U.S. patent application number 14/192372 was filed with the patent office on 2014-08-21 for alpha-glucanase and oral care composition containing the same.
This patent application is currently assigned to DANISCO US INC.. The applicant listed for this patent is Danisco US Inc.. Invention is credited to Steven Kim, Suzanne Lantz, Michael Pepsin.
Application Number | 20140234231 14/192372 |
Document ID | / |
Family ID | 40765534 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234231 |
Kind Code |
A1 |
Kim; Steven ; et
al. |
August 21, 2014 |
ALPHA-GLUCANASE AND ORAL CARE COMPOSITION CONTAINING THE SAME
Abstract
Isolated .alpha.-glucanases from Hypocrea tawa, Trichoderma
reesei, and Trichoderma konilangbra are described, as well as oral
care compositions containing the same. The oral care composition
may be employed to prevent or reduce dental plaque.
Inventors: |
Kim; Steven; (Fremont,
CA) ; Lantz; Suzanne; (San Carlos, CA) ;
Pepsin; Michael; (Castro Valley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Danisco US Inc. |
Palo Alto |
CA |
US |
|
|
Assignee: |
DANISCO US INC.
Palo Alto
CA
|
Family ID: |
40765534 |
Appl. No.: |
14/192372 |
Filed: |
February 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12937362 |
May 26, 2011 |
8709386 |
|
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PCT/US2009/040013 |
Apr 9, 2009 |
|
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14192372 |
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61044316 |
Apr 11, 2008 |
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Current U.S.
Class: |
424/50 ; 435/200;
435/252.31; 435/252.35; 435/254.11; 435/254.2; 435/254.3;
435/254.4; 435/254.5; 435/254.6; 435/254.7; 435/320.1;
536/23.2 |
Current CPC
Class: |
C12N 9/2405 20130101;
A61Q 11/00 20130101; C12Y 302/01059 20130101; A61K 8/66
20130101 |
Class at
Publication: |
424/50 ;
536/23.2; 435/200; 435/320.1; 435/254.6; 435/254.2; 435/252.31;
435/252.35; 435/254.5; 435/254.11; 435/254.3; 435/254.7;
435/254.4 |
International
Class: |
A61K 8/66 20060101
A61K008/66; C12N 9/24 20060101 C12N009/24; A61Q 11/00 20060101
A61Q011/00 |
Claims
1. An isolated .alpha.-glucanase comprising an amino acid sequence
that is: a) at least 99% identical to the mature Hypocrea tawa
.alpha.-glucanase (amino acid residues 38-635 of SEQ ID NO: 1); or
b) at least 85% identical to the mature Trichoderma konilangbra
.alpha.-glucanase (amino acid residues 38-627 of SEQ ID NO: 3).
2. An isolated polynucleotide encoding an isolated
.alpha.-glucanase of claim 1.
3. A recombinant nucleic acid comprising the isolated
polynucleotide of claim 2.
4. A vector comprising the recombinant nucleic acid of claim 3.
5. A host cell comprising the recombinant nucleic acid of claim
3.
6. A cell culture comprising: a) growth medium; and b) a population
of cells of claim 5.
7. A method of producing protein comprising: maintaining the
culture of cells of claim 6 under conditions suitable for
production of said isolated .alpha.-glucanase.
8. The method of claim 7, further comprising harvesting said
.alpha.-glucanase from said growth medium.
9. A method comprising receiving an isolated .alpha.-glucanase
selected from the following: a) .alpha.-glucanase having an amino
acid sequence that is at least 99% identical to that of mature
Hypocrea tawa .alpha.-glucanase (amino acid residues 38-635 of SEQ
ID NO: 1); b) .alpha.-glucanase having an amino acid sequence that
is at least 85% identical to that of mature Trichoderma reesei
.alpha.-glucanase (amino acid residues 38-622 of SEQ ID NO: 2); or
c) .alpha.-glucanase having an amino acid sequence that is at least
85% identical to that of mature Trichoderma konilangbra
.alpha.-glucanase (amino acid residues 38-627 of SEQ ID NO: 3); and
admixing said isolated .alpha.-glucanase with an orally acceptable
excipient to make an oral care composition.
10. The method of claim 9, further comprising: packaging said oral
care composition.
11. The method of claim 1, wherein the .alpha.-glucanase is not
identical to Trichoderma reesei .alpha.-glucanase (amino acid
residues 38-622 of SEQ ID NO: 2).
12. An oral care composition comprising: a) an orally acceptable
excipient; and b) an isolated .alpha.-glucanase of claim 1.
13. An oral care composition comprising: an orally acceptable
excipient; and an isolated .alpha.-glucanase selected from the
following: a) .alpha.-glucanase having an amino acid sequence that
is at least 99% identical to that of mature Hypocrea tawa
.alpha.-glucanase (amino acid residues 38-635 of SEQ ID NO: 1); b)
.alpha.-glucanase having an amino acid sequence that is at least
85% identical to that of mature Trichoderma reesei
.alpha.-glucanase (amino acid residues 38-622 of SEQ ID NO: 2); or
c) .alpha.-glucanase having an amino acid sequence that is at least
85% identical to that of mature Trichoderma konilangbra
.alpha.-glucanase (amino acid residues 38-627 of SEQ ID NO: 3).
14. The composition of claim 13, wherein the .alpha.-glucanase is
not identical to Trichoderma reesei .alpha.-glucanase (amino acid
residues 38-622 of SEQ ID NO: 2).
15. The oral care composition of claim 12, wherein said
.alpha.-glucanase is present in said composition at a concentration
of 0.0001% to 5% by weight of said composition.
16. The oral care composition of claim 12, further comprising a
second enzyme.
17. The oral care composition of claim 16, wherein said second
enzyme is a deaminase, esterase, glycosidase, lipase, oxidase,
peroxidase, protease, urease or cellulase.
18. The oral care composition of claim 12, wherein said composition
is formulated as a toothpaste.
19. The oral care composition of claim 12, wherein said composition
comprises at least one of a thickener, a surfactant, a humectant,
and an abrasive.
20. A method comprising: contacting the oral care composition of
claim 12 with a tooth under conditions suitable for activity of
said .alpha.-glucanase.
21. The method of claim 20, wherein said contacting is performed
using a toothbrush.
22. The method of claim 20, wherein said method results in
prevention and/or reduction in dental plaque.
Description
PRIORITY
[0001] The present application is a divisional of U.S. patent
application Ser. No. 12/937,362, filed May 26, 2011, now U.S. Pat.
No. 8,709,386, which is a U.S. National Phase Application of
International Application No. PCT/US2009/040013, filed Apr. 9,
2009, which claims priority to U.S. Provisional Application Ser.
No. 61/044,316, filed on Apr. 11, 2008, which are incorporated by
reference in their its entirety.
SEQUENCE LISTING
[0002] The sequence listing submitted via EFS, in compliance with
37 C.F.R. .sctn.1.52(e), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
"31149US-D1_SequenceListing.txt" created on Feb. 21, 2014, which is
53,951 bytes in size.
BACKGROUND
[0003] The formation of dental plaque leads to dental caries,
gingival inflammation, periodontal disease, and eventually tooth
loss. Dental plaque is a mixture of bacteria, epithelial cells,
leukocytes, macrophages, and other oral exudate. The bacteria
produce highly branched polysaccharides, which, together with
micro-organisms from the oral cavity, form an adhesive matrix for
the continued proliferation of dental plaque.
[0004] As dental plaque continues to accumulate, rock-hard white or
yellowish deposits arise. These deposits are called calcified
plaque, calculus, or tartar, and are formed in the saliva from
plaque and minerals, e.g., calcium.
[0005] There is an ongoing need for new ways to prevent and/or
reduce dental plaque and associated tooth decay.
SUMMARY
[0006] In one aspect, an isolated .alpha.-glucanase is provided,
comprising an amino acid sequence that is (a) at least 99%
identical to the mature Hypocrea tawa .alpha.-glucanase (amino acid
residues 38-635 of SEQ ID NO: 1); or (b) at least 85% identical to
the mature Trichoderma konilangbra .alpha.-glucanase (amino acid
residues 38-627 of SEQ ID NO: 3).
[0007] In another aspect, an isolated polynucleotide encoding a
subject .alpha.-glucanase, and recombinant nucleic acid containing
the isolated polynucleotide is provided. A vector and a host cell
containing the recombinant nucleic acid are also provided.
[0008] In another aspect, a cell culture is provided. In some
embodiments, the cell culture contains a growth medium and a
population of the above-described host cells. The cell culture may
be used to produce a subject .alpha.-glucanase by maintaining the
cell culture under conditions suitable for production of the
isolated .alpha.-glucanase. If the .alpha.-glucanase is secreted,
it may be harvested from the growth medium.
[0009] In another aspect, a method of producing protein is
provided, comprising maintaining the culture of cells described
above under conditions suitable for production of the isolated
.alpha.-glucanase. In some embodiments, the method further
comprises harvesting the .alpha.-glucanase from the growth
medium.
[0010] In another aspect, a method is provided, comprising
receiving an isolated .alpha.-glucanase selected from the
following: (a) .alpha.-glucanase having an amino acid sequence that
is at least 99% identical to that of mature Hypocrea tawa
.alpha.-glucanase (amino acid residues 38-635 of SEQ ID NO: 1); (b)
.alpha.-glucanase having an amino acid sequence that is at least
85% identical to that of mature Trichoderma reesei
.alpha.-glucanase (amino acid residues 38-622 of SEQ ID NO: 2); or
(c) .alpha.-glucanase having an amino acid sequence that is at
least 85% identical to that of mature Trichoderma konilangbra
.alpha.-glucanase (amino acid residues 38-627 of SEQ ID NO: 3); and
admixing the isolated .alpha.-glucanase with an orally acceptable
excipient to make an oral care composition. In some embodiments,
the .alpha.-glucanase is not identical to Trichoderma reesei
.alpha.-glucanase (amino acid residues 38-622 of SEQ ID NO: 2). In
some embodiments, the method further comprises packaging the oral
care composition.
[0011] In another aspect, an oral care composition is provided,
comprising (a) an orally acceptable excipient; and (b) an isolated
.alpha.-glucanase.
[0012] In a related aspect, an oral care composition is provided,
comprising: an orally acceptable excipient and an isolated
.alpha.-glucanase selected from the following: (a)
.alpha.-glucanase having an amino acid sequence that is at least
99% identical to that of mature Hypocrea tawa .alpha.-glucanase
(amino acid residues 38-635 of SEQ ID NO: 1); (b) .alpha.-glucanase
having an amino acid sequence that is at least 85% identical to
that of mature Trichoderma reesei .alpha.-glucanase (amino acid
residues 38-622 of SEQ ID NO: 2); or (c) .alpha.-glucanase having
an amino acid sequence that is at least 85% identical to that of
mature Trichoderma konilangbra .alpha.-glucanase (amino acid
residues 38-627 of SEQ ID NO: 3). In some embodiments, the
.alpha.-glucanase is not identical to Trichoderma reesei
.alpha.-glucanase (amino acid residues 38-622 of SEQ ID NO: 2).
[0013] In some embodiments, the .alpha.-glucanase is present in the
composition at a concentration of 0.0001% to 5% by weight of the
composition.
[0014] In some embodiments, the oral care composition further
comprises a second enzyme. In particular embodiments, the second
enzyme is a deaminase, esterase, glycosidase, lipase, oxidase,
peroxidase, protease, urease or cellulase.
[0015] In some embodiments, the composition is formulated as a
toothpaste, although other oral care formulations are envisioned.
In some embodiments, the composition comprises at least one of a
thickener, a surfactant, a humectant, and an abrasive, for
example.
[0016] In another aspect, a method is provided, in which the
isolated .alpha.-glucanase is received, and then admixing with an
orally acceptable excipient to make an oral care composition is
provided. The oral care composition may be packaged.
[0017] In yet another aspect, a method comprising contacting a
subject oral care composition with a tooth under conditions
suitable for activity of the .alpha.-glucanase is provided. The
contacting may be performed using, e.g., a toothbrush. In
particular cases, the method results in prevention and/or reduction
in dental plaque.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows an amino acid sequence alignment of
.alpha.-glucanases from Hypocrea tawa (SEQ ID NO: 1), Trichoderma
reesei (SEQ ID NO: 2); Trichoderma konilangbra (SEQ ID NO: 3) and
T. harzianum (SEQ ID NO: 4), and a consensus sequence (SEQ ID NO:
5) based on the alignment. Various features of the
.alpha.-glucanases are indicated, such as the signal sequence, the
catalytic domain, the linker domain, and the glucan-binding
domain.
[0019] FIG. 2 is a table that summarizes the results of HPLC
analysis of the insoluble glucan hydrolysates obtained following
overnight incubation with various cell-free culture solutions.
[0020] FIG. 3 shows the activity of supernatants from cultures of
T. reesei expressing the putative alph.alpha.-1,3-glucanases from
T. reesei, H. tawa, and T. konilangbra at pH 4.5 and at pH 6.0. The
native alpha-glucanases were not deleted from the host strain. The
glucan hydrolysis reactions were loaded based on culture volume,
not protein content.
DEFINITIONS
[0021] Unless defined otherwise herein, all technical and
scientific terms should be given their ordinary meaning as used in
the art. The following terms are defined for clarity. Other
definitions may appear elsewhere in the specification.
[0022] As used herein, the term ".alpha.-glucanase" refers to an
enzyme that hydrolyses 1,3-.alpha.-D-glucosidic linkages in a
polysaccharide. The .alpha.-glucanases s described herein have an
activity described as EC 3.2.1.59, according to IUBMB enzyme
nomenclature, and can hydrolyse insoluble glucan. The systematic
name for an .alpha.-glucanase is 1,3(1,3;1,4)-.alpha.-D-glucan
3-glucanohydrolase. This enzyme may be referred to as
1,3-.alpha.-glucanase in certain publications. Note that the
present enzymes may have other activities in addition to
1,3-.alpha.-D-glucosidic activity, including but not limited to
1,2-.alpha.-D-glucosidic activity.
[0023] As used herein, the term "oral care composition" refers to
an admixture of ingredients, which in the ordinary course of usage
is not intentionally swallowed for purposes of systemic
administration of particular therapeutic agents, but is rather
retained in the oral cavity for a time sufficient to contact dental
surfaces and/or oral tissues for purposes of delivering a
beneficial agent to the oral activity. An oral composition may be
in the form of toothpaste, dentifrice, tooth powder, tooth gel,
subgingival gel, mouthrinse, denture product, mouthspray, lozenge,
oral tablet, chewing gum, or the like. The oral composition may
also be incorporated onto strips or films for direct application or
attachment to oral surfaces.
[0024] Unless otherwise specified, the term "dentrifice" refers to
paste, gel, solid or liquid oral care composition formulation.
Examples of dentrifices are toothpaste, tooth gel, and tooth
powder. A dentrifice may be a single phase composition or may be a
combination of two or more separate compositions. A dentrifice may
be in any desired form, such as deep striped, surface striped,
multilayered, having the gel surrounding the paste, or any
combination thereof. Each composition in a dentrifice comprising
two or more separate compositions may be contained in a physically
separated compartment of a dispenser and dispensed
side-by-side.
[0025] As used herein, the term "orally acceptable carrier" refers
to a safe and effective material for use in an oral care
composition. Such materials include fluoride ion sources,
anticalculus agents, buffers, abrasive polishing materials,
peroxide sources, alkali metal bicarbonate salts, thickening
materials, humectants, water, surfactants, titanium dioxide, flavor
system, sweetening agents, xylitol, coloring agents, and mixtures
thereof.
[0026] As used herein, the terms "tooth" or "teeth" refers to
natural teeth as well as artificial teeth or dental prosthesis.
[0027] As used herein, the term "enamel" refers to the part of a
tooth that is normally visible and is composed of mostly minerals,
including hydroxylapatite. Enamel encompasses naturally-occurring
enamels in teeth of humans and animals as well as enamel-like
substance used to replace damaged or missing teeth parts, including
resins and porcelains used for such purposes.
[0028] As used herein, the terms "tartar" and "calculus" are used
interchangeably to refer to mineralized dental plaque deposits.
[0029] As used herein, the term "glycocalyx" refers to
extracellular polymeric material produced by some bacteria,
epithelial cells, and other cells, which forms a coating on the
surface of teeth and serves as a matrix for the attachment of
plaque.
