Note: Descriptions are shown in the official language in which they were submitted.
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CLEANING COMPOSITIONS COMPRISING TRANSGLUCOSIDASE
FIELD OF THE INVENTION
[01] The present invention provides compositions comprising a transglucosidase
enzyme and
a natural gum polysaccharide, wherein the natural gum polysaccharide is a
substrate for the
transglucosidase enzyme. The present invention also provides methods for using
a
transglucosidase enzyme to degrade natural gum polysaccharide. In some
preferred
embodiments, the compositions and methods find use in cleaning applications.
BACKGROUND OF THE INVENTION
[02] Detergent and other cleaning compositions often include a complex
combination of
active ingredients. For example, many 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. However, in spite of the complexity of
current detergents,
there are many stains that are difficult to remove. Thus, there remains a need
in the art for
compositions and methods for the effective cleaning of various stains.
SUMMARY OF THE INVENTION
[03] The present invention provides compositions comprising a transglucosidase
enzyme and
a natural gum polysaccharide, wherein the natural gum polysaccharide is a
substrate for the
transglucosidase enzyme. The present invention also provides methods for using
a
transglucosidase enzyme to degrade natural gum polysaccharide. In some
preferred
embodiments, the compositions and methods find use in cleaning applications.
[04] In some embodiments, the compositions comprise natural gum
polysaccharides such as
xanthan gums. In some further embodiments, the transglucosidase enzyme has an
activity
defined as EC 2.4.1.24, according to IUBMB nomenclature. In some additional
embodiments,
the transglucosidase enzyme has an amino acid sequence that is at least about
90% identical to
an Aspergillus transglucosidase enzyme. In some still further embodiments, the
composition
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further comprises at least one surfactant. In some additional embodiments, the
natural gum
polysaccharide is present as a stain on an object. In some particularly
preferred embodiments,
the object is a fabric.
[05] The present invention also provides methods comprising combining a
transglucosidase
enzyme with at least one natural gum polysaccharide to degrade the natural gum
polysaccharide.
In some embodiments, the transglucosidase enzyme has an activity defined as EC
2.4.1.24,
according to IUBMB nomenclature. In some further embodiments, the natural gum
polysaccharide is a xanthan gum.
[06] The present invention also provides cleaning methods comprising:
contacting an object
soiled with a natural gum polysaccharide with a cleaning composition
comprising a
transglucosidase enzyme; and maintaining the object and cleaning composition
under conditions
sufficient to effect degradation of the natural gum polysaccharide and thereby
clean the object.
In some embodiments, the object is soiled with a food that contains at least
one natural gum
polysaccharide. In some further embodiments, the natural gum polysaccharide
comprises a
xanthan gum. In some still further embodiments, the object is selected from
fabrics and hard
surfaces. In some additional embodiments, the transglucosidase enzyme has an
amino acid
sequence that is at least about 90% identical to an Aspergillus
transglucosidase enzyme. In some
embodiments, the cleaning composition further comprises at least one
surfactant. In some
further embodiments, the 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. In some
yet additional
embodiments, the cleaning composition has a pH of about pH 5.0 to about pH
11.5. In some
still further embodiments, the cleaning composition comprises the
transglucosidase enzyme at a
concentration in the range of about 0.01 ppm to about 100 ppm.
[07] In some embodiments, the present invention provides compositions
comprising: a
transglucosidase enzyme; and at least one natural gum polysaccharide; wherein
the natural gum
polysaccharide is a substrate for the transglucosidase enzyme. In some
embodiments, the natural
gum polysaccharide comprises a xanthan and/or guar gum. However, it is not
intended that the
present invention be limited to any particular gum polysaccharide. In some
preferred
embodiments, the natural gum polysaccharide is present as a stain on an object
(e.g., a fabric),
while in other embodiments, it is free in solution. In some embodiments, the
transglucosidase
enzyme comprises an amino acid sequence that is at least about 70% identical
to, at least about
80% identical to, at least about 85% identical to, at least about 90%
identical to, at least about
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95% identical to, or at least about 98% identical to an Aspergillus
transglucosidase. In some
embodiments, the compositions further comprise at least one surfactant and/or
other cleaning
agent.
[08] The present invention also provides methods that include combining a
transglucosidase
enzyme with at least one natural gum polysaccharide to degrade the natural gum
polysaccharide.
