U.S. patent application number 11/740076 was filed with the patent office on 2008-06-19 for endoglucanases.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Lene Nonboe Andersen, Torben Henriksen, Michiko Ihara, Markus Sakari Kauppinen, Lene Lange, Soren Flensted Lassen, Ruby Ilum Nielsen, Martin Schulein, Shinobu Takagi.
Application Number | 20080145912 11/740076 |
Document ID | / |
Family ID | 27545143 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080145912 |
Kind Code |
A1 |
Schulein; Martin ; et
al. |
June 19, 2008 |
Endoglucanases
Abstract
The present invention relates to enzyme preparations consisting
essentially of an enzyme which has cellulytic activity and
comprises a first amino acid sequence having the following sequence
TABLE-US-00001 (SEQ ID NO: 79) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12 Trp Xaa 13 14 and a second
amino acid sequence having the following sequence TABLE-US-00002
Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5 wherein, at position
3 of the first sequence, the amino acid is Trp, Tyr or Phe; at
position 4 of the first sequence, the amino acid is Trp, Tyr or
Phe; at position 8 of the first sequence, the amino acid is Arg,
Lys or His; at positions 9, 10, 12 and 14, respectively, of the
first sequence, and at position 4 of the second sequence, the amino
acid is any of the 20 naturally occurring amino acid residues with
the provisos that, in the first amino acid sequence, (i) when the
amino residue at position 12 is Ser, then the amino acid residue at
position 14 is not Ser, and (ii) when the amino residue at position
12 is Gly, then the amino acid residue at position 14 is not Ala,
performs very well in industrial applications such as laundry
compositions, for biopolishing of newly manufactured textiles, for
providing an abraded look of cellulosic fabric or garment, and for
treatment of paper pulp. Further, the invention relates to DNA
constructs encoding such enzymes, a method for providing a gene
encoding for such enzymes, a method of producing the enzymes,
enzyme preparations containing such enzymes, and the use of these
enzymes for a number of industrial applications.
Inventors: |
Schulein; Martin;
(Copenhagen, DK) ; Henriksen; Torben; (Copenhagen,
DK) ; Andersen; Lene Nonboe; (Allerod, DK) ;
Lassen; Soren Flensted; (Kobenhavn N, DK) ;
Kauppinen; Markus Sakari; (Kobenhavn N, DK) ; Lange;
Lene; (Valby, DK) ; Nielsen; Ruby Ilum;
(Farum, DK) ; Takagi; Shinobu; (Ichikawa-shi,
JP) ; Ihara; Michiko; (Chiba-shi, JP) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27545143 |
Appl. No.: |
11/740076 |
Filed: |
April 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10965499 |
Oct 14, 2004 |
7226773 |
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11740076 |
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10007521 |
Dec 10, 2001 |
6855531 |
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10965499 |
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09229911 |
Jan 13, 1999 |
6387690 |
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10007521 |
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08651136 |
May 21, 1996 |
6001639 |
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09229911 |
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PCT/DK96/00105 |
Mar 18, 1996 |
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08651136 |
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Current U.S.
Class: |
435/197 ;
435/263; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12Y 302/01004 20130101;
D21C 5/005 20130101; A61K 38/00 20130101; C07K 2319/00 20130101;
D21H 21/10 20130101; D21H 17/005 20130101; Y02W 30/40 20150501;
Y02W 30/43 20150501; C05F 17/20 20200101; D06P 5/02 20130101; Y02P
20/145 20151101; D06P 5/158 20130101; C05F 11/08 20130101; C11D
3/386 20130101; C12N 9/2437 20130101; C12P 19/14 20130101; D06M
16/003 20130101 |
Class at
Publication: |
435/197 ;
536/23.2; 435/320.1; 435/263 |
International
Class: |
C12N 9/18 20060101
C12N009/18; C07H 21/04 20060101 C07H021/04; C12N 15/00 20060101
C12N015/00; D06M 16/00 20060101 D06M016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 1995 |
DK |
0272/95 |
Aug 8, 1995 |
DK |
0885/95 |
Aug 8, 1995 |
DK |
0886/95 |
Aug 8, 1995 |
DK |
0887/95 |
Aug 8, 1995 |
DK |
0888/95 |
Feb 12, 1996 |
DK |
0137/96 |
Claims
1. An enzyme preparation consisting essentially of an enzyme which
has cellulytic activity and comprises a first amino acid sequence
having the following sequence TABLE-US-00060 (SEQ ID NO: 79) Thr
Arg Xaa Xaa Asp Cys Cys Xaa Xaa Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11
12 Trp Xaa 13 14
and a second amino acid sequence having the following sequence
TABLE-US-00061 Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5
wherein (a) the amino acid residue at position 3 of the first
sequence is Trp, Tyr or Phe; (b) the amino acid residue at position
4 of the first sequence is Trp, Tyr or Phe; (c) the amino acid
residue at position 8 of the first sequence is Arg, Lys or His; and
(d) the amino acid residues at positions 9, 10, 12 and 14 of the
first sequence and at position 4 of the second sequence are
independently any of the 20 naturally occurring amino acid
residues, provided that, in the first amino acid sequence, (i) when
the amino acid residue at position 12 is Ser, then the amino acid
residue at position 14 is not Ser, and (ii) when the amino acid
residue at position 12 is Gly, then the amino acid residue at
position 14 is not Ala.
2-10. (canceled)
11. An enzyme preparation consisting essentially of an enzyme
having cellulytic activity and being obtainable from a strain
belonging to Hymenomycetes (Basidiomycota) which enzyme comprises
an amino acid sequence selected from the group consisting of the
sequences TABLE-US-00062 Xaa Thr Arg Xaa Phe Asp Xaa (SEQ ID NO:
105) 1 2 3 4 5 6 7; Xaa Thr Arg Xaa Tyr Asp Xaa (SEQ ID NO: 106) 1
2 3 4 5 6 7; and Xaa Thr Arg Xaa Trp Asp Xaa (SEQ ID NO: 107) 1 2 3
4 5 6 7
wherein (a) Xaa at position 4 is Trp, Tyr or Phe; and (b) Xaa at
positions 1 and 7 is independently any of the 20 naturally
occurring amino acid residues.
12-65. (canceled)
66. A DNA construct comprising a DNA sequence encoding an enzyme
exhibiting cellulytic activity, which DNA sequence comprises (a)
the DNA sequence of SEQ ID NO: 1, and/or the DNA sequence
obtainable from the plasmid in Saccharomyces cerevisiae DSM 9770,
or (b) an analogue of the DNA sequence of SEQ ID NO: 1 or the DNA
sequence obtainable from the plasmid in Saccharomyces cerevisiae
DSM 9770, which (i) is homologous, preferably at least 70%
homologous, with the DNA sequence of SEQ ID NO: 1 and/or the DNA
sequence obtainable from the plasmid in Saccharomyces cerevisiae
DSM 9770, (ii) hybridizes under the conditions described herein
with the same nucleotide probe as the DNA sequence of SEQ ID NO: 1
and/or the DNA sequence obtainable from the plasmid in
Saccharomyces cerevisiae DSM 9770, (iii) encodes a polypeptide
which is homologous preferably at least 65% homologous, with the
polypeptide encoded by a DNA sequence comprising the DNA sequence
of SEQ ID NO: 1 and/or the DNA sequence obtainable from the plasmid
in Saccharomyces cerevisiae DSM 9770, (iv) encodes a polypeptide
which is immunologically reactive with an antibody raised against
the purified endoglucanase encoded by the DNA sequence of SEQ ID
NO: 1 or obtainable from the plasmid in Saccharomyces cerevisiae,
DSM 9770.
67. The DNA construct of claim 66, in which the DNA sequence is
isolated from or produced on the basis of a DNA library of a strain
belonging to the family Chaetomiaceae, preferably to the genus
Myceliophthora, in particular a strain of M. thermophila,
especially M. thermophila, CBS 117.65.
68-83. (canceled)
84. A recombinant expression vector comprising a DNA construct of
claim 66.
85. A cell comprising a DNA construct of claim 66.
86. A cell comprising a recombinant expression vector of claim
84.
87-88. (canceled)
89. A method of producing an enzyme exhibiting endoglucanase
activity, comprising culturing a cell of claim 85 under conditions
permitting the production of the enzyme, and recovering the enzyme
from the culture.
90. (canceled)
91. A method of providing colour clarification of laundry, which
method comprising treating the laundry with a soaking, washing or
rinsing liquor comprising an enzyme preparation of claim 1.
92-96. (canceled)
97. A laundry composition comprising the enzyme preparation of
claim 1, and a compound selected from the group consisting of a
surfactant, a builder compound, and a fabric softening agent.
98. The laundry composition of claim 97, which further comprises
one or more enzymes selected from the group consisting of
proteases, amylases, lipases, cellulases, xylanases, peroxidases
and laccases.
99. The composition of claim 97, wherein the surfactant is a
nonionic, anionic, cationic, zwitterionic, ampholytic or amphoteric
surfactant.
100. The composition of claim 97, wherein the fabric softening
agent is a cationic or nonionic softening agent, preferably a
quaternary ammonium compound, and which optionally further
comprises one or more compounds selected from a surfactant an
electrolyte, a buffer, an antioxidant and a liquid carrier.
101-105. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/965,499 filed on Oct. 14, 2004, which is a continuation of U.S.
application no. 10/007,521 filed Dec. 10, 2001, now U.S. Pat. No.
6,855,531, which is a continuation of U.S. application no.
09/229,911 filed Jan. 13, 1999, now U.S. Pat. No. 6,387,690, which
is a divisional of U.S. application Ser. No. 08/651,136 filed May
21, 1996, now U.S. Pat. No. 6,001,639, which is a continuation of
international application no. PCT/DK96/00105 filed Mar. 18, 1996,
which claims priority under 35 U.S.C. 119 of Danish application
nos. 0272/95, 0885/95, 0886/95, 0887/95, 0888/95, and 0137/96 filed
Mar. 17, 1995, Aug. 8, 1995, Aug. 8, 1995, Aug. 8, 1995, Aug. 8,
1995 and Feb. 12, 1996, respectively, the contents of which are
fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel enzyme preparations
comprising an enzyme exhibiting endoglucanase activity which
performs very well in industrial applications such as laundry
compositions, for biopolishing of newly manufactured textiles, for
providing an abraded look of cellulosic fabric or garment, and for
treatment of paper pulp. Further, the invention relates to DNA
constructs encoding such enzymes, a method for providing a gene
encoding for such enzymes, a method of producing the enzymes,
enzyme preparations containing such enzymes, and the use of these
enzymes for a number of industrial applications.