[0030] As used herein, the term "plaque" refers to a biofilm that
forms on the surface of a tooth (or of teeth). The microorganisms
that form the biofilm are mostly bacteria, including but not
limited to Streptococcus mutans, Streptococcus anaerobes,
Fusobacterium spp., and Actinobacteria spp. Plaque may form on, be
supported by, or be part of a glycocalyx.
[0031] The microorganisms present in dental plaque are all
naturally present in the oral cavity, and are normally harmless.
However, failure to remove plaque by regular tooth brushing means
that they are allowed to build up in a thick layer. Those
microorganisms nearest the tooth surface convert to anaerobic
respiration; it is in this state that they start to produce
acids.
[0032] As used herein, the term "recombinant" refers to a
polynucleotide or polypeptide that does not occur in, is not
secreted by, or has an altered expression pattern in, a wild type
host cell. Recombinant polypeptides and polynucleotides have
respective sequences that are different from the wild-type
sequence, have different temporal or spacial expression patterns
from wild type polypeptides and polynucleotides, and/or are
expressed at different levels than wild type polypeptides and
polynucleotides. 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 a recombinant polypeptide.
[0033] As used herein, 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.
[0034] As used herein, the terms "protein" and "polypeptide" are
used interchangeably to refer to a chain of amino acids linked by
peptide bonds. Unless otherwise specified polypeptides are written
in the standard N-terminal to C-terminal direction.
[0035] As used herein, 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,
although the structures of many signal sequence are known. The
mature form of the extracellular protein lacks the signal sequence,
which is cleaved off during the secretion process.
[0036] As used herein, a "coding sequence" is a DNA segment that
encodes a polypeptide.
[0037] As used herein, the term "nucleic acid" encompasses DNA,
RNA, hybrids, and synthetic or chemically-modified nucleic acids,
whether single-stranded or double-stranded. The terms "nucleic
acid" and "polynucleotide" are used interchangeably to refer to a
chain of nucleosides linked by phosphodiester, sulfodiester, or
similar bonds. Unless otherwise specified polynucleotides are
written in the standard 5' to 3' direction.
[0038] As used herein, 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.
[0039] As used herein, an "expression vector" refers to a DNA
construct comprising a protein-coding region that is operably
linked to a suitable control sequence 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 the
termination of transcription and translation.
[0040] As used herein, a "promoter" is a regulatory sequence that
initiates transcription of a downstream nucleic acid.
[0041] As used herein, the term "operably linked" refers to an
arrangement of elements that allows the elements to function in a
described or apparent manner. For example, a promoter is operably
linked to a coding sequence if it controls the transcription of the
sequence.
[0042] As used herein, the term "selective/selectable marker"
refers to a protein capable of expression in a host that allows for
ease of selection of those cells containing an introduced nucleic
acid or vector. Examples of selectable markers include, but are not
limited to, proteins that confer resistance 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.
[0043] As used herein, the term "derived from" encompasses the
terms "originated from," "obtained" or "obtainable from," and
"isolated from".
[0044] As used herein, a "non-pathogenic" organism is an organism
that is not pathogenic (i.e., disease or disorder-causing) to
humans.
[0045] As used herein, the terms "recovered," "isolated," and
"separated" refer to a protein, cell, nucleic acid or amino acid
that is removed from at least one component with which it is
naturally associated.
[0046] As used herein, the terms "transformed," "stably
transformed," and "transgenic," used in reference to a cell, means
that the cell has a non-native (e.g., heterologous) nucleic acid
sequence integrated into its genome or as an episomal plasmid that
is maintained over multiple generations.
[0047] 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.
[0048] As used herein, 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).
[0049] As used herein, the term "compatible" means that the
components of a specified composition are capable of being
commingled (admixed) without interaction in a manner which would
substantially reduce the stability and/or efficacy of a component
in the composition.
[0050] As used herein, the term "lozenge" includes but is not
limited to: breath mints, troches, pastilles, microcapsules, and
fast-dissolving solid forms including freeze dried forms (cakes,
wafers, thin films, tablets) and compressed tablets.
[0051] As used herein, the term "fast-dissolving solid form" means
that a solid dosage form dissolves in less than about 60 seconds,
less than about 15 seconds, or less than about 5 seconds, after
placing the solid dosage form in the oral cavity or a container
containing dental prosthetics.
[0052] Numeric ranges are inclusive of the numbers defining the
range. All percentages and ratios used herein are by weight of the
specific oral composition and not of the overall oral formulation
that is delivered, unless otherwise specified. The singular
articles "a," an," and the," include the plural unless otherwise
specified or apparent from context.
[0053] Headings are provided for ease of reading and should not be
construed as limitations. The description included under one
heading generally applies to the document as a whole, unless
otherwise specified or apparent from context.
[0054] Exemplary material and methods are described, although other
methods and materials may result in similar or equivalent results.
All patents and publications, including all sequences disclosed
within such patents and publications, are expressly incorporated by
reference.
DETAILED DESCRIPTION
[0055] Described are compositions and methods relating to
polypeptides having .alpha.-glucanase activity. The compositions
and methods are useful for reducing or preventing the formation of
plaque, and for reducing or preventing the underlying physiological
conditions that promote the formation of plaque.
A. Polypeptides, Polynucleotides, and Host Cells
[0056] One aspect of the present compositions and methods relates
to an isolated .alpha.-glucanase. In some embodiments, the
.alpha.-glucanase comprises an amino acid sequence that is at least
98% identical to (e.g., at least 99% or 99.5% identical to) the
amino acid sequence of the mature Hypocrea tawa .alpha.-glucanase
(amino acid residues 38-635 of SEQ ID NO: 1). In particular
embodiments, the .alpha.-glucanase comprises the amino acid
sequence of the mature H. tawa .alpha.-glucanase (amino acid
residues 38-635 of SEQ ID NO: 1).
[0057] In some embodiments, the .alpha.-glucanase comprises an
amino acid sequence that is at least 85% identical to (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to) the amino acid sequence of the mature Trichoderma konilangbra
.alpha.-glucanase (amino acids 38-627 of SEQ ID NO: 3). In
particular embodiments, the .alpha.-glucanase comprises the amino
acid sequence of the mature T. konilangbra .alpha.-glucanase (amino
acids 38-627 of SEQ ID NO: 3).
[0058] In some embodiments, the .alpha.-glucanase comprises an
amino acid sequence that is at least 85% identical to (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to) the amino acid sequence of the mature Trichoderma reesei
.alpha.-glucanase (amino acids 38-622 of SEQ ID NO: 2). In
particular embodiments, the .alpha.-glucanase comprises an amino
acid sequence that is not identical to the amino acid sequence of
the mature T. reesei .alpha.-glucanase (amino acids 38-622 of SEQ
ID NO: 2).
[0059] In some embodiments, the .alpha.-glucanase is similar to but
not identical to a wild type .alpha.-glucanase. For example, in
some embodiments, the .alpha.-glucanase has an amino acid sequence
that is at least 98% identical to, but not identical to, the mature
H. tawa .alpha.-glucanase (amino acid residues 38-635 of SEQ ID NO:
1). Likewise, in certain embodiments, the .alpha.-glucanase may
have an amino acid sequence that at least 85% identical to, but not
identical to, the mature T. reesei or T. konilangbra
.alpha.-glucanase (amino acids 38-622 of SEQ ID NO: 2 or amino
acids 38-627 of SEQ ID NO: 3, respectively).
[0060] The amino acid sequences for over 50 different
.alpha.-glucanases are known and have been deposited in NCBI's
Genbank database, including those from Aspergillus niger (accession
no.: XP.sub.--001390909.1; GID: 145236523), Penicillium
purpurogenum (accession no.: AAF27912.1; GID: 6752866), Emericella
nidulans (accession no.: CAC48025.1; GID: 15072711) and
Cryptococcus neoformans (accession no.: AAW47079.1; GID: 57230770).
These Genbank accessions 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. An entry describing a domain that
is conserved in .alpha.-glucanases has been deposited as pfam03659
in NCBI's Conserved Domain Database (Marchler-Bauer et al. CDD: a
conserved domain database for interactive domain family analysis.
(2007) Nucleic Acids Res. 35:D237-40). The sequence of a
.alpha.-glucanase from S. pombe, as well as a discussion of the
structure of .alpha.-glucanases is found in Fuglsang et al. ((2000)
J. Biol. Chem. 275:2009-18).
[0061] Guidance for which amino acids can be changed to produce an
active variant of the wild-type .alpha.-glucanases of H. tawa, T.
reesei and T. konilangbra that retains .alpha.-glucanase activity
can be obtained, for example, by aligning the amino acid sequences
of those .alpha.-glucanase proteins, identifying amino acids that
are at identical positions in the proteins but are different
between the proteins, and transferring those amino acids from one
protein to the other. Exemplary sequence alignments are shown in
FIG. 1, and in Fuglsang et al. (supra).
[0062] A variant polypeptide may include conservative amino acid
substitutions that preserve the general charge,
hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid
being substituted, while imparting other beneficial biochemical
properties on the polypeptide. Non-limiting examples of
conservative substitutions include those between the following
groups: Gly/Ala, Val/Ile/Leu, Lys/Arg, Asn/Gln, Glu/Asp,
Ser/Cys/Thr and Phe/Trp/Tyr. These and other conservative
substitutions are shown in the Table, below.
TABLE-US-00001 Original Amino Acid Code Conservative Substitution
Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys,
D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn
Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic
Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys,
S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,
Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn,
D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp
Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L
D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,
D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn
Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val
Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D- Trp,
Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline
P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or
L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,
allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,
D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met
[0063] Alternatively, the amino acid substitutions are not
conservative and change the general charge,
hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid
being substituted.
[0064] Assays for evaluating .alpha.-glucanase activity are
described in a variety of publications, including: Fuglsang et al.
(supra), Inoue et al. ((1988) Carbohydr. Res. 182:277-86),
Ait-Lahsen et al. ((2001) Appl. Environ. Microbiol. 67:5833-9), and
Sumitomo et al. ((2007) Biochim. Biophys. Acta. 1770:716-24).
[0065] Also provided is an isolated polynucleotide encoding an
.alpha.-glucanas as described, and a recombinant nucleic acid
containing the isolated polynucleotide. Given that the genetic code
is known, such a polynucleotide can be readily designed based on
the amino acid sequence. In one embodiment, the isolated
polynucleotide has a nucleotide sequence that is at least 70%
identical to (e.g., at least 80% identical to, at least 90%
identical to, at least 95% identical to, at least 98% identical
to), or may hybridize under stringent conditions to, the nucleotide
sequence of a wild type H. tawa .alpha.-glucanase gene or coding
sequence (e.g., SEQ ID NOs: 29 and 30, respectively), a wild type
T. reesei .alpha.-glucanase gene or coding sequence (e.g., SEQ ID
NOs: 31 and 32, respectively), or a wild type T. konilangbra
.alpha.-glucanase gene or coding sequence (e.g., SEQ ID NOs: 33 and
34, respectively). In certain embodiments, the coding sequence of
the .alpha.-glucanase is codon optimized for expression of the
.alpha.-glucanase in the host cell used. Since codon usage tables
listing the usage of each codon in many host cells, including
Trichoderma reesei and various other yeast and bacterial host cells
are known in the art (see, e.g., Nakamura et al. (2000) Nucl. Acids
Res. 28:292) or readily derivable, such nucleic acids can be
readily designed given the amino acid sequence of a
.alpha.-glucanase to be expressed.
[0066] An expression vector and a host cell containing the
recombinant nucleic acid are also provided. In certain embodiments,
the host cell is bacterial (e.g., a Bacillus sp. or Streptomyces
sp. host cell) or filamentous fungal host cell that, in certain
cases, may be non-pathogenic, i.e., non-pathogenic to humans. In
particular embodiments, the cells may be filamentous fungal cells
of a strain that has a history of use for production of proteins
that has GRAS status, i.e., a Generally Recognized as Safe, by the
FDA.
[0067] In particular embodiments, the subject fungal cell may be a
cell of the following species: Trichoderma, (e.g., Trichoderma
reesei (previously classified as T. longibrachiatum and currently
also known as Hypocrea jecorina), Trichoderma viride, Trichoderma
koningii, and Trichoderma harzianum)); Penicillium spp., Humicola
spp. (e.g., Humicola insolens and Humicola grisea); Chrysosporium
spp. (e.g., C. lucknowense), Gliocladium spp., Aspergillus spp.
(e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans,
Aspergillus kawachi, Aspergillus aculeatus, Aspergillus japonicus,
Aspergillus sojae, and Aspergillus awamori), Fusarium spp.,
Neurospora spp., Hypocrea spp., or Emericella spp. (see, also,
Innis et al. (1985) Science 228:21-26), among others. In some
embodiments, subject fungal cells may be strains of Aspergillus
niger which include ATCC 22342, ATCC 44733, ATCC 14331 and strains
derived therefrom. In some embodiments, a host cell may be one
wherein native genes have been deleted or inactivated. For example,
genes corresponding to protease genes or genes corresponding to
cellulase genes may be deleted or inactivated.
[0068] The above described nucleic acid may be present in the
nuclear genome of the host cell or may be present in a plasmid that
replicates in the host cell, for example, a transient expression
vector, a shuttle vector, an artificial chromosome, and the
like.
[0069] In particular embodiments, the .alpha.-glucanase may be
produced by expressing a fusion protein containing a signal
sequence operably linked to the .alpha.-glucanase in a fungal host
cell. In such embodiments, the .alpha.-glucanase may be secreted
into culture medium, where it can be harvested. The signal sequence
of the fusion protein may be any signal sequence that facilitates
protein secretion from the host cell. The signal sequence employed
may be endogenous or non-endogenous to the 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. or Aspergillus
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, e.g., Saarelainen
(1997) Appl. Environ. Microbiol. 63:4938-40). In a particular
embodiment, an .alpha.-glucanase may be secreted using its own
(i.e., the endogenous) signal sequence.
[0070] It follows that in some embodiments an .alpha.-glucanase is
produced by introducing a nucleic acid into a host cell, which
nucleic acid comprises a signal sequence-encoding portion operably
linked to a .alpha.-glucanase-encoding portion, where translation
of the nucleic acid produces a fusion protein comprising an
.alpha.-glucanase portion having an N-terminal signal sequence for
secretion of the .alpha.-glucanase portion from the host cell.
[0071] 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 host cell. Suitable carrier proteins include those of T.
reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase II
(Ce16A, or CBHII) (see, e.g., Paloheimo et al. (2003) Appl.
Environ. Microbiol. 69:7073-82), 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 .alpha.-glucanase in
operable linkage may therefore be employed.
[0072] In addition to a coding sequence, the nucleic acid may
further contain other elements that are necessary for expression of
the .alpha.-glucanase 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) Mol Cell Biol.
4:2306-15); Boel et al. (1984) EMBO J. 3:1097-102; and Boel et al.
(1984) EMBO J. 3:1581-85), Aspergillus nidulans anthranilate
synthase genes, Aspergillus oryzae TAKA amylase genes, or A.
nidulans trpC (Punt et al. (1987) Gene 56:117-24). The promoter
and/or terminator may be native or non-endogenous to the
Trichoderma sp. host cell.
[0073] A culture of host cells (i.e., a composition containing a
population of host cells and growth media) is also provided. The
growth medium of the culture may contain the .alpha.-glucanase
described above. In certain embodiments, the cell culture may
contain growth medium and a population of the above-described
cells. The cell culture may be used to produce a subject
.alpha.-glucanase by maintaining the cell culture under conditions
suitable for production of the isolated .alpha.-glucanase. If the
.alpha.-glucanase is secreted, it may be harvested from the growth
medium.
[0074] Methods of expressing proteins in filamentous fungi,
including methods in which cells are engineered to produce secreted
protein include those described in U.S. Pat. Nos. 6,022,725 and
6,268,328, and in published U.S. Pat. App. Nos. 20060041113,
20060040353, 20060040353, and 20050208623, which are incorporated
herein by reference. In addition, general methods for the
transformation of Aspergillus strains are disclosed in Cao et al.
(2000) Protein Sci. 9:991-1001) and Yelton et al. (1984) Proc.
Natl. Acad. Sci. USA 81:1470-74) and general methods for the
transformation of Trichoderma strains are disclosed in Nevalainen
et al. (1992) "The Molecular Biology of Trichoderma and its
Application to the Expression of Both Homologous and Heterologous
Genes" in Molecular Industrial Mycology, Eds. Leong and Berka,
Marcel Dekker Inc., NY, pp 129-48).
[0075] If it is secreted in to culture medium, the
.alpha.-glucanase 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. 808153; 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 .alpha.-glucanase 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.
B. Oral Care Compositions
[0076] An aspect of the present compositions and methods relates to
an oral care composition. The oral care composition contains one or
more of the described .alpha.-glucanases and an orally acceptable
carrier. Each of the one or more .alpha.-glucanases may be present
in the composition at a concentration in the range of 0.0001% to 5%
(e.g., 0.0001% to 0.0005%, 0.0005% to 0.001%, 0.001% to 0.005%,
0.005% to 0.01%, 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5%
to 1%, or 1% to 5%) by weight, although concentrations outside of
this range are envisioned. The oral care composition may be made by
a method that includes admixing the .alpha.-glucanase with an
orally acceptable excipient. In certain cases, this method may
further include packinging the oral care composition.
[0077] The oral care composition may be in the form of, e.g., a
dentifrice, toothpaste, tooth powder, topical oral gel, mouthrinse,
denture product, mouthspray, lozenge, oral tablet, or chewing gum.
The oral composition may also be incorporated onto strips, films,
floss, or tape for direct application or attachment to oral
surfaces.
[0078] An orally acceptable carrier may comprise one or more
compatible solid or liquid filler diluents or encapsulating
substances, which are suitable for topical oral administration.
Suitable carriers or excipients include the usual and conventional
components of dentifrices (including non-abrasive gels and gels for
subgingival application), mouth rinses, mouth sprays, chewing gums,
and lozenges (including breath mints) as more fully described
hereinafter. The present oral care compositions in aqueous form may
optimally have a pH ranging from about 4.0 to about 10.0, e.g.,
from about 5.0 to about 8.0.
[0079] In some embodiments, the carrier is selected based on the
manner in which the way the composition is to be introduced into
the oral cavity. For example, if a toothpaste (including tooth
gels, etc.) is to be used, then a "toothpaste carrier" may be
chosen (comprising e.g., abrasive materials, surfactants, binders,
humectants, flavoring and sweetening agents, etc.) as disclosed in
e.g., U.S. Pat. No. 3,988,433, to Benedict. If a mouthrinse is to
be used, then a "mouthrinse carrier" may be chosen (comprising
e.g., water, flavoring and sweetening agents, etc.), as disclosed
in e.g., U.S. Pat. No. 3,988,433 to Benedict. If a mouth spray is
to be used, then a "mouth spray carrier" may be chosen or if a
lozenge is to be used, then a "lozenge carrier" may be chosen
(e.g., a candy base). If a chewing gum is to be used, a "chewing
gum carrier" may be chosen (comprising e.g., gum base, flavoring
and sweetening agents). If a sachet is to be used, then a "sachet
carrier" may be chosen (e.g., sachet bag, flavoring and sweetening
agents). If a subgingival gel is to be used (for delivery of
actives into the periodontal pockets or around the periodontal
pockets), then a "subgingival gel carrier" may be chosen. Other
useful carriers suitable for the preparation of compositions of the
present invention are well known in the art. Their selection may
depend on secondary considerations like taste, cost, shelf
stability, the desire for a sugar or salt-free composition, and the
like.
[0080] In some embodiments, the composition may be in the form of a
non-abrasive gel, e.g., a subgingival gel, which may be aqueous or
non-aqueous. Aqueous gels generally include a thickening agent
(from about 0.1% to about 20%), a humectant (from about 10% to
about 55%), a flavoring agent (from about 0.04% to about 2%), a
sweetening agent (from about 0.1% to about 3%), a coloring agent
(from about 0.01% to about 0.5%), and the balance water. In certain
cases, the composition may comprise an anticaries agent (from about
0.05% to about 0.3% as fluoride ion), and an anticalculus agent
(from about 0.1% to about 13%).
[0081] In other embodiments, the composition may also be in the
form of a dentifrice, such as a toothpaste, tooth gel or tooth
powder. Components of such toothpaste and tooth gels may include
one or more of a dental abrasive (from about 5% to about 50%), a
surfactant (from about 0.5% to about 10%), a thickening agent (from
about 0.1% to about 5%), a humectant (from about 10% to about 55%),
a flavoring agent (from about 0.04% to about 2%), a sweetening
agent (from about 0.1% to about 3%), a coloring agent (from about
0.01% to about 0.5%) and water (from about 2% to about 45%). Such
toothpaste or tooth gel may also include one or more of an
anticaries agent (from about 0.05% to about 0.3% as fluoride ion),
and an anticalculus agent (from about 0.1% to about 13%). Tooth
powder may contain substantially all non-liquid components.
[0082] One exemplary dentifrice composition is described in U.S.
Pat. No. 6,238,648, and has the following formulation (w/w):
TABLE-US-00002 Glycerin 14.0 Polyethylene Glycol 300 4.5 Silica
21.5 Tetrasodium Pyrophosphate 4.5 Water 23.5 Xanthan Gum 0.3
Carboxymethyl Cellulose 0.5 Sodium Fluoride 0.2 Flavor 1.0 Sodium
Lauryl Sulfate (27.9% Solution) 4.5 Sodium Saccharin 0.4 Titanium
Dioxide 0.4 Sodium Bicarbonate 0.9 Sodium Carbonate, Anhydrous 1.4
Poloxamer 407 1.8 Xylitol 10.0 Propylene Glycol 10.6 TOTAL
100.00
[0083] Another exemplary dentifrice composition is described in
U.S. Pat. No. 5,578,295, and has the following formulation
(w/w):
TABLE-US-00003 Triclosan diphosphate 1 Sorbitol 33 Saccharin 0.46
Silica 22 NaF 0.243 Glycerin 9 NaOH (50%) 0.2 Carbopol 0.2 Keltrol
0.6 TiO.sub.2 0.5 Sodium alkyl sulphate (28% soln.) 4 PEG 6 3
FD&C Blue #1 (1% soln) 0.05 Flavor 1.1 Water q.s.
[0084] In some embodiments, the composition is a mouthwash,
including mouth spray. Components of such mouthwashes and mouth
sprays typically include one or more of water (from about 45% to
about 95%), ethanol (from about 0% to about 25%), a humectant (from
about 0% to about 50%), a surfactant (from about 0.01% to about
7%), a flavoring agent (from about 0.04% to about 2%), a sweetening
agent (from about 0.1% to about 3%), and a coloring agent (from
about 0.001% to about 0.5%). Such mouthwashes and mouth sprays may
also include one or more of an anticaries agent (from about 0.05%
to about 0.3% as fluoride ion), and an anticalculus agent (from
about 0.1% to about 3%).
[0085] In certain embodiments, the composition may be dental
solutions including irrigation fluids. Components of such dental
solutions generally include one or more of water (from about 90% to
about 99%), preservative (from about 0.01% to about 0.5%),
thickening agent (from 0% to about 5%), flavoring agent (from about
0.04% to about 2%), sweetening agent (from about 0.1% to about 3%),
and surfactant (from 0% to about 5%).
[0086] Chewing gum compositions typically include one or more of a
gum base (from about 50% to about 99%), a flavoring agent (from
about 0.4% to about 2%) and a sweetening agent (from about 0.01% to
about 20%).
[0087] Lozenges may include discoid-shaped solids comprising a
therapeutic agent in a flavored base. The base may be a hard sugar
candy, glycerinated gelatin or combination of sugar with sufficient
mucilage to give it form. These dosage forms are generally well
known in the art.
[0088] In another embodiment, the invention provides a dental
implement impregnated with the composition provided herein. The
dental implement may comprise an implement for contact with teeth
and other tissues in the oral cavity, the implement being
impregnated with a composition comprising an oxidase with
polyethyleneimine or sorbitol. The dental implement may be in the
form of impregnated fibers including dental floss or tape, chips,
strips, films, toothpicks, and polymer fibers.
[0089] Exemplary materials that may be present in an orally
acceptable carrier are described below.
[0090] Abrasives
[0091] Dental abrasives include many different materials. The
material selected may be compatible within the composition of
interest and may not excessively abrade dentin. Suitable abrasives
may include, for example, silicas including gels and precipitates,
insoluble sodium polymetaphosphate, hydrated alumina, calcium
carbonate, dicalcium orthophosphate dihydrate, calcium
pyrophosphate, tricalcium phosphate, calcium polymetaphosphate, and
resinous abrasive materials such as particulate condensation
products of urea and formaldehyde.
[0092] One class of abrasives for use in the compositions is a
particulate thermo-setting polymerized resin. Suitable resins
include, for example, melamines, phenolics, ureas, melamine-ureas,
melamine-formaldehydes, urea-formaldehyde,
melamine-urea-formaldehydes, cross-linked epoxides, and
cross-linked polyesters.
[0093] Silica dental abrasives of various types may be selected
because of their benefits dental cleaning and polishing performance
without unduly abrading tooth enamel or dentine. The silica
abrasive polishing materials, as well as other abrasives, may have
an average particle size ranging between about 0.1 to about 30
microns, or from about 1 to about 15 microns. The abrasive can be
precipitated silica or silica gels such as the silica xerogels.
[0094] Mixtures of abrasives may also be used. The total amount of
abrasive in dentifrice compositions may range from about 6% to
about 70% by weight. Toothpastes may contain from about 10% to
about 50% of abrasives, by weight of the composition. Solution,
mouth spray, mouthwash and non-abrasive gel compositions may
contain no abrasive.
[0095] Surfactants
[0096] The present compsotions may also contain a surfactant, e.g.,
a sarcosinate surfactant, isethionate surfactant or taurate
surfactant. In certain embodiments, the composition may contain
alkali metal or ammonium salts of these surfactants. In certain
cases, the composition may contain sodium and potassium salts of
the following: lauroyl sarcosinate, myristoyl sarcosinate,
palmitoyl sarcosinate, stearoyl sarcosinate and oleoyl sarcosinate.
Other suitable compatible surfactants may be used in place of or in
combination with these surfactants.
[0097] Suitable anionic surfactants include the water-soluble salts
of alkyl sulfates having from 10 to 18 carbon atoms in the alkyl
radical and the water-soluble salts of sulfonated monoglycerides of
fatty acids having from 10 to 18 carbon atoms. Sodium lauryl
sulfate and sodium coconut monoglyceride sulfonates are examples of
anionic surfactants of this type. Mixtures of anionic surfactants
may also be utilized.
[0098] Suitable cationic surfactants include derivatives of
aliphatic quaternary ammonium compounds having one long alkyl chain
containing from about 8 to 18 carbon atoms such as lauryl
trimethylammonium chloride; cetyl pyridinium chloride; cetyl
trimethylammonium bromide;
di-isobutylphenoxyethyl-dimethylbenzylammonium chloride; coconut
alkyltrimethylammonium nitrite; cetyl pyridinium fluoride; etc. In
certain cases, surfactant compounds may be the quaternary ammonium
fluorides with detergent properties. Some cationic surfactants may
act as germicides in the composition.
[0099] Suitable nonionic surfactants include compounds produced by
the condensation of alkylene oxide groups (hydrophilic in nature)
with an organic hydrophobic compound which may be aliphatic or
alkylaromatic in nature. Examples include the Pluronics,
polyethylene oxide condensates of alkyl phenols, products derived
from the condensation of ethylene oxide with the reaction product
of propylene oxide and ethylene diamine, ethylene oxide condensates
of aliphatic alcohols, long chain tertiary amine oxides, long chain
tertiary phosphine oxides, long chain dialkyl sulfoxides and
mixtures of such materials.
[0100] Suitable zwitterionic synthetic surfactants include
derivatives of aliphatic quaternary ammonium, phosphonium, and
sulfonium compounds, in which the aliphatic radicals can be
straight chain or branched, and wherein one of the aliphatic
substituents contains from about 8 to 18 carbon atoms and one
contains an anionic water-solubilizing group, e.g., carboxy,
sulfonate, sulfate, phosphate or phosphonate.
[0101] Suitable betaine surfactants include decyl betaine or
2-(N-decyl-N,N-dimethylammonio)acetate, coco betaine or
2-(N-coco-N,N-dimethyl ammonio)acetate, myristyl betaine, palmityl
betaine, lauryl betaine, cetyl betaine, cetyl betaine, stearyl
betaine, etc. Amidobetaines are exemplified by cocoamidoethyl
betaine, cocoamidopropyl betaine, lauramidopropyl betaine and the
like. In certain embodiments, the betaines in the composition are
cocoamidopropyl betaine or lauramidopropyl betaine.
[0102] Surfactants may be present at a concentration in the range
of about 0.1% to about 2.5%, from about 0.3% to about 2.5%, or from
about 0.5% to about 2.0% by weight of the total composition.
[0103] Anti-Plaque Agent
[0104] The compositions may also include an anti-plaque agent, such
as a synthetic anionic polymer, e.g., polyacrylate or copolymers of
maleic anhydride or acid and methyl vinyl ether as well as
polyamino propane sulfonic acid (AMPS), zinc citrate trihydrate,
polypeptides (such as polyaspartic and polyglutamic acids), and
mixtures thereof.
[0105] Chelating Agents
[0106] The compositions may include a chelating agent. Chelating
agents include tartaric acid and pharmaceutically-acceptable salts
thereof, citric acid and alkali metal citrates and mixtures
thereof. Chelating agents may complex calcium found in the cell
walls of the bacteria. Chelating agents may also disrupt plaque by
removing calcium from the calcium bridges which help hold this
biomass intact. A chelating agent that may result in tooth
demineralization should not be used.
[0107] In some embodiments, alkali metal citrates (e.g., sodium and
potassium citrate) are present in the compositions. In certain
cases, chelating agents include a citric acid/alkali metal citrate
combination. In other cases, alkali metal salts of tartaric acid
may be used. Other agents include disodium tartrate, dipotassium
tartrate, sodium potassium tartrate, sodium hydrogen tartrate and
potassium hydrogen tartrate. The tartaric acid salt chelating agent
may be used alone or in combination with other optional chelating
agents. In certain embodiments, these chelating agents have a
calcium binding constant of about 10.sup.1 to 10.sup.5 to provide
improved cleaning with reduced plaque formation.
[0108] Another group of chelating agents is the anionic polymeric
polycarboxylates. Such materials are well known in the art, being
employed in the form of their free acids, partially or fully
neutralized water soluble alkali metal (e.g. potassium and
preferably sodium) or ammonium salts. In certain cases, composition
contain 1:4 to 4:1 copolymers of maleic anhydride or acid with
another polymerizable ethylenically unsaturated monomer, such as
methyl vinyl ether (methoxyethylene) having an average molecular
weight (AMW) of about 30,000 to about 1,000,000.
[0109] Other operative polymeric polycarboxylates may include those
such as the 1:1 copolymers of maleic anhydride with ethyl acrylate,
hydroxyethyl methacrylate, N-vinyl-2-pyrrolidone, or ethylene, and
1:1 copolymers of acrylic acid with methyl or hydroxyethyl
methacrylate, methyl or ethyl acrylate, isobutyl vinyl ether or
N-vinyl-2-pyrrolidone.
[0110] Additional operative polymeric polycarboxylates may be
copolymers of maleic anhydride with styrene, isobutylene or ethyl
vinyl ether, polyacrylic, polyitaconic and polymaleic acids, and
sulfoacrylic oligomers of AMW as low as 1,000.
[0111] The amounts of chelating agent may be about 0.1% to about
2.5%, about 0.5% to about 2.5%, or from about 1.0% to about
2.5%.
[0112] Fluoride Source
[0113] In certain embodiments, a water-soluble fluoride compound
may be present in an oral care composition in an amount sufficient
to give a fluoride ion concentration in the composition at
25.degree. C., from about 0.0025% to about 5.0% by weight, or about
0.005% to about 2.0% by weight. A wide variety of fluoride
ion-yielding materials may be employed as sources of soluble
fluoride in the present compositions. Representative fluoride ion
sources may include stannous fluoride, sodium fluoride, potassium
fluoride, sodium monofluorophosphate and many others. In certain
cases, the subject composition contain stannous fluoride and sodium
fluoride, as well as mixtures thereof.
[0114] Teeth Whitening Actives and Teeth Color Modifying
Substances
[0115] A teeth whitening agent and/or teeth color-modifying
substance may also be present in the oral care compositions. These
substances are suitable for modifying the color of the teeth. These
substances may comprise particles that when applied on the tooth
surface modify that surface in terms of absorption and, or
reflection of light. Such particles may provide an appearance
benefit when a film containing such particles is applied over the
surfaces of a tooth or teeth.
[0116] Particles include pigments and colorants routinely used in
the cosmetic arts. There are no specific limitations as to the
pigment and, or colorant used in the present composition. Pigments
and colorants include inorganic white pigments, inorganic colored
pigments, pearling agents, filler powders and the like. Specific
examples may be selected from the group consisting of talc, mica,
magnesium carbonate, calcium carbonate, magnesium silicate,
aluminum magnesium silicate, silica, titanium dioxide, zinc oxide,
red iron oxide, brown iron oxide, yellow iron oxide, black iron
oxide, ferric ammonium ferrocyanide, manganese violet, ultramarine,
nylon powder, polyethylene powder, methacrylate powder, polystyrene
powder, silk powder, crystalline cellulose, starch, titanated mica,
iron oxide titanated mica, bismuth oxychloride, and mixtures
thereof. In certain embodiments, titanium dioxide, bismuth
oxychloride, zinc oxide, or mixtures thereof are used.
[0117] The pigments may be used as opacifiers and colorants. These
pigments may be used as treated particles, or as the raw pigments
themselves. Typical pigment levels may be selected for the
particular impact that is desired by the consumer. For example, for
teeth that are particularly dark or stained one may use pigments in
sufficient amount to lighten the teeth. On the other hand, where
individual teeth or spots on the teeth are lighter than other
teeth, pigments to darken the teeth may be useful. The levels of
pigments and colorants may be used in the range of about 0.05% to
about 20%, from about 0.10% to about 15%, or from about 0.25% to
about 10% of the composition.
[0118] Thickening Agents
[0119] In certain embodiments, such as toothpaste or gels, some
thickening material may provide a consistency of the composition,
active release characteristics upon use, shelf stability, and
stability of the composition, etc. Thickening agents include
carboxyvinyl polymers, carrageenan, hydroxyethyl cellulose,
laponite and water soluble salts of cellulose ethers such as sodium
carboxymethylcellulose and sodium carboxymethyl hydroxyethyl
cellulose. Natural gums, such as gum karaya, xanthan gum, gum
arabic, and gum tragacanth, may also be used. Colloidal magnesium
aluminum silicate or finely divided silica may be used as part of
the thickening agent to further improve texture.
[0120] Thickening or gelling agents may include a class of
homopolymers of acrylic acid crosslinked with an alkyl ether of
pentaerythritol or an alkyl ether of sucrose, or carbomers, or
mixtures thereof.
[0121] Copolymers of lactide and glycolide monomers having a
molecular weight in the range of from about 1,000 to about 120,000
(number average), may be used in the subject composition such as a
"subgingival gel."
[0122] Thickening agents may be used in an amount from about 0.1%
to about 15%, about 2% to about 10%, or from about 4% to about 8%,
by weight of the total toothpaste or gel composition. Higher
concentrations may be used for chewing gums, lozenges (including
breath mints), sachets, non-abrasive gels and subgingival gels.
[0123] Humectants
[0124] In certain embodiments, topical, oral carrier of the subject
composition may include a humectant. The humectant may serve to
keep the subject compositions from hardening upon exposure to air,
to give compositions a moist feel to the mouth, and, for particular
humectants, to impart desirable sweetness of flavor to toothpaste
compositions. The humectant, on a pure humectant basis, may
comprise from about 0% to about 70%, or about 5% to about 25%, by
weight of the compositions herein. Suitable humectants for use in
compositions of the subject invention include edible polyhydric
alcohols such as glycerin, sorbitol, xylitol, butylene glycol,
polyethylene glycol, and propylene glycol. In certain cases, the
humectant is sorbitol and/or glycerin.
[0125] Flavoring and Sweetening Agents
[0126] Flavoring agents may also be added to the compositions.
Suitable flavoring agents include oil of wintergreen, oil of
peppermint, oil of spearmint, clove bud oil, menthol, anethole,
methyl salicylate, eucalyptol, cassia, 1-menthyl acetate, sage,
eugenol, parsley oil, oxanone, alpha-irisone, marjoram, lemon,
orange, propenyl guaethol, cinnamon, vanillin, thymol, linalool,
cinnamaldehyde glycerol acetal known as CGA, and mixtures thereof.
Flavoring agents may be used in the compositions at levels of from
about 0.001% to about 5%, by weight of the composition.
[0127] Suitable sweetening agents include sucrose, glucose,
saccharin, dextrose, levulose, lactose, mannitol, sorbitol,
fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame,
D-tryptophan, dihydrochalcones, acesulfame, cyclamate salts, sodium
cyclamate or sodium saccharin, and mixtures thereof. A composition
may contain from about 0.1% to about 10% of these agents, or from
about 0.1% to about 1%, by weight of the composition.
[0128] In addition to flavoring and sweetening agents, coolants,
salivating agents, warming agents, and numbing agents may be used
as optional ingredients in the subject composition. These agents
may be present in the compositions at a level of from about 0.001%
to about 10%, or from about 0.1% to about 1%, by weight of the
composition.
[0129] The coolant may be any of a wide variety of materials.
Included among such materials are carboxamides, menthol, ketals,
diols, and mixtures thereof. Exemplary coolants are paramenthan
carboxyamide agents such as N-ethyl-p-menthan-3-carboxamide,
N,2,3-trimethyl-2-isopropylbutanamide, and mixtures thereof. Other
coolants may be selected from the group consisting of menthol,
3-1-menthoxypropane-1,2-diol, menthone glycerol acetal, and menthyl
lactate. The terms menthol and menthyl include dextro- and
levorotatory isomers of these compounds and racemic mixtures,
thereof.
[0130] Warming agents include capsicum and nicotinate esters, such
as benzyl nicotinate. Numbing agents may include benzocaine,
lidocaine, clove bud oil, and ethanol.
[0131] Alkali Metal Bicarbonate Salt
[0132] The compositions may also include an alkali metal
bicarbonate salt. Alkali metal bicarbonate salts may be soluble in
water and unless stabilized, may release carbon dioxide in an
aqueous system. Sodium bicarbonate, also known as baking soda, may
be present as the alkali metal bicarbonate salt. In certain
embodiments, the composition contains from about 0.5% to about 30%,
about 0.5% to about 15%, or from about 0.5% to about 5% of an
alkali metal bicarbonate salt.
[0133] Miscellaneous Carriers
[0134] Water employed in the preparation of the oral care
compositions may be of low ion content and free of organic
impurities, and may be present in an amount of about 5% to about
70%, or from about 20% to about 50%, by weight, of an aqueous
composition. These amounts of water may include free water that is
added, plus water that is introduced with other agents or
carriers.
[0135] Poloxamers may be employed in the compositions. The
poloxamer may be classified as a nonionic surfactant. It may
function as an emulsifying agent, binder, or stabilizer, or perform
a related function. Poloxamers include difunctional block-polymers
terminating in primary hydroxyl groups with molecular weights
ranging from 1,000 to above 15,000.
[0136] Other emulsifying agents that may be used in the
compositions include polymeric emulsifiers. Predominantly high
molecular weight polyacrylic acid polymers may be useful as
emulsifiers.
[0137] Titanium dioxide may also be added to the composition.
Titanium dioxide is a white powder which may add opacity to the
compositions. Titanium dioxide may comprise from about 0.25% to
about 5% by weight of the composition.
[0138] The pH of the composition is preferably adjusted through the
use of one or more buffering agents. Buffering agents refer to
agents that can be used to adjust the pH of the compositions in a
range of about pH 4.0 to about pH 10.0. Buffering agents include
monosodium phosphate, trisodium phosphate, sodium hydroxide, sodium
carbonate, sodium acid pyrophosphate, citric acid, and sodium
citrate. Buffering agents may be administered at a level of from
about 0.5% to about 10%, by weight of the present compositions. In
certain embodiments, the pH of dentifrice compositions may be
measured from a 3:1 aqueous slurry of dentifrice, e.g., 3 parts
water to 1 part dentifrice.
[0139] Other agents that may be used in the present compositions
include dimethicone copolyols selected from alkyl- and
alkoxy-dimethicone copolyols, such as C.sub.12 to C.sub.20 alkyl
dimethicone copolyols and mixtures thereof. In certain cases, the
compositions contain cetyl dimethicone copolyol. The dimethicone
copolyol may be present in a level of from about 0.01% to about
25%, about 0.1% to about 5%, or from about 0.5% to about 1.5% by
weight. The dimethicone copolyols may aid in providing positive
tooth feel benefits.
[0140] Other Active Agents
[0141] The present oral care composition may also include other
active agents, such as antimicrobial agents. Included among such
agents are water insoluble non-cationic antimicrobial agents such
as halogenated diphenyl ethers, phenolic compounds including phenol
and its homologs, mono and poly-alkyl and aromatic halophenols,
resorcinol and its derivatives, bisphenolic compounds and
halogenated salicylanilides, benzoic esters, and halogenated
carbanilides. Water soluble antimicrobials include quaternary
ammonium salts and bis-biquamide salts, among others. An additional
water soluble antimicrobial agent is triclosan monophosphate.
Quaternary ammonium agents include those in which one or two of the
substitutes on the quaternary nitrogen has a carbon chain length
(typically alkyl group) from about 8 to about 20, or from about 10
to about 18 carbon atoms while the remaining substitutes (typically
alkyl or benzyl group) have a lower number of carbon atoms, such as
from about 1 to about 7 carbon atoms, such as methyl or ethyl
groups. Dodecyl trimethyl ammonium bromide, tetradecylpyridinium
chloride, domiphen bromide, N-tetradecyl-4-ethyl pyridinium
chloride, dodecyl dimethyl (2-phenoxyethyl)ammonium bromide, benzyl
dimethylstearyl ammonium chloride, cetyl pyridinium chloride,
quaternized
5-amino-1,3-bis(2-ethyl-hexyl)-5-methyl-hexahydropyrimidine,
benzalkonium chloride, benzethonium chloride and methyl
benzethonium chloride are exemplary of quaternary ammonium
antibacterial agents. Other compounds are
bis[4-(R-amino)-1-pyridinium]alkanes. Other antimicrobials, such as
copper bisglycinate, copper glycinate, zinc citrate, and zinc
lactate, may also be included.
[0142] In addition to an .alpha.-glucanase, the present oral care
compositions may also contain one or more other enzymes that have
carbohydrate hydrolysis, antimicrobial, or teeth whitening
activity. Such enzymes include, but are not limited to, a
deaminase, an esterase, a glycosidase, glucanhydrolase, a
dextrinase, an amylase, a transglucosidase, a cellulase, a
hemicellulase, a lipase, an oxidase, a peroxidase, a protease, and
a urease.
C. Method of Use
[0143] Another aspect of the present compositions and method is a
method of contacting a tooth surface with a composition comprising
an alpha-glucanase to reduce or prevent tooth decay, or to reduce
or prevent the underlying causes of tooth decay.
[0144] In some embodiments, the method involves contacting a tooth
surface with a composition comprising an alpha-glucanase to
hydrolyze a glycocalyx present on the tooth surface. According to
this embodiment, the 1,3-.alpha.-D-glucosidase activity of the
alpha-glucanase, and/or other activities of the alpha-glucanase,
hydrolizes polysaccharides, glucans, mannans, and/or adhesive
molecules produced by plaque bacteria, thereby decreasing the
ability of plaque to adhere to the tooth surface, reducing plaque
formation or reducing the levels of existing plaque. Such
polysaccharides, glucans, mannans, and/or adhesive molecules may be
present in what is conventionally referred to as the
glycocalyx.
[0145] In some embodiments, the method involves contacting a tooth
surface with a composition comprising an alpha-glucanase to dewater
the tooth surface by disrupting the polysaccharides, glucans,
mannans, and/or adhesive molecules produced by bacteria in the
mouth, thereby reducing film formation or bacterial adhesion,
and/or preventing the accumulation of bacterial acids and other
substances that damage the tooth surface.
[0146] The method may include contacting the tooth surface with one
or more alpha-glucanases, which may be formulated as described,
above. The methods may include contacting the tooth surface with an
additional enzyme, which may be present in the same formulation or
a different formulation. In some embodiments, the enzyme is a
deaminase, an esterase, a glycosidase, glucanhydrolase, a
dextrinase, an amylase, a transglucosidase, a cellulase, a
hemicellulase, a lipase, an oxidase, a peroxidase, a protease, and
a urease. The additional enzyme may also be an additional
alpha-glucanase.
[0147] Tooth and other personal care compositions and their
mechanisms of action are described in detail in Lad. R. (ed.)
"Biotechnology in Personal Care", Cosmetic Science and Technology
Series, Vol. 29, Taylor and Francis Group, New York, N.Y., USA,
2006. This reference is indicative of the state of the art, as is
incorporated herein.
[0148] In addition to oral care applications, the present
compositions and methods can be adapted for the prevention or
removal of biofilims in a large number of other situtations, for
example, in cooling water equipment, in drinking water equipment,
in food products and food handling equipment, on (or in) medical
implants, in paper and textile manufacturing and processing, in oil
refinery and mining equipment, on the hulls of ships and boats, in
chemical manufacturing, in swimming pools, aquariums, and ponds,
and the like. In such cases, alpha glucanases can be used to
disrupt polysaccharide components present in the biofilm, thereby
reducing the attachment and/or adhesion of microorganism to
surfaces. The disruption of such polysaccharides also results in
dewatering, which reduces or prevents the formation of a
microenvironment suitable for the growth and propagation of
microorganism on a surface.
[0149] Other aspects and embodiments of the present compositions
and methods will be apparent to the skilled person in view of the
disclosure.
EXPERIMENTAL
[0150] The following examples are offered to illustrate the present
compositions and methods, and advantages thereof, and should not be
construed as limiting their scope.
Example 1
Identification of Candidate Fungal .alpha.-1,3-glucanases (EC
3.2.1.59)
[0151] Several candidate fungi, including Hypocrea tawa,
Trichoderma reesei, Trichoderma konilangbra, and Trichoderma
harzianum were grown in culture in defined media with 15% maltose
(28.degree. C., 7 days, 150 rpm agitation). Supernatants were
harvested by sterile filtration, concentrated, and desalted for
glucan (and dextran) hydrolysis activity screening. The desalted
culture supernatants (5% by volume) were added to 0.2% washed
insoluble glucan in 100 mM phosphate buffer, pH 6.3 (or 100 mM
acetate buffer, pH 4.5). The reaction mixtures were incubated
overnight at 37-40 C..degree.. The mixtures were visually inspected
for solubilization of the insoluble glucan, and the supernatants
were analyzed by HPLC for soluble hydrolysis products. For HPLC
analysis, the reaction supernatant was diluted 10-fold into 10 mM
NaOH, and 10 .mu.l was then injected into an Agilent 1100 HPLC
equipped with electrochemical detection. Mono- and disaccharides
were eluted with a NaOH/sodium acetate gradient on a PA1 anion
exchange column. The components of unknown mixtures were identified
based on previously run standards. The supernatants from the
Trichoderma reesei, Trichoderma konilangbra, and Hypocrea tawa
resulted in the most solubilization of glucan (see, e.g., FIG. 2).
The .alpha.-1,3-glucanases of H. tawa, T. reesei, and T.
konilangbra were selected for cloning, expression, and
characterization. Putative T. reesei .alpha.-1,3-glucanase
sequences were identified in the genome sequence (JGI) by
homology.
Example 2
Isolation of Genomic DNA
[0152] Fungal cultures of T. reesei, T. konilangbra, and H. tawa
were prepared by adding 30 mL of sterile YEG broth to three 250 mL
baffled Erlenmeyer shaking flasks in the biological hood. A
1.times.1 inch square was cut and removed from each respective
fungal culture plate using a sterile plastic loop and placed into
the appropriate culture flask. The inoculated flasks were then
placed into the 28.degree. C. shaking incubator to grow
overnight.
[0153] The T. reesei, T. konilangbra, H. tawa cultures were removed
from the shaking incubator and the contents of each flask were
poured into separate sterile 50 mL Sarstedt tubes. The Sarstedt
tubes were placed in a table-top centrifuge and spun at 4,500 rpm
for 10 minutes to pellet the fungal mycelia. The supernatants were
discarded and a large loopful of each mycelial sample was
transferred to a separate tube containing lysing matrix (FastDNA).
Genomic DNA was extracted from the harvested mycelia using the
FastDNA kit (Qbiogene), according to the manufacturer's protocol
for algae, fungi and yeast. The homogenization time (Mini
BeadBeater-8) was 25 seconds. The amount and quality of genomic DNA
extracted was determined by gel electrophoresis.
Example 3
Obtaining alpha-glucanase polypeptides by PCR
[0154] A. T. reesei
[0155] Putative .alpha.-1,3 glucanase genes were identified in the
T. reesei genome (JGI) by homology. PCR primers for T. reesei were
designed based on the putative homolog DNA sequences. Degenerate
PCR primers were designed for T. konilangbra or H. tawa based on
the putative T. reesei protein sequences and other published
.alpha.-1,3 glucanase protein sequences.
TABLE-US-00004 T. reesei specific PCR primers: (SEQ ID NO: 6)
SK592: 5'-CACCATGTTTGGTCTTGTCCGC (SEQ ID NO: 7) SK593:
5'-TCAGCAGTACTGGCATGCTG
[0156] The PCR conditions used to amplify the putative .alpha.-1,3
glucanase from genomic DNA extracted from T. reesei strain RL-P37
were as follows: 1. 94.degree. C. for 2 minutes, 2. 94.degree. C.
for 30 seconds, 3. 56.degree. C. for 30 seconds, 4. 72.degree. C.
for 3 minutes, 5. return to step 2 for 24 cycles, 6. 4.degree. C.
indefinitely. Reaction samples contained 2 .mu.L of RL-P37 genomic
DNA, 10 .mu.L of the 10.times. buffer, 2 .mu.L 10 mM dNTPs mixture,
1 .mu.L primers SK592 and SK593 at 20 .mu.M, 1 .mu.L of the Pfu
Ultra and 83 .mu.L distilled water.
B. T. konilangbra and H. tawa
[0157] Initial PCR reactions used degenerate primers designed from
protein alignments of several homologous sequences. A primary set
of degenerate primers, designed to anneal near the 5' and 3' ends,
were used in the first PCR reaction to amplify similar sequences to
that of an .alpha.-1,3 glucanase.
Degenerate Primers for Initial Cloning:
TABLE-US-00005 [0158] H. tawa and T. konilangbra: (SEQ ID NO: 8)
MA1F: GTNTTYTGYCAYTTYATGAT (SEQ ID NO: 9) MA2F:
GTNTTYTGYCAYTTYATGATHGGNAT (SEQ ID NO: 10) MA4F:
GAYTAYGAYGAYGAYATGCARCG (SEQ ID NO: 11) MA5F:
GTRCAYTTRCAIGGICCIGGIGGRCARTANCC (SEQ ID NO: 12) MA6R:
YTCICCIGGNAGNGGRCANCCRTT (SEQ ID NO: 13) MA7R:
RCARTAYTGRCAIGCYGTYGGYGGRCARTA
[0159] The products of these PCR reactions were then used in a
nested PCR, using primers designed to attach within the product of
the initial PCR fragment, under the same amplification
conditions.
Specific Primers for Initial Cloning:
TABLE-US-00006 [0160] T. konilangbra: (SEQ ID NO: 14) TP1S:
CCCCCTGGCCAAGTATGTGT (SEQ ID NO: 15) TP2A: GTACGCAAAGTTGAGCTGCT
(SEQ ID NO: 16) TP3S: AGCACATCGCTGATGGATAT (SEQ ID NO: 17) TP3A:
AAGTATACGTTGCTTCCGGC (SEQ ID NO: 18) TP4S: CTGACGATCGGACTRCACGT
(SEQ ID NO: 19) TP4A: GGTTGTCGACGTAGAGCTGT H. tawa: (SEQ ID NO: 20)
HP2A: ACGATCGGCAGAGTCATAGG (SEQ ID NO: 21) HP3S:
ATCGGATTGCATGTCACGAC (SEQ ID NO: 22) HP3A: TACATCCAGACCGTCACCAG
(SEQ ID NO: 23) HP4S: ACGTTTGCTCTTGCGGTATC (SEQ ID NO: 24) HP4A:
TCATTATCCCAGGCCTAAAA
[0161] Gel electrophoresis of the PCR products was used to
determine whether fragments of expected size were amplified. Single
nested PCR products of the expected size were purified using the
QIAquick PCR purification kit (Qiagen). In addition, expected size
products were excised and extracted from agarose gels containing
multiple product bands and purified using the QIAquick Gel
Extraction kit (Qiagen).
Example 4
Transformation/Isolate Screening/Plasmid Extraction
[0162] PCR products were inserted into cloning vectors using the
Invitrogen Zero Blunt.RTM. TOPO.RTM. PCR Cloning Kit, according to
the manufacturer's specifications. The vector was then transformed
into One.RTM.Shot Top 10 chemically competent E. coli cells
(Invitrogen), according to the manufacturer's recommendation and
then spread onto LB plates containing 50 ppm of Kanamycin. These
plates were incubated in the 37.degree. C. incubator overnight.
[0163] To select transformants that contained the vector and DNA
insert, colonies were selected from the plate for crude plasmid
extraction. 50 .mu.L of DNA Extraction Solution (100 mM NaCl, 10 mM
EDTA, 2 mM Tris pH 7) was added to clean 1.5 mL Eppendorf tubes. In
the biological hood, 7-10 individual colonies of each TOPO.RTM.
transformation clone were numbered, picked and resuspended in the
extraction solution. In the chemical hood, 50 .mu.L of Phenol:
Chloroform: Isoamyl alcohol was added to each sample and vortexed
thoroughly. Tubes were microcentrifuged at maximum speed for 5
minutes, after which 20 .mu.L of the top aqueous layer was removed
and placed into a clean PCR tubes. 1 .mu.L of RNase 2 mg/mL was
then added, and samples were mixed and incubated at 37.degree. C.
for 30 minutes. The entire sample volume was then run on a gel to
determine the presence of the insert in the TOPO.RTM. vector based
on difference in size to an empty vector. Once the transformant
colonies had been identified, those clones was scraped from the
plate, and used to inoculate separate 15 mL tubes containing 5 ml
of LB/Kanamycin medium (0.0001%). The cultures were placed in the
37.degree. C. shaking incubator overnight.
[0164] Samples were removed from the incubator and centrifuged for
6 minutes at 6,000 rpm using the Sorval centrifuge. The QIAprep
Spin Miniprep kit (Qiagen) and protocol were used to isolate the
plasmid DNA, which was then digested to confirm the presence of the
insert. The restriction enzyme used was dependent on the sites
present in and around the insert sequence. Gel electrophoresis was
used to determine fragment size. Appropriate DNA samples were
submitted for sequencing (Sequetech, Mountain View, Calif.).
Example 5
Cloning the 3' and 5' Ends
[0165] All DNA fragments were sequenced. Sequences were aligned and
compared to determine nucleotide and amino acid identities using
Align X and ContigExpress.RTM. (Vector NTI.RTM. suite, Invitrogen).
Specific primers were designed to amplify the 3' and 5' portions of
each incomplete fragment from H. tawa and T. konilangbra by
extending outward from the known sequence. At least three specific
primers, each nested within the amplified product of the previous
primer set, were designed for each template. Amplification of the
5' and 3' sequences was performed using the nested primer sets with
the LA PCR In vitro Cloning Kit (TaKaRa BIO Inc.).
[0166] Fresh genomic DNA was prepared for this amplification.
Cultures of T. konilangbra and H. tawa were prepared by inoculating
30 mL of YEG broth with a 1 square inch section of the appropriate
sporulated fungal plate culture in 250 mL baffled Erlenmeyer
flasks. The flasks were incubated in the 28.degree. C. shaking
incubator overnight. The cultures were harvested by centrifugation
in 50 mL Sarstedt tubes at 4,500 rpm for 10 minutes. The
supernatant was discarded and the mycelia were stored overnight in
a -80.degree. C. freezer. The frozen mycelia were then placed into
a coffee grinder along with a few pieces of dry ice. The grinder
was run until the entire mixture had a powder-like consistency. The
powder was then air dried and transferred to a sterile 50 mL
Sarstedt tube containing 10 mL of Easy-DNA.TM. Kit Solution A
(Invitrogen) and the manufacturer's protocol was followed. The
concentration of the genomic DNA collected from the extraction was
measured using the NanoDrop spectrophotometer.
[0167] The LA PCR In vitro Cloning Kit cassettes were chosen based
on the absence of a particular restriction site within the known
DNA sequences, and the manufacturer's instructions were followed.
For first PCR run, 1 .mu.L of the ligation DNA sample was diluted
in 33.5 .mu.L of sterilized distilled water. Different primers were
used depending on the sample and the end fragment desired. For the
5' ends, primers HP4A and TP3A were used for H. tawa and T.
konilangbra respectively, while for the 3' ends primers HP4S and
TP3S were used for H. tawa and T. konilangbra. The PCR mixture was
prepared by adding 34.5 .mu.L diluted ligation DNA solution, 5
.mu.L of 10.times. LA Buffer II (Mg.sup.+2), 8 .mu.L dNTPs mixture,
1 .mu.L cassette primer 1, 1 .mu.L specific primer I (depending on
sample and end fragment), and 0.5 .mu.L TaKaRa LA Taq. The PCR
tubes were then placed in a thermocycler following the listed
protocol: 1. 94.degree. C. for 10 minutes, 2. 94.degree. C. for 30
seconds, 3. 55.degree. C. for 30 seconds, 4. 72.degree. C. for 4
minutes, return to step 2. 30 times, 4.degree. C. indefinitely.
[0168] A second PCR reaction was prepared by taking 1 .mu.L of the
first PCR reaction and diluting the sample in sterilized distilled
water to a dilution factor of 1/10,000. A second set of primers
nested within the first amplified region were used to amplify the
fragment isolated in the first PCR reaction. Primers HP3A and TP4A
were used to amplify toward the 5' end of H. tawa and T.
konilangbra respectively, while primers HP3S and TP4S were used to
amplify toward the 3' end. The diluted DNA was added to the PCR
reaction containing 33.5 .mu.L distilled sterilized water, 5 .mu.L
10.times. LA Buffer II (Mg.sup.+2), 8 .mu.L dNTPs mixture, 1 .mu.L
of cassette primer 2, 1 .mu.L of specific primer 2 (dependent on
sample and fragment end), 0.5 .mu.L, TaKaRa LA Taq, and mixed
thoroughly before the PCR run. The PCR protocol was the same as the
first reaction, without the initial 94.degree. C. for 10 minutes.
After the reaction was complete, the sample was run by gel
electrophoresis to determine size and number of fragments isolated.
If a single band was present, the sample was purified and sent for
sequencing. If no fragment was isolated, a third PCR reaction was
performed using the previous protocol for a nested PCR reaction.
After running the amplified fragments by gel electrophoresis, the
brightest band was excised, purified, and sent for sequencing.
Example 6
Analysis of Sequence Alignments
[0169] Sequences were obtained and analyzed using the Vector NTI
suite, including Align X, and ContigExpress. Each respective end
fragment sequence was aligned to the previously obtained fragments
of H. tawa and T. konilangbra to obtain the entire gene sequence.
Nucleotide alignments with T. harzianum and T. reesei sequences
revealed the translation start and stop points of the gene of
interest in both H. tawa and T. konilangbra. After the entire gene
sequence was identified, specific primers were designed to amplify
the entire gene from the genomic DNA. Primers were designed as
described earlier, with the exception of adding CACC nucleotide
sequence before the translational starting point, for GATEWAY.RTM.
cloning (Invitrogen).
[0170] Primers for Final Cloning:
TABLE-US-00007 T. konilangbra: (SEQ ID NO: 25) T1FS:
caccatgctaggcattctccg (SEQ ID NO: 26) T1FA: tcagcagtattggcatgccg H.
tawa: (SEQ ID NO: 27) H1FS: CACCATGTTGGGCGTTTTTCG (SEQ ID NO: 28)
H1FA: CTAGCAGTATTGRCATGCCG
[0171] The PCR protocol was followed as previously described with
the exception of altering the annealing temperature to 55.degree.
C. After a single band was isolated and viewed through gel
electrophoresis, the amplified fragment was purified as described
earlier and used in the pENTR/D TOPO.RTM. (Invitrogen)
transformation, according to the manufacturer's instructions.
Chemically competent E. coli were then transformed as previously
described, and transferred to LB plates containing 50 ppm of
kanamycin. Following 37.degree. C. incubation overnight,
transformants containing the plasmid and insert were selected after
crude DNA extraction and plasmid size analysis, as previously
described. The selected transformants were scraped from the plate
and used to inoculate a fresh 15 mL tube containing 5 ml of
LB/Kanamycin medium (0.0001%). Cultures were placed in the
37.degree. C. shaking incubator overnight. Cells were harvested by
centrifugation and the plasmid DNA extracted as previously
described. Plasmid DNA was digested to confirm the presence of the
insert sequence, and then submitted for sequencing. The LR Clonase
reaction (Gateway Cloning, Invitrogen) was used, according to
manufacturer's instructions, to directionally transfer the insert
from the pENTR/D vector into the destination vector. The
destination vector is designed for expression of a gene of
interest, in T. reesei, under control of the CBH1 promoter and
terminator, with A. niger acetamidase for selection.
Example 7
Biolistic Transformation
[0172] A T. reesei spore suspension was spread onto the center
.about.6 cm diameter of an acetamidase transformation plate (150
.mu.L of a 5.times.10.sup.7-5.times.10.sup.8 spore/mL suspension).
The plate was then air dried in a biological hood. The stopping
screens (BioRad 165-2336) and the macrocarrier holders (BioRad
1652322) were soaked in 70% ethanol and air dried. DriRite
desiccant was placed in small Petri dishes (6 cm Pyrex) and
overlaid with Whatman filter paper. The macrocarrier holder
containing the macrocarrier (BioRad 165-2335) was placed flatly on
top of the filter paper and the Petri dish lid replaced.
[0173] A tungsten particle suspension was prepared by adding 60 mg
tungsten M-10 particles (microcarrier, 0.7 micron, BioRad #1652266)
to an Eppendorf tube. 1 mL ethanol (100%) was added. The tungsten
was vortexed in the ethanol solution and allowed to soak for 15
minutes. The Eppendorf tube was microfuged briefly at maximum speed
to pellet the tungsten. The ethanol was decanted and washed three
times with sterile distilled water. After the water wash was
decanted the third time, the tungsten was resuspended in 1 mL of
sterile 50% glycerol.
[0174] The transformation reaction was prepared by adding 25 .mu.L
suspended tungsten to a 1.5 mL Eppendorf tube for each
transformation. Subsequent additions were made in order, 2 .mu.L
DNA pTrex3g expression vectors, 25 .mu.L 2.5M CaCl.sub.2, 10 .mu.L
0.1M spermidine. The reaction was vortexed continuously for 5-10
minutes, keeping the tungsten suspended. The Eppendorf tube was the
microfuged briefly and decanted. The tungsten pellet was washed
with 200 .mu.L of 70% ethanol, microfuged briefly to pellet and
decanted. The pellet was washed with 200 .mu.L of 100% ethanol,
microfuged briefly to pellet, and decanted. The tungsten pellet was
resuspended in 24 .mu.L 100% ethanol. The Eppendorf tube was placed
in an ultrasonic water bath for 15 seconds and 8 .mu.L aliquots
were transferred onto the center of the desiccated macrocarriers.
The macrocarriers were left to dry in the desiccated Petri
dishes.
[0175] A Helium tank was turned on to 1500 psi. 1100 psi rupture
discs (BioRad 165-2329) were used in the Model PDS-1000/He
Biolistic Particle Delivery System (BioRad).
[0176] When the tungsten solution was dry, a stopping screen and
the macrocarrier holder were inserted into the PDS-1000. An
acetamidase plate, containing the target T. reesei spores, was
placed 6 cm below the stopping screen. A vacuum of 29 inches Hg was
pulled on the chamber and held. The He Biolistic Particle Delivery
System was fired. The chamber was vented and the acetamidase plate
removed for incubation at 28.degree. C. until colonies appeared (5
days).
TABLE-US-00008 Modified amdS Biolistic agar (MABA) per liter Part
I, make in 500 ml dH.sub.2O 1000x salts 1 ml Noble agar 20 g pH to
6.0, autoclave Part II, make in 500 ml dH.sub.2O Acetamide 0.6 g
CsCl 1.68 g Glucose 20 g KH.sub.2PO.sub.4 15 g
MgSO.sub.4.cndot.7H.sub.2O 0.6 g CaCl.sub.2.cndot.2H.sub.2O 0.6 g
pH to 4.5, 0.2 micron filter sterilize; leave in 50.degree. C. oven
to warm, add to agar, mix, pour plates. Stored at room
temperature.
TABLE-US-00009 1000x Salts per liter FeSO.sub.4.cndot.7H.sub.2O 5 g
MnSO.sub.4.cndot.H.sub.2O 1.6 g ZnSO.sub.4.cndot.7H.sub.2O 1.4 g
CoCl.sub.2.cndot.6H.sub.2O 1 g Bring up to 1 L dH.sub.2O. 0.2
micron filter sterilize
Example 8
Expression of .alpha.-1,3 glucanases by T. reesei Transformants
[0177] A 1 cm.sup.2 agar plug was used to inoculate Proflo seed
media. Cultures were incubated at 28.degree. C., with 200 rpm
shaking. On the second day, a 10% transfer was aseptically made
into Production media. The cultures were incubated at 28.degree.
C., with 200 rpm shaking. On the third day, cultures were harvested
by centrifugation. Supernatants were sterile-filtered (0.2 .mu.m
PES) and stored at 4.degree. C. Analysis by SDS-PAGE identified
clones expressing the respective alpha-glucanase genes.
Example 9
Preparation of Insoluble Glucan Substrate
[0178] Four sterile flasks containing BHI (brain heart infusion)
broth were inoculated with Streptococcus sobrinus (ATCC 27607),
from a BHI plate. The cultures were incubated at 37.degree. C. for
24 hrs, static, after which they were visibly turbid. The
supernatants (containing the S. sobrinus glucosyltransferases) were
harvested by centrifugation (15 minutes, 10,000 rpm). The
supernatants were pooled and sterile-filtered (0.22 .mu.m) to
remove any remaining cells. Aseptically, sucrose was added to a
final concentration of 5%, which induces the glucosyltransferases
to produce glucan polymer. The culture supernatant plus sucrose was
sealed, mixed, and stored at 37.degree. C. incubator for 24-48 hrs
(static). A fluffy precipitate formed and was harvested by
centrifugation (10,000 rpm, 15 minutes). The supernatant was
decanted, leaving the precipitate, which was washed with dI water
three times, lastly in a tared Eppendorf tube. The glucan was dried
in a SpeedVac, and the Eppendorf weight was recorded after
drying.
Example 10
PAHBAH Reducing Sugars Assay to Measure the Activity of Cloned
Alpha-Glucanases
[0179] Transformed T. reesei culture supernatants, buffer and
insoluble glucan were distributed into a round-bottom 96-well plate
in the amounts listed below. Two types of buffers (pH 4.5 and pH
6.0) were used to compare activity levels. After the samples were
distributed, the 96-well plate was sealed and placed into a shaking
37.degree. C. incubator overnight.
[0180] Glucan Hydrolysis Mixture:
TABLE-US-00010 50 .mu.L culture supernatant 50 .mu.L of a 28 mg/mL
glucan solution 5 .mu.L 1M Citrate buffer (pH 4.5 or 6.0) 105
.mu.L
[0181] PAHBAH solution:
TABLE-US-00011 0.5 g Sodium potassium tartrate 0.15 g
p-hydroxybenzoic acid hydrazide (PAHBAH) 10 mL 2% NaOH
[0182] Following incubation, 150 .mu.L, of freshly made PAHBAH
(p-hydroxybenzoic acid hydrazide) reagent was transferred into 0.2
mL PCR tubes along with 20 .mu.L of each hydrolysis mixture. These
tubes were then lightly mixed and placed into a thermocycler, where
they were heated to 99.degree. C. for 30 minutes. The PCR tubes
were removed and 150 .mu.L, of each sample was transferred to a
fresh 96-well plate. The absorbance of each sample was measured at
410 nm. Results of this assay are shown in FIG. 3.
Sequence CWU 1
1
341635PRTHypocrea tawa 1Met Leu Gly Val Phe Arg Arg Leu Gly Leu Gly
Ser Leu Ala Ala Ala 1 5 10 15 Ala Leu Ser Ser Leu Gly Thr Ala Ala
Pro Ala Asn Val Ala Ile Arg 20 25 30 Ser Leu Glu Glu Arg Ala Ser
Ser Ala Asp Arg Leu Val Phe Cys His 35 40 45 Phe Met Ile Gly Ile
Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp 50 55 60 Asp Asp Met
Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 65 70 75 80 Asn
Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr Ala Tyr 85 90
95 Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile Ser Phe Asp Phe
100 105 110 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys
Ile Ala 115 120 125 Gln Tyr Ala Asn Arg Pro Ala Gln Leu Tyr Val Asp
Asn Arg Pro Phe 130 135 140 Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp
Val Asn Ala Leu Arg Asn 145 150 155 160 Ala Ala Gly Ser Asn Val Tyr
Phe Val Pro Asn Phe His Pro Gly Gln 165 170 175 Ser Ser Pro Ser Asn
Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185 190 Asn Asp Gly
Asn Asn Lys Ala Pro Lys Gln Gly Gln Thr Val Thr Val 195 200 205 Ala
Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr 210 215
220 Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val
225 230 235 240 Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Ala Leu
Ile Tyr Asn 245 250 255 Arg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro
Met Val Glu Ile Val 260 265 270 Thr Trp Asn Asp Tyr Gly Glu Ser His
Tyr Val Gly Pro Leu Lys Ser 275 280 285 Lys His Phe Asp Asp Gly Asn
Ser Lys Trp Val Asn Asp Met Pro His 290 295 300 Asp Gly Phe Leu Asp
Leu Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn 305 310 315 320 Arg Asp
Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln Leu Val Tyr 325 330 335
Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr 340
345 350 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met
Gly 355 360 365 Arg Pro Asp Gly Trp Gln Thr Met Asp Asp Thr Val Tyr
Val Ala Ala 370 375 380 Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr
Ser Gly Gly Thr Thr 385 390 395 400 Gln Thr Phe Gln Gly Asn Ala Gly
Ala Asn Leu Phe Gln Ile Pro Ala 405 410 415 Ser Ile Gly Gln Gln Lys
Phe Ala Leu Thr Arg Asn Gly Gln Thr Val 420 425 430 Phe Ser Gly Thr
Ser Leu Met Asp Ile Thr Asn Val Cys Ser Cys Gly 435 440 445 Ile Tyr
Asn Phe Asn Pro Tyr Val Gly Thr Ile Pro Ala Gly Phe Asp 450 455 460
Asp Pro Leu Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His 465
470 475 480 Val Thr Thr Cys Gln Ala Lys Pro Ser Leu Gly Thr Asn Pro
Pro Val 485 490 495 Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser
Thr Thr Arg Ala 500 505 510 Ser Ser Pro Pro Val Ser Ser Thr Arg Val
Ser Ser Pro Pro Val Ser 515 520 525 Ser Pro Pro Val Thr Ser Arg Thr
Ser Ser Ser Pro Pro Pro Pro Ala 530 535 540 Ser Ser Thr Pro Ser Ser
Gly Gln Val Cys Val Ala Gly Thr Val Ala 545 550 555 560 Asp Gly Glu
Ser Gly Asn Tyr Ile Gly Leu Cys Gln Phe Ser Cys Asn 565 570 575 Tyr
Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala 580 585
590 Pro Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro
595 600 605 Gly Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys
Asn His 610 615 620 Asn Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys 625
630 635 2622PRTTrichoderma reesei 2Met Phe Gly Leu Val Arg Arg Leu
Gly Val Gly Ala Leu Val Ala Ala 1 5 10 15 Ala Leu Ser Ser Leu Ala
Ala Ala Ala Pro Ala Asn Val Ala Ile Arg 20 25 30 Ser Leu Glu Glu
Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His 35 40 45 Phe Met
Ile Gly Ile Cys Gly Asp Arg Thr Ser Ser Thr Asp Tyr Asp 50 55 60
Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 65
70 75 80 Asn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Asn Phe
Ala Tyr 85 90 95 Asp Ala Ala Asp Arg Ala Gly Met Lys Val Phe Ile
Ser Phe Asp Phe 100 105 110 Asn Trp Trp Ser Pro Gly Asn Ala Ala Gly
Val Gly Gln Lys Ile Ala 115 120 125 Gln Tyr Ala Ser Arg Pro Ala Gln
Leu Tyr Val Asp Asn Arg Pro Phe 130 135 140 Ala Ser Ser Phe Ala Gly
Asp Gly Leu Asp Val Asn Thr Leu Arg Asn 145 150 155 160 Ala Ala Gly
Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gln 165 170 175 Ser
Ser Pro Ser Thr Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185
190 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln Asn Val Thr Val
195 200 205 Ala Asp Gly Asp Asn Ser Tyr Arg Ser Trp Leu Ala Gly Lys
Pro Tyr 210 215 220 Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe
Gly Pro Glu Val 225 230 235 240 Ser Tyr Ser Lys Asn Trp Val Phe Pro
Gly Gly Ser Leu Trp Tyr Asp 245 250 255 Arg Trp Gln Asp Val Leu Arg
Gln Gly Phe Glu Met Val Glu Ile Val 260 265 270 Thr Trp Asn Asp Tyr
Gly Glu Ser His Tyr Thr Gly Pro Leu Glu Ser 275 280 285 Arg His Tyr
Asp Asp Gly Asn Ser Lys Trp Thr Asn Asp Met Pro His 290 295 300 Asp
Gly Phe Leu Asp Leu Ala Lys Pro Phe Ile Ala Ala Tyr Lys Asn 305 310
315 320 Arg Asp Thr Asp Val Ala Pro Tyr Ile Gln Asn Glu Gln Leu Ile
Tyr 325 330 335 Trp Tyr Arg Arg Asn Leu Lys Gly Leu Asp Cys Asp Ala
Thr Asp Thr 340 345 350 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly
Asn Tyr Phe Met Gly 355 360 365 Arg Pro Asp Gly Trp Gln Thr Met Asp
Asp Thr Val Tyr Val Val Ala 370 375 380 Leu Leu Lys Ser Ala Gly Thr
Val Thr Val Thr Ser Gly Gly Ala Thr 385 390 395 400 Gln Thr Phe Gln
Gly Thr Ala Gly Ala Asn Leu Phe Glu Val Pro Ala 405 410 415 Asn Leu
Gly Gln Gln Lys Phe Ala Leu Ser Arg Asn Gly Gln Thr Val 420 425 430
Phe Ser Ser Thr Ser Leu Met Asp Ile Thr Asn Val Cys Pro Cys Gly 435
440 445 Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Val Pro Ala Gly Phe
Asp 450 455 460 Asp Pro Leu Gly Pro Asp Gly Leu Ala Ser Leu Thr Ile
Gly Leu His 465 470 475 480 Val Thr Thr Cys Gln Ala Lys Pro Ser Leu
Gly Thr Asn Pro Pro Ile 485 490 495 Thr Ser Gly Pro Gly Ser Ser Val
Pro Val Ser Thr Pro Pro Gly Ser 500 505 510 Thr Thr Arg Phe Ser Ser
Thr Pro Val Ser Ser Arg Ser Ser Ser Ser 515 520 525 Thr Pro Pro Val
Ser Thr Pro Pro Pro Gly Gln Val Cys Val Ala Gly 530 535 540 Thr Val
Ala Asp Gly Gln Ser Gly Asn Tyr Ile Gly Leu Cys Asn Phe 545 550 555
560 Ser Cys Asn Phe Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala
565 570 575 Tyr Gly Ala Pro Ile Asn Pro Pro Ala Thr Asn Gly Arg Asn
Gly Cys 580 585 590 Pro Leu Pro Gly Glu Asp Asp Ser Tyr Leu Gly Leu
Cys Ser Phe Ser 595 600 605 Cys Asn His Asn Tyr Cys Pro Pro Thr Ala
Cys Gln Tyr Cys 610 615 620 3627PRTTrichoderma konilangbra 3Met Leu
Gly Ile Leu Arg Arg Leu Ala Leu Gly Ala Leu Ala Ala Ala 1 5 10 15
Ala Leu Ser Pro Leu Val Val Ala Ala Pro Ala Asn Val Ala Ile Arg 20
25 30 Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys
His 35 40 45 Phe Met Ile Gly Ile Cys Gly Asp Arg Gly Ser Ser Thr
Asp Tyr Asp 50 55 60 Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile
Asp Ala Phe Ala Leu 65 70 75 80 Asn Ile Gly Val Asp Gly Tyr Thr Asp
Gln Gln Leu Asn Phe Ala Tyr 85 90 95 Asp Ala Ala Asp Arg Ala Gly
Met Lys Val Phe Ile Ser Phe Asp Phe 100 105 110 Asn Trp Trp Ser Pro
Gly Asn Ala Val Gly Val Gly Gln Lys Ile Ala 115 120 125 Gln Tyr Ala
Ser Arg Pro Ala Gln Leu Tyr Val Asp Asn Arg Pro Phe 130 135 140 Ala
Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu Arg Asn 145 150
155 160 Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly
Gln 165 170 175 Ser Ser Pro Ser Thr Ile Asp Gly Ala Leu Asn Trp Met
Ala Trp Asp 180 185 190 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly
Arg Asn Val Thr Val 195 200 205 Ala Asp Gly Asp Asn Ser Tyr Arg Ser
Trp Leu Gly Gly Lys Pro Tyr 210 215 220 Leu Ala Pro Val Ser Pro Trp
Phe Phe Thr His Phe Gly Pro Glu Val 225 230 235 240 Ser Phe Ser Lys
Asn Trp Val Phe Pro Gly Gly Ser Leu Leu Tyr Asp 245 250 255 Arg Trp
Gln Asp Val Leu Arg Gln Gly Pro Glu Met Val Glu Ile Ile 260 265 270
Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Thr Gly Pro Leu Lys Ser 275
280 285 Arg His Tyr Asp Asp Gly Asn Ser Lys Trp Thr Asn Asp Met Pro
His 290 295 300 Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe Ile Ala Ala
Tyr Lys Asn 305 310 315 320 Arg Asp Thr Asn Val Ala Arg Tyr Val Gln
Ser Asp Gln Leu Val Tyr 325 330 335 Trp Tyr Arg Arg Thr Leu Lys Gly
Leu Asp Cys Asp Ala Thr Asp Thr 340 345 350 Thr Ser Asn Arg Pro Ala
Asn Asn Ala Ser Gly Asn Tyr Phe Met Gly 355 360 365 Arg Pro Asp Gly
Trp Gln Thr Met Asp Asp Thr Val Tyr Val Val Ala 370 375 380 Leu Leu
Thr Ala Ala Gly Thr Val Thr Val Thr Ser Gly Gly Ala Thr 385 390 395
400 Gln Thr Phe Gln Gly Thr Ala Gly Ala Asn Leu Phe Glu Val Pro Ala
405 410 415 Asn Leu Gly Gln Gln Lys Phe Ala Leu Ser Arg Asn Gly Gln
Thr Val 420 425 430 Phe Ser Ser Thr Ser Leu Met Asp Ile Ala Asn Val
Cys Pro Cys Gly 435 440 445 Leu Tyr Asn Phe Asn Pro Tyr Val Gly Thr
Val Pro Pro Gly Phe Asp 450 455 460 Asp Pro Leu Gln Ala Asp Gly Leu
Ala Ser Leu Thr Ile Gly Leu His 465 470 475 480 Val Thr Thr Cys Gln
Ala Arg Pro Ser Leu Gly Thr Asn Pro Pro Ile 485 490 495 Thr Ser Gly
Pro Gly Ser Ser Val Pro Ala Ser Thr Thr Arg Ser Thr 500 505 510 Ser
Pro Pro Gly Ser Thr Ser Arg Phe Ser Ser Thr Pro Val Ser Ser 515 520
525 Arg Ser Ile Ser Ser Thr Pro Pro Val Ser Thr Pro Pro Pro Gly Gln
530 535 540 Val Cys Val Ala Gly Thr Val Ala Asp Gly Gln Ser Gly Asn
Tyr Ile 545 550 555 560 Gly Leu Cys Asn Phe Ser Cys Asn Phe Gly Tyr
Cys Pro Pro Gly Pro 565 570 575 Cys Lys Cys Thr Ala Phe Gly Ala Pro
Ile Asn Pro Pro Ala Thr Asn 580 585 590 Gly Arg Asn Gly Cys Pro Leu
Pro Gly Glu Asp Asp Ser Tyr Leu Gly 595 600 605 Leu Cys Ser Phe Ser
Cys Asn His Asn Tyr Cys Pro Pro Thr Ala Cys 610 615 620 Gln Tyr Cys
625 4634PRTTrichoderma harzianum 4Met Leu Gly Val Val Arg Arg Leu
Gly Leu Gly Ala Leu Ala Ala Ala 1 5 10 15 Ala Leu Ser Ser Leu Gly
Ser Ala Ala Pro Ala Asn Val Ala Ile Arg 20 25 30 Ser Leu Glu Glu
Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His 35 40 45 Phe Met
Ile Gly Ile Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp 50 55 60
Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 65
70 75 80 Asn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr
Ala Tyr 85 90 95 Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile
Ser Phe Asp Phe 100 105 110 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly
Val Gly Gln Lys Ile Ala 115 120 125 Gln Tyr Ala Ser Arg Pro Ala Gln
Leu Tyr Val Asp Asn Arg Pro Phe 130 135 140 Ala Ser Ser Phe Ala Gly
Asp Gly Leu Asp Val Asn Ala Leu Arg Ser 145 150 155 160 Ala Ala Gly
Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gln 165 170 175 Ser
Ser Pro Ser Asn Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185
190 Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln Thr Val Thr Val
195 200 205 Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys
Pro Tyr 210 215 220 Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe
Gly Pro Glu Val 225 230 235 240 Ser Tyr Ser Lys Asn Trp Val Phe Pro
Gly Gly Pro Leu Ile Tyr Asn 245 250 255 Arg Trp Gln Gln Val Leu Gln
Gln Gly Phe Pro Met Val Glu Ile Val 260 265 270 Thr Trp Asn Asp Tyr
Gly Glu Ser His Tyr Val Gly Pro Leu Lys Ser 275 280 285 Lys His Phe
Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met Pro His 290 295 300 Asp
Gly Phe Leu Asp Leu Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn 305 310
315 320 Arg Asp Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln Leu Val
Tyr 325 330 335 Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala
Thr Asp Thr 340 345 350 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly
Asn Tyr Phe Met Gly 355 360 365 Arg Pro Asp Gly Trp Gln Thr Met Asp
Asp Thr Val Tyr Val Ala Ala 370 375 380 Leu Leu Lys Thr Ala Gly Ser
Val Thr Val Thr Ser Gly Gly Thr Thr 385 390 395 400 Gln Thr Phe Gln
Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile Pro Ala 405
410 415 Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln Thr
Val 420 425 430 Phe Ser Gly Thr Ser Leu Met Asp Ile Thr Asn Val Cys
Ser Cys Gly 435 440 445 Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile
Pro Ala Gly Phe Asp 450 455 460 Asp Pro Leu Gln Ala Asp Gly Leu Phe
Ser Leu Thr Ile Gly Leu His 465 470 475 480 Val Thr Thr Cys Gln Ala
Lys Pro Ser Leu Gly Thr Asn Pro Pro Val 485 490 495 Thr Ser Gly Pro
Val Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala 500 505 510 Ser Ser
Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser 515 520 525
Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro Ala Ser 530
535 540 Ser Thr Pro Pro Ser Gly Gln Val Cys Val Ala Gly Thr Val Ala
Asp 545 550 555 560 Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys Gln Phe
Ser Cys Asn Tyr 565 570 575 Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys
Thr Ala Phe Gly Ala Pro 580 585 590 Ile Ser Pro Pro Ala Ser Asn Gly
Arg Asn Gly Cys Pro Leu Pro Gly 595 600 605 Glu Gly Asp Gly Tyr Leu
Gly Leu Cys Ser Phe Ser Cys Asn His Asn 610 615 620 Tyr Cys Pro Pro
Thr Ala Cys Gln Tyr Cys 625 630 5633PRTArtificial Sequencesynthetic
consensus sequence 5Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala
Leu Ala Ala Ala 1 5 10 15 Ala Leu Ser Ser Leu Gly Xaa Ala Ala Pro
Ala Asn Val Ala Ile Arg 20 25 30 Ser Leu Glu Glu Arg Ala Ser Ser
Ala Asp Arg Leu Val Phe Cys His 35 40 45 Phe Met Ile Gly Ile Xaa
Gly Asp Arg Gly Ser Ser Xaa Asp Tyr Asp 50 55 60 Asp Asp Met Gln
Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu 65 70 75 80 Asn Ile
Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Xaa Xaa Ala Tyr 85 90 95
Asp Xaa Ala Asp Arg Xaa Gly Met Lys Val Phe Ile Ser Phe Asp Phe 100
105 110 Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys Ile
Ala 115 120 125 Gln Tyr Ala Ser Arg Pro Ala Gln Leu Tyr Val Asp Asn
Arg Pro Phe 130 135 140 Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val
Asn Ala Leu Arg Asn 145 150 155 160 Ala Ala Gly Ser Asn Val Tyr Phe
Val Pro Asn Phe His Pro Gly Gln 165 170 175 Ser Ser Pro Ser Xaa Ile
Asp Gly Ala Leu Asn Trp Met Ala Trp Asp 180 185 190 Asn Asp Gly Asn
Asn Lys Ala Pro Lys Pro Gly Gln Xaa Val Thr Val 195 200 205 Ala Asp
Gly Asp Asn Xaa Tyr Xaa Xaa Trp Leu Gly Gly Lys Pro Tyr 210 215 220
Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val 225
230 235 240 Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Ser Leu Ile
Tyr Xaa 245 250 255 Arg Trp Gln Xaa Val Leu Xaa Gln Gly Phe Xaa Met
Val Glu Ile Val 260 265 270 Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr
Xaa Gly Pro Leu Lys Ser 275 280 285 Xaa His Xaa Asp Asp Gly Asn Ser
Lys Trp Xaa Asn Asp Met Pro His 290 295 300 Asp Gly Phe Leu Asp Leu
Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn 305 310 315 320 Arg Asp Thr
Asp Xaa Xaa Lys Tyr Val Gln Asn Glu Gln Leu Val Tyr 325 330 335 Trp
Tyr Arg Arg Asn Leu Lys Xaa Leu Asp Cys Asp Ala Thr Asp Thr 340 345
350 Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly
355 360 365 Arg Pro Asp Gly Trp Gln Thr Met Asp Asp Thr Val Tyr Val
Xaa Ala 370 375 380 Leu Leu Lys Thr Ala Gly Xaa Val Thr Val Thr Ser
Gly Gly Xaa Thr 385 390 395 400 Gln Thr Phe Gln Gly Xaa Ala Gly Ala
Asn Leu Phe Xaa Xaa Pro Ala 405 410 415 Xaa Xaa Gly Gln Gln Lys Phe
Ala Leu Xaa Arg Asn Gly Gln Thr Val 420 425 430 Phe Ser Xaa Thr Ser
Leu Met Asp Ile Thr Asn Val Cys Xaa Cys Gly 435 440 445 Ile Tyr Asn
Phe Asn Pro Tyr Val Gly Thr Xaa Pro Ala Gly Phe Asp 450 455 460 Asp
Pro Leu Gln Ala Asp Gly Leu Xaa Ser Leu Thr Ile Gly Leu His 465 470
475 480 Val Thr Thr Cys Gln Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro
Xaa 485 490 495 Thr Ser Gly Pro Xaa Ser Ser Xaa Pro Ala Ser Xaa Thr
Xaa Arg Ala 500 505 510 Ser Ser Pro Pro Xaa Ser Xaa Thr Arg Xaa Ser
Ser Xaa Pro Val Ser 515 520 525 Ser Xaa Xaa Xaa Arg Xaa Ser Ser Ser
Xaa Pro Pro Xaa Xaa Xaa Ser 530 535 540 Thr Pro Pro Xaa Gly Gln Val
Cys Val Ala Gly Thr Val Ala Asp Gly 545 550 555 560 Xaa Ser Gly Asn
Tyr Ile Gly Leu Cys Xaa Phe Ser Cys Asn Xaa Gly 565 570 575 Tyr Cys
Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro Ile 580 585 590
Xaa Pro Pro Ala Xaa Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly Glu 595
600 605 Xaa Asp Xaa Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn
Tyr 610 615 620 Cys Pro Pro Thr Ala Cys Gln Tyr Cys 625 630
622DNAArtificial Sequencesynthetic oligonucleotide 6caccatgttt
ggtcttgtcc gc 22720DNAArtificial Sequencesynthetic oligonucleotide
7tcagcagtac tggcatgctg 20820DNAArtificial Sequencesynthetic
oligonucleotide 8gtnttytgyc ayttyatgat 20926DNAArtificial
Sequencesynthetic oligonucleotide 9gtnttytgyc ayttyatgat hggnat
261023DNAArtificial Sequencesynthetic oligonucleotide 10gaytaygayg
aygayatgca rcg 231132DNAArtificial Sequencesynthetic
oligonucleotide 11gtrcayttrc anggnccngg nggrcartan cc
321224DNAArtificial Sequencesynthetic oligonucleotide 12ytcnccnggn
agnggrcanc crtt 241330DNAArtificial Sequencesynthetic
oligonucleotide 13rcartaytgr cangcygtyg gyggrcarta
301420DNAArtificial Sequencesynthetic oligonucleotide 14ccccctggcc
aagtatgtgt 201520DNAArtificial Sequencesynthetic oligonucleotide
15gtacgcaaag ttgagctgct 201620DNAArtificial Sequencesynthetic
oligonucleotide 16agcacatcgc tgatggatat 201720DNAArtificial
Sequencesynthetic oligonucleotide 17aagtatacgt tgcttccggc
201820DNAArtificial Sequencesynthetic oligonucleotide 18ctgacgatcg
gactrcacgt 201920DNAArtificial Sequencesynthetic oligonucleotide
19ggttgtcgac gtagagctgt 202020DNAArtificial Sequencesynthetic
oligonucleotide 20acgatcggca gagtcatagg 202120DNAArtificial
Sequencesynthetic oligonucleotide 21atcggattgc atgtcacgac
202220DNAArtificial Sequencesynthetic oligonucleotide 22tacatccaga
ccgtcaccag 202320DNAArtificial Sequencesynthetic oligonucleotide
23acgtttgctc ttgcggtatc 202420DNAArtificial Sequencesynthetic
oligonucleotide 24tcattatccc aggcctaaaa 202521DNAArtificial
Sequencesynthetic oligonucleotide 25caccatgcta ggcattctcc g
212620DNAArtificial Sequencesynthetic oligonucleotide 26tcagcagtat
tggcatgccg 202721DNAArtificial Sequencesynthetic oligonucleotide
27caccatgttg ggcgtttttc g 212820DNAArtificial Sequencesynthetic
oligonucleotide 28ctagcagtat tgrcatgccg 20292145DNAHypocrea tawa
29atgttgggcg tttttcgccg cctcgggctc ggctcccttg ccgccgcagc tctgtcttct
60ctcggcactg ccgctcccgc caatgttgct attcggtctc tcgaggaacg tgcttcttct
120gccgaccgtc tcgtattctg tcacttcatg gttagtgttt atttacgaag
tatcagaatc 180aggactaaca tggcattttc atgacagatt ggtattgttg
gtgaccgtgg cagctcggca 240gactatgatg atgatatgca acgtgccaaa
gccgctggca ttgacgcctt cgctctgaat 300atcggcgttg acggctatac
cgaccagcag cttgggtatg cctatgactc tgccgatcgt 360aatggcatga
aagtcttcat ttcattcgat ttcaattggt ggagccccgg caatgcagtt
420ggtgttggcc agaagattgc gcagtatgcc aaccgtcccg cccagctata
tgtcgataac 480cgtccattcg cctcttcctt cgctggtgac ggtctggatg
taaatgcgtt gcgcaatgct 540gcaggctcca acgtttactt tgtgcccaac
ttccaccctg gtcaatcttc tccctcaaac 600attgacggcg ccctgaactg
gatggtaagt tgcaactgca gagctgagag taggaaagca 660aactgatgtg
tttttaggcc tgggataatg atggaaacaa caaggcaccc aagcaaggcc
720agacagtcac ggtggcagac ggcgacaacg cctacaagaa ttggttaggt
ggcaagcctt 780acctagcacc tgtctcacct tggtttttca cccatttcgg
ccccgaagtt tcatattcca 840agaactgggt tttcccaggt ggtgctctga
tctataaccg gtggcaacag gtcttgcagc 900aaggcttccc catggttgag
attgttacat ggaatgacta cggcgagtct cactacgtcg 960gcccacttaa
gtctaagcat ttcgacgatg gcaactccaa atgggtcaat gatatgcccc
1020atgatggatt cctggatctt tcaaagccgt tcattgctgc ttataagaac
agggatactg 1080acatctccaa gtatgttcag aatgagcagc ttgtctactg
gtaccgccgc aacttaaagg 1140cactggactg cgacgccacc gacaccacct
ctaaccgccc ggctaataat ggaagcggta 1200attactttat gggacgccct
gatggttggc aaaccatgga tgatactgtt tatgttgccg 1260cacttctcaa
gactgccggt tctgttacgg tcacgtctgg cggcaccact caaacgttcc
1320agggcaacgc cggagccaac ctcttccaaa tcccagccag catcggccag
caaaagtttg 1380ctctaactcg taacggtcag accgtcttta gcggaacctc
attgatggat atcaccaacg 1440tttgctcttg cggtatctac aacttcaacc
catatgtggg taccattcct gccggcttcg 1500acgaccctct tcaggctgac
ggtcttttct ctttgaccat cggattgcat gtcacgactt 1560gtcaggccaa
gccatctctt ggaaccaatc ctcctgtcac ttctggccct gtgtcctcgc
1620ttccagcttc ctccactacc cgcgcatcct cgcctcctgt ttcttcaact
cgcgtctctt 1680ccccccctgt ctcttcccct ccagttactt ctcgcacctc
ttcttctcct ccccctccgg 1740ccagcagcac gccgtcatcg ggtcaggttt
gcgttgccgg aaccgttgct gacggcgagt 1800ctggcaacta catcggcctg
tgccaattca gctgcaagta ggttgccccc atacccctta 1860cttgttttct
taactaatcc tttgtagcta cggttactgc ccaccaggac cgtgcaagtg
1920caccgccttt ggcgctccca tctcgccacc ggcaagcaat gggcgcaatg
gctgccctct 1980accgggcgaa ggagatggtt atctgggcct gtgcagtttc
agttgtaacc ataattactg 2040ccccccaacg gcatgccaat actgctagaa
gggtgggcgc gccgacccag ctttcttgta 2100caaagttggc attataagaa
agcattgctt atcaatttgt tgcaa 2145301908DNAHypocrea tawa 30atgttgggcg
tttttcgccg cctcgggctc ggctcccttg ccgccgcagc tctgtcttct 60ctcggcactg