In some embodiments, the methods comprise: contacting an object soiled with a
natural gum
polysaccharide with a cleaning composition comprising a transglucosidase
enzyme; and
maintaining the object and cleaning composition together under conditions
sufficient to effect
degradation of the natural gum polysaccharide and thereby clean the object.
[09] In some embodiments, the object (e.g., a fabric) is soiled with a food
that contains at
least one natural gum polysaccharide (e.g., a food that contains at least one
xanthan and/or guar
gum). In some embodiments, the cleaning composition further comprises at least
one surfactant,
other cleaning agents, and/or at least one additional enzyme (e.g., a
protease, amylase, cellulase,
lipase, cutinase, oxido-reductase, or the like), for the degradation of other
stain components. In
some embodiments, the cleaning compositions have a pH in the range of about pH
5.0 to about
pH 11.5, and the transglucosidase enzyme is present in the cleaning
composition at a
concentration in the range of about 0.01 ppm to about m.
[010] In some embodiments, the methods provided herein result in more
efficient removal of
stains that contain natural gum polysaccharides than equivalent methods that
do not employ a
transglucosidase enzyme.
[011] In some further embodiments, the present invention provides an isolated
enzyme
containing an Aspergillus transglucosidase produced by a Trichoderma reesei
host cell. The
present invention also provides cleaning compositions (e.g., laundry
detergents), containing the
enzyme are provided, as well as well as a method of using the enzyme for
cleaning an object
(e.g., a fabric).
DESCRIPTION OF THE DRAWINGS
[012] 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.
[013] Figure 1 provides a vector map of pTrex3(AGL51 M).
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[014] Figure 2 provides the nucleotide sequence of an expression cassette of
pTrex3(AGL51M) (SEQ ID NO:1).
[015] Figure 3 provides a graph showing the enzymatic activity of
transglucosidase on 0.2%
xanthan, as measured by a reducing sugar assay.
[016] Figure 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).
[017] Figure 5 provides a graph of a dose response experiment, showing that
Trip-TG is active
on salad dressing soiled microswatches at a concentration of I to 5 ppm in
AATCC HDL.
[018] Figure 6 provides a graph showing Trip-TG cleaning activity on salad
dressing
microswatches in heavy duty solid detergent (HDD).
[019] Figure 7 provides a graph showing Trip-TG cleaning activity on marmalade
stain in a
Tergotometer assay using 0.15% AATCC HDL.
DESCRIPTION OF THE INVENTION
[020] The present invention provides compositions comprising a
transglucosidase enzyme and
a natural gum polysaccharide, wherein the natural gum polysaccharide is a
substrate for the
transglucosidase enzyme. The present invention also provides methods for using
a
transglucosidase enzyme to degrade natural gum polysaccharide. In some
preferred
embodiments, the compositions and methods find use in cleaning applications.
[021] 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.
[022] As used herein, 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
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of those included limits are also included in the invention.
[023] 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 patents and publications mentioned herein are incorporated
herein by
5 reference to disclose and describe the methods and/or materials, including
sequences, in
connection with which the publications are cited. Any GENBANK database
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.
[024] 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.
[025] 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. Although many methods and materials similar or equivalent
to those
described herein find use in the practice or testing of the present invention,
some of 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.
Definitions
[026] 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.
[027] 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.
[028] The term "recombinant" refers to a polynucleotide or polypeptide that
does not naturally
occur in a host cell. In some embodiments, recombinant molecules contain two
or more
naturally-occurring sequences that are linked together in a way that does not
occur naturally. A
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recombinant cell contains a recombinant polynucleotide or polypeptide.
[029] 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.
[030] The terms "protein" and "polypeptide" are used interchangeably herein,
in context.
[031] 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.
[032] A "coding sequence" is a DNA segment that encodes a polypeptide.
[033] 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, in context.
[034] A "vector" refers to a polynucleotide designed to introduce nucleic
acids into one or
more host cells. Suitable vectors autonomously replicate in different host
cells and include:
cloning vectors, expression vectors, shuttle vectors, plasmids, phage
particles, cassettes and the
like.
[035] An "expression vector" as used heiein 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. In some embodiments, such
control sequences
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.
[036] A "promoter" is a regulatory sequence that initiates transcription of a
downstream
nucleic acid.
[037] 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.
[038] The term "selective marker" refers to a protein capable of expression in
a host that allows
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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.
[039] The term "derived" encompasses the terms "originated from," "obtained,"
"obtainable
from," and "isolated from" and refers to the source of a sequence and/or
protein.
[040] A "non-pathogenic" organism is an organism that is not pathogenic to
human and/or
other animals.
[041] 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.
[042] 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.
[043] 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.
[044] The term "introduced" in the context of inserting a nucleic acid
sequence into a cell,
refers to "transfection," "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).
[045] 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 to those
of skill in the art. One example of high stringency conditions include
hybridization at about
42 C in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ug/ml
denatured
carrier DNA followed by washing two times in 2X SSC and 0.5% SDS at room
temperature and
two additional times in 0.1 X SSC and 0.5% SDS at 42 C.
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[046] 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 and/or
fabric to be
cleaned, and the desired form of the composition for the cleaning conditions
during use.
[047] It is intended that the terms include, but are not limited to detergent
compositions (e.g.,
liquid, gel, 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).
[048] 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.
[049] 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,
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caking inhibitors, masking agents, enzyme activators, antioxidants, and/or
solubilizers, etc.
[050] As used herein, "enhanced performance" in a cleaning composition is
defined as
increasing cleaning (e.g., removal and/or decolorization) of stains
(particularly those comprising
natural gum polysaccharide-related stains such as chocolate cream, salad
dressing, guar, etc.), as
determined by usual evaluation after a standard wash cycle.
[051] 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.
[052] As used herein, "dishwashing composition" refers to all forms for
compositions for
cleaning dishes, including but not limited to gel, granular and liquid forms.
[053] As used herein, "fabric cleaning composition" refers to all forms of
detergent
compositions for cleaning fabrics, including but not limited to, gel,
granular, liquid and bar
forms.
[054] 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.
[055] 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).
[056] 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.
[057] 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.
[058] The term "transglucosidase" refers to an enzyme that transfers an a-D-
glucosyl residue in
a 1,4-a-D-glucan to the primary hydroxy group of glucose, free or combined in
a 1,4-a-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-a-D-glucan:1,4-a-D-glucan(D-glucose) 6-a-D-glucosyltransferase. This
enzyme may be
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referred to as a-glucosidase in certain publications.
[059] 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 stained with foodstuffs
containing a natural
5 gum polysaccharide. In some embodiments, the stain exhibits a visible color,
while in other
embodiments, it does not exhibit a visible color.
[060] 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,
10 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 Xanthomonas campestris), are examples of natural gum
polysaccharides. Other
natural gum polysaccharides include, but are not limited to agar (E406),
alginic acid (E400), (3-
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 (E40 1), spruce gum, and tara gum
(E417).
[061] 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, (3-galactosidase and a-
galactosidase.
[062] 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 and/or during hand washing of laundry. Likewise, the working
pH of a
dishwashing detergent is the pH of that detergent as it is being used in a
dishwasher and/or
during hand washing of dishes. In some embodiments, detergents that are in
concentrated or
solid form are diluted or dissolved before the pH of that detergent is at its
working pH.
[063] 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 and/or during hand washing of laundry. 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 and/or during hand washing of
dishes. In some
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embodiments, detergents that are in concentrated or solid form are diluted or
dissolved before
the concentration of an enzyme in a detergent is at its working concentration.
DETAILED DESCRIPTION OF THE INVENTION
[064] The present invention provides compositions comprising a
transglucosidase enzyme and
a natural gum polysaccharide, wherein the natural gum polysaccharide is a
substrate for the
transglucosidase enzyme. The present invention also provides methods for using
a
transglucosidase enzyme to degrade natural gum polysaccharide. In some
preferred
embodiments, the compositions and methods find use in cleaning applications.
Indeed the
present invention provides various compositions containing transglucosidase
enzymes, as well
as methods of using the compositions.
Transglucosidase Enzyme-Containinti Compositions
[065] As noted above, the present invention provides compositions comprising
transglucosidase. In some embodiments, the composition comprises at least one
transglucosidase
enzyme, and a natural gum polysaccharide, where the natural gum polysaccharide
is a substrate
for the transglucosidase enzyme.
[066] 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 some
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.
[067] Any suitable transglucosidase enzyme finds use in the present invention
(See e.g., Pazur
et al., Carbohydr. Res. 1986 149:137-47 [1986]; and Nakamura et al., J.
Biotechnol., 53:75-84
[1997]). In some embodiments, the transglucosidase enzyme that find use in the
present
invention are commercially available (e.g., including but not limited to
enzymes obtained from
Megazyme, Wicklow, Ireland; or Danisco US Inc., Genencor Division, Palo Alto,
CA). In some
embodiments, the enzyme is an Aspergillus niger transglucosidase produced in
Trichoderma
reesei cells. In some additional embodiments, the transglucosidase is a wild
type fungal
transglucosidase (e.g., including but not limited to 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),
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BAA08125.1 (GID:1054565; Aspergillus oryzae), XP_001210809.1 (GID:115492363;
Aspergillus terreus), XP_001271891.1 (GID:121707620; Aspergillus clavatus),
XP_001266999.1 (GID:119500484; Neosartoryafischeri), XP_751811.1
(GID:70993928;
Aspergillusfumigatus), XP_659621.1 (GID:67523121; Aspergillus nidulans),
XP_001216899.1
(GID:115433524; Aspergillus terreus) and XP_001258585.1 (GID:119473371;
Neosartorya
frscheri)), or a variant thereof that has an amino acid sequence that is at
least about 70%
identical, at least about 80% identical, at least about 85% identical, at
least about 90% identical,
at least about 95% identical, or at least about 98% identical to a wild type
fungal
transglucosidase.
[068] In some preferred embodiments, the enzyme is generally present in the
composition at a
concentration in the range of about 0.01 ppm (parts per million, w/v) to about
100 ppm (e.g.,
about 0.01 ppm to about 0.05 ppm, about 0.05 ppm to about 0.1 ppm, about 0.1
ppm to about 0.5
ppm, about 0.5 ppm to about 1 ppm, about 1 ppm to about 5 ppm, about 5 ppm to
about 10 ppm,
or about I Oppm to about 100ppm).
[069] In some preferred embodiments, the composition is a cleaning
composition. In some
particularly preferred embodiments, the composition further comprises at least
one surfactant,
cleaning agents, and/or other fabric care agents described in greater detail
below.
[070] In some embodiments, the natural gum polysaccharide is in an aqueous
solution, while in
other embodiments it is affixed to an object (e.g., as a stain that is present
on the surface of an
object such as a fabric or hard surface). In some embodiments, the natural gum
polysaccharide is
in dried form. Since natural gum polysaccharides are commonly employed in a
variety of
foodstuffs, in some embodiments, the object is soiled with a foodstuff that
contains a natural
gum polysaccharide. Exemplary foodstuffs that contain a natural gum
polysaccharide include,
but are not limited to 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. In some embodiments, the natural gum
polysaccharide is present in
the foodstuff at a concentration of about 0.1% to about 1.5%, about 0.1% to
about 0.5%, about
0.5% to about 1.0%, about 1.0%, or about 1.5%.
[071 ] In some preferred embodiments, the transglucosidase is produced using
conventional
methods. For example, in some embodiments, it is 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 Actinomycetes), or eukaryotic hosts (e.g.,
Trichoderma,
Aspergillus, Saccharomyces, and Pichia).
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13
[072] In some embodiments, the transglucosidase enzyme is produced by
expressing a fusion
protein containing a signal sequence operably linked to the transglucosidase
enzyme in a T.
reesei host cell. In some of these embodiments, the transglucosidase enzyme is
secreted into
culture medium, from which it is harvested. Any signal sequence finds use in
suitable fusion
proteins, such that protein secretion from the Trichoderma host cell is
facilitated. In some
embodiments, the signal sequence is endogenous, while in other embodiments it
is non-
endogenous to the Trichoderma host cell and, in some embodiments, it is 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,
a-amylase,
aspartyl proteases, glucoamylase, mannanase, glycosidase and barley
endopeptidase B (See e.g.,
Saarelainen, Appl. Environ. Microbiol., 63: 4938-4940 [1997]). In some
embodiments, and as
further described in the Examples section of this disclosure, the
transglucosidase is secreted
using its own signal sequence (i.e., the AGLI, AGL2 or AGL3 signal sequences).
[073] In some embodiments, therefore, the transglucosidase is produced using a
nucleic acid
that comprises: 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.
