encapsulated in dextrin
soluble polymer.
Protease . Proteolytic enzyme sold under the tradename Savinase,
Alcalase, Durazym by Novo Nordisk A/S, Maxacal, Maxapem sold
by Gist-Brocades and proteases described in patents W091/06637
and/or W095/10591 and/or EP 251,446.
Amylase . Amylolytic enzyme prepared by the present invention method
having hydrolytic activity 50% faster than that of the
amylase enzyme having SEQ. ID. 1.
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Lipase . Lipolytic enzyme sold under the tradename Lipolase,
Lipolase Ultra by Novo Nordisk A/S
Cellulase . Cellulytic enzyme sold under the tradename Carezyme,
Celluzyme and/or Endolase by Novo Nordisk A/S.
CMC . Sodium carboxymethyl cellulose.
HEDP . 1,1-hydroxyethane diphosphonic acid.
DETPMP . Diethylene triamine penta (methylene phosphoric acid),
marketed by Monsanto under the Trade name bequest 2060.
PVNO . Poly(4-vinylpyridine~N-Oxide.
PVPVI . Poly (4-vinylpyridine~N-oxide/copolymer of vinyl-imidazole
and vinyl-pyrrolidone.
Brightener 1 . Disodium 4,4'-bis(2-sulphostyryl)biphenyl.
Brightener 2 . Disodium 4,4'-bis(4-anilino-6-morpholino-1.3.5-triazin-2-yl)
stilbene-2:2'-disulfonate.
Silicone antifoam . Polydimethylsiloxane foam controller with siloxane-
oxyalkylene copolymer as dispersing agent with a ratio of
said foam controller to said dispersing agent of 10:1 to
100:1.
Granular Suds . 12% Silicone/silica, 18% stearyl alcoho1,70% starch in
Suppressor granular form
SRP 1 . Sulfobenzoyl or sodium isethionate end capped esters with
oxyethylene oxy and terephtaloyl backbone.
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' 42
SItP 2 . Diethoxylated poly ( 1,2 propylene terephtalate) short block
polymer.
Sulphate . Anhydrous sodium sulphate.
HMWPEO . High molecular weight polyethylene oxide
Example 1
The following detergent formulations, according to the present invention are
prepared, where I
and III are phosphorus-containing detergent compositions, and II is a zeolite-
containing
detergent composition:
I II III
Blown Powder:
STPP 24.0 - 24.0
Zeolite A - 24.0 -
C45AS 9.0 6.0 13.0
MA/AA 2.0 4.0 2.0
LAS 6.0 8.0 I 1.0
TAS 2.0 - -
Silicate 7.0 3.0 3.0
CMC 1.0 1.0 0.5
Brightener 2 0.2 0.2 0.2
Soap 1.0 '~ 1.0 1.0
DETPMP 0.4 0.4 0.2
Spray On
C45E7 2.5 2.5 2.0
C25E3 2.5 2.5 2.0
Silicone antifoam 0.3 0.3 0.3
Perfume 0.3 0.3 0.3
Dry additives:
Carbonate 6.0 13.0 15.0
PB4 18.0 I 8.0 10.0
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' 43
PB 1 4.0 4.0 0
TAED 3.0 3.0 1.0
Photoactivated bleach0.02 0.02 0.02
Protease 0.01 0.01 0.01
Lipase 0.009 0.009 0.009
Amylase 0.002 0.003 0.01
Dry mixed sodium 3.0 3.0 5.0
sulfate
Balance (Moisture 100.0 100.0 100.0
&
Miscellaneous)
Density (g/litre) 630 670 670
ExamFile 22
The following nil bleach-containing detergent formulations of particular use
in the washing of
colored clothing, according to the present inventibn are prepared:
I II III
Blown Powder
Zeolite A 15.0 15.0 -
Sodium sulfate 0.0 5.0 -
LAS 3.0 3.0 -
DETPMP 0.4 0.5 -
CMC 0.4 0.4 -
MA/AA 4.0 4.0 -
Agglomerates
C45AS '~ . - 11.0
LAS 6.0 5.0 -
TAS 3.0 2.0 -
Silicate 4.0 4.0 -
Zeolite A 10.0 15.0 13.0
CMC - - 0.5
MA/AA - - 2.0
Carbonate 9.0 7.0 7.0
Spray On
Perfume 0.3 0.3 0.5
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44
C45E7 4.0 4.0 4.0
C25E3 2.0 2.0 2.0
Dry additives
MA/AA - - 3.0
NaSKS-6 - - 12.0
Citrate 10.0 - 8.0
Bicarbonate 7.0 3.0 5.0
Carbonate 8.0 5.0 7.0
PVPVI/PVNO 0.5 0.5 0.5
Protease 0.026 0.016 0.047
Lipase 0.009 -- 0.009
Amylase 0.005 0.05 0.005
Cellulase 0.006 0.006 --
Silicone antifoam 5.0 5.0 5.0
Dry additives
Sodium sulfate 0.0 9.0 0.0
Balance (Moisture and 100.0 100.0 100.0
Miscellaneous)
Density (g/litre) 700 700 700
Ex~m~
The following detergent formulations, according to the present invention are
prepared:
I II III IV
LAS 20.0 14.0 24.0 22.0
QAS 0.7 1.0 - 0.7
TFAA - 1.0 - -
C25E5/C45E7 - 2.0 - 0.5
C45E3S - 2.5 - -
STPP 30.0 18.0 30.0 22.0
Silicate 9.0 5.0 10.0 8.0
Carbonate 13.0 7.5 - 5.0
Bicarbonate - 7.5 - -
DETPMP 0.7 1.0 - -
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SRP 1 0.3 0.2 - 0.1
MA/AA 2.0 . 1.5 2.0 1.0
CMC 0.8 0.4 0.4 0.2
Protease 0.008 0.01 0.026 0.026
Amylase 0.007 0.004 0.005 0.002
Lipase 0.004 -- -- 0.002
Cellulase 0.0015 0.0005 - -
Photoactivated 70ppm 45ppm - l Oppm
bleach
Brightener 1 0.2 0.2 0.08 0.2
PB 1 6.0 2.0 - -
NOBS 2.0 1.0 - -
Balance (Moisture100 100 . 100 100
and
Miscellaneous)
Example 4
The following liquid detergent formulations, according to the present
invention are prepared:
I II III IV V VI VII VIII
LAS 10.0 13.0 9.0 - 25.0 - - -
C25AS 4.0 I.0 2.0 10.0 - 13.0 18.0 15.0
C25E3S 1.0 - - 3.0 - 2.0 2.0 4.0
C25E7 6.0 8.0 13.0 2.5 - - 4.0 4.0
TFAA - - - 4.5 - 6.0 8.0 8.0
QAS - - - - 3.0 1.0 - -
TPKFA 2.0 - 13.0 2.0 - 15.0 7.0 7.0
'~
Rapeseed fatty- - - 5.0 - - 4.0 4.0
acids
Citric 2.0 3.0 1.0 1.5 I.0 1.0 1.0 1.0
Dodecenyl/ 12.0 10.0 - - 15.0 - - -
tetradecenyl
succinic
acid
Oleic acid 4.0 2.0 1.0 - 1.0 - - -
Ethanol 4.0 4.0 7.0 2.0 7.0 2.0 3.0 2.0
1,2 Propanediol4.0 4.0 2.0 7.0 6.0 8.0 10.0 13.-
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WO 99/20768 PCTlIB98/01615
46
Mono Ethanol - - - 5.0 - - 9.0 9.0
Amine
Tri Ethanol - - 8 - - - - -
Amine
NaOH (pH) 8.0 8.0 7.6 7.7 8.0 7.5 8.0 8.2
Ethoxylated 0.5 - 0.5 0.2 - - 0.4 0.3
tetraethylene
pentamine
DETPMP 1.0 1.0 0.5 1.0 2.0 1.2 1.0 -
SItP 2 0.3 - 0.3 0.1 - - 0.2 0.1
PVNO - _ _ - _ _ _ 0.10
Protease .005 .005 .004 .003 0.08 .005 .003 .006
Lipase - .002 - .0002 - - .003 .003
Amylase .002 .002 .005 .004 .002 .008 .005 .005
Cellulase - - - .0001 - - .0004 .0004
Boric acid 0.1 0.2 - 2:0 1.0 1.5 2.5 2.5
Na formate - - 1.0 - - - - -
Ca chloride - 0.015 - 0.01 - - - -
Bentonite - - - - 4.0 4.0 - -
clay
Suspending - - - - 0.6 0.3 - -
clay
SD3
Balance Moisture100 100 100 100 100 100 100 100
and Miscellaneous
Gxamye ~ ' .
Granular fabric detergent compositions which provide "softening through the
wash" capability
are prepared in accord with the present invention
I II
45AS - 10.0
LAS 7.6 -
68AS 1.3 -
45E7 4.0 -
25E3 - S.0
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47
Coco-alkyl-dimethyl hydroxy-1.4 1.0
ethyl ammonium chloride
Citrate 5.0 3.0
Na-SKS-6 - 11.0
Zeolite A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4
PB 1 15.0 -
Percarbonate - 15.0
TAED 5. 0 S.0
Smectite clay 10.0 5.0
HMWPEO - 0.1
Protease 0.02 0.01
Lipase 0.02 0.01
Amylase 0.01 0.005
Cellulase 0.001 -
Silicate 3.0 5.0
Carbonate 10.0 10.0
Granular suds suppressor 1.0 4.0
CMC 0.2 0.1
Water/minors Up to 100%
Ex~mpl~
Syndet bar fabric detergent compositions are prepared in accord with the
present invention
I II III IV
C26 AS 20.00 20.00 20.00 20.00
CFAA 5.0 5.0 5.0 5.0
LAS (C11-13) 10.0 10.0 10.0 10.0
Sodium carbonate 25.0 25.0 25.0 25.0
Sodium pyrophosphate 7.0 7.0 7.0 7.0
STPP 7.0 7.0 7.0 7.0
Zeolite A 5.0 5.0 5.0 5.0
CMC 0.2 0.2 0.2 0.2
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48
Polyacrylate (MW 1400) 0.2 0.2 0.2 0.2
Coconut monethanolamide5.0 5.0 5.0 5.0
Amylase 0.01 0.02 0.005 0.01
Protease 0.3 - 0.5 0.05
Brightener, perfume 0.2 0.2 0.2 0.2
CaS04 1.0 1.0 1.0 1.0
MgS04 1.0 1.0 1.0 1.0
Water 4.0 4.0 4.0 4.0
Filler* : balance to
100%
*Can be selected from convenient materials such as CaC03, talc, clay
(Kaolinite, Smectite),
silicates, and the like.
In addition to the above examples, the amylase of the present invention can be
formulated
into any suitable laundry detergent composition, non-limiting examples of
which are described
in U.S. 5,679,630 Baeck et al., issued October 21, 1997; U.S. 5,565,145 Watson
et al., issued
October 15, 1996; U.S. 5,478,489 Fredj et al., issued December 26, 1995; U.S.
5,470,507 Fredj
et al., issued November 28, 1995; U.S. 5,466,802 Panandiker et al., issued
November 14, 1995;
U.S. 5,460,752 Fredj et al., issued October 24, 1995; U.S. 5,458,810 Fredj et
al., issued October
17, 1995; U.S. 5,458,809 Fredj et al., issued October 17, 1995; U.S. 5,288,431
Huber et al.,
issued February 22, 1994 all of which are incorporated herein by reference.
The compositions of the present invention can be suitably prepared by any
process
chosen by the formulator, non-limiting examples of which are described in U.S.
5,691,297
Nassano et al., issued November 11, 1997; U.S. 5,574,005 Welch et al., issued
November 12,
1996; U.S. 5,569,645 Dinniwell et al., issued October 29, 1996; U.S. 5,565,422
Del Greco et al.,
issued October IS, 1996; U.S. 5,516,448 Capeci et al., issued May 14, 1996;
U.S. 5,489,392
Capeci et al., issued February 6, 1996; U.S. 5,486,303 Capeci et al., issued
January 23, 1996 all
of which are incorporated herein by reference.
CA 02307324 2000-04-17
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1
(1) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO: 1
His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15
Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser
20 25 30
Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp
35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 90
Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly
85 90 95
Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp
100 105 110
Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
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2
115 120 125
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg
165 170 175
Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu
180 185 190
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His
195 200 205
Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn
210 215 220
Thr Leu Asn Leu Asp Gly Phe Arg Lle Asp Ala Val Lys His Ile Lys
225 230 235 240
Tyr Ser Phe Thr Arg Asp Trp. Leu Thr His Val Arg Asn Ala Thr Gly
245 250 255
Lys c3lu Mat Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala
260 265 270
Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp
275 280 285
VaT Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn
290 295 300
Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pry
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3
305 310 315 320
Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu
325 330 335
Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp
385 360 365
Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Lle
370 375 380
Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn
405 410 415
Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro
420 425 430
Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val
435 440 445
Trp~ His Asp Ile Thr.Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala
450 455 460
Asg Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp
465 470 475 480
Val Lys Arg