ccgctcccgc caatgttgct attcggtctc tcgaggaacg tgcttcttct
120gccgaccgtc tcgtattctg tcacttcatg attggtattg ttggtgaccg
tggcagctcg 180gcagactatg atgatgatat gcaacgtgcc aaagccgctg
gcattgacgc cttcgctctg 240aatatcggcg ttgacggcta taccgaccag
cagcttgggt atgcctatga ctctgccgat 300cgtaatggca tgaaagtctt
catttcattc gatttcaatt ggtggagccc cggcaatgca 360gttggtgttg
gccagaagat tgcgcagtat gccaaccgtc ccgcccagct atatgtcgat
420aaccgtccat tcgcctcttc cttcgctggt gacggtctgg atgtaaatgc
gttgcgcaat 480gctgcaggct ccaacgttta ctttgtgccc aacttccacc
ctggtcaatc ttctccctca 540aacattgacg gcgccctgaa ctggatggcc
tgggataatg atggaaacaa caaggcaccc 600aagcaaggcc agacagtcac
ggtggcagac ggcgacaacg cctacaagaa ttggttaggt 660ggcaagcctt
acctagcacc tgtctcacct tggtttttca cccatttcgg ccccgaagtt
720tcatattcca agaactgggt tttcccaggt ggtgctctga tctataaccg
gtggcaacag 780gtcttgcagc aaggcttccc catggttgag attgttacat
ggaatgacta cggcgagtct 840cactacgtcg gcccacttaa gtctaagcat
ttcgacgatg gcaactccaa atgggtcaat 900gatatgcccc atgatggatt
cctggatctt tcaaagccgt tcattgctgc ttataagaac 960agggatactg
acatctccaa gtatgttcag aatgagcagc ttgtctactg gtaccgccgc
1020aacttaaagg cactggactg cgacgccacc gacaccacct ctaaccgccc
ggctaataat 1080ggaagcggta attactttat gggacgccct gatggttggc
aaaccatgga tgatactgtt 1140tatgttgccg cacttctcaa gactgccggt
tctgttacgg tcacgtctgg cggcaccact 1200caaacgttcc agggcaacgc
cggagccaac ctcttccaaa tcccagccag catcggccag 1260caaaagtttg
ctctaactcg taacggtcag accgtcttta gcggaacctc attgatggat
1320atcaccaacg tttgctcttg cggtatctac aacttcaacc catatgtggg
taccattcct 1380gccggcttcg acgaccctct tcaggctgac ggtcttttct
ctttgaccat cggattgcat 1440gtcacgactt gtcaggccaa gccatctctt
ggaaccaatc ctcctgtcac ttctggccct 1500gtgtcctcgc ttccagcttc
ctccactacc cgcgcatcct cgcctcctgt ttcttcaact 1560cgcgtctctt
ccccccctgt ctcttcccct ccagttactt ctcgcacctc ttcttctcct
1620ccccctccgg ccagcagcac gccgtcatcg ggtcaggttt gcgttgccgg
aaccgttgct 1680gacggcgagt ctggcaacta catcggcctg tgccaattca
gctgcaacta cggttactgc 1740ccaccaggac cgtgcaagtg caccgccttt
ggcgctccca tctcgccacc ggcaagcaat 1800gggcgcaatg gctgccctct
accgggcgaa ggagatggtt atctgggcct gtgcagtttc 1860agttgtaacc
ataattactg ccccccaacg gcatgccaat actgctag 1908312063DNATrichoderma
reesei 31atgtttggtc ttgtccgccg actcggggtc ggcgcccttg tcgccgcagc
cctttcctcc 60ctcgctgccg ccgcgccagc caacgtcgcc atccgctccc tcgaggaacg
ggctagcagc 120gcagatagac ttgtgttttg ccactttatg gtgcgtttgt
ccgccccaag agcattgaaa 180attcagtaat cactgacacg ccttactcgt
gatgctagat tgggatatgt ggtgatcgca 240cctccagtac cgattatgat
gatgatatgc agcgagccaa ggccgcgggc attgacgcct 300ttgcccttaa
cattggtgtc gacggataca cggaccagca gctcaacttt gcctacgacg
360ccgctgatcg cgccgggatg aaggtgttca tctcctttga cttcaactgg
tggagccccg 420gcaacgcggc aggcgtcggc cagaagattg cccaatatgc
gtcgcggccc gcacagctct 480acgtcgacaa ccgtcccttt gcatcgtcgt
ttgccggtga cggccttgac gtgaatacgc 540tgcggaatgc ggccggcagc
aacgtgtact ttgtgcccaa cttccacccc gggcagtcgt 600cgccgtccac
catcgacggg gctctgaact ggatggtacg tctgggcgtt gtggctcgag
660gataaagcaa agaccaagta ctcatgcgct gacacgctcc acaggcctgg
gacaacgacg 720gcaacaacaa ggcccccaag cccggccaaa acgtcacagt
cgccgacggc gacaactcct 780accgcagctg gctcgccggc aagccctacc
tcgcccccgt ctcgccctgg ttcttcaccc 840acttcggccc agaggtatcg
tacagtaaga actgggtctt ccctggcggc tccctgtggt 900acgaccgctg
gcaggacgtg ctgcgccagg gcttcgagat ggtcgaaatc gtcacctgga
960acgattacgg tgagagccac tacacggggc ccctggaaag tcgacactat
gacgacggaa 1020actcgaaatg gaccaacgac atgccgcacg acggcttcct
ggacctggcg aagccattca 1080ttgccgcgta caagaaccgc gacacggacg
tggcgcccta catccagaat gagcagctga 1140tctactggta tcggcggaat
ctcaaggggc tggactgcga cgcgaccgac acgacgtcga 1200accgcccggc
gaacaacggc agcggcaact acttcatggg tcggcccgac gggtggcaga
1260cgatggacga cacggtgtat gtggtggcgc tgctcaagag cgcgggcacg
gtgacggtga 1320cgtcgggcgg cgccacgcag acgttccagg gcaccgccgg
cgccaacctg ttcgaggtcc 1380cagccaacct tgggcagcag aagtttgccc
tgtcccgcaa cgggcagacc gtcttcagca 1440gcacgtcgct gatggatatc
accaatgtgt gcccgtgcgg catctacaac ttcaacccgt 1500atgtcgggac
tgtgcccgct ggctttgacg acccgctcgg gcccgatggc cttgcttctt
1560tgacaatcgg actgcacgtc acgacttgtc aggccaagcc gtcgctgggg
accaacccgc 1620ccatcacttc cggccccggc tcctcggtgc ccgtttccac
tccgcccggt tccacgaccc 1680gcttctcgtc aacgccggtt tcatctcgct
ccagctcgtc cacgccgccg gttagcacgc 1740cgccgcctgg ccaagtctgt
gtcgccggta cggtggctga cggccagtcc ggcaactata 1800ttggcctctg
caacttcagc tgcaagtaag ttaccccatg tctcatgacg atgtatctcc
1860gacaccagct aacgtttgcc agcttcgggt actgtccccc cggaccctgc
aagtgcactg 1920cctacggcgc tccgatcaac ccaccagcaa cgaatgggcg
aaatgggtgc cccttgcctg 1980gagaagacga tagttatctg ggcctctgca
gcttcagctg caaccacaat tactgtccgc 2040caacagcatg ccagtactgc tga
2063321869DNATrichoderma reesei 32atgtttggtc ttgtccgccg actcggggtc
ggcgcccttg tcgccgcagc cctttcctcc 60ctcgctgccg ccgcgccagc caacgtcgcc
atccgctccc tcgaggaacg ggctagcagc 120gcagatagac ttgtgttttg
ccactttatg attgggatat gtggtgatcg cacctccagt 180accgattatg
atgatgatat gcagcgagcc aaggccgcgg gcattgacgc ctttgccctt
240aacattggtg tcgacggata cacggaccag cagctcaact ttgcctacga
cgccgctgat 300cgcgccggga tgaaggtgtt catctccttt gacttcaact
ggtggagccc cggcaacgcg 360gcaggcgtcg gccagaagat tgcccaatat
gcgtcgcggc ccgcacagct ctacgtcgac
420aaccgtccct ttgcatcgtc gtttgccggt gacggccttg acgtgaatac
gctgcggaat 480gcggccggca gcaacgtgta ctttgtgccc aacttccacc
ccgggcagtc gtcgccgtcc 540accatcgacg gggctctgaa ctggatggcc
tgggacaacg acggcaacaa caaggccccc 600aagcccggcc aaaacgtcac
agtcgccgac ggcgacaact cctaccgcag ctggctcgcc 660ggcaagccct
acctcgcccc cgtctcgccc tggttcttca cccacttcgg cccagaggta
720tcgtacagta agaactgggt cttccctggc ggctccctgt ggtacgaccg
ctggcaggac 780gtgctgcgcc agggcttcga gatggtcgaa atcgtcacct
ggaacgatta cggtgagagc 840cactacacgg ggcccctgga aagtcgacac
tatgacgacg gaaactcgaa atggaccaac 900gacatgccgc acgacggctt
cctggacctg gcgaagccat tcattgccgc gtacaagaac 960cgcgacacgg
acgtggcgcc ctacatccag aatgagcagc tgatctactg gtatcggcgg
1020aatctcaagg ggctggactg cgacgcgacc gacacgacgt cgaaccgccc
ggcgaacaac 1080ggcagcggca actacttcat gggtcggccc gacgggtggc
agacgatgga cgacacggtg 1140tatgtggtgg cgctgctcaa gagcgcgggc
acggtgacgg tgacgtcggg cggcgccacg 1200cagacgttcc agggcaccgc
cggcgccaac ctgttcgagg tcccagccaa ccttgggcag 1260cagaagtttg
ccctgtcccg caacgggcag accgtcttca gcagcacgtc gctgatggat
1320atcaccaatg tgtgcccgtg cggcatctac aacttcaacc cgtatgtcgg
gactgtgccc 1380gctggctttg acgacccgct cgggcccgat ggccttgctt
ctttgacaat cggactgcac 1440gtcacgactt gtcaggccaa gccgtcgctg
gggaccaacc cgcccatcac ttccggcccc 1500ggctcctcgg tgcccgtttc
cactccgccc ggttccacga cccgcttctc gtcaacgccg 1560gtttcatctc
gctccagctc gtccacgccg ccggttagca cgccgccgcc tggccaagtc
1620tgtgtcgccg gtacggtggc tgacggccag tccggcaact atattggcct
ctgcaacttc 1680agctgcaact tcgggtactg tccccccgga ccctgcaagt
gcactgccta cggcgctccg 1740atcaacccac cagcaacgaa tgggcgaaat
gggtgcccct tgcctggaga agacgatagt 1800tatctgggcc tctgcagctt
cagctgcaac cacaattact gtccgccaac agcatgccag 1860tactgctga
1869332294DNATrichoderma konilangbra 33ggctccgcgg ccgccccctt
caccatgcta ggcattctcc gccgtctcgc gctcggcgcc 60ctcgccgccg cggccctctc
tcctctcgtc gtcgccgcgc ctgccaatgt cgccatccgc 120tccctcgagg
aacgggcgag tagcgcagac aggctcgtgt tctgccactt catggtacgt
180gtggctgccc gaaaaggata cggcgtcatc ccgatagcaa cctgggctgt
caccgcagca 240cccgatgtta caaccactga cgtgccggcc tcgttgtaga
ttgggatatg cggtgatcgc 300ggctccagca ctgattatga cgacgatatg
caaagggcca aggcagcggg catcgacgcg 360tttgcgttga acattggcgt
cgatggatac acggaccagc agctcaactt tgcgtacgac 420gccgccgacc
gcgccgggat gaaggtgttc atctccttcg acttcaactg gtggagcccc
480ggcaacgcag taggcgtcgg ccagaagatt gcccaatacg cgtcgcggcc
cgcacagctc 540tacgtcgaca accggccctt tgcgtcgtcg tttgccggcg
atggccttga cgtgaatgcg 600ctgcgcaacg ccgccggaag caacgtatac
tttgtgccca acttccaccc cgggcagtcc 660tccccgtcaa ccatcgacgg
ggccctcaac tggatggtac gtttggacgt tgcgagtcaa 720ggagaatccg
cagaaaaagg ctgacagctg aggccaatgt gctcatgtgc tgacaygccg
780gacaggcctg ggacaatgac ggaaacaaca aggcccccaa gcccggccgc
aacgtcaccg 840tcgccgacgg cgacaactcg taccgtagct ggctgggcgg
caagccctac ctggcccccg 900tttcgccctg gttcttcacc cacttcggcc
ccgaggtttc cttcagcaag aactgggtct 960tcccgggcgg ctcgctcctc
tacgaccgct ggcaggacgt gctgcrccag ggccccgaaa 1020tggtcgagat
catcacctgg aacgattacg gtgagagcca ctacaccggg cccctcaaaa
1080gtcgccacta tgacgacgga aactcgaaat ggaccaacga catgccgcac
gacggattcc 1140tggacctgtc gaaaccgttt atagcggcgt acaagaaccg
cgacacgaac gtggcacggt 1200acgtccagtc cgaccagctc gtctactggt
acagaaggac gctcaagggg ctggactgcg 1260acgcgactga cacgacgtca
aaccggcccg cgaacaacgc cagcggcaac tacttcatgg 1320gccggcccga
cgggtggcag acgatggacg acaccgtgta cgtggtggcg ctrctcacgg
1380ccgcgggaac tgtgacggtg acgtccggcg gggccaccca gacgttccag
ggcaccrccg 1440gagccaacct gttcgaggtc ccggccaacc tgggccagca
gaagtttgcc ctgtcccgca 1500acgggcagac cgtcttcagc agcacatcgc
tgatggatat cgctaatgtg tgcccgtgcg 1560gcctctacaa cttcaacccg
tatgtcggga ctgtcccgcc cggttttgac gacccgctgc 1620aggctgatgg
ccttgcgtcg ctgacgatcg gactgcacgt cacgacctgt caggccagac
1680cctccctggg aacgaacccg cccatcactt ccggccccgg ctcctcggtg
cccgcttcaa 1740ccacccgctc gacttctccg cccggttcca cgagccgctt
ctcgtcgacc ccggtttcgt 1800cccgctccat ctcttcgacg ccaccggtca
gcacgccgcc ccctggccaa gtatgtgtgg 1860ccggcacagt cgctgacggc
cagtcgggca actatattgg cctatgcaac ttcagctgca 1920agtaagtcgt
cccacgttcc gtcatgatgt ctctgcctca gctaacatgt gccagcttcg
1980gctactgtcc tcccggccct tgcaagtgca ccgcctttgg cgctcccatc
aacccaccgg 2040cgaccaatgg gcgaaacgga tgccccttgc ctggagagga
tgatagttac ttgggcctct 2100gcagcttcag ttgcaaccat aactactgcc
ctccgacggc atgccaatac tgctgaaagg 2160gtgggcgcgc cgacccagct
ttcttgtaca aagttggcat tataagaaag cattgcttat 2220caatttgttg
caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
2280ctgatatccc ctat 2294341884DNATrichoderma konilangbra
34atgctaggca ttctccgccg tctcgcgctc ggcgccctcg ccgccgcggc cctctctcct
60ctcgtcgtcg ccgcgcctgc caatgtcgcc atccgctccc tcgaggaacg ggcgagtagc
120gcagacaggc tcgtgttctg ccacttcatg attgggatat gcggtgatcg
cggctccagc 180actgattatg acgacgatat gcaaagggcc aaggcagcgg
gcatcgacgc gtttgcgttg 240aacattggcg tcgatggata cacggaccag
cagctcaact ttgcgtacga cgccgccgac 300cgcgccggga tgaaggtgtt
catctccttc gacttcaact ggtggagccc cggcaacgca 360gtaggcgtcg
gccagaagat tgcccaatac gcgtcgcggc ccgcacagct ctacgtcgac
420aaccggccct ttgcgtcgtc gtttgccggc gatggccttg acgtgaatgc
gctgcgcaac 480gccgccggaa gcaacgtata ctttgtgccc aacttccacc
ccgggcagtc ctccccgtca 540accatcgacg gggccctcaa ctggatggcc
tgggacaatg acggaaacaa caaggccccc 600aagcccggcc gcaacgtcac
cgtcgccgac ggcgacaact cgtaccgtag ctggctgggc 660ggcaagccct
acctggcccc cgtttcgccc tggttcttca cccacttcgg ccccgaggtt
720tccttcagca agaactgggt cttcccgggc ggctcgctcc tctacgaccg
ctggcaggac 780gtgctgcrcc agggccccga aatggtcgag atcatcacct
ggaacgatta cggtgagagc 840cactacaccg ggcccctcaa aagtcgccac
tatgacgacg gaaactcgaa atggaccaac 900gacatgccgc acgacggatt
cctggacctg tcgaaaccgt ttatagcggc gtacaagaac 960cgcgacacga
acgtggcacg gtacgtccag tccgaccagc tcgtctactg gtacagaagg
1020acgctcaagg ggctggactg cgacgcgact gacacgacgt caaaccggcc
cgcgaacaac 1080gccagcggca actacttcat gggccggccc gacgggtggc
agacgatgga cgacaccgtg 1140tacgtggtgg cgctrctcac ggccgcggga
actgtgacgg tgacgtccgg cggggccacc 1200cagacgttcc agggcaccrc
cggagccaac ctgttcgagg tcccggccaa cctgggccag 1260cagaagtttg
ccctgtcccg caacgggcag accgtcttca gcagcacatc gctgatggat
1320atcgctaatg tgtgcccgtg cggcctctac aacttcaacc cgtatgtcgg
gactgtcccg 1380cccggttttg acgacccgct gcaggctgat ggccttgcgt
cgctgacgat cggactgcac 1440gtcacgacct gtcaggccag accctccctg
ggaacgaacc cgcccatcac ttccggcccc 1500ggctcctcgg tgcccgcttc
aaccacccgc tcgacttctc cgcccggttc cacgagccgc 1560ttctcgtcga
ccccggtttc gtcccgctcc atctcttcga cgccaccggt cagcacgccg
1620ccccctggcc aagtatgtgt ggccggcaca gtcgctgacg gccagtcggg
caactatatt 1680ggcctatgca acttcagctg caacttcggc tactgtcctc
ccggcccttg caagtgcacc 1740gcctttggcg ctcccatcaa cccaccggcg
accaatgggc gaaacggatg ccccttgcct 1800ggagaggatg atagttactt
gggcctctgc agcttcagtt gcaaccataa ctactgccct 1860ccgacggcat
gccaatactg ctga 1884
* * * * *