[074] In some embodiments, the fusion protein further contains, 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,
but are not limited to
those of T. reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase
II (Ce16A, or
CBHII) (See e.g., Paloheimo et al., Appl. Environ. Microbiol., 69: 7073-7082
[2003]) or T.
reesei cellobiohydrolase I (CBHI). In some embodiments, the carrier protein is
a truncated T.
reesei CBH 1 protein that includes the CBH 1 core region and part of the CBH I
linker region.
Thus, in some embodiments, 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 are employed.
[075] In some embodiments, the coding sequence of the transglucosidase is
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., 28:292 [2000]) or readily derivable,
such nucleic acids
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14
can be readily designed giving the amino acid sequence of a transglucosidase
to be expressed.
[076] In some embodiments, in addition to a coding sequence, the nucleic acid
further contains
other elements that are necessary for expression of the transglucosidase
enzyme in the host cell.
For example, in some embodiments, the nucleic acid contains a promoter for
transcription of the
coding sequence, and a transcriptional terminator. Exemplary promoters that
find use in T.
reesei include, but are not limited to the T. reesei cbhl, cbh2, egll, egl2,
eg5, xlnl and xln2
promoters, or a hybrid or truncated version thereof. For example, in some
embodiments, the
promoter is a T. reesei cbhl promoter. Suitable terminators include the T.
reesei cbhl, cbh2,
egll, egl2, eg5, xlnl and xln2 terminators, and many others, including, for
example, the
terminators from A. niger or A. awamori glucoamylase genes, as known to those
of skill in the
art, as well as Aspergillus nidulans anthranilate synthase genes, Aspergillus
oryzae TAKA
amylase genes, orA. nidulans trpC (Punt et al., Gene 56:117-124 [1987]). In
some
embodiments, the promoter and/or terminator are native to the Trichoderma sp.
host cell, while
in other embodiments, they are non-endogenous to the Trichoderma sp. host
cell.
[077] In some embodiments in which a T. reesei host cell is employed for
expression of the
transglucosidase enzyme, the cell is genetically modified to reduce expression
of secreted
proteins that are endogenous to the cell. In some embodiments, the cell
comprises one or more
native genes, particularly genes that encode secreted proteins, that have been
deleted or
inactivated. For example, in some embodiments, one or more protease-encoding
genes (e.g., an
aspartyl protease-encoding gene; See, Berka et al, Gene 86:153-162 [1990]; and
US Pat. No.
6,509,171) or cellulase-encoding genes are deleted or inactivated. In some
embodiments, the
Trichoderma sp. host cell is a T. reesei host cell containing inactivating
deletions in the cbhl,
cbh2 and egll, and egl2 genes, as described in WO 05/001036. In some
embodiments, the
above-described nucleic acid is present in the nuclear genome of the
Trichoderma sp. host cell,
while in other embodiments, it is present in a plasmid that replicates in the
Trichoderma host
cell,.
[078] Any suitable method for introducing nucleic acids into Trichoderma host
cells finds use
in the present invention (e.g., electroporation, nuclear microinjection,
transduction, transfection,
lipofection-mediated and DEAE-dextrin mediated transfection, incubation with
calcium
phosphate DNA precipitate, high velocity bombardment with DNA-coated
microprojectiles, and
protoplast fusion). Indeed, general transformation techniques are well known
in the art (See e.g.,
Campbell et al., Curr. Genet., 16:53-56 [1989]; WO 05/001036; US Pat. No.
6,022,725; US Pat.
No. 6,103,490; US Pat No. 6,268,328; and published U.S. Patent Appln Ser. Nos.
20060041113,
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WO 2008/118382 PCT/US2008/003788
20060040353, 20060040353 and 20050208623, which publications are incorporated
herein by
reference). In some embodiments, the preparation of Trichoderma for
transformation includes
the preparation of protoplasts from fungal mycelia. (See e.g.,, Campbell et
al., supra). In some
embodiments, the mycelia are obtained from germinated vegetative spores.
5 [079] In some embodiments, once it is secreted into culture medium, the
transglucosidase
enzyme is recovered using any suitable, convenient method (e.g., by
precipitation,
centrifugation, affinity, filtration or any other method known in the art. For
example, affinity
chromatography (Tilbeurgh et al., FEBS Lett., 16:215 [1984]); ion-exchange
chromatographic
methods (Goyal et al., Biores. Technol., 36:37 [1991]; Fliess et al., Eur. J.
Appl. Microbiol.,
10 Biotechnol. 17:314 [1983]; Bhikhabhai et al., J. Appl. Biochem., 6:336
[1984]; and Ellouz et al.,
Chromatogr., 396:307 [1987]), including ion-exchange using materials with high
resolution
power (Medve et al., J. Chromatogr. A 808:153 [1998]; hydrophobic interaction
chromatography (Tomaz and Queiroz, J. Chromatogr. A 865:123 [1999]; two-phase
partitioning
(Brumbauer et al., Bioseparation 7:287 [1999]); ethanol precipitation; reverse
phase HPLC;
15 chromatography on silica or on a cation-exchange resin such as DEAE;
chromatofocusing; SDS-
PAGE; ammonium sulfate precipitation; or gel filtration (e.g.,using Sephadex G-
75), find use.
In some embodiments, the transglucosidase is used without purification from
the other
components of the culture medium. In some of these embodiments, the culture
medium is
simply concentrated and then used without further purification of the protein
from the
components of the growth medium, while in other embodiments, it is used
without any further
modification or treatment.
[080] The present invention also provides methods for degrading a natural gum
polysaccharides. In some embodiments, the methods include combining (e.g.,
mixing) a
transglucosidase enzyme with a natural gum polysaccharide under conditions
such that the
natural gum polysaccharide is degraded. 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
[081 ] In addition to the above-described transglucosidase-containing
compositions, the present
invention also provides cleaning methods. These methods generally include:
contacting an
object soiled with at least one natural gum polysaccharide with a cleaning
composition
comprising a transglucosidase enzyme; and maintaining the object and cleaning
composition
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together under conditions sufficient to effect degradation of the natural gum
polysaccharide and
thereby clean the object.
[082] In some preferred embodiments, the cleaning composition employed in
these methods is
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. Various
formulations for cleaning compositions find use in the present invention,
including, but not
limited to those described in great detail in W00001826, incorporated by
reference herein.
[083] In some embodiments, the subject cleaning composition (e.g., laundry
detergent)
comprises from about 1% to about 80% (e.g., about 5% to about 50% (by weight))
of at least
one surfactant (e.g., non-ionic surfactant, cationic surfactant, anionic
surfactant, and/or a
zwitterionic surfactant, or any mixture thereof, such as a mixture of anionic
and nonionic
surfactants). Exemplary surfactants include, but are not limited to: 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 find use in
various
detergents, particularly laundry detergents, are described in U.S. Patent Nos.
3,664,961,
3,919,678, 4,222,905, and 4,239,659, each of which is incorporated by
reference herein.
[084] In some embodiments, the subject cleaning composition is solid (e.g.,
powder or tablet
form), liquid, or a gel. In some preferred embodiments, the composition
further comprises at
least one buffer (e.g., sodium carbonate, or sodium bicarbonate), detergent
builder, bleach,
bleach activator, enzyme, 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 and/or perfume. In some embodiments, the cleaning
composition is
combined with a detergent before use as a laundry additive.
[085] In some embodiments, the cleaning compositions further contain at least
one non-starch
food polysaccharide degrading enzyme (e.g., hemicellulase, mannanase,
pectinase, xylanase, or
pectate lyase) and, optionally, one or more other enzymes, (e.g., proteases,
such as a subtilisin
protease and/or SSI protein; lipase, amylase, cellulase, cutinase, lipase,
oxidoreductase, etc.), for
the removal of other stains.
[086] A wide variety of other ingredients useful in detergent cleaning
compositions find use in
the compositions provided herein, including, but not limited to other active
ingredients, carriers,
hydrotropes, processing aids, dyes or pigments, solvents for liquid
formulations, etc. In
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embodiments in which an additional increment of sudsing is desired, suds
boosters such as the
CIo -C16 alkolamides are incorporated into the compositions, typically at
about 1% to about 10%
of the composition by weight.
[087] In some embodiments, detergent compositions comprise water and other
solvents as
carriers. Low molecular weight primary or secondary alcohols exemplified by
methanol,
ethanol, propanol, and isopropanol find use as such carriers. In some
embodiments, 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) also find use.
In some further
embodiments, the compositions comprise from about 5% to about 90%, typically
from about
10% to about 50% of such carriers.
[088] In some preferred embodiments, the detergent compositions provided
herein are
formulated such that during use in aqueous cleaning operations, the wash water
has 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 some
embodiments, the cleaning
compositions are automatic dishwashing detergents that have working pHs in the
range of about
pH 9.0 to about pH 11.5, about pH 9.0 to about pH 9.5, about pH 9.5 to about
pH 10.0, about pH
10.0 to about pH 10.5, about pH 10.5 to about pH 11.0, or about pH 11.0 to
about pH 11.5. In
some other embodiments, the cleaning compositions are liquid laundry
detergents that have
working pHs in the range of about pH 7.5 to about pH 8.5, about pH 7.5 to
about pH 8.0, or
about pH 8.0 to about pH 8.5. In some other embodiments, the cleaning
compositions are solid
laundry detergents that have working pHs in the range of about pH 9.5 to about
pH 10.5, about
pH 9.5 to about pH 10.0, or about pH 10.0 to about pH 10.5.
[089] Various bleaching compounds, such as the percarbonates, perborates and
the like, also
find use in the various compositions provided herein, typically at levels from
about 1% to about
15% by weight. If desired, such compositions 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.
[090] 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,
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various suds suppressors, especially silicones and secondary alcohols, various
fabric softeners,
especially smectite clays, and the like, also find use in various
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 that find use
in compositions
provided by the present invention.
[0911 In some embodiments, enzyme stabilizers also find use in the cleaning
compositions
provided herein. Such stabilizers include, but are not limited to 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%).
[092] In some embodiments, the hard surface cleaning compositions and/or
fabric cleaning
compositions are formulated to include various builders at levels from about
5% to about 50%
by weight. Typical builders include, but are not limited to 1-10 micron
zeolites,
polycarboxylates such as citrate and oxydisuccinates, layered silicates,
phosphates, and the like.
Other conventional builders are listed in standard formularies, and find use
in the present
invention.
[093] Other optional ingredients include chelating agents, clay soil
removal/anti redeposition
agents, polymeric dispersing agents, bleaches, brighteners, suds suppressors,
solvents and
aesthetic agents.
[094] The cleaning methods provided by the present invention are 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 some
preferred
embodiments, in comparison to an otherwise equivalent method that does not
contain a
transglucosidase enzyme, the cleaning compositions of the present invention
are more effective
at stain removal. Using a standard reflectometer-based assay, for example, the
subject method
removes and/or discolors at least about 20%, at least about 40%, at least
about 60%, at least
about 80% or, in some embodiments, at least about 90% more stain than an
equivalent method
that does not employ a transglucosidase enzyme.
[095] 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.
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EXPERIMENTAL
[096] 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
[097] A nucleic acid encoding the mature transglucosidase enzyme of A. niger
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 Figure 1. The
transglucosidase protein
encoded by this vector was operably linked to the CBH1 signal sequence to
facilitate its
secretion into the growth medium. The transglucosidase coding sequence was
flanked by the T.
reesei cbhl promoter and terminator. The nucleotide sequence of the expression
cassette of
pTrex3(AGL51M ) vector is set forth in Figure 2.
[098] The 7.57 kb XbaI-Xbal fragment of the plasmid pTrex3(AGL51M ) was
purified by
agarose gel electrophoresis and used to transform the spores of T. reesei by
electroporation. The
electroporation parameters were as follows: voltage - 16 kV/cm, capacitance -
25 F , resistance
- 50 Q. 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 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/1; cesium
chloride 1.68 g/l; glucose 20 g/l; potassium dihydrogen phosphate 15 g/l;
magnesium sulfate
heptahydrate 0.6 g/1; calcium chloride dihydrate 0.6 g/l; iron (II) sulfate
5mg/1; zinc sulfate 1.4
mg/1; cobalt (II) chloride 1 mg/l; manganese (II) sulfate 1.6 mg/1; agar 20
g/1; 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.
[099] Isolates showing stable growth on selective medium were used to
inoculate 5 ml of
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lactose defined medium (See e.g., WO 2005/001036, p. 60) in 20x 175 mm test
tubes. The tubes
were fixed in a rotary shaker at about 45 angle and shaken at 200 rpm and 28
C for 4-5 days.
Electrophoretic analysis of the culture supernatants demonstrated the presence
of a new protein
band of approximately expected molecular weight.
5 [0100] 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-a-glucoside and I mg/ml BSA. After 30 min
incubation at
C the reaction was terminated by the addition of an equal volume I M sodium
carbonate and
OD405 was recorded. Typically, transformants expressed 1-2U transglucosidase
activity
10 (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
15 [0101 ] Hydrolytic activity by enzymes on xanthan gum was measured by the
reducing sugar
assay using the PAHBAH (para-hydroxybenzoic acid hydrazide) reagent, as known
in the art
(See, Lever et al., Anal. Biochem., 47: 273 [1972]). 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
20 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.
[0102] The assay was performed as follows in a 24 well microplate (COSTAR
3526, Corning
25 Incorporated, Corning, N.Y.): one ml of buffer was added to well 1, one ml
of buffer plus
enzyme was added to we112, 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 I to 1000.
After all reagents were added, a plastic cover was place over the microplate
and the cover and
30 plate intersection was wrapped tightly with several layers of Parafilm
(Pechiney Plastic Packing,
Menasha, WI) to prevent evaporation. The reaction plate was incubated for 1 to
16 hr, at 37 C
on a shaker rotating at 100 rpm.
[0103] Reducing sugar activity was measured using an Eppendorf Mastercycler
Gradient
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21
(Eppendorf Scientific, Westbury, NY) 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 99C followed by cooling at 4C 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, NY) and read by a Spectra Max 250 Plate
Reader
(Molecular Devices, Sunnyvale, CA) at 405 nm against a blank of distilled
water.
[0104] 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.
[0105] Figure 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 6Ø
EXAMPLE 3
Transglucosidase Removes Soil from Stained Patches
[0106] 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 C with 100rpm 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
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22
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.
[0107] The graphs provided in Figure 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.
[0108] Figure 5 shows the results obtained in a Trip-TG dose response
microswatch experiment.
Trip-TG removed salad dressing soils at I ppm in 0.15% AATCC heavy duty
liquid..
[0109] Figure 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% copolymer or other
additives.
[0110] This experiment showed transglucosidase soil removal activity in both
heavy duty
liquids (HDL) and in heavy duty solids (HDD).
EXAMPLE 4
Tergotometer Analysis of the Cleaning Activity of Transglucosidase
[0111] Tergotometer studies used a 6 pot Tergotometer Model 7243S (U. S.
Testing, Co. Inc.
Hoboken, N. J.) maintained at 30 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 6gpg hardness (diluted from stock 15000 gpg hardness
solution containing
1.735 M calcium chloride and 0.67 M magnesium chloride) and 25mM Hepes buffer
pH 7.4
After a 30 min wash cycle, the swatches were washed three times in 1.5 1 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. Figure 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.
[0112] The above examples demonstrate that transglucosidase effectively
degrades xanthan, and
removes certain soils from cotton swatches.
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23
[0113] All patents and publications mentioned in the specification are
indicative of the levels of
those skilled in the art to which the invention pertains. All patents and
publications are herein
incorporated by reference to the same extent as if each individual publication
was specifically
and individually indicated to be incorporated by reference.
[0114] Those of skill in the art readily appreciate that the present invention
is well adapted to
carry out the objects and obtain the ends and advantages mentioned, as well as
those inherent
therein. The compositions and methods described.herein are representative of
preferred
embodiments, are exemplary, and are not intended as limitations on the scope
of the invention. It
is readily apparent to one skilled in the art that varying substitutions and
modifications may be
made to the invention disclosed herein without departing from the scope and
spirit of the
invention.
[0115] The invention illustratively described herein suitably may be practiced
in the absence of
any element or elements, limitation or limitations which is not specifically
disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of
limitation, and there is no intention that in the use of such terms and
expressions of excluding
any equivalents of the features shown and described or portions thereof, but
it is recognized that
various modifications are possible within the scope of the invention. Thus, it
should be
understood that although the present invention has been specifically disclosed
by preferred
embodiments and optional features, modification and variation of the concepts
herein disclosed
may be resorted to by those skilled in the art, and that such modifications
and variations are
considered to be within the scope of this invention.
[0116] The invention has been described broadly and generically herein. Each
of the narrower
species and subgeneric groupings falling within the generic disclosure also
form part of the
invention. This includes the generic description of the invention with a
proviso or negative
limitation removing any subject matter from the genus, regardless of whether
or not the excised
material is specifically recited herein.