BACKGROUND OF THE INVENTION
[0003] Cellulases or cellulytic enzymes are enzymes involved in
hydrolysis of cellulose. In the hydrolysis of native cellulose, it
is known that there are three major types of cellulase enzymes
involved, namely cellobiohydrolase (1,4-beta-D-glucan
cellobiohydrolase, EC 3.2.1.91), endo-beta-1,4-glucanase
(endo-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and
beta-glucosidase (EC 3.2.1.21).
[0004] Cellulases are synthesized by a large number of
microorganisms which include fungi, to actinomycetes, mycobacteria
and true bacteria but also by plants. Especially endoglucanases of
a wide variety of specificities have been identified.
[0005] A very important industrial use of cellulytic enzymes is the
use for treatment of cellulosic textile or fabric, e.g., as
ingredients in detergent compositions or fabric softener
compositions, for bio-polishing of new fabric (garment finishing),
and for obtaining a "stone-washed" look of cellulose containing
fabric, especially denim, and several methods for such treatment
have been suggested, e.g., in GB-A-1 368 599, EP-A-0 307 564 and
EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244,
PCT/DK95/000108 and PCT/DK95/00132.
[0006] Another important industrial use of cellulytic enzymes is
the use for treatment of paper pulp, e.g., for improving the
drainage or for deinking of recycled paper.
[0007] Especially the endoglucanases (EC No. 3.2.1.4) constitute an
interesting group of hydrolases for the mentioned industrial uses.
Endoglucanases catalyses endo hydrolysis of 1,4-beta-D-glycosidic
linkages in cellulose, cellulose derivatives (such as carboxy
methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1,4
bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or
xyloglucans and other plant material containing cellulosic parts.
The authorized name is endo-1,4-beta-D-glucan 4-glucano hydrolase,
but the abbreviated term endoglucanase is used in the present
specification. Reference can be made to T.-M. Enveri, "Microbial
Cellulases" in W. M. Fogarty, Microbial Enzymes and Biotechnology,
Applied Science Publishers, p. 183-224 (1983); Methods in
Enzymology, Vol. 160, p. 200-391 (1988) (edited by Wood, W. A. and
Kellogg, S. T.); Beguin, P., "Molecular Biology of Cellulose
Degradation", Annu. Rev. Microbiol. Vol. 44, pp. 219-248 (1990);
Beguin, P. and Aubert, J-P., "The biological degradation of
cellulose", FEMS Microbiology Reviews Vol. 13, pp. 25-58 (1994);
Henrissat, B., "Cellulases and their interaction with cellulose",
Cellulose, Vol. 1, pp. 169-196 (1994).
[0008] Fungal endoglucanases have been described in numerous
publications, especially those derived from species as, e.g.,
Fusarium oxysporum, Trichoderma reesei, Trichoderma
longibrachiatum, Aspergillus aculeatus, Neocallimastix patriciarum,
and, e.g., from species of the genera Piromyces, Humicola,
Myceliophthora, Geotricum, Penicillium, Irpex, Coprinus.
[0009] For example, fungal endoglucanases have been described by
Sheppard, P. O., et al., "The use of conserved cellulase
family-specific sequences to clone Cellulase homologue cDNAs from
Fusarium oxysporum, Gene, Vol. 15, pp. 163-167 (1994), Saloheimo,
A., et al., "A novel, small endoglucanase gene, egl5, from
Trichoderma reesei isolated by expression in yeast", Molecular
Microbiology Vol. 13(2), pp. 219-228 (1994); van Arsdell, J. N. et
al., Cloning, characterization, and expression in Saccharomyces
cerevisiae of endoglucanase I from Trichoderma reesei,
Bio/Technology 5, 60-64 (1987); Penttila, M. et al., "Homology
between cellulase genes of to Trichoderma reesei: complete
nucleotide sequence of the endoglucanase I gene", Gene 45: 253-263
(1986), Saloheimo, M. et al, "EGIII, a new endoglucanase from
Trichoderma reesei the characterization of both gene and enzyme",
Gene 63: 11-21 (1988), Gonzales, R., et al., "Cloning, sequence
analysis and yeast expression of the egl1 gene from Trichoderma
longibrachiatum", Appl. Microbiol. Biotechnol., Vol. 38, pp.
370-375 (1992); Ooi, T. et al. "Cloning and sequence analysis of a
cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus", Curr.
Genet., Vol. 18, pp. 217-222 (1990); Ooi, T. et al, "Complete
nucleotide sequence of a gene coding for Aspergillus aculeatus
cellulase (FI-CMCase)", Nucleic Acids Research, Vol. 18, No. 19, p.
5884 (1990); Xue, G. et al., "Cloning and expression of multiple
cellulase cDNAs from the anaerobic rumen fungus Neocallimastix
patriciarum in E. coli", J. Gen. Microbiol., Vol. 138, pp 1413-1420
(1992); Xue, G. et al., "A novel polysaccharide hydrolase cDNA
(celD) from Neocallimastix patriciarum encoding three
multi-functional catalytical domains with high endoglucanase,
cellobiohydrolase and xylanase activities", 3. Gen. Microbiol.,
Vol. 138, pp. 2397-2403 (1992), Zhou, L. et al., "Intronless celB
from the anaerobic fungus Neocallimastix patriciarum encodes a
modular family A endoglucanase", Biochem. J., Vol. 297, pp. 359-364
(1994); Dalboge, H. and Heldt-Hansen. H. P. "A novel method for
efficient expression cloning of fungal enzyme genes", Mol. Gen.
Genet., Vol. 243, pp. 253-260 (1994); Ali, B. R. S. et al.,
"Cellulases and hemicellulases of the anaerobic fungus Piromyces
constitute a multiprotein cellulose-binding complex and are encoded
by multigene families", "FEMS Microbiol. Lett., Vol. 125, No. 1,
pp. 15-21 (1995). Further, the DNA Data Bank of Japan (DDBJ
database publicly available at Internet) comprises two DNA
sequences cloned from Penicillium janthinellum encoding
endoglucanases (cloned by A. Koch and G. Mernitz, respectively) and
a DNA sequence cloned from Humicola grisea var thermoidea encoding
an endoglucanase (cloned by T. Uozumi). Two endoglucanases from
Macrophomina phaseolina have been cloned and sequenced, see Wang,
H. Y. and Jones, R. W. "Cloning, characterization and functional
expression of an endoglucanase-encoding gene from the
phytopathogenic fungus Macrophomina phaseolina" in Gene,
158:125-128, 1995, and Wang, H. Y. and Jones, R. W.: "A unique
endoglucanase-encoding gene cloned from the phytopathogenic fungus
Macrophomina phaseolina" in Applied And Environmental Microbiology,
61:2004-2006, 1995. One of these endoglucanases shows high homology
to the egl3 endoglucanase from the fungus Trichoderma reesei, the
other shows homology to the egl1 from the microbial phytopathogen
Pseudomonas solanacearum indicating that both endoglucanases belong
to family 5 of glycosyl hydrolases (B. Henrissat, Biochem J 280;
309-316 (1991)). Filament-specific expression of a cellulase gene
in the dimorphic fungus Ustilago maydis is disclosed in
Schauwecker, F. et al. (1995).
[0010] WO 91/17243 (Novo Nordisk A/S) discloses a cellulase
preparation consisting of a homogenous endoglucanase component
immunoreactive with an antibody raised against a highly purified 43
kDa endoglucanase derived from Humicola insolens, DSM 1800; WO
91/17244 (Novo Nordisk A/S) discloses a new (hemi)cellulose
degrading enzyme, such as an endoglucanase, a cellobiohydrolase or
a beta-glucosidase, which may be derived from fungi other than
Trichoderma and Phanerochaete; WO 93/20193 discloses an
endoglucanase derivable from Aspergillus aculeatus; WO 94/21801
(Genencor Inc.) concerns a cellulase system isolated from
Trichoderma longibrachiatum exhibiting endoglucanase activity, WO
94/26880 (Gist Brocades N. V.) discloses an isolated mixture of
cellulose degrading enzymes, which preferable are obtained from
Trichoderma Aspergillus or Disporotrichum, comprising
endoglucanase, cellobiohydrolase, and xyloglucanase activity; and
WO 95/02043 (Novo Nordisk A/S) describes an enzyme with
endoglucanase activity derived from Trichoderma harzianum, which
can be used for a number of purposes including e.g., degradation or
modification of plant cell walls.
[0011] It is also known that cellulases may or may not have a
cellulose binding domain (a CBD). The CBD enhances the binding of
the enzyme to a cellulose containing fiber and increases the
efficacy of the catalytic active part of the enzyme.
[0012] There is an ever existing need for providing novel cellulase
enzyme preparations which may be used for applications where
cellulase preferably an endoglucanase, activity is desirable.
[0013] The object of the present invention is to provide novel
enzyme preparations having substantial cellulytic activity at acid,
neutral or alkaline conditions and improved performance in paper
pulp processing, textile treatment, laundry processes or in animal
feed; preferably novel cellulases, more preferably well-performing
endoglucanases, which are contemplated to be producible or produced
by recombinant techniques.
SUMMARY OF THE INVENTION
[0014] Surprisingly, it has been found that a group of
endoglucanases having certain unique characteristics perform very
well in those industrial applications for which endoglucanases are
conventionally used. These unique characteristics can be described
in terms of conserved regions of the amino acid sequence of the
enzyme protein and the inventors have found that cellulytic
enzymes, i.e., enzymes exhibiting cellulytic activity, having
certain conserved regions are very effective, e.g., in the
treatment of laundry, in the treatment of newly manufactured
textile, in the treatment of papermaking pulp.
[0015] Accordingly, in its first aspect the present invention
relates to an enzyme preparation consisting essentially of an
enzyme having cellulytic activity and comprising a first amino acid
sequence consisting of 14 amino acid residues having the following
sequence
TABLE-US-00003 (SEQ ID NO: 79) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12 Trp Xaa 13 14
and a second amino acid sequence consisting of 5 amino acid
residues having the following sequence
TABLE-US-00004 Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5
wherein
[0016] at position 3 of the first sequence, the amino acid is Trp,
Tyr or Phe;
[0017] at position 4 of the first sequence, the amino acid is Trp,
Tyr or Phe;
[0018] at position 8 of the first sequence, the amino acid is Arg,
Lys or His;
[0019] at positions 9, 10, 12 and 14, respectively, of the first
sequence, and at position 4 of the second sequence, the amino acid
is any of the 20 naturally occurring amino acid residues with the
provisos that, in the first amino acid sequence, (i) when the amino
residue at position 12 is Ser, then the amino acid residue at
position 14 is not Ser, and (ii) when the amino residue at position
12 is Gly, then the amino acid residue at position 14 is not
Ala.
[0020] This surprising finding of clearly recognisable conserved
regions, in spite of rather prominent variations found within
well-performing endoglucanase enzymes, is a result of studies of a
number of fungal DNA sequences encoding for specific amino acid
sequences of enzymes having significant cellulytic, especially
endoglucanase, activities.
[0021] Based on this finding, a novel molecular method tailored to
screen specifically for genomic DNA or cDNA characterized by
encoding the enzymes of the invention has been developed. As tools
for these three sets of degenerated primers were constructed.
Accordingly, in its second aspect, the invention relates to a
method for providing a gene encoding for cellulytic enzymes having
the above conserved regions.
[0022] By using this method, i.e., the set of primers for a PCR
screening on genomic DNA, it was surprisingly found that DNA
encoding for said enzymes can be found from a broad range of fungi,
belonging to taxonomically very different organisms and inhabiting
ecologically very different niches.
[0023] Further, by using this method it has been possible to find
DNA sequences encoding for the core regions (catalytically active
regions or domains) of said enzymes without any attached cellulose
binding domain (CBD) which core regions of enzymes would not have
been selected by using conventional performance based screening
approaches. The inventors have verified experimentally that the
linking of a CBD region to a core region enzyme (comprising the
catalytically active region or domain of the enzyme) of the present
invention results in a significantly improved performance, e.g., a
fifty times higher performance., of the multiple domain enzyme.
[0024] Accordingly, the present invention provides novel
cellulases, especially endoglucanases, having improved performance
in industrial applications, either in their native form, or homo-
or heterologously produced.
[0025] In further aspects, the present invention relates to novel
cellulytic enzyme preparations which are derivable from
taxonomically specific phyli, classes, orders, families, genera,
and species; e.g., from Basidiomycotous Hymenomycetes, Zygomycota,
Chytridiomycota; or from the classes Discomycetes,
Loculoascomycetes, Plectomycetes; Archaeascomycetes,
Hemiascomycetes or from the orders Diaportales, Xylariales,
Trichosphaeriales, Phyllachorales; or from the families Nectriaeae,
Sordariaceae, Chaetomiaceae, Ceratostomaceae, Lasiosphaeriaceae; or
from the genera Cylindrocarpon, Gliocladium, Volutella,
Scytalidium, Acremonium, or from the species Fusarium lycopersici,
Fusarium passiflora, Fusarium solani, Fusarium anguioides, Fusarium
poae, Humicola nigrescens, Humicola grisea, especially such
consisting essentially of an enzyme comprising an amino acid
sequence selected from the group consisting of the sequences
TABLE-US-00005 (SEQ ID NOS: 105-107) Xaa Thr Arg Xaa Phe Asp Xaa 1
2 3 4 5 6 7; Xaa Thr Arg Xaa Tyr Asp Xaa 1 2 3 4 5 6 7; and Xaa Thr
Arg Xaa Trp Asp Xaa 1 2 3 4 5 6 7
wherein Xaa at position 4 is Trp, Tyr or Phe and Xaa at positions 1
and 7 is any of the 20 naturally occurring amino acid residues.
[0026] More specifically, the enzyme preparation of the invention
is preferably obtainable from the taxonomically specific phyli,
classes, orders, families, genera, and species mentioned above
which all produce endoglucanases comprising a first peptide
consisting of 13 amino acid residues having the following
sequence
TABLE-US-00006 (SEQ ID NO: 108) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12 Trp 13
and a second peptide consisting of 5 amino acid residues having the
following sequence
TABLE-US-00007 Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5
wherein, at position 3 of the first sequence, the amino acid is
Trp, Tyr or Phe; at position 4 of the first sequence, the amino
acid is Trp, Tyr or Phe; at position 8 of the first sequence, the
amino acid is Arg, Lys or His; at positions 9, 10, and 12,
respectively, of the first sequence, and at position 4 of the
second sequence, the amino acid is any of the 20 naturally
occurring amino acid residues.
[0027] In yet further aspects, the present invention provides DNA
constructs comprising a DNA sequence encoding an enzyme exhibiting
endoglucanase activity, which DNA sequence comprises the DNA
sequence of SEQ ID NOS: 1, 7, 9, 11, 13, 15, 21, and 25,
respectively, or analogues thereof.
[0028] The present invention also relates to a recombinant
expression vector comprising a DNA construct of the invention, to a
cell comprising a DNA construct or a recombinant expression vector
of the invention; to a method of producing an enzyme, e.g., a
recombinant enzyme, of the invention; to a method of providing
colour clarification of laundry by using the enzyme of the
invention to a laundry composition comprising the enzyme of the
invention, to uses of the enzyme of the invention for degradation
or modification of plant material, e.g., cell walls, for treatment
of fabric, textile or garment, for treatment of paper pulp; and to
an enzyme preparation which is enriched in an enzyme of the present
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A, 1B and 1C show an alignment of the deduced encoded
amino acid sequences of Acremonium sp, (I) (SEQ ID NO: 8),
Volutella colletotrichoides (SEQ ID NO: 22), Crinipellis scabella
(SEQ ID NO: 16), Acremonium sp. (II) (SEQ ID NO: 10),
Myceliophthora thermophila (SEQ ID NO: 2), Thielavia terrestris
(SEQ ID NO: 12), Macrophomina phaseolina (SEQ ID NO: 14). The
Pileup program (Feng and Doolittle, 1987) (GCG package, version
8.0) was used to create the best alignment. Identical residues in
at least four sequences (boxed) are indicated around the
corresponding amino acids.
[0030] FIGS. 2A, B and C illustrate the taxonomic classification
within the Fungal Kingdom of all the microorganisms disclosed
herein as being capable of producing said enzyme preparations and
enzymes of the invention.
[0031] The taxonomic classification used herein builds primarily on
the system used in the NIH Data Base (Entrez, version spring 1996)
available on the internet.
[0032] Regarding classification of organisms which are not included
in the Entrez data base the following generally available and world
wide accepted reference books have been used:
[0033] For Ascomycetes: Eriksson, O. E. & Hawksworth, D. L.:
Systema Ascomycetum vol 12 (1993).
[0034] For Basidiomycetes: Julich, W.: Higher Taxa of
Basidiomycetes, Bibliotheca Mycologia 85, 485 pp (1981).
[0035] For Zygomycetes: O'Donnell, K.: Zygomycetes in culture,
University of Georgia, US, 257 pp (1979).
[0036] General Mycological Reference Books;
Hawksworth, D. L., Kirk, P. M., Sutton, B. C. and Pegler, D. N.:
Dictionary of the fungi, International Mycological Institute, 616
pp (1995); Von Arx, J. A.: The genera of fungi sporulating in
culture, 424 pp (1981),
[0037] The taxonomic implacement of the genus Humicola has until
recently remained unclear. However, studies of 18SRNA of a wide
selection of Sordariales has given strong indications of referring
Humicola to the order Sordariales (Taylor, Clausen & Oxenboll,
unpublished). Further these data suggests Humicola along with
Scytalidium to be only rather distantly related to the families
Sordariaceae, Chaetomiaceae, Ceratostomataceae, and
Lasiosphaeriaceae. In accordance with the above Humicola and
Scytalidium are here placed within the order Sordariales, with
unclassified Family.
[0038] FIGS. 3A and 38 show an alignment of the deduced partial
amino acid sequences derived from a selection of 26 of the 46
microorganisms described in Example 5 (SEQ ID NOS: 40, 30, 38, 74,
64, 32, 52, 62, 66, 28, 34, 68, 76, 72, 46, 54, 42, 36, 48, 44, 78,
58, 50, 60, and 56).
DETAILED DESCRIPTION OF THE INVENTION
[0039] In the present context, the term "the 20 naturally occurring
amino acid residues" denotes the amino acid residues usually found
in proteins and conventionally known as alanine (Ala or A), valine
(Val or V), leucine (Leu or L), isoleucine (Ile or I), proline (Pro
or P), phenylalanine (Phe or F), tryptophan (Trp or W), methionine
(Met or M), glycine (Gly or G), serine (Ser or S), threonine (Thr
or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or
N), glutamine (Gln or Q), aspartic acid (Asp or D), glutamic acid
(Glu or E), lysine (Lys or K), arginine (Arg or R), and histidine
(H is or H).
[0040] According to the present invention there is provided novel
well-performing endoglucanases comprising conserved amino acid
sequence regions, especially a first amino acid sequence consisting
of 14 amino acid residues having the following sequence
TABLE-US-00008 (SEQ ID NO: 79) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12 Trp Xaa 13 14
and a second amino acid sequence consisting of 5 amino acid
residues having the following sequence
TABLE-US-00009 Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5
wherein
[0041] at position 3 of the first sequence, the amino acid is Trp,
Tyr or Phe;
[0042] at position 4 of the first sequence, the amino acid is Trp,
Tyr or Phe;
[0043] at position 8 of the first sequence, the amino acid is Arg,
Lys or His;
[0044] at positions 9, 10, 12 and 14, respectively, of the first
sequence, and at position 4 of the second sequence, the amino acid
is any of the 20 naturally occurring amino acid residues with the
provisos that, in the first amino acid sequence, (i) when the amino
residue at position 12 is Ser, then the amino acid residue at
position 14 is not Ser, and (ii) when the amino residue at position
12 is Gly, then the amino acid residue at position 14 is not
Ala.
[0045] Preferably, the enzyme of the invention is of microbial
origin, i.e., obtainable from a microorganism such as a fungus.
[0046] In a preferred embodiment, the amino acid residue at
position 9 of the first sequence is selected from the group
consisting of proline, threonine, valine, alanine, leucine,
isoleucine, phenylalanine, glycine, cysteine, asparagine,
glutamine, tyrosine, serine, methionine and tryptophan, preferably
from the group consisting of proline and threonine.
[0047] In another preferred embodiment, the amino acid residue at
position 10 of the first sequence is selected from the group
consisting of proline, threonine, valine, alanine, leucine,
isoleucine, phenylalanine, glycine, cysteine, asparagine,
glutamine, tyrosine, serine, methionine and tryptophan, preferably
serine.
[0048] In yet another preferred embodiment, the amino acid residue
at position 12 of the first sequence is selected from the group
consisting of proline, threonine, valine, alanine, leucine,
isoleucine, phenylalanine, glycine, cysteine, asparagine,
glutamine, tyrosine, serine, methionine and tryptophan, preferably
from the group consisting of alanine and glycine.
[0049] In yet another preferred embodiment, the amino acid residue
at position 14 of the first sequence is selected from the group
consisting of proline, threonine, valine, alanine, leucine,
isoleucine, phenylalanine, glycine, cysteine, asparagine,
glutamine, tyrosine, serine, methionine, tryptophan, glutamic acid
and aspartic acid, preferably from the group consisting of proline,
threonine, serine, alanine, glutamic acid and aspartic acid.
[0050] Preferably, the amino acid residue at position 4 of the
second sequence is selected from the to group consisting of
proline, threonine, valine, alanine, leucine, isoleucine,
phenylalanine, glycine., cysteine, asparagine, glutamine, tyrosine,
serine, methionine, tryptophan, glutamic acid and aspartic acid,
more preferably from the group consisting of alanine, glycine, and
glutamine.
[0051] Examples of more preferred embodiments are such wherein, in
the first sequence, the amino acid residue at position 3 is
tyrosine; or the amino acid residue at position 4 is tryptophan; or
the amino acid residue at position 8 is lysine.
[0052] In an especially preferred embodiment, the enzyme of the
invention has a first sequence comprising the amino acid
sequence
TABLE-US-00010 (SEQ ID NO: 79) Thr Arg Tyr Trp Asp Cys Cys Lys Pro
Ser Cys Ala 1 2 3 4 5 6 7 8 9 10 11 12 Trp 13,
or the amino acid sequence
TABLE-US-00011 (SEQ ID NO: 79) Thr Arg Tyr Trp Asp Cys Cys Lys Thr
Ser Cys Ala 1 2 3 4 5 6 7 8 9 10 11 12 Trp 13,
or the amino acid sequence
TABLE-US-00012 (SEQ ID NO: 79) Thr Arg Tyr Trp Asp Cys Cys Lys Pro
Ser Cys Gly 1 2 3 4 5 6 7 8 9 10 11 12 Trp 13.
[0053] In a second aspect, the present invention provides a method
for providing a microbial strain comprising a gene encoding such an
enzyme which method comprises hybridization, e.g., PCR
amplification, under standard conditions with an oligonucleotide
derived from any of the conserved regions, illustrated in FIG.
1.
[0054] A useful oligonucleotide comprises a nucleotide sequence
encoding at least a pentapeptide comprised in a peptide selected
from the group consisting of
TABLE-US-00013 (SEQ ID NO: 79) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12 Trp Xaa 13 14
the amino acid at position 3 or 4 being Trp, Tyr or Phe; the amino
acid in position 8 being Arg, Lys or His; the amino acid at
positions 9, 10, 12 and 14, respectively, being any of the 20
naturally occurring amino acid residues; and b.
TABLE-US-00014 Trp Cys Cys Xaa Cys Tyr (SEQ ID NO. 81) 1 2 3 4 5
6
the amino acid at position 4 being any of the 20 naturally
occurring amino acid residues; and c.
TABLE-US-00015 (SEQ ID NO: 82) Xaa Pro Gly Gly Gly Xaa Gly Xaa Phe
1 2 3 4 5 6 7 8 9
the amino acid at position 1 being Met or Ile; the amino acid at
positions 6 and 8, respectively, is Leu, Ile or Val; and d.
TABLE-US-00016 (SEQ ID NO: 83) Gly Cys Xaa Xaa Arg Xaa Asp Trp Xaa
1 2 3 4 5 6 7 8 9
the amino acid at position 3 being any of the 20 naturally
occurring amino acid residues; the amino acid at positions 4 and 6,
respectively, being Trp, Tyr or Phe; and the amino acid at position
9 being Phe or Met;
[0055] The useful oligonucleotides also comprise nucleotide
sequences complementary to the sequences mentioned.
[0056] In a preferred embodiment of the method of the invention,
the oligonucleotide corresponds to a PCR primer selected from the
PCR primers
TABLE-US-00017 sense: (SEQ ID NO: 84)
5'-CCCCAAGCTTACI.sup.A/.sub.CGITA.sup.C/.sub.TTGGGA.sup.C/.sub.TTG.sup.C/.-
sub.TTG.sup.C/.sub.TAA.sup.A/.sub.G .sup.A/.sub.CC-3' antsense 1:
(SEQ ID NO: 85)
5'-CTAGTCTAGATA.sup.A/.sub.GCAIGC.sup.A/.sub.GCA.sup.A/.sub.GCACC-3';
antisense 2: (SEQ ID NO: 86)
CTAGTCTAGAAAIA.sup.A/.sub.G/.sup.TICCI.sup.A/.sup.C/.sup.GICCICCIGG-3';
antisense 3: (SEQ ID NO: 87)
5'-CTAGTCTAGAIAACCA.sup.A/.sub.GTCA.sup.A/.sub.G.sup.A/.sub.TAIC.sup.G/.su-
b.TCC-3.
[0057] In a third aspect, the present invention provides an enzyme
preparation which essentially consists of an enzyme having
cellulytic activity and having the conserved regions found by the
inventors, i.e., which comprises a peptide consisting of 7 amino
acid residues having the following sequence (SEQ ID NOS:
105-107)
TABLE-US-00018 Xaa Thr Arg Xaa Phe Asp Xaa 1 2 3 4 5 6 7; Xaa Thr
Arg Xaa Tyr Asp Xaa 1 2 3 4 5 6 7; and Xaa Thr Arg Xaa Trp Asp Xaa
1 2 3 4 5 6 7
wherein Xaa at position 4 is Trp, Tyr or Phe; and Xaa at positions
1 and 7 is any of the 20 naturally occurring amino acid
residues.
[0058] This enzyme is obtainable from a strain belonging to
Basidiomycotous Hymenomycetes (see FIG. 2), more preferably to the
group consisting of the orders Agaricales, Auriculariales, and
Aphyllophorales, even more preferably to the group consisting of
the families Exidiaceae, Tricholomataceae, Coprinaceae,
Schizophyllaceae, Bjerkanderaceae and Polyporaceae, especially to
the group consisting of the genera Exidia, Crinipellis, Fomes,
Panaeolus, Trametes, Schizophyllum, and Spongipellis.
[0059] Specific examples are endoglucanases obtainable from a
strain belonging to the group consisting of the species Exidia
glandulosa, Crinipellis scabella, Fomes fomentarius, and
Spongipellis sp., more specific examples being Exidia glandulosa,
CBS 277.96, Crinipellis scabella, CBS 280.96, Fomes fomentarius,
CBS 276.96, and Spongipellis sp., CBS 283.96.
[0060] Exidia glandulosa was deposited at Centraalbureau voor
Schimmelcultures, Oosterstraat 1, Postbus 273, NL-3740 AG Baarn,
the Netherlands, on 12 Mar., 199, under the deposition number CBS
277.96; Crinipellis scabella was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
280.96, Fomes fomentarius was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
276.96, and Spongipellis sp. was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
283.96, all deposited under the Budapest Treaty.
[0061] The enzyme preparation of the invention is also obtainable
from a strain belonging to Chytridiomycota, preferably from a
strain belonging to the class of Chytridiomycetes, more preferably
belonging to the group consisting of the order Spizellomycetales,
even more preferably to the family Spizellomycetaceae, especially
belonging to the genus Rhizophlyctis. A specific example is a
strain belonging to the species Rhizophlyctis rosea, more
specifically to Rhizophlyctis rosea, CBS 282.96.
[0062] Rhizophlyctis rosea was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar. 1996, under the deposition number CBS
282.96, under the Budapest Treaty.
[0063] The enzyme preparation of the invention is also obtainable
from a strain belonging to Zygomycota, preferably belonging to the
class Zygomycetes, more preferably to the order Mucorales, even
more preferably to the group of families consisting of Mucoraceae
and Thamnidiaceae, especially belonging to the group consisting of
the genera Rhizomucor, Phycomyces and Chaetostylum. Specific
examples are strains belonging to the genera Rhizomucor pusillus,
Phycomyces nitens, and Chaetostylum fresenii more specifically to
Rhizomucor pusillus, IFO 4578, and Phycomyces nitens, IFO 4814 and
Chaetostylum freseni, NRRL 2305.
[0064] Further, the enzyme preparation of the invention is also
obtainable from a strain belonging to the group consisting of
Archaeascomycetes, Discomycetes, Hemiascomycetes,
Loculoascomycetes, and Plectomycetes, preferably belonging to the
group consisting of the orders Pezizales, Rhytismatales,
Dothideales, and Eurotiales. Especially, the enzyme is obtainable
from a strain belonging the group consisting of the families
Cucurbitariaceae, Ascobolaceae. Rhytismataceae, and Trichocomaceae,
preferably belonging the group consisting of the genera Diplodia,
Microsphaeropsis, Ulospora, Macrophomina, Ascobolus, Saccobolus,
Penicillium, and Thermomyces. Specific examples are enzymes
obtainable from a strain belonging the group consisting of the
species Diplodia gossypina, Microsphaeropsis sp., Ulospora
bilgramii, Aureobasidium sp. Macrophomina phaseolina, Ascobolus
stictoides, Saccobolus diutellus, Peziza, Pencillium verruculosum,
Penicillium chrysogenum, and Thermomyces verrucosus, more
specifically Diplodia gossypina, CBS 274.96, Ulospora bilgramii
NKBC 1444, Macrophomina phaseolina, CBS 281.96, Saccobolus
dilutellus, CBS 275.96, Penicillium verruculosum, ATCC 62396,
Penicillium chrysogenum, ATCC 9480, and Thermomyces verrucosus, CBS
285.96.
[0065] Diplodia gossypina was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
274.96, Macrophomina phaseolina was deposited at Centraalbureau
voor Schimmelcultures on 12 Mar., 1996, under the deposition number
CBS 281.96, Saccobolus dilutellus was deposited at Centraalbureau
voor Schimmelcultures on 12 Mar., 1996, under the deposition number
CBS 275.96; Thermomyces verrucosus was deposited at Centraalbureau
voor Schimmelcultures on 12 Mar., 1996, under the deposition number
CBS 285.96; all under the Budapest Treaty.
[0066] Yet further, the enzyme is obtainable from a strain
belonging to the group consisting of the orders Diaportales,
Xylariales, Trichosphaeriales and Phyllachorales, preferably from a
strain belonging to the group consisting of the families
Xylariaceae, Vaisaceae, and Phyllachoraceae, more preferably
belonging to the genera Diaporthe, Colletotrichum, Nigrospora,
Xylaria, Nodulisporum and Poronia. Specific examples are the
species Diaporthe syngenesia, Colletotrichum lagenarium, Xylaria
hypoxylon, Nigrospora sp., Nodulisporum sp., and Poronia to
punctata, more specifically Diaporthe syngenesia. CBS 278.96,
Colletotrichum lagenarium, ATCC 52609, Nigrospora sp., CBS 272.96,
Xylaria hypoxylon, CBS 284.96.
[0067] Diaporthe syngenesia was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
278.96, Nigrospora sp. was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
272.96, Xylaria hypoxylon was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
284.96, all under the Budapest Treaty.
[0068] The enzyme is also obtainable from the unidentified fungal,
mitosporic, coleomycetous deposited at Centraalbureau voor
Schimmelcultures on 12 Mar. 1996, under the deposition numbers CBS
270.96, CBS 271.96 and CBS 273.96, respectively, under the Budapest
Treaty.
[0069] The enzyme is also obtainable from a strain belonging to the
group consisting of the genera Cylindrocarpon, Gliocladium,
Nectria, Volutella, Sordaria, Scytalidium Thielavia, Syspastospora,
Cladorrhinum, Chaetomium, Myceliphthora and Acremonium, especially
from a strain belonging to the group consisting of the species
Cylindrocarpon sp., Nectria pinea, Volutella colletotrichoides,
Sordaria fimicola, Sordaria macrospora, Thielavia terrestris,
Thielavia thermophila, Syspastospora boninensis, Cladorrhinum
foecundissimum, Chaetomium murorum, Chaetomium virescens,
Chaetomium brasiliensis, Chaetomium cunicolorum, Myceliophthora
thermophila, Gliocladium catenulatum, Scytalidium thermophila, and
Acremonium sp., more specifically from Nectria pinea, CBS 279.96.
Volutella colletotrichoides, CBS 400.58, Sodaria fimicola, ATCC
52644, Sordaria macrospora, ATCC 60255, Thielavia terrestris, NRRL
8126, Thielavia thermophila, CCBS174.70, Chaetomium murorum, CBS
163.52, Chaetomium virescens, CBS 547.75, Chaetomium brasiliensis,
CBS 122.65, Chaetomium cunicolorum, CBS 799.83, Syspastospora
boninensis NKBC 1515, Cladorrhinum foecundissimum, ATCC 62373,
Myceliophthora thermophila, CBS 117.65, Scytalidium thermophila,
ATCC 28085, Gliocladium catenulatum, ATCC 10523, and Acremonium
sp., CBS 478.94.
[0070] Nectria pinea was deposited at Centraalbureau voor
Schimmelcultures on 12 Mar., 1996, under the deposition number CBS
279.96, and Acremonium Chaetomium sp. was deposited on 28 Sep. 1994
under the deposition number CBS 478.94, both according to the
Budapest Treaty.
[0071] The enzyme is also obtainable from a strain belonging to the
group consisting of the species Fusarium solani, Fusarium
anguioides, Fusarium poae, Fusarium oxysporum sp. lycopersici,
Fusarium oxysporum ssp. passiflora, Humicola nigrescens and
Humicola grisea, especially Fusarium oxysporum ssp lycopersici CBS
645.78, Fusarium oxysporum ssp passiflora, CBS 744.79, Fusarium
solani, IMI 107.511, Fusarium anguioides, IFO 4467, Fusarium poae,
ATCC 60883, Humicola nigrescens, CBS 819.73 and Humicola grisea,
ATCC 22726. It is to be noted that Humicola grisea is different
from Humicola grisea var. thermoidea.
[0072] In a preferred embodiment, the enzyme preparation of the
invention is derived from the disclosed classes, orders, families,
genera and species and essentially consists of an enzyme comprising
a first peptide consisting of 13 amino acid residues having the
following sequence
TABLE-US-00019 (SEQ ID NO: 79) Thr Arg Xaa Xaa Asp Cys Cys Xaa Xaa
Xaa Cys Xaa 1 2 3 4 5 6 7 8 9 10 11 12
and a second peptide consisting of 5 amino acid residues having the
following sequence
TABLE-US-00020 Trp Cys Cys Xaa Cys (SEQ ID NO: 80) 1 2 3 4 5
wherein, at position 3 of the first sequence, the amino acid is
Trp, Tyr or Phe; at position 4 of the first sequence, the amino
acid is Trp, Tyr or Phe; at position 8 of the first sequence, the
amino acid is Arg, Lys or His; at positions 9, 10, and 12,
respectively, of the first sequence, and at position 4 of the
second sequence, the amino acid is any of the 20 naturally
occurring amino acid residues.
[0073] Preferably, the amino acid residue at position 9 of the
first sequence is selected from the group consisting of proline,
threonine, valine, alanine, leucine, isoleucine, phenylalanine,
glycine, cysteine, asparagine, glutamine, tyrosine, serine,
methionine and tryptophan, more preferably from the group
consisting of proline and threonine; the amino acid residue at
position 10 of the first sequence which is selected from the group
consisting of proline, threonine, valine, alanine, leucine,
isoleucine, phenylalanine, glycine, cysteine, asparagine,
glutamine, tyrosine, serine, methionine and tryptophan, preferably
serine; the amino acid residue at position 12 of the first sequence
is selected from the group consisting of proline, threonine,
valine, alanine, leucine, isoleucine, phenylalanine, glycine,
cysteine, asparagine, glutamine, tyrosine, serine, methionine and
tryptophan, preferably from the group consisting of alanine and
glycine; and the amino acid residue at position 4 of the second
sequence is selected from the group consisting of proline,
threonine, valine, alanine, leucine, isoleucine, phenylalanine,
glycine, cysteine, asparagine, glutamine, tyrosine, serine,
methionine, tryptophan, glutamic acid and aspartic acid, more
preferably from the group consisting of alanine, glycine, and
glutamine.
[0074] In further aspects, the present invention provides a DNA
construct comprising a DNA sequence encoding an enzyme exhibiting
endoglucanase activity, which DNA sequence comprises
[0075] a) the DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21,
or 25, respectively, or the DNA sequence obtainable from the
plasmid in Saccharomyces cerevisiae DSM 9770, DSM 10082, DSM 10080.
DSM 10081, Escherichia coli, DSM 10512, DSM 10511, DSM 10571, DSM
10576, respectively; or
[0076] b) an analogue of the DNA sequence of SEQ ID NO: 1, 7, 9,
11, 13, 15, 21, or 25, respectively, or the DNA sequence obtainable
from the plasmid in Saccharomyces cerevisiae DSM 9770, DSM 10082,
DSM 10080, DSM 10081, Escherichia coli, DSM 10512, DSM 10511, DSM
10571, DSM 10576, respectively, which
[0077] i) is homologous with the DNA sequence of SEQ ID NO: 1, 7,
9, 11, 13, 15, 21, or 25, respectively, or the DNA sequence
obtainable from the plasmid in Saccharomyces cerevisiae DSM 9770,
DSM 10082, DSM 10080, DSM 10081 Escherichia coli DSM 10512, DSM
10511, DSM 10571, DSM 10576, respectively,
[0078] ii) hybridizes with the same oligonucleotide probe as the
DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25
respectively, or the DNA sequence obtainable from the plasmid in
Saccharomyces cerevisiae DSM 9770, DSM 10082, DSM 10080, DSM 10081,
Escherichia coli, DSM 10512, DSM 10511, DSM 10571, DSM 10576,
respectively,
[0079] iii) encodes a polypeptide which is homologous with the
polypeptide encoded by a DNA sequence comprising the DNA sequence
of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25 respectively, or the
DNA sequence obtainable from the plasmid in Saccharomyces
cerevisiae DSM 9770, DSM 10082, DSM 10080 DSM 10081, Escherichia
coli, DSM 10512, DSM 10511, DSM 10571, DSM 10576, respectively,
[0080] iv) encodes a polypeptide which is immunologically reactive
with an antibody raised against the purified endoglucanase encoded
by the DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25,
respectively, or the DNA sequence obtainable from the plasmid in
Saccharomyces cerevisiae DSM 9770, DSM 10082, DSM 10080, DSM 10081,
Escherichia coli, DSM 10512, DSM 10511, DSM 10571, DSM 10576,
respectively.
[0081] Escherichia coli DSM 10512 was deposited under the Budapest
Treaty on 2 Feb., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0082] Escherichia coli DSM 10511 was deposited under the Budapest
Treaty on 2 Feb., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0083] Escherichia coli DSM 10571 was deposited under the Budapest
Treaty on 6 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0084] Escherichia coli DSM 10576 was deposited under the Budapest
Treaty on 12 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, to
D-38124 Braunschweig, Germany).
[0085] Escherichia coli DSM 10583 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0086] Escherichia coli DSM 10584 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0087] Escherichia coli DSM 10585 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder leg 16, D-38124
Braunschweig, Germany).
[0088] Escherichia coli DSM 10586 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0089] Escherichia coli DSM 10587 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder leg 16, D-38124
Braunschweig, Germany).
[0090] Escherichia coli DSM 10588 was deposited under the Budapest
Treaty on 13 Mar., 1996, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0091] Saccharomyces cerevisiae DSM 9770 was deposited under the
Budapest Treaty on 24 Feb., 1995, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0092] Saccharomyces cerevisiae DSM 10082 was deposited under the
Budapest Treaty on 30 Jun., 1995, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0093] Saccharomyces cerevisiae DSM 10080 was deposited under the
Budapest Treaty on 30 Jun., 1995, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0094] Saccharomyces cerevisiae DSM 10081 was deposited under the
Budapest Treaty on 30 Jun., 1995, at DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 16, D-38124
Braunschweig, Germany).
[0095] The DNA construct of the invention relating to SEQ ID NO: 1
can be isolated from or produced on the basis of a DNA library of a
strain of Myceliophthora, in particular a strain of M. thermophila,
especially M. thermophila, CBS 117.65.
[0096] The DNA constructs of the invention relating to SEQ ID NOS:
7 and 9 can be isolated from or produced on the basis of a DNA
library of a strain of Acrermonium, especially Acremonium sp., CBS
478.94.
[0097] The DNA construct of the invention relating to SEQ ID NO: 11
can be isolated from or produced on the basis of a DNA library of a
strain of Thielavia in particular a strain of Thielavia terrestris,
especially Thielavia terrestris, NRRL 8126.
[0098] The DNA construct of the invention relating to SEQ ID NO-13
can be isolated from or produced on the basis of a DNA library of a
strain of Macrophomina, in particular a strain of M. phaseolina,
especially M. phaseolina, CBS 281.96
[0099] The DNA construct of the invention relating to SEQ ID NO: 15
can be isolated from or produced on the basis of a DNA library of a
strain of Crinipellis, in particular a strain of C. scabella,
especially C. scabella, CBS 280.96.
[0100] The DNA construct of the invention relating to SEQ ID NO: 25
can be isolated from or produced on the basis of a DNA library of a
strain of Sordaria, in particular a strain of Sordaria
fimicola.
[0101] In the present context, the "analogue" of the DNA sequence
of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25, respectively, is
intended to indicate any DNA sequence encoding an enzyme exhibiting
endoglucanase activity, which has any or all of the properties
i)-iv). The analogous DNA sequence
[0102] a) may be isolated from another or related (e.g., the same)
organism producing the enzyme with endoglucanase activity on the
basis of the DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or
25, respectively, e.g., using the procedures described herein; the
homologue may be an allelic variant of the DNA sequence comprising
the DNA sequences shown herein, i.e., an alternative form of a gene
that arises through mutation; mutations can be silent (no change in
the encoded enzyme) or may encode enzymes having altered amino acid
sequence; the homologue of the present DNA sequence may also be a
genus or species homologue, i.e., encoding an enzyme with a similar
activity derived from another species,
[0103] b) may be constructed on the basis of the DNA sequences of
SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25, respectively, e.g., by
introduction of nucleotide substitutions which do not give rise to
another amino acid sequence of the endoglucanase encoded by the DNA
sequence, but which correspond to the codon usage of the host
organism intended for production of the enzyme, or by introduction
of nucleotide substitutions which may give rise to a different
amino acid sequence. However, in the tatter case amino acid changes
are preferably of a minor nature, that is conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein, small deletions, typically of one to about
30 amino acid; small amino- or carboxyl-terminal extensions, such
as an amino-terminal methionine residue, a small tinker peptide of
up to about 20 residues, or a small extension that facilitates
purification, such as a poly-histidine tract, an antigenic epitope
or a binding domain. See in general Ford et al., Protein Expression
and Purification 2: 95-107, 1991, Examples of conservative
substitutions are within the group of basic amino acids (such as
arginine, lysine, histidine), acidic amino acids (such as glutamic
acid and aspartic acid), polar amino acids (such as glutamine and
asparagine), hydrophobic amino acids (such as leucine, isoleucine,
valine), aromatic amino acids (such as phenylalanine, tryptophan,
tyrosine) and small amino acids (such as glycine, alanine, serine,
threonine, methionine).
[0104] It will be apparent to persons skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acids essential to the activity of the polypeptide encoded by
the DNA construct of the invention, and therefore preferably not
subject to substitution, may be identified according to procedures
known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science 244:
1081-1085 (1989)). In the latter technique mutations are introduced
at every residue in the molecule, and the resultant mutant
molecules are tested for biological (i.e., endoglucanase) activity
to identify amino acid residues that are critical to the activity
of the molecule. Sites of substrate-enzyme interaction can also be
determined by analysis of crystal structure as determined by such
techniques as nuclear magnetic resonance, crystallography or
photoaffinity labeling, See, for example, de Vos et al., Science
255: 306-312 (1992); Smith et al. J. Mol. Biol. 224: 899-904
(1992); /Wlodaver et al., FEBS Lett. 309: 59-64 (1992).
[0105] The endoglucanase encoded by the DNA sequence of the DNA
construct of the invention may comprise a cellulose binding domain
(CBD) existing as an integral part of the encoded enzyme, or a CBD
from another origin may be introduced into the endoglucanase enzyme
thus creating an enzyme hybride. In this context, the term
"cellulose-binding domain" is intended to be understood as defined
by Peter Tomme et al. "Cellulose-Binding Domains, Classification
and Properties" in "Enzymatic Degradation of Insoluble
Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS
Symposium Series, No. 618, 1996. This definition classifies more
than 120 cellulose-binding domains (CBDs) into 10 families (I-X),
and it demonstrates that CBDs are found in various enzymes such as
cellulases, xylanases, mannanases, arabinofuranosidases, acetyl
esterases and chitinases. CBDs have also been found in algae, e.g.,
the red alga Porphyra purpurea as a non-hydrolytic
polysaccharide-binding protein, for reference see Peter Tomme et
al., supra. However, most of the CBDs are from cellulases and
xylanases, CBDs are found at the N or C termini of proteins or are
internal, Enzyme hybrids are known in the art, see, e.g., WO
90/00609 and WO 95/16782, and may be prepared by transforming into
a host cell a DNA construct comprising at least a fragment of DNA
encoding the cellulose-binding domain ligated, with or without a
linker, to a DNA sequence encoding the enzyme of interest and
growing the host cell to express the fused gene. Enzyme hybrids may
be described by the following formula,
CBD-MR-X,
wherein CBD is the N-terminal or the C-terminal region of an amino
acid sequence corresponding to at least the cellulose-binding
domain; MR is the middle region (the linker), and may be a bond, or
a short linking group preferably of from about 2 to about 100
carbon atoms, more preferably of from 2 to 40 carbon atoms; or is
preferably from about 2 to about 100 amino acids, more preferably
of from 2 to 40 amino acids; and X is an N-terminal or C-terminal
region of a polypeptide encoded by the DNA sequence of the
invention.
[0106] The homology referred to in i) above is determined as the
degree of identity between the two sequences indicating a
derivation of the first sequence from the second, The homology may
suitably be determined by means of computer programs known in the
art such as GAP provided in the GCG program package (Needleman, S.
B. and Wunsch, C. D., Journal of Molecular Biology, 48: 443-453,
1970). Using GAP with the following settings for DNA sequence
comparison: GAP creation penalty of 5.0 and GAP extension penalty
of 0.3, the coding region of the DNA sequence exhibits a degree of
identity preferably of at least 60%, more preferably at least 65%,
more preferably at least 70%, even more preferably at least 80%,
especially at least 90%, with the coding region of the DNA sequence
of SEQ ID NO: 1, 7, 9, 11, 13, 15, or 21, respectively, or the DNA
sequence obtainable from the plasmid in Saccharomyces cerevisiae,
DSM 9770, DSM 10082, DSM 10080, DSM 10081, Escherichia coli DSM
10512, DSM 10511, DSM 10571, or DSM 10576, respectively.
[0107] The hybridization referred to in ii) above is intended to
indicate that the analogous DNA sequence hybridizes to the same
probe as the DNA sequence encoding the endoglucanase enzyme under
certain specified conditions which are described in detail in the
Materials and Methods section hereinafter. The oligonucleotide
probe to be used is the DNA sequence corresponding to the
endoglucanase encoding part of the DNA sequence of SEQ ID NO: 1, 7,
9, 11, 13, 15 or 21, respectively, or the DNA sequence obtainable
from the plasmid in Saccharomyces cerevisiae, DSM 9770, DSM 10082,
DSM 10080, DSM 10081, Escherichia coli, DSM 10512, DSM 10511, DSM
10571 or DSM 10576, respectively.
[0108] The homology referred to in iii) above is determined as the
degree of identity between the two sequences indicating a
derivation of the first sequence from the second. The homology may
suitably be determined by means of computer programs known in the
art such as GAP provided in the GCG program package (Needleman, S.
B. and Wunsch, C. D., Journal of Molecular Biology, 48: 443-453,
1970). Using GAP with the following settings for polypeptide
sequence comparison: GAP creation penalty of 3.0 and GAP extension
penalty of 0.1 the polypeptide encoded by an analogous DNA sequence
exhibits a degree of identity preferably of at least 55%, more
preferably at least 80%, more preferably at least 858%, even more
preferably at least 70%, more preferably at least 80%, especially
at least 90%, with the enzyme encoded by a DNA construct comprising
the DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21, or 25,
respectively, or the DNA sequence obtainable from the plasmid in
Saccharomyces cerevisiae, DSM 9770, DSM 10082, DSM 10080, DSM
10081, Escherichia coli, DSM 10512, DSM 10511, DSM 10571 or DSM
10576, respectively.
[0109] In connection with property iv) above it is intended to
indicate an endoglucanase encoded by a DNA sequence isolated from
strain Saccharomyces cerevisiae, DSM 9770. DSM 10082, DSM 10080:
DSM 10081, Escherichia coli, DSM 10512, DSM 10511: DSM 10571 or DSM
105768 respectively, and produced in a host organism transformed
with said DNA sequence or the corresponding endoglucanase naturally
produced by Myceliophthora thermophila, Acremonium sp., Thielavia
terrestis, Macrophomina phaseolina, Crinipellis scabella, Volutella
colletotrichoides, or Sordaria fimicola, respectively. The
immunological reactivity may be determined by the method described
in the Materials and Methods section below.
[0110] In further aspects the invention relates to an expression
vector harbouring a DNA construct of the invention, a cell
comprising the DNA construct or expression vector and a method of
producing an enzyme exhibiting endoglucanase activity which method
comprises culturing said cell under conditions permitting the
production of the enzyme, and recovering the enzyme from the
culture.
[0111] In a still further aspect the invention relates to an enzyme
exhibiting endoglucanase activity, which enzyme
[0112] a) is encoded by a DNA construct of the invention
[0113] b) produced by the method of the invention, and/or
[0114] c) is immunologically reactive with an antibody raised
against a purified endoglucanase encoded by the DNA sequence of SEQ
ID NO: 1, 7, 9, 11, 13, 15, or 21, respectively, or the DNA
sequence obtainable from the plasmid in Saccharomyces cerevisiae,
DSM 9770, DSM 10082, DSM 10080, DSM 10081, Escherichia coli, DSM
10512, DSM 10511, DSM 10571 or DSM 10576, respectively.
[0115] The endoglucanase mentioned in c) above may be encoded by
the DNA sequence isolated from the strain Saccharomyces cerevisiae,
DSM 9770, DSM 10082, DSM 10080, DSM 10081, Escherichia coli, DSM
10512, DSM 10511, DSM 10571 or DSM 10576, respectively, and
produced in a host organism transformed with said DNA sequence or
the corresponding endoglucanase naturally produced by
Myceliophthora thermophila, Acremonium sp., Thielavia terrestris,
Macrophomina phaseolina, Crinipellis scabella, Volutella
colletotrichoides or Sordaria fimicola, respectively.
[0116] Generally, in the present context the term "enzyme" is
understood to include a mature protein or a precursor form thereof
as well to a functional fragment thereof which essentially has the
activity of the full-length enzyme. Furthermore, the term "enzyme"
is intended to include homologues of said enzyme.
[0117] Homologues of the present enzyme may have one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein, small deletions, typically of one to about
30 amino acids; small amino- or carboxyl-terminal extensions, such
as an amino-terminal methionine residue, a small linker peptide of
up to about 20-25 residues, or a small extension that facilitates
purification, such as a poly-histidine tract, an antigenic epitope
or a binding domain. See in general Ford et al., Protein Expression
and Purification 2: 95-107 (1991). Examples of conservative
substitutions are within the group of basic amino acids (such as
arginine, lysine, histidine), acidic amino acids (such as glutamic
acid and aspartic acid), polar amino acids (such as glutamine and
asparagine), hydrophobic amino acids (such as leucine, isoleucine,
valine), aromatic amino acids (such as phenylalanine, tryptophan,
tyrosine) and small amino acids (such as glycine, alanine, serine,
threonine, methionine).
[0118] It will be apparent to persons skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active enzyme.
Amino acids essential to the activity of the enzyme of the
invention, and therefore preferably not subject to substitution,
may be identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham, 1989). In the latter technique mutations are
introduced at every residue in the molecule, and the resultant
mutant molecules are tested for cellulytic activity to identify
amino acid residues that are critical to the activity of the
molecule. Sites of ligand-receptor interaction can also be
determined by analysis of crystal structure as determined by such
techniques as nuclear magnetic resonance, crystallography or
photoaffinity labelling. See, for example, de Vos et al., 1992;
Smith et al., 1992, Wlodaver et al., 1992.
[0119] The homologue may be an allelic variant, i.e., an
alternative form of a gene that arises through mutation, or an
altered enzyme encoded by the mutated gene, but having
substantially the same activity as the enzyme of the invention.
Hence mutations can be silent (no change in the encoded enzyme) or
may encode enzymes having altered amino acid sequence.
[0120] The homologue of the present enzyme may also be a genus or
species homologue, i.e. an enzyme with a similar activity derived
from another species.
[0121] A homologue of the enzyme may be isolated by using the
procedures described herein.
Molecular Screening and, Cloning by Polymerase Chain Reaction
(PCR)
[0122] Molecular screening for DNA sequences of the invention may
be carried out by polymerase chain reaction (PC R) using genomic
DNA or double-stranded cDNA isolated from a suitable source, such
as any of the herein mentioned organisms, and synthetic
oligonucleotide primers prepared on the basis of the DNA sequences
or the amino acid sequences disclosed herein. For instance, it
suitable oligonucleotide primers may be the primers described in
the Materials and Methods section.
[0123] In accordance with well-known procedures, the PCR fragment
generated in the molecular screening may be isolated and subcloned
into a suitable vector. The PCR fragment may be used for screening
DNA libraries by, e.g., colony or plaque hybridization.
Expression Cloning in Yeast
[0124] The DNA sequence of the invention encoding an enzyme
exhibiting endoglucanase activity may be isolated by a general
method involving [0125] cloning, in suitable vectors, a DNA library
from a suitable source, such as any of the herein mentioned
organisms [0126] transforming suitable yeast host cells with said
vectors, [0127] culturing the host cells under suitable conditions
to express any enzyme of interest encoded by a clone in the DNA
library, [0128] screening for positive clones by determining any
endoglucanase activity of the enzyme produced by such clones, and
[0129] isolating the enzyme encoding DNA from such clones.
[0130] The general method is further disclosed in WO 94/14953 the
contents of which are hereby incorporated by reference. A more
detailed description of the screening method is given in Example 1
below.
[0131] The DNA sequence coding for the enzyme may for instance be
isolated by screening a cDNA library of Macrophomina phaseolina.
Crinipellis scabella, Sordaria fimicola or Volutella
colletotrichoides, and selecting for clones expressing the
appropriate enzyme activity (i.e., endoglucanase activity) or from
Escherichia coli DSM 10512 deposited under the Budapest Treaty on 2
Feb. 1996, at DSM (Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Mascheroder Weg 16, D-38124 Braunschweig,
Germany), or from Escherichia coli DSM 10511 deposited under the
Budapest Treaty on 2 Feb. 1996, at DSM, or from Escherichia coli
DSM 10576, deposited under the Budapest Treaty on 12 Mar. 1996, at
DSM; or from Escherichia coli DSM 10571 deposited under the
Budapest Treaty on 6 Mar. 1996, at DSM; or by screening a cDNA
library of Myceliphthora thermophila, CBS 117.65, Acremonium sp.,
CBS 478.94 or Thielavia terrestris, NRRL 8126, and selecting for
clones expressing the appropriate enzyme activity (i.e.,
endoglucanase activity) or from Saccharomyces cerevisiae DSM 9770
deposited under the Budapest Treaty on 24 Feb. 1995, at DSM
(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Mascheroder Weg 16, D-38124 Braunschweig, Germany), or from
Saccharomyces cerevisiae DSM 10082 deposited under the Budapest
Treaty on 30 Jun., 1996, at DSM, from Saccharomyces cerevisiae DSM
10080 deposited under the Budapest Treaty on 30 Jun. 1995, or from
Saccharomyces cerevisiae DSM 10081 deposited under the Budapest
Treaty on 30 Jun. 1995, at DSM. The appropriate DNA sequence may
then be isolated from the clone by standard procedures, e.g., as
described in Example 1.
Nucleic Acid Construct
[0132] As used herein the term "nucleic acid construct" is intended
to indicate any nucleic acid molecule of cDNA, genomic DNA,
synthetic DNA or RNA origin. The term "construct" is intended to
indicate a nucleic acid segment which may be single or
double-stranded, and which may be based on a complete or partial
naturally occurring nucleotide sequence encoding an enzyme of
interest. The construct may optionally contain other nucleic acid
segments.
[0133] The nucleic acid construct encoding the enzyme of the
invention may suitably be of genomic or cDNA origin, for instance
obtained by preparing a genomic or cDNA library and screening for
DNA sequences coding for all or part of the enzyme by hybridization
using synthetic oligonucleotide probes in accordance with standard
techniques (cf. Sambrook et al., 1989).
[0134] The nucleic acid construct encoding the enzyme may also be
prepared synthetically by established standard methods, e.g., the
phosphoramidite method described by Beaucage and Caruthers (1981),
or the method described by Matthes et al. (1984). According to the
phosphoramidite method, oligonucleotides are synthesized, e.g., in
an automatic DNA synthesizer, purified, annealed, ligated and
cloned in suitable vectors.
[0135] Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed genomic
and cDNA origin prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0136] The nucleic acid construct may also be prepared by
polymerase chain reaction using specific primers, for instance as
described in U.S. Pat. No. 4,683,202 or Saiki et al. (1988).
[0137] The nucleic acid construct is preferably a DNA construct
which term will be used exclusively in this specification and
claims,
Recombinant Vector
[0138] A recombinant vector comprising a DNA construct encoding the
enzyme of the invention may be any vector which may conveniently be
subjected to recombinant DNA procedures, and the choice of vector
will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e., a vector which exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g., a plasmid. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0139] The vector is preferably an expression vector in which the
DNA sequence encoding the enzyme of the invention is operably
linked to additional segments required for transcription of the DNA
In general, the expression vector is derived from plasmid or viral
DNA, or may contain elements of both. The term, "operably linked"
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in a promoter and proceeds through the DNA sequence coding for the
enzyme.
[0140] The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
[0141] Examples of suitable promoters for use in yeast host cells
include promoters from yeast glycolytic genes (Hitzeman et al., J.
Biol. Chem. 255: 12073-12080 (1980); Alber and Kawasaki, J. Mol.
Appl. Gen. 1: 419-434 (1982)) or alcohol dehydrogenase genes (Young
et al., in Genetic Engineering of Microorganisms for Chemicals
(Hollaender et at, eds.), Plenum Press, New York, 1982), or the
TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature
304: 652-654 (1983)) promoters.
[0142] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, to the ADH3 promoter (McKnight et
a<. The EMBO J. 4: 2093-2099 (1985)) or the tpiA promoter.
Examples of other useful promoters are those derived from the gene
encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase, A. niger neutral alpha-amylase, A. niger acid stable
alpha-amylase, A. niger or A. awamori glucoamylase (gluA),
Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae
triose phosphate isomerase or A. nidulans acetamidase. Preferred
are the TAKA-amylase and gluA promoters.
[0143] Examples of suitable promoters for use in bacterial host
cells include the promoter of the Bacillus stearothermophilus
maltogenic amylase gene, the Bacillus licheniformis alpha-amylase
gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus
subtilis alkaline protease gen, or the Bacillus pumilus xylosidase
gene, or by the phage Lambda P.sub.R or P.sub.L promoters or the E.
coli lac, trp, or tac promoters.
[0144] The DNA sequence encoding the enzyme of the invention may
also, if necessary, be operably connected to a suitable
terminator.
[0145] The recombinant vector of the invention may further comprise
a DNA sequence enabling the vector to replicate in the host cell in
question.
[0146] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the host cell,
such as the gene coding for dihydrofolate reductase (DHFR) or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 1985, pp. 125-130). For filamentous fungi, selectable
markers include amdS pyrG, argB, niaD, sC.
[0147] To direct an enzyme of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
may be provided in the recombinant vector. The secretory signal
sequence is joined to the DNA sequence encoding the enzyme in the
correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the enzyme, The
secretory signal sequence may be that normally associated with the
enzyme or may be from a gene encoding another secreted protein.
[0148] For secretion from yeast cells, the secretory signal
sequence may encode any signal peptide which ensures efficient
direction of the expressed enzyme into the secretory pathway of the
cell. The signal peptide may be a naturally occurring signal
peptide, or a functional part thereof, or it may be a synthetic
peptide. Suitable signal peptides have been found to be the
alpha-factor signal peptide (cf. U.S. Pat. No. 4,870,008), the
signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et
al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase
signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp.
887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the
yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani
et al., Yeast 6, 1990, pp. 127-137).
[0149] For efficient secretion in yeast, a sequence encoding a
leader peptide may also be inserted downstream of the signal
sequence and upstream of the DNA sequence encoding the enzyme, The
function of the leader peptide is to allow the expressed enzyme to
be directed from the endoplasmic reticulum to the Golgi apparatus
and further to a secretory vesicle for secretion into the culture
medium (i.e., exportation of the enzyme across the cell wall or at
least through the cellular membrane into the periplasmic space of
the yeast cell). The leader peptide may be the yeast alpha-factor
leader (the use of which is described in, e.g., U.S. Pat. No.
4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529).
Alternatively, the leader peptide may be a synthetic leader
peptide, which is to say a leader peptide not found in nature.
Synthetic leader peptides may, for instance, be constructed as
described in WO 89102463 or WO 92/11378.
[0150] For use in filamentous fungi, the signal peptide may
conveniently be derived from a gene encoding an Aspergillus sp.
amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase
or protease, a Humicola lanuginosa lipase. The signal peptide is
preferably derived from a gene encoding A. oryzae TAKA amylase, A.
niger neutral alpha-amylase, A. niger acid-stable amylase, or A.
niger glucoamylase.
[0151] The procedures used to ligate the DNA sequences coding for
the present enzyme, the promoter and optionally the terminator
and/or secretory signal sequence, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., cit.).
Host Cells
[0152] The DNA sequence encoding the present enzyme introduced into
the host cell may be either homologous or heterologous to the host
in question. If homologous to the host cell, i.e., produced by the
host cell in nature, it will typically be operably connected to
another promoter sequence or, if applicable, another secretory
signal sequence and/or terminator sequence than in its natural
environment. The term "homologous" is intended to include a cDNA
sequence encoding an enzyme native to the host organism in
question. The term "heterologous" is intended to include a DNA
sequence not expressed by the host cell in nature. Thus, the DNA
sequence may be from another organism, or it may be a synthetic
sequence.
[0153] The host cell into which the DNA construct or the
recombinant vector of the invention is introduced may be any cell
which is capable of producing the present enzyme and includes
bacteria, yeast, fungi and higher eukaryotic cells.
[0154] Examples of bacterial host cells which, on cultivation, are
capable of producing the enzyme of the invention are gram-positive
bacteria such as strains of Bacillus, such as strains of B.
subtilis, B. licheniformis, B. lentus, B. brevis, B.
stearothermophilus, B. alkalophilus, B. amyloliquefaciens. B.
coagulans, B. circulans, B. lautus, B. megatherium or B.
thuringiensis, or strains of Streptomyces, such as S. lividans or
S. murinus, or gram-negative bacteria such as Echerichia coli. The
transformation of the bacteria may be effected by protoplast
transformation or by using competent cells in a manner known per se
(cf. Sambrook et al., supra).
[0155] When expressing the enzyme in bacteria such as E. coli, the
enzyme may be retained in the cytoplasm, typically as insoluble
granules (known as inclusion bodies), or may be directed to the
periplasmic space by a bacterial secretion sequence. In the former
case, the cells are lysed and the granules are recovered and
denatured after which the enzyme is refolded by diluting the
denaturing agent. In the latter case, the enzyme may be recovered
from the periplasmic space by disrupting the cells, e.g., by
sonication or osmotic shock, to release the contents of the
periplasmic space and recovering the enzyme.
[0156] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosacharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous enzymes therefrom are described, e.g., in
U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos.
4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are
hereby incorporated by reference. Transformed cells are selected by
a phenotype determined by a selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient, e.g., leucine. A preferred vector for use in yeast is the
POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequence
encoding the enzyme of the invention may be preceded by a signal
sequence and optionally a leader sequence, e.g., as described
above. Further examples of suitable yeast cells are strains of
Kluyveromyces such as K. lactis, Hansenula, e.g., H. polymorpha, or
Pichia, e.g., P. pastoris (cf. Gleeson et al., S. Gen. Microbiol.
132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).
[0157] Examples of other fungal cells are cells of filamentous
fungi, e.g., Aspergillus spp., Neurospora spp., Fusarium spp. or
Trichoderma spp., in particular strains of A. oryzae, A. nidulans,
A. niger, or Fusarium graminearum. The use of Aspergillus spp. for
the expression of proteins is described in, e.g., EP 272 277, ER
230 023. The transformation of F. oxysporum may, for instance, be
carried out as described by Malardier et al., 1989, Gene 78;
147-156.
[0158] When a filamentous fungus is used as the host cell, it may
be transformed with the DNA construct of the invention,
conveniently by integrating the DNA construct in the host
chromosome to obtain a recombinant host cell. This integration is
generally considered to be an advantage as the to DNA sequence is
more likely to be stably maintained in the cell. Integration of the
DNA constructs into the host chromosome may be performed according
to conventional methods, e.g., by homologous or heterologous
recombination.
[0159] The transformed or transfected host cell described above is
then cultured in a suitable nutrient medium under conditions
permitting the expression of the present enzyme, after which the
resulting enzyme is recovered from the culture.
[0160] The medium used to culture the cells may be any conventional
medium suitable for growing the host cells, such as minimal or
complex media containing appropriate supplements. Suitable media
are available from commercial suppliers or may be prepared
according to published recipes (e.g., in catalogues of the American
Type Culture Collection). The enzyme produced by the cells may then
be recovered from the culture medium by conventional procedures
including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.,
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g., ion exchange chromatography, ge filtration
chromatography, affinity chromatography, or the like, dependent on
the type of enzyme in question.
[0161] In a still further aspect, the present invention relates to
a method of producing an enzyme according to the invention, wherein
a suitable host cell transformed with a DNA sequence encoding the
enzyme is cultured under conditions permitting the production of
the enzyme, and the resulting enzyme is recovered from the
culture.
Enzyme Screening Driven by Taxonomy as Well as Ecology,
[0162] A powerful tool like the molecular screening disclosed
herein, designed to detect and select said type of interesting
enzymes, can still not stand on its own. In order to maximize the
chances of making interesting discoveries the molecular screening
approach was in the present investigation combined with careful
selection of which fungi to screen. The selection was done through
a thorough insight in the identification of fungi, in taxonomical
classification and in phylogenetic relationships.
[0163] A taxonomic hot spot for production of cellulytic enzymes
can further only be fully explored if also the ecological approach
is included. Thorough knowledge about the adaptation to various
substrates (especially saprotrophic, necrotrophic or biotrophic
degradation of plant materials) are prerequisites for designing an
intelligent screening and for managing a successful selection of
strains and ecological niches to be searched.
[0164] Both the taxonomy and the ecological approach disclosed
herein aim at maximizing discovery of said enzymes in the molecular
screening program. However, still several hundreds (or if all
preliminary work is included) several thousand fungi have been
brought in culture in order to detect the 53 hits of said type of
cellulytic enzyme here reported.
[0165] The screening and cloning may be carried out using the
following:
Materials and Methods
List of Organisms:
[0166] Saccharomyces cerevisiae, DSM 9770, DSM 10082, DSM 10080,
DSM 10081, or Escherichia coli, DSM 10512, DSM 10511, DSM 10571,
DSM 10576, respectively, containing the plasmid comprising the full
length DNA sequence, coding for the endoglucanase of the invention,
in the shuttle vector pYES 2.0.
[0167] Escherichia coli DSM 10583, 10584, 10585, 10586, 10587, and
10588.
[0168] Diplcodia gossypina Cooke
Deposit of Strain, Acc No: CBS 274.96
Classification: Ascomycota, Loculoascomycetes, Dothideales,
Cucurbitariaceae
[0169] Ulospora bilgramii (Hawksw. et at) Hawksw. et al.,
Acc No of strain: NKBC 1444, Nippon University, (Prof. Tubaki
collection) Classification: Ascomycota, Loculoascomycetes,
Dothideales, (family unclassified)
[0170] Microsphaeropsis sp.
Isolated from: Leaf of Camellia japonica (Theaceae, Guttiferales),
grown in Kunming Botanical garden, Yunnan Province, China
Classification: Ascomycota, Loculoascomycetes, Dothideales, (family
unclassified)
[0171] Macrophomina phaseolina (Tassi) Goidannich
Syn: Rhizoctonia bataticola
Deposit of Strain, Acc No.: CBS 281.96
[0172] Isolated from seed of Glycine max (Leguminosa), cv CMM 60,
grown in Thailand, 1990
Classification: Ascomycota, Discomycetes, Rhytismatales,
Rhytismataceae
[0173] Ascobolus stictoideus Speg.
Isolated from goose dung, Svalbard, Norway
Classification: Ascomycota, Discomycetes, Pezizales,
Ascobolaceae
[0174] Saccobolus dilutellus (Fuck.) Sacc.
Deposit of strain: Acc No CBS 275.96
Classification: Ascomycota, Discomycetes, Pezizales,
Ascobolaceae
[0175] Penicillium verruculosum Peyronel
Ex on Acc No of species. ATCC 62396
Classification, Ascomycota, Plectomycetes, Eurotiales,
Trichocomaceae
[0176] Penicillium chrysogenum Thom
Acc No of Strain: ATCC 9480
Classification: Ascomycota, Plectomycetes, Eurotiales,
Trichocomaceae
[0177] Thermomyces verrucosus Pugh et al
Deposit of Strain, Acc No.: CBS 285.96
[0178] Classification: