U.S. patent application number 11/728060 was filed with the patent office on 2008-09-25 for cleaning compositions comprising transglucosidase.
Invention is credited to Hugh C. McDonald, Ayrookaran J. Poulose, Jayarama K. Shetty.
Application Number | 20080229514 11/728060 |
Document ID | / |
Family ID | 39773235 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080229514 |
Kind Code |
A1 |
Poulose; Ayrookaran J. ; et
al. |
September 25, 2008 |
Cleaning compositions comprising transglucosidase
Abstract
Provided herein is a composition comprising: a) a
transglucosidase enzyme; and b) a natural gum polysaccharide,
wherein said natural gum polysaccharide is a substrate for said
transglucosidase enzyme. A method of using a transglucosidase
enzyme to a degrade natural gum polysaccharide is also provided.
The composition and method may be employed in cleaning
applications.
Inventors: |
Poulose; Ayrookaran J.;
(Belmont, CA) ; McDonald; Hugh C.; (Carlsbad,
CA) ; Shetty; Jayarama K.; (Pleasanton, CA) |
Correspondence
Address: |
GENENCOR INTERNATIONAL, INC.
925 PAGE MILL ROAD
Palo Alto
CA
94304-1013
US
|
Family ID: |
39773235 |
Appl. No.: |
11/728060 |
Filed: |
March 22, 2007 |
Current U.S.
Class: |
8/138 ; 510/281;
510/392; 510/470 |
Current CPC
Class: |
D06L 4/40 20170101; C11D
3/38636 20130101; D06L 1/14 20130101; C12N 9/107 20130101; C11D
3/222 20130101 |
Class at
Publication: |
8/138 ; 510/281;
510/392; 510/470 |
International
Class: |
D06L 1/02 20060101
D06L001/02; D06L 1/14 20060101 D06L001/14 |
Claims
1. A composition comprising: a) transglucosidase enzyme; and b)
natural gum polysaccharide; wherein said natural gum polysaccharide
is a substrate for said transglucosidase enzyme.
2. The composition of claim 1, wherein said natural gum
polysaccharide is xanthan gum.
3. The composition of claim 1, wherein said transglucosidase enzyme
has an activity defined as EC 2.4.1.24, according to IUBMB
nomenclature.
4. The composition of claim 1, wherein said transglucosidase enzyme
has an amino acid sequence that is at least 90% identical to an
Aspergillus transglucosidase enzyme.
5. The composition of claim 1, wherein said composition further
comprises a surfactant.
6. The composition of claim 1, wherein said natural gum
polysaccharide is present as a stain on an object.
7. The composition of claim 6, wherein said object is a fabric.
8. A method comprising combining a transglucosidase enzyme with a
natural gum polysaccharide to degrade said natural gum
polysaccharide.
9. The method of claim 8, wherein said transglucosidase enzyme has
an activity defined as EC 2.4.1.24, according to IUBMB
nomenclature.
10. The method of claim 8, wherein said natural gum polysaccharide
is xanthan gum.
11. A cleaning method comprising: a) contacting an object soiled
with a natural gum polysaccharide with a cleaning composition
comprising a transglucosidase enzyme; and b) maintaining said
object and cleaning composition under conditions sufficient to
effect degradation of said natural gum polysaccharide and thereby
clean said object.
12. The cleaning method of claim 11, wherein said object is soiled
with a food that contains said natural gum polysaccharide.
13. The cleaning method of claim 11, wherein said natural gum
polysaccharide is a xanthan gum.
14. The cleaning method of claim 11, wherein said object is
selected from fabrics and hard surfaces.
15. The cleaning method of claim 11, wherein said transglucosidase
enzyme has an amino acid sequence that is at least 90% identical to
an Aspergillus transglucosidase enzyme.
16. The cleaning method of claim 11, wherein said cleaning
composition further comprises a surfactant.
17. The cleaning method of claim 11, wherein said cleaning
composition further comprises at least one enzyme selected from
proteases, amylases, cellulases, lipases, cutinases, mannanases,
pectinases, pectate lyases and oxido-reductases, for the
degradation of other stain components.
18. The cleaning method of claim 11, wherein said cleaning
composition has a pH of pH 5.0 to pH 11.5.
19. The cleaning method of claim 11, wherein said cleaning
composition comprises said enzyme at a concentration in the range
of 0.01 ppm to 100 ppm.
Description
BACKGROUND
[0001] Detergent and other cleaning compositions often include a
complex combination of active ingredients. For example, certain
cleaning products contain a surfactant system, enzymes for
cleaning, bleaching agents, builders, suds suppressors,
soil-suspending agents, soil-release agents, optical brighteners,
softening agents, dispersants, dye transfer inhibition compounds,
abrasives, bactericides, and perfumes. Despite the complexity of
current detergents, there are many stains that are difficult to
remove.
[0002] This disclosure relates to the use of transglucosidase as a
cleaning agent.
SUMMARY OF THE INVENTION
[0003] Certain aspects of this disclosure relate to a composition
comprising: a) a transglucosidase enzyme; and b) a natural gum
polysaccharide; wherein the natural gum polysaccharide is a
substrate for the transglucosidase enzyme. The natural gum
polysaccharide may be, for example, a xanthan or guar gum. The
natural gum polysaccharide is present as a stain on an object,
e.g., a fabric, or free in solution, for example. In certain
embodiments, the transglucosidase enzyme may be an amino acid
sequence that is at least 70% identical to, e.g., at least 80%
identical to, at least 85% identical to, at least 90% identical to,
at least 95% identical to, or at least 98% identical to an
Aspergillus transglucosidase. The composition may further comprise
a surfactant or other cleaning agent. Methods that include
combining a transglucosidase enzyme with a natural gum
polysaccharide to degrade the natural gum polysaccharide are also
provided.
[0004] A cleaning method is also provided. In general terms, the
cleaning method includes: a) contacting an object soiled with a
natural gum polysaccharide with a cleaning composition comprising a
transglucosidase enzyme; and b) maintaining the object and cleaning
composition together under conditions sufficient to effect
degradation of the natural gum polysaccharide and thereby clean the
object.
[0005] In particular embodiments, the object (which may be a
fabric, for example) may be soiled with a food that contains the
natural gum polysaccharide, e.g., a food that contains a xanthan or
guar gum. The cleaning composition may contain a surfactant, other
cleaning agents, or another enzyme, e.g., a protease, amylase,
cellulase, lipase, cutinase, oxido-reductase, or the like, for the
degradation of other stain components. The cleaning composition
used in the method may have a pH in the range of pH 5.0 to pH 11.5,
and the transglucosidase enzyme may be present in the cleaning
composition at a concentration in the range of 0.01 ppm to 100
ppm.
[0006] In certain cases, use of the subject method results in more
efficient removal of stains that contain natural gum
polysaccharides than equivalent methods that do not employ a
transglucosidase enzyme.
[0007] In a particular embodiment, an isolated enzyme containing an
Aspergillus transglucosidase produced by a Trichoderma reesei host
cell is provided. Cleaning compositions, e.g., laundry detergents,
containing the same are provided, as well as well as a method of
using the same for cleaning an object, e.g., a fabric.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Certain aspects of the following detailed description are
best understood when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures:
[0009] FIG. 1 shows a vector map of pTrex3(AGL51M).
[0010] FIG. 2 shows the nucleotide sequence of an expression
cassette of pTrex3(AGL51M) (SEQ ID NO:1).
[0011] FIG. 3 is a graph showing the enzymatic activity of
transglucosidase on 0.2% xanthan, as measured by a reducing sugar
assay.
[0012] FIG. 4 presents graphs showing the cleaning performance of
Trip-TG on guar soiled microswatches (top panel) and salad dressing
soiled microswatches (bottom panel) in 50 mM Hepes buffer (pH 7.4)
and in AATCC HDL Detergent (pH 7.4).
[0013] FIG. 5 is a graph of a dose response experiment showing that
Trip-TG is active on salad dressing soiled microswatches at a
concentration of 1 to 5 ppm in AATCC HDL.
[0014] FIG. 6 is a graph showing Trip-TG cleaning activity on salad
dressing microswatches in heavy duty solid detergent (HDD).
[0015] FIG. 7 is a graph showing Trip-TG cleaning activity on
marmalade stain in a Tergotometer assay using 0.15% AATCC HDL.
DEFINITIONS
[0016] All patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference in their entirety. Unless
defined otherwise herein, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs (See
e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 2D ED., John Wiley and Sons, New York [1994]; and Hale and
Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial,
NY [1991], both of which provide one of skill with a general
dictionary of many of the terms used herein). Although many methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention, the
preferred methods and materials are described herein. Numeric
ranges are inclusive of the numbers defining the range, as well as
every number in between.
[0017] As used herein and in the appended claims, the singular "a",
"an" and "the" includes the plural reference unless the context
clearly dictates otherwise. Thus, for example, reference to a "host
cell" includes a plurality of such host cells.
[0018] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively. The
headings provided herein are not limitations of the various aspects
or embodiments of the invention that can be had by reference to the
specification as a whole. Accordingly, the terms defined
immediately below are more fully defined by reference to the
Specification as a whole.
[0019] The term "recombinant" refers to a polynucleotide or
polypeptide that does not naturally occur in a host cell. A
recombinant molecule may contain two or more naturally-occurring
sequences that are linked together in a way that does not occur
naturally. A recombinant cell contains a recombinant polynucleotide
or polypeptide.
[0020] The term "heterologous" refers to elements that are not
normally associated with each other. For example, if a host cell
produces a heterologous protein, that protein is not normally
produced in that host cell. Likewise, a promoter that is operably
linked to a heterologous coding sequence is a promoter that is
operably linked to a coding sequence that it is not usually
operably linked to in a wild-type host cell. The term "homologous",
with reference to a polynucleotide or protein, refers to a
polynucleotide or protein that occurs naturally in a host cell.
[0021] The terms "protein" and "polypeptide" are used
interchangeably herein.
[0022] A "signal sequence" is a sequence of amino acids present at
the N-terminal portion of a protein which facilitates the secretion
of the mature form of the protein from the cell. The definition of
a signal sequence is a functional one. The mature form of the
extracellular protein lacks the signal sequence, which is cleaved
off during the secretion process.
[0023] A "coding sequence" is a DNA segment that encodes a
polypeptide.
[0024] The term "nucleic acid" encompasses DNA, RNA, single
stranded or double stranded and chemical modifications thereof. The
terms "nucleic acid" and "polynucleotide" are used interchangeably
herein.
[0025] A "vector" refers to a polynucleotide designed to introduce
nucleic acids into one or more host cells. Vectors can autonomously
replicate in different host cells and include: cloning vectors,
expression vectors, shuttle vectors, plasmids, phage particles,
cassettes and the like.
[0026] An "expression vector" as used herein means a DNA construct
comprising a protein-coding region that is operably linked to a
suitable control sequence that is capable of effecting expression
of the protein in a suitable host cell. Such control sequences may
include a promoter to effect transcription, an optional operator
sequence to control transcription to produce mRNA, a sequence
encoding suitable ribosome binding sites on the mRNA, and enhancers
and other sequences which control termination of transcription and
translation.
[0027] A "promoter" is a regulatory sequence that initiates
transcription of a downstream nucleic acid.
[0028] The term "operably linked" refers to an arrangement of
elements that allows them to be functionally related. For example,
a promoter is operably linked to a coding sequence if it controls
the transcription of the sequence.
[0029] The term "selective marker" refers to a protein capable of
expression in a host that allows for ease of selection of those
hosts containing an introduced nucleic acid or vector. Examples of
selectable markers include, but are not limited to, antimicrobials
(e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that
confer a metabolic advantage, such as a nutritional advantage on
the host cell.
[0030] The term "derived" encompasses the terms "originated from",
"obtained" or "obtainable from", and "isolated from".
[0031] A "non-pathogenic" organism is an organism that is not
pathogenic to humans.
[0032] The terms "recovered", "isolated", and "separated" as used
herein refer to a protein, cell, nucleic acid or amino acid that is
removed from at least one component with which it is naturally
associated.
[0033] As used herein, the terms "transformed", "stably
transformed" and "transgenic" used in reference to a cell means the
cell has a non-native (e.g., heterologous) nucleic acid sequence
integrated into its genome or as an episomal plasmid that is
maintained through multiple generations.
[0034] As used herein, the term "expression" refers to the process
by which a polypeptide is produced based on the nucleic acid
sequence of a gene. The process includes both transcription and
translation.
[0035] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means "transfection", or
"transformation" or "transduction" and includes reference to the
incorporation of a nucleic acid sequence into a eukaryotic or
prokaryotic cell wherein the nucleic acid sequence may be
incorporated into the genome of the cell (e.g., chromosome,
plasmid, plastid, or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0036] The term "hybridization" refers to the process by which a
strand of nucleic acid joins with a complementary strand through
base pairing as known in the art. A nucleic acid is considered to
be "Selectively hybridizable" to a reference nucleic acid sequence
if the two sequences specifically hybridize to one another under
moderate to high stringency hybridization and wash conditions.
Moderate and high stringency hybridization conditions are known
(see, e.g., Ausubel, et al., Short Protocols in Molecular Biology,
3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular
Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring
Harbor, N.Y.). One example of high stringency conditions include
hybridization at about 42 C in 50% formamide, 5.times.SSC, 5.times.
Denhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA
followed by washing two times in 2.times.SSC and 0.5% SDS at room
temperature and two additional times in 0.1.times.SSC and 0.5% SDS
at 42.degree. C.
[0037] As used herein, "cleaning composition" and "cleaning
formulation" refer to a composition that finds use in the removal
of undesired compounds (e.g., a stain) from items to be cleaned,
such as fabric, dishes, contact lenses, other solid substrates,
hair (shampoos), skin (soaps and creams), teeth (mouthwashes,
toothpastes), etc. The term encompasses any materials/compounds
selected for the particular type of cleaning composition desired in
the form of the product (e.g., liquid, gel, granule, or spray
composition), as long as the composition is compatible with the
subject enzyme in the composition. The specific selection of
cleaning composition materials are readily made by considering the
surface, item or fabric to be cleaned, and the desired form of the
composition for the cleaning conditions during use.
[0038] It is intended that the terms include, but are not limited
to detergent compositions (e.g., liquid and/or solid laundry
detergents and fine fabric detergents; hard surface cleaning
formulations, such as for glass, wood, ceramic and metal counter
tops and windows; carpet cleaners; oven cleaners; fabric
fresheners; fabric softeners; and textile and laundry pre-spotters,
as well as dish detergents).
[0039] The term "cleaning composition" as used herein includes,
unless otherwise indicated, granular, tablet or powder-form
all-purpose or heavy-duty washing agents, especially cleaning
detergents; liquid, gel or paste-form all-purpose washing agents,
especially the so-called heavy-duty liquid (HDL) types; liquid
fine-fabric detergents; hand dishwashing agents or light duty
dishwashing agents, including those of the high-foaming type;
machine dishwashing agents, including the various tablet, granular,
liquid and rinse-aid types for household and institutional use;
liquid cleaning and disinfecting agents, including antibacterial
hand-wash types, cleaning bars, mouthwashes, denture cleaners, car
or carpet shampoos, bathroom cleaners; hair shampoos and
hair-rinses; shower gels and foam baths; metal cleaners; as well as
cleaning auxiliaries such as bleach additives and "stain-stick",
pre-treat or laundry additive types.
[0040] As used herein, the terms "detergent composition" and
"detergent formulation" are used in reference to compositions that
are formulated for use in a wash medium for the cleaning of soiled
objects. In particular embodiments, the term is used in reference
to laundering fabrics and/or garments (e.g., "laundry detergents").
In alternative embodiments, the term refers to other detergents,
such as those used to clean dishes, cutlery, etc. (e.g.,
"dishwashing detergents"). It is not intended that the present
invention be limited to any particular detergent formulation or
composition. Indeed, it is intended that in addition to the subject
enzyme, a detergent composition may also contain surfactants,
transferase(s), hydrolytic enzymes, oxido reductases, builders,
bleaching agents, bleach activators, bluing agents and fluorescent
dyes, caking inhibitors, masking agents, enzyme activators,
antioxidants, and/or solubilizers, etc.
[0041] As used herein, "enhanced performance" in a cleaning
composition is defined as increasing cleaning (e.g., removal and/or
decolorization) of stains (particularly those of, e.g., natural gum
polysaccharide-related stains such as those of chocolate cream,
salad dressing or guar, etc.), as determined by usual evaluation
after a standard wash cycle.
[0042] As used herein the term "hard surface cleaning composition,"
refers to detergent compositions for cleaning hard surfaces such as
floors, walls, tile, bath and kitchen fixtures, and the like. Such
compositions are provided in any form, including but not limited to
solids, liquids, emulsions, etc.
[0043] As used herein, "dishwashing composition" refers to all
forms for compositions for cleaning dishes, including but not
limited to granular and liquid forms.
[0044] As used herein, "fabric cleaning composition" refers to all
forms of detergent compositions for cleaning fabrics, including but
not limited to, granular, liquid and bar forms.
[0045] As used herein, "textile" refers to woven fabrics, as well
as staple fibers and filaments suitable for conversion to or use as
yarns, woven, knit, and non-woven fabrics. The term encompasses
yarns made from natural, as well as synthetic (e.g., manufactured)
fibers.
[0046] As used herein, "textile materials" is a general term for
fibers, yarn intermediates, yarn, fabrics, and products made from
fabrics (e.g., garments and other articles).
[0047] As used herein, "fabric" encompasses any textile material.
Thus, it is intended that the term encompass garments, as well as
fabrics, yarns, fibers, non-woven materials, natural materials,
synthetic materials, and any other textile material.
[0048] As used herein, "effective amount of transglucosidase"
refers to the quantity of transglucosidase enzyme necessary to
achieve the enzymatic activity required in the specific application
(e.g., cleaning composition, etc.). Such effective amounts are
readily ascertained by one of ordinary skill in the art and are
based on many factors, such as the particular enzyme variant used,
the cleaning application, the specific composition of the cleaning
composition, and whether a liquid or dry (e.g., granular, bar)
composition is required, and the like.
[0049] The term "transglucosidase" refers to an enzyme that
transfers an .alpha.-D-glucosyl residue in a 1,4-.alpha.-D-glucan
to the primary hydroxy group of glucose, free or combined in a
1,4-.alpha.-D-glucan. The transglucosidase described herein has an
activity described as EC 2.4.1.24, according to IUBMB enzyme
nomenclature. The systematic name for the transglucosidase
described herein is 1,4-.alpha.-D-glucan:
1,4-.alpha.-D-glucan(D-glucose) 6-.alpha.-D-glucosyltransferase.
This enzyme may be referred to as .alpha.-glucosidase in certain
publications.
[0050] The term "soiled object" refers to an object, e.g., a fabric
or dish, that is soiled, e.g., stained, with a second composition.
Encompassed by the term "soiled object" are dirty fabrics, such as
dirty clothing, linens, and fabrics that are stained with
foodstuffs containing a natural gum polysaccharide. In certain
embodiments, the stain may or may not have a visible color.
[0051] The term "natural gum polysaccharide" refers to a non-starch
polysaccharide of natural origin that is capable of causing a large
viscosity increase in solution at low concentration. Such
polysaccharides are commonly employed in the food industry and are
used as thickening agents, gelling agents, emulsifiers and
stabilizers in many foodstuffs, e.g., sauces, creams, dairy
products, ice creams, mousses, milkshakes, salad dressings, etc.
Guar gum (food additive E412), an edible thickening agent extracted
from the leguminous guar bean shrub, and xanthan gum (food additive
E415), a polysaccharide that is produced by fermentation of glucose
or sucrose, e.g., by the Xanthomonas campestris bacterium, are
examples of natural gum polysaccharides. Other natural gum
polysaccharides include: agar (E406), alginic acid (E400),
.beta.-glucan, carrageenan (E407), chicle gum, dammar gum, gellan
gum (E418), glucomannan (E425), gum arabic (E414), gum ghatti, gum
tragacanth (E413), karaya gum (E416), locust bean gum (E410),
mastic gum, sodium alginate (E401), spruce gum, and tara gum
(E417).
[0052] The term "non-starch food polysaccharide degrading enzyme"
refers to an enzyme that degrades non-starch food polysaccharides.
Exemplary enzymes include, but are not limited to, hemicellulase,
mannanase, pectinase, xylanase, .beta.-galactosidase and
.alpha.-galactosidase.
[0053] The term "working pH" refers to the pH of a detergent during
its use. For example, the working pH of a laundry detergent is the
pH of the detergent when it is used to wash fabrics in a washing
machine. Likewise, the working pH of a dishwashing detergent is the
pH of that detergent as it is being used in a dishwasher.
Detergents that are in concentrated or solid form may be diluted or
dissolved before the pH of that detergent is at its working pH.
[0054] The term "working concentration" refers to the concentration
of an enzyme in a detergent during its use. For example, the
working concentration of an enzyme in a laundry detergent is the
concentration of that enzyme when the laundry detergent is used to
wash fabrics in a washing machine. Likewise, the working
concentration of an enzyme in a dishwashing detergent is the
concentration of that enzyme in the detergent as it is being used
in a dishwasher. Detergents that are in concentrated or solid form
may be diluted or dissolved before the concentration of an enzyme
in a detergent is at its working concentration.
[0055] Other definitions of terms may appear throughout the
specification.
DETAILED DESCRIPTION
[0056] Various compositions containing a transglucosidase enzyme
are provided, as well as methods of using the same.
[0057] Before the exemplary embodiments are described in more
detail, it is to be understood that this invention is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0058] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0059] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, exemplary methods and materials are now
described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. Any
Genbank accessions recited herein are incorporated by reference in
their entirety, including the nucleic acid and protein sequences
therein and the annotation of those sequences, as of the earliest
filing date of this patent application.
[0060] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Transglucosidase Enzyme-Containing Compositions
[0061] As noted above, a composition comprising a transglucosidase
enzyme is provided. In certain embodiments, the composition may
comprise a) a transglucosidase enzyme, and b) a natural gum
polysaccharide, where the natural gum polysaccharide is a substrate
for the transglucosidase enzyme.
[0062] As noted above, the transglucosidase enzyme generally has an
activity defined as EC 2.4.1.24, according to IUBMB enzyme
nomenclature, which activity transfers glucosyl residues in certain
glucans to the primary hydroxy group of glucose. In certain
embodiments, the enzyme may also have an activity that degrades
natural gum polysaccharide, e.g., xanthan and
galactomannan-containing polysaccharides such as guar gum or lima
bean gum, by clipping off sugar side chains or cleaving internal
bonds to break the polysaccharide backbone.
[0063] Transglucosidase enzymes that may be employed in the subject
compositions are generally described in Barker et al (Studies of
Aspergillus niger. Part II. Transglycosidation by Aspergillus
niger. J. Chem. Soc. 1953 3588-3593); Pazur et al (The glycoprotein
nature and antigenicity of a fungal D-glucosyltransferase.
Carbohydr. Res. 1986 149:137-47) and Nakamura et al (Cloning and
sequencing of an alpha-glucosidase gene from Aspergillus niger and
its expression in A. nidulans J. Biotechnol. 1997 53:75-84). In
particular embodiments, the transglucosidase enzyme that may be
employed may be purchased from Megazyme (Co. Wicklow, Ireland) or
Genencor Int. (Palo Alto, Calif.) under the trade name
Transglucosidase L-500.TM.. In particular embodiments, in
particular embodiments, the enzyme may be an Aspergillus niger
transglucosidase enzyme produced in Trichoderma reesei cells. In
certain cases, the transglucosidase enzyme may be a wild type
fungal transglucosidase (e.g., a fungal transglucosidase having an
amino acid sequence deposited in NCBI's Genbank database as
accession numbers: D45356 (GID:2645159; Aspergillus niger),
BAD06006.1 (GID:4031328; Aspergillus awamori), BAA08125.1
(GID:1054565; Aspergillus oryzae), XP.sub.--001210809.1
(GID:115492363; Aspergillus terreus), XP.sub.--001271891.1
(GID:121707620; Aspergillus clavatus), XP.sub.--001266999.1
(GID:119500484; Neosartorya fischeri), XP.sub.--751811.1
(GID:70993928; Aspergillus fumigatus), XP.sub.--659621.1
(GID:67523121; Aspergillus nidulans), XP.sub.--001216899.1
(GID:115433524; Aspergillus terreus) and XP.sub.--001258585.1
(GID:119473371; Neosartorya fischeri), or a variant thereof that
has an amino acid sequence that is at least 70% identical, e.g., at
least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, or at least 98% identical to a
wild type fungal transglucosidase. The enzyme is generally present
in the composition at a concentration in the range of 0.01 ppm
(parts per million, w/v) to 100 ppm, e.g., 0.01 ppm to 0.05 ppm,
0.05 ppm to 0.1 ppm, 0.1 ppm to 0.5 ppm, 0.5 ppm to 1 ppm, 1 ppm to
5 ppm, 5 ppm to 10 ppm, or 10 ppm to 100 ppm for example.
[0064] In particular embodiments, the composition may be a cleaning
composition and, as such, the composition may further comprise a
surfactant, other cleaning agents, and/or other fabric care agents
described in greater detail below.
[0065] The natural gum polysaccharide may be in an aqueous
solution, or affixed to an object, e.g., as a stain that is present
on the surface of an object, e.g., a fabric. In particular
embodiments, the natural gum polysaccharide may be in dried form.
Since natural gum polysaccharides are commonly employed in a
variety of foodstuffs, the object may be, in particular
embodiments, soiled with a foodstuff that contains a natural gum
polysaccharide. Exemplary foodstuffs that contain a natural gum
polysaccharide include, e.g., sauces, creams, dairy products, ice
creams, mousses, milkshakes, salad dressings, fruit juice
beverages, canned fruit, jelly, soy sauce, oyster sauce, packaged
meats, cheese, and bakery products. The natural gum polysaccharide
may be present in the foodstuff at a concentration of 0.1% to 1.5%,
e.g., 0.1% to 0.5%, 0.5% to 1.0% or 1.0% or 1.5%, for example.
[0066] The subject transglucosidase enzyme may be produced using
conventional methods. For example, it may be secreted into the
periplasm (e.g., by Gram-negative organisms, such as E. coli), or
into the extracellular space e.g., by Gram-positive organisms,
(such as Bacillus and Actinomyces), or eukaryotic hosts (e.g.,
Trichoderma, Aspergillus, Saccharomyces, and Pichia).
[0067] In particular embodiments, the subject transglucosidase
enzyme may be produced by expressing a fusion protein containing a
signal sequence operably linked to the transglucosidase enzyme in a
T. reesei host cell. In these embodiments, the transglucosidase
enzyme is secreted into culture medium, where it can be harvested.
The signal sequence of a subject fusion protein may be any signal
sequence that facilitates protein secretion from the Trichoderma
host cell. The signal sequence employed may be endogenous or
non-endogenous to the Trichoderma host cell and, in certain
embodiments, may be a signal sequence of a protein that is known to
be highly secreted from a Trichoderma sp. host cell. Such signal
sequence include, but are not limited to: the signal sequence of
cellobiohydrolase I, cellobiohydrolase II, endoglucanases I,
endoglucanases II, endoglucanases III, .alpha.-amylase, aspartyl
proteases, glucoamylase, mannanase, glycosidase and barley
endopeptidase B (see Saarelainen, Appl. Environ. Microbiol. 1997
63: 4938-4940), for example. In a particular embodiment, and as
further described in the Examples section of this disclosure, the
transglucosidase may be secreted using its own signal sequence
(i.e., the AGL1, AGL2 or AGL3 signal sequences, as described in
Margolles-Clark et al supra).
[0068] In certain embodiments, therefore, the transglucosidase may
be produced using a nucleic acid which may comprise: a signal
sequence-encoding nucleic acid operably linked to an
transglucosidase-encoding nucleic acid, where translation of the
nucleic acid produces a fusion protein comprising an
transglucosidase portion having an N-terminal signal sequence for
secretion of the transglucosidase portion from a Trichoderma host
cell.
[0069] In particular embodiments, the fusion protein may further
contain, in addition to a signal sequence, a "carrier protein" that
is a portion of a protein that is endogenous to and highly secreted
by the T. reesei sp. host cell. Suitable carrier proteins include
those of T. reesei mannanase I (Man5A, or MANI), T. reesei
cellobiohydrolase II (Cel6A, or CBHII) (see, e.g., Paloheimo et al
Appl. Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T.
reesei cellobiohydrolase I (CBHI). In one embodiment, the carrier
protein is a truncated T. reesei CBH1 protein that includes the
CBH1 core region and part of the CBH1 linker region. A nucleic acid
encoding a fusion protein containing, from amino-terminus to
carboxy-terminus, a signal sequence, a carrier protein and a
subject phytase in operable linkage may therefore be employed.
[0070] In certain embodiments, the coding sequence of the
transglucosidase may be codon optimized for expression of the
transglucosidase in the host cell used. Since codon usage tables
listing the usage of each codon in many host cells, including
Trichoderma reesei, are known in the art (see, e.g., Nakamura et
al, Nucl. Acids Res. 2000 28: 292) or readily derivable, such
nucleic acids can be readily designed giving the amino acid
sequence of a transglucosidase to be expressed.
[0071] In addition to a coding sequence, the nucleic acid may
further contain other elements that are necessary for expression of
the transglucosidase enzyme in the host cell. For example, the
nucleic acid may contain a promoter for transcription of the coding
sequence, and a transcriptional terminator. Exemplary promoters
that may be employed in T. reesei include the T. reesei cbh1, cbh2,
egl1, egl2, eg5, xln1 and xln2 promoters, or a hybrid or truncated
version thereof. For example, the promoter may be a T. reesei cbh1
promoter. Suitable terminators include the T. reesei cbh1, cbh2,
egl1, egl2, eg5, xln1 and xln2 terminators, and many others,
including, for example, the terminators from A. niger or A. awamori
glucoamylase genes (Nunberg et al. (1984) supra, and Boel et al.,
(1984) supra), Aspergillus nidulans anthranilate synthase genes,
Aspergillus oryzae TAKA amylase genes, or A. nidulans trpC (Punt et
al., (1987) Gene 56:117-124). The promoter and/or terminator may be
native or non-endogenous to the Trichoderma sp. host cell.
[0072] If a T. reesei host cell is employed for expression of the
transglucosidase enzyme, the cell may be genetically modified to
reduce expression of secreted proteins that are endogenous to the
cell. In one embodiment, the cell may contain one or more native
genes, particularly genes that encode secreted proteins, that have
been deleted or inactivated. For example, one or more
protease-encoding genes (e.g. an aspartyl protease-encoding gene;
see Berka et al, Gene 1990 86:153-162 and U.S. Pat. No. 6,509,171)
or cellulase-encoding genes may be deleted or inactivated. In one
embodiment, the Trichoderma sp. host cell may be a T. reesei host
cell contain inactivating deletions in the cbh1, cbh2 and egl1, and
egl2 genes, as described in WO 05/001036. The above-described
nucleic acid may be present in the nuclear genome of the
Trichoderma sp. host cell or may be present in a plasmid that
replicates in the Trichoderma host cell, for example.
[0073] A nucleic acid may be introduced into the Trichoderma host
cell using any one of a number of different techniques, e.g.,
electroporation, nuclear microinjection, transduction,
transfection, (e.g., lipofection mediated and DEAE-Dextrin mediated
transfection), incubation with calcium phosphate DNA precipitate,
high velocity bombardment with DNA-coated microprojectiles, and
protoplast fusion. General transformation techniques are known in
the art (See, e.g., Ausubel et al., (1987), supra, chapter 9; and
Sambrook (1989) supra, and Campbell et al., (1989) Curr. Genet.
16:53-56). See also WO 05/001036; U.S. Pat. No. 6,022,725; U.S.
Pat. No. 6,103,490; U.S. Pat. No. 6,268,328; and published U.S.
patent applications 20060041113, 20060040353, 20060040353 and
20050208623, which publications are incorporated herein by
reference. In one embodiment, the preparation of Trichoderma for
transformation includes the preparation of protoplasts from fungal
mycelia. (See, Campbell et al., (1989) Curr. Genet. 16:53-56). In
certain embodiments, the mycelia are obtained from germinated
vegetative spores.
[0074] Once it is secreted in to culture medium, the
transglucosidase enzyme may be recovered by any convenient method,
e.g., by precipitation, centrifugation, affinity, filtration or any
other method known in the art. For example, affinity chromatography
(Tilbeurgh et al., (1984) FEBS Lett. 16:215); ion-exchange
chromatographic methods (Goyal et al., (1991) Biores. Technol.
36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol.
17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and
Ellouz et al., (1987) Chromatography 396:307), including
ion-exchange using materials with high resolution power (Medve et
al., (1998) J. Chromatography A 808:153; hydrophobic interaction
chromatography (Tomaz and Queiroz, (1999) J. Chromatography A
865:123; two-phase partitioning (Brumbauer, et al., (1999)
Bioseparation 7:287); ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
or gel filtration using, e.g., Sephadex G-75, may be employed. In
particular embodiments, the transglucosidase may be used without
purification from the other components of the culture medium. In
these embodiments, the culture medium may simply be concentrated,
for example, and then used without further purification of the
protein from the components of the growth medium, or used without
any further modification.
[0075] A method of degrading a natural gum polysaccharide is also
provided. The method includes combining (e.g., mixing) a
transglucosidase enzyme with a natural gum polysaccharide to
degrade the natural gum polysaccharide. Conditions suitable for the
activity of the transglucosidase enzymes (e.g., the temperature
range, pH range, and other reaction components suitable for the
activity of a transglucosidase enzyme) are known in the art.
Cleaning Methods
[0076] In addition to the above-described
transglucosidase-containing compositions, cleaning methods are also
provided. These methods generally include: a) contacting an object
soiled with a natural gum polysaccharide with a cleaning
composition comprising a transglucosidase enzyme; and b)
maintaining the object and cleaning composition together under
conditions sufficient to effect degradation of the natural gum
polysaccharide and thereby clean the object.
[0077] The cleaning composition employed in this method may be,
e.g., a fabric cleaning composition (e.g., a laundry detergent), a
surface cleaning composition, a dish cleaning composition, an
automatic dishwasher detergent composition or the like.
Formulations for exemplary cleaning compositions are described in
great detail in WO0001826, which is incorporated by reference
herein.
[0078] In a particular embodiment, the subject cleaning composition
(e.g., laundry detergent) may contain from about 1% to 80%, e.g.,
5% to 50% (by weight) of surfactant, which may be a non-ionic
surfactant, cationic surfactant, an anionic surfactant or a
zwitterionic surfactant, or any mixture thereof, e.g., a mixture of
anionic and nonionic surfactants. Exemplary surfactants include:
alkyl benzene sulfonate (ABS), including linear alkyl benzene
sulfonate and linear alkyl sodium sulfonate, alkyl phenoxy
polyethoxy ethanol (e.g., nonyl phenoxy ethoxylate or nonyl
phenol), diethanolamine, triethanolamine and monoethanolamine.
Exemplary surfactants that may be present in detergents,
particularly laundry detergents, are described in U.S. Pat. Nos.
3,664,961, 3,919,678, 4,222,905, and 4,239,659.
[0079] The subject cleaning composition may be in solid (e.g., in
powder or tablet form), liquid form or gel, and may further contain
a buffer such as sodium carbonate, sodium bicarbonate, or detergent
builder, bleach, bleach activator, an enzyme, an enzyme stabilizing
agent, suds booster, suppresser, anti-tarnish agent, anti-corrosion
agent, soil suspending agent, soil release agent, germicide, pH
adjusting agent, non-builder alkalinity source, chelating agent,
organic or inorganic filler, solvent, hydrotrope, optical
brightener, dye or perfumes. The subject cleaning composition may,
in certain embodiments, be combined with a detergent before use as
a laundry additive.
[0080] In certain embodiments, the subject cleaning composition may
contain a further non-starch food polysaccharide degrading enzyme,
e.g., hemicellulase, mannanase, pectinase, xylanase, or pectate
lyase and, optionally, one or more other enzymes such as a protease
for instance a subtilisin protease and/or SSI protein, or a lipase,
amylase, cellulase, cutinase, lipase, oxidoreductase, etc., for the
removal of other stains.
[0081] A wide variety of other ingredients useful in detergent
cleaning compositions can be included in the compositions herein,
including: other active ingredients, carriers, hydrotropes,
processing aids, dyes or pigments, solvents for liquid
formulations, etc. If an additional increment of sudsing is
desired, suds boosters such as the C.sub.10-C.sub.16 alkolamides
can be incorporated into the compositions, typically at about 1% to
about 10% of the composition by weight.
[0082] A detergent composition can contain water and other solvents
as carriers. Low molecular weight primary or secondary alcohols
exemplified by methanol, ethanol, propanol, and isopropanol are
suitable. Monohydric alcohols are preferred for solubilizing
surfactants, but polyols, such as those containing from about 2 to
about 6 carbon atoms and from about 2 to about 6 hydroxy groups
(e.g., 1,3-propanediol, ethylene glycol, glycerine, and
1,2-propanediol) can also be used. The compositions may contain
from about 5% to about 90%, typically from about 10% to about 50%
of such carriers.
[0083] The detergent compositions herein can be formulated such
that during use in aqueous cleaning operations, the wash water may
have a pH between about 5.0 and about 11.5. Finished products,
thus, are typically formulated at this range. Techniques for
controlling pH at recommended usage levels include the use of
buffers, alkalis, acids, etc., and are well known to those skilled
in the art. In particular embodiments, the cleaning composition may
be an automatic dishwashing detergent that has a working pH in the
range of pH 9.0 to pH 11.5, e.g., pH 9.0 to pH 9.5, pH 9.5 to pH
10.0, pH 10.0 to pH 10.5, pH 10.5 to pH 11.0 or pH 11.0 to pH 11.5.
In certain other embodiments, the cleaning composition may be a
liquid laundry detergent that has a working pH in the range of pH
7.5 to pH 8.5, e.g., pH 7.5 to pH 8.0 or pH 8.0 to pH 8.5. In other
embodiments, the cleaning composition may be a solid laundry
detergent that has a working pH in the range of pH 9.5 to pH 10.5,
e.g., pH 9.5 to pH 10.0 or pH 10.0 to pH 10.5.
[0084] Various bleaching compounds, such as the percarbonates,
perborates and the like, can be used in such compositions,
typically at levels from about 1% to about 15% by weight. If
desired, such compositions can also contain bleach activators such
as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and
the like, which are also known in the art. Usage levels typically
range from about 1% to about 10% by weight.
[0085] Various soil release agents, especially of the anionic
oligoester type, various chelating agents, especially the
aminophosphonates and ethylenediaminedisuccinates, various clay
soil removal agents, especially ethoxylated tetraethylene
pentamine, various dispersing agents, especially polyacrylates and
polyasparatates, various brighteners, especially anionic
brighteners, various suds suppressors, especially silicones and
secondary alcohols, various fabric softeners, especially smectite
clays, and the like can all be used in such compositions at levels
ranging from about 1% to about 35% by weight. Standard formularies
and published patents contain multiple, detailed descriptions of
such conventional materials.
[0086] Enzyme stabilizers may also be used in the cleaning
compositions. Such stabilizers include propylene glycol (preferably
from about 1% to about 10%), sodium formate (preferably from about
0.1% to about 1%) and calcium formate (preferably from about 0.1%
to about 1%).
[0087] When formulating the hard surface cleaning compositions and
fabric cleaning compositions of the present invention, the
formulator may wish to employ various builders at levels from about
5% to about 50% by weight. Typical builders include the 1-10 micron
zeolites, polycarboxylates such as citrate and oxydisuccinates,
layered silicates, phosphates, and the like. Other conventional
builders are listed in standard formularies.
[0088] Other optional ingredients include chelating agents, clay
soil removal/anti redeposition agents, polymeric dispersing agents,
bleaches, brighteners, suds suppressors, solvents and aesthetic
agents.
[0089] The cleaning method described herein is more effective at
removal of certain stains, e.g., stains from foodstuffs containing
natural gum polysaccharides, than equivalent methods that do not
employ a transglucosidase enzyme. In particular embodiments, in
comparison to an otherwise equivalent method that does not contain
a transglucosidase enzyme, the subject cleaning composition is more
effective at stain removal. Using a standard reflectometer-based
assay, for example, the subject method removes and/or discolors at
least 20%, at least 40%, at least 60%, at least 80% or, in certain
embodiments, at least 90% more stain than an equivalent method that
does not employ a transglucosidase enzyme.
[0090] In order to further illustrate the present invention and
advantages thereof, the following specific examples are given with
the understanding that they are being offered to illustrate the
present invention and should not be construed in any way as
limiting its scope.
EXAMPLES
[0091] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Expression of A. niger Transglucosidase in T. reesei
[0092] A nucleic acid encoding the mature transglucosidase enzyme
of A. niger protein was amplified from the genomic DNA of A. niger
by PCR and cloned into the vector pTrex3 to make pTrex3(AGL51M).
pTrex3(AGL51M) is illustrated in FIG. 1. The transglucosidase
protein encoded by this vector is operably linked to the CBH1
signal sequence to facilitate its secretion into the growth medium.
The transglucosidase coding sequence is flanked by the T. reesei
cbh1 promoter and terminator. The nucleotide sequence of the
expression cassette of pTrex3(AGL51M) vector is set forth in FIG.
2.
[0093] The 7.57 kb XbaI-XbaI fragment of the plasmid pTrex3(AGL51M)
was purified by agarose gel electrophoresis and used to transform
the spores of the T. reesei Morph 1.1. pyr.sup.+ strain by
electroporation. The electroporation parameters were as follows:
voltage--16 kV/cm, capacitance--25 .mu.F, resistance--50.OMEGA..
Electroporation was carried out using a suspension of freshly
harvested T. reesei spores suspended in ice-cold 1.2 M sorbitol.
Following electroporation, the spores were incubated overnight in a
rotary shaker (30.degree. C., 200 rpm) in a medium containing 1 M
sorbitol, 0.3% glucose, 0.3% Bacto peptone and 0.15% yeast extract.
The germinating spores were plated on a selective medium containing
acetamide as a sole source of nitrogen (acetamide 0.6 g/l; cesium
chloride 1.68 g/l; glucose 20 g/l; potassium dihydrogen phosphate
15 g/l; magnesium sulfate heptahydrate 0.6 g/l; calcium chloride
dihydrate 0.6 g/l; iron (II) sulfate 5 mg/l; zinc sulfate 1.4 mg/l;
cobalt (II) chloride 1 mg/l; manganese (II) sulfate 1.6 mg/l; agar
20 g/l; pH 4.25). Transformant colonies appeared in about 1 week.
Individual transformants were transferred onto fresh acetamide
selective plates and allowed to grow for 3-4 days.
[0094] Isolates showing stable growth on selective medium were used
to inoculate 5 ml of Lactose defined medium (WO 2005/001036, p. 60)
in 20.times.175 mm test tubes. The tubes were fixed in a rotary
shaker at about 45.degree. angle and shaken at 200 rpm and
28.degree. C. for 4-5 days. Electrophoretic analysis (not shown) of
the culture supernatants demonstrated the presence of a new protein
band of approximately expected molecular weight.
[0095] The production of transglucosidase activity by the
transformants was also confirmed using an enzymatic assay. The
assay was carried out in 100 mM sodium acetate buffer, pH 4.5,
containing 4 mM para-nitrophenyl-.alpha.-glucoside and 1 mg/ml BSA.
After 30 min incubation at 30.degree. C. the reaction was
terminated by the addition of an equal volume 1 M sodium carbonate
and OD.sub.405 was recorded. Typically, transformants expressed 1-2
U transglucosidase activity (expressed as micromoles of
para-nitrophenol liberated per min) per ml of culture broth. In
untransformed controls, the activity was below detection limit.
Example 2
Transglucosidase Degrades Xanthan Gum
[0096] Hydrolytic activity by enzymes on xanthan gum was measured
by the reducing sugar assay using the PAHBAH (para-hydroxybenzoic
acid hydrazide) reagent (Lever et al, Anal. Biochem. 1972 47: 273).
Xanthan gum (CAS 111 38-66-2) was purchased from Sigma Chemicals,
St. Louis Mo. and dissolved in 50 mM sodium acetate buffer pH 6.0
at a concentration of 0.2%. For some experiments AATCC standard
heavy duty liquid detergent (AATCC HDL 2003 without brightner, Test
Fabrics, Inc. West Pittston, Pa.) was added at 1.5 ml per liter
(0.15%). The AATCC HDL liquid detergent contained 12% linear alkyl
sulfonates, 8% alcohol ethoxylates, 8% propanediol, 1.2% citric
acid, 4% fatty acid and 4% sodium hydroxide with the balance being
water.
[0097] The assay was performed as follows in a 24 well microplate
(COSTAR 3526, Corning Incorporated, Corning, N.Y.): one ml of
buffer was added to well 1, one ml of buffer plus enzyme was added
to well 2, one ml of buffer and substrate to well 3, and one ml of
buffer, plus substrate and enzyme was added to well 4. For
statistical purposes, each well may be set up 2 to 4 times. Enzymes
to be tested are usually diluted in reaction buffer from 1 to 10 to
1 to 1000. After all reagents were added, a plastic cover was place
over the microplate and the cover and plate intersection was
wrapped tightly with several layers of Parafilm (Pechiney Plastic
Packing, Menasha, Wis.) to prevent evaporation. The reaction plate
was incubated for 1 to 16 hr, at 37 C on a shaker rotating at 100
rpm.
[0098] Reducing sugar activity was measured using an Eppendorf
Mastercycler Gradient (Eppendorf Scientific, Westbury, N.Y.)
thermal cycler and 0.2 ml disposable PCR (polymerase chain
reaction) strip tubes and caps purchased from VWR International,
West Chester, Pa. Reducing sugar reagent was prepared as follows:
to 10 ml of 2% sodium hydroxide in distilled water, add 0.15 g of
sodium potassium tartrate tetrahydrate (Rochelle Salt, Sigma
Chemical Co.) and 0.15 g of parahydroxybenzoic acid hydrazide
(H-9882, Sigma Chemical Co.). The solution called PAHBAH reagent
was swirled to solubilize all ingredients and put on ice in the
dark until used. This reagent was made fresh daily. Immediately
before sample analysis, 0.160 ml of PAHBAH reagent was added to
each tube of a PCR strip followed by 5 to 20 ul of enzyme samples
and controls. All tubes were capped tightly, placed in the thermal
cycler, and incubated for 15 min at 99 C followed by cooling at 4 C
for at least 15 min. After cooling, strip tube caps were removed
and 0.15 ml of each sample was placed in a 96 well flat bottomed
microplate (COSTAR 9017, Corning Inc. Corning, N.Y.) and read by a
Spectra Max 250 Plate Reader (Molecular Devices, Sunnyvale, Calif.)
at 405 nm against a blank of distilled water.
[0099] Each enzyme sample was analyzed as follows: the optical
density (OD) of the control sample was subtracted from the OD of
the buffer sample and this value was added to the substrate buffer
control. The O.D. of the enzyme plus substrate reaction was
compared to the sum of the substrate and sample controls.
[0100] FIG. 3 shows that transglucosidases produced in Trichoderma
(Trip-TG) and in Aspergillus (Mega-TG) showed significant reducing
sugar activity on xanthan gum in 50 mM acetate buffer plus AATCC
heavy duty liquid detergent at pH 6.0.
Example 3
Transglucosidase Removes Soil from Stained Patches
[0101] Salad dressing with pigment (STC CFT CS-6) and guar-pigment
(STC CFT CS-43) soiled cotton swatches were obtained from Test
Fabrics, Inc. West Pittston, Pa., USA. Swatches for the microplate
assay were cut into 15 mm circles (disks) with textile Punch Press
Model B equipped with a 5/8'' die cutter. Single swatch disks were
placed into each well of a 24-well microplate (Costar 3526). One
(1) ml of washing solution, containing per liter, 1.5 ml AATCC HDL
detergent, 50 mM Hepes buffer, and 6 to 60 ppm enzyme diluted in 50
mM Hepes buffer pH 7.4 was added to each well. The microplate was
covered with a plastic lid and aluminum foil and incubated at
37.degree. C. with 100 rpm gentle rotation for 4-16 hr. The plates
were removed from the shaker and the detergent solution was removed
by aspiration. Each microplate well was washed three (3) times with
1.5 ml of Dulbecco's PBS pH 7.3 and three (3) times with 1.5 ml of
distilled water. Each disk was removed from its well and dried
overnight between sheets of paper towels and not exposed to direct
light. Disks were inspected visually and then analyzed with a
Minolta Reflectometer CR-200 calibrated on a standard white tile.
The average L values with standard deviations were calculated.
[0102] The graphs of FIG. 4 show that Trip-TG (diluted 1/50 in 50
mM Hepes buffer) removes soil from both salad dressing stains and
guar-pigment stains in 50 mM Hepes buffer pH 7.4 and in 50 mM Hepes
buffer pH 7.4 plus 0.15% AATCC HDL.
[0103] FIG. 5 shows the results obtained in a Trip-TG dose response
microswatch experiment. Trip-TG removed salad dressing soils at 1
ppm in 0.15% AATCC heavy duty liquid.
[0104] FIG. 6 shows that Trip-TG removed salad dressing soil in a
microswatch cleaning experiment in 0.1% North American AATCC-1993
HDD standard without brightner. This detergent contained 18% linear
alkyl sulfonates, 25% Zeolite A, 18% soda ash, 0.5% sodium
silicate, 22% sodium sulfate, 10% moisture, and a 6.3% n hole for
copolymer or other additives.
[0105] This experiments showed transglucosidase soil removal
activity in both heavy duty liquids (HDL) and in heavy duty solids
(HDD).
Example 4
Analysis of Cleaning Activity of Transglucosidase Enzyme Using the
Tergotometer
[0106] Tergotometer studies used a 6 pot Tergotometer Model 7243S
(U.S. Testing, Co. Inc. Hoboken, N.J.) maintained at 30.degree. C.
Agitation speed was set to 100 rpm. Cotton swatches (5 per each
tergotometer pot) obtained from Warwick Equest Limited, Consett,
County Durham, England, stained with circles of foodstuffs were
added to 1 liter of 0.15% AATCC HDL detergent containing 6 gpg
hardness (diluted from stock 15000 gpg hardness solution containing
1.735 M calcium chloride and 0.67 M magnesium chloride) and 25 mM
Hepes buffer pH 7.4 After a 30 min wash cycle, the swatches were
washed three times in 1.5 l of cold tap water, spun for 7 min in a
spin cycle to remove excess water, and dried overnight at room
temperature. Percent soil release (% SRI) was calculated by
standard methods after analysis of each stain by reflectometer.
FIG. 7 shows that transglucosidase significantly cleaned marmalade
stain compared to a no enzyme control or a control with an
unrelated protein, bovine serum albumin (BSA-50, Fraction V,
Immunoglobulin and Protease Free) obtained from Rockland
Immunochemicals, Gilbertsville, Pa.
[0107] The above examples demonstrate that transglucosidase
effectively degrades xanthan, and removes certain soils from cotton
swatches.
[0108] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as should
not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
that are obvious to those skilled in the art and/or related fields
are intended to be within the scope of the present invention.
Sequence CWU 1
1
114879DNAArtificial Sequencesynthetic pTrex3 expression cassette
1tctagagttg tgaagtcggt aatcccgctg tatagtaata cgagtcgcat ctaaatactc
60cgaagctgct gcgaacccgg agaatcgaga tgtgctggaa agcttctagc gagcggctaa
120attagcaaaa ggctatgaga aattctggag acggcttgtt gaatcatggc
gttccattct 180tcgacaagca aagcgttccg tcgcagtagc aggcactcat
tcccgaaaaa actcggagat 240tcctaagtag cgagaaccgg aataatataa
taggcaatac attgagttgc ctcgacggtt 300gcaatgcagg ggtactgagc
ttggacataa ctgttccgta ccccacctct tctcaacctt 360tggcgtttcc
ctgattcagg tcccgtacaa gtcgtaatca ctattaaccc agactgaccg
420gacgtgtttt gcccttcatt tggagaaata atgtcattgc gatgtgtaat
ttgcctgctt 480gaccgactgg ggctgttcga agcccaatta ggattgttat
ccgaactctg ctcgtagagg 540catgttgtga atctgtgtcg ggcaggacac
gcctcgaagg ttcacggcaa gggaaaccac 600cgatagcagt gtctagtagc
aacctgtaaa gcgcatgcag catcactgga aaatacaaac 660caatggctaa
aagtacataa gttaatgcct aaagaagtca tataccagcg gctaataatt
720gtacaatcaa gtggctaaac gtaccgtaat ttgccaaggc tttggggttg
cagaagcaac 780ggcaaagccc cacttcccca cgtttgtttc ttcactcagt
ccaatctcag ctggtgatcc 840cccaattggg tcgcttgttt gttccggtga
agtgaaagaa gacgaggtag aatgtctgac 900tcggagcgtt ttgcatacaa
ccaagggcag tgatggaaga cagtgaaatg ttgacattca 960aggagtattt
agccagggat gcttgagtgt atcgtgtaag gaggtttgtt gccgatcgac
1020gaatactgta tagtcacttc tgatgaagtg gtccatattg aaatgtaagt
cggcactgaa 1080caggcaaaag attgagttga aactgcctaa gatctcgggc
cctcgggcct tcggctttgg 1140gtgacatgtt tgtgctccgg gcaaatgcaa
agtgtggtag gatcgaacac actgctgcct 1200ttaccaagca gctgagggta
tgtgataggc aaatgttcag gggccactgc atggtttcga 1260aagaaagaga
gcttagccaa gaacaatagc cgataaagat agcctcatta aacggaatga
1320gctagtaggc aaagtcagcg aatgtgtata tataaaggtt cgaggtccgt
gcctccctca 1380tgctctccca tctactctca actcagatcc tccaggagac
ttgtacacca tcttttgagg 1440cacagaaacc caatagtcaa ccgcggactg
cgcatcatgt atcggaagtt ggccgtcatc 1500tcggccttct tggcacagct
cgtgttccac cactgcccct tcgcagccgc agtttaccat 1560tcctgcttcc
gcagatgtcg gtgcgcagct gattgccaac atcgatgatc ctcaggctgc
1620cgacgcgcag tcggtttgtc gggctacaag gttcaaaagt gcagcacaat
tcacgtggat 1680tcactgccag tcttcagctc gcgggcaggc catgtaacgt
atacggcaca gatgttgagt 1740ccttgacact gtctgtggag taccagattc
ggatcgacga atattcagat tctccccact 1800catgttgact ccacaaacgc
ttcttggtac tttctttcgg aaaacctggt ccccagaccc 1860aaggcttccc
tcaatgcatc tgtatcccag acgacctttt tgtgtatggt caaatgagcc
1920gtcgttcaat ttcaaggtga tccgaaaggc tacaggcgac gcgcttttca
gtacagaagg 1980cactgtgctc gtatatgaga atcagttcat cgaattttga
ccgcgctccc tgagaatata 2040acttgtatgg ccttggggag catatcacgc
aattccgcct ccagagaaat gctaatctga 2100ccatatatcc ttcggatgat
ggaacaccta ttgaccagtg agtctgatat cccgcccgtt 2160cttctggttc
tactcttgaa acttactcgt cctagaaacc tctacggcca acatcccttc
2220tatctggata caagatatta caaaggagat aggcagaatg ggtcttatat
cccgtcaaaa 2280gcagcgggct gatgcctcgc aagattatat ctccctctct
catggcgtgt ttctgaggaa 2340ctctcatgga cttgagatac tcctccggtc
tcaaaaattg atctggcgga ccctagtgga 2400ggaatcgatc tcacttctac
tcaggccccg ccccggccga tgttaccagg caatatctta 2460ccagcactgt
gggattaccg gccatgcagc aatacaacac tcttggattc caccaatgtc
2520gtggggctac aacaactggt ggatctggcg gacgttgttg cgaactttga
gaagtttgag 2580atcccgttgg aatatatctg gtgcgtattg tactggttta
tggtatctca aaacagtcta 2640acaggcatta ggaccgatat tgactactgc
acggatatcg caactttgac aacgatcaac 2700atcgcttttc ctacagtgag
ggcgatgaat ttctcagcaa gctacatgag agtggacgct 2760actatgtacc
catgttgatg cggcgctcta cattctaatc ccgaaaatgc ctctgatgcg
2820taagtgtcta gtgacaaatt atattactgc ctgtatgcta attagcgata
cagatacgct 2880acgtatgaca gaggagctgg gacgacgtct tcctcaagaa
tccgatggta gcctctatat 2940tggagccgtt tggccaggat atacagtctt
ccccgattgg catcatccca aggcagttga 3000cttctgggct aacgagcttg
ttatcggtcg aagaaagtgg cgttcgatgt gtgtggtacg 3060acatgtctga
agtttcatcc ttctgtgtcg ggagctgtgg cacaggtaac ctgactctga
3120acccggcaca cccatcgttt cttctccccg ggagcctggt gatatcatat
atgataccca 3180gaggctttca atatcaccaa cgctacagag gcggcgtcag
cttcggcggg agcttccagt 3240caggctgcag caaccgcgac caccacgtcg
acttcggatc atatctgcgg acaacgccca 3300cgctggtgtc cgcaatgttg
agcacccacc ctatgtgatc aaccatgacc aagaaggcca 3360tgatctcagt
gtccatgcgg tgtcgccgaa tgcaacgcat gttatggtgt tgaggagtat
3420gatgtgcacg tctctacgga catcaaggat tgaacgctac ctaccaaggt
ctgcttgagg 3480tctggtctca taagcggcgg ccatttatta ttggccgctc
aaccttcgcg gctctggcaa 3540atgggcaggc cactgggcgg cgacaactat
tccaaatggt ggtccatgta ctactccatc 3600tcgcaagccc tctccttctc
acttttcggc attccgatgt ttggtgcgga caccttgggt 3660ttaacggaaa
ctccgatgag gagtctgcaa ccgatggatg caactgtccg cattcttccc
3720attctaccga aaccacaatg agctctccac aatcccacag gagccttatc
ggtgggcttc 3780tttattgaag caaccaagtc cgccatgaga ttcggtacgc
catcctacct tacttttata 3840cgttgtttga cctggcccac accacgggct
ccactgtaat gcgcgcactt tcctgggaat 3900tccctaagac ccaacattgg
ctgcggttga gactcaatca tggttgggcc ggccatcatg 3960gtggtcccgg
tattggagcc tctggtcaat acggtcaagg gcgtattccc aggagttgga
4020catggcgaag tgtgtacgat tggtacaccc aggctgcagt tgatcgaagc
ccggggtcaa 4080cacgaccatt tcggcaccat tgggccacat cccagtttat
gtacgaggtg gaaacatctt 4140gccgatgcaa gagccggcat gaccactcgt
gaagcccggc aaaccccgtg gctttgctag 4200ctgcactagg aagcaatgga
accgcgtcgg ggcagctcta tctcgatgat ggagagagca 4260tctaccccaa
tgccaccctc catgtgactt cacggcatcg cggtcaagcc tgcgctcgcg
4320gctcaaggaa gatggaaaga gaggaacccg cttgctaatg tgacggtgct
cggagtgaac 4380aaggagccct ctgcggtgac cctgaatgga cggccgtatt
tcccgggtct gtcacgtaca 4440attctcgtcc caggttctct ttgttggggg
gctgcaaaac ttgacgaagg gcggcgcatg 4500ggcggaaaac tgggtattgg
aatggtagtg ggcgcgcgcg cgccagctcc gtgcgaaagc 4560ctgacgcacc
ggagattctt ggtgagcccg tatcatgacg gcggcgggag ctacatggcc
4620ccgggtgatt tatttttttt gtatctactt ctgacccttt tcaatatacg
gtcaactcat 4680ctttcactgg agatgcggct gcttggtatt gcgatgttgt
cagcttggca aattgtggct 4740ttcgaaaaca caaaacgatt ccttagtagc
catgcatttt aagataacga atagaagaaa 4800gaggaaatta aaaaaaaaaa
aaaaacaaca tcccgttcat aacccgtaga atcgccgctc 4860ttcgtgtatc
ccagtacca 4879
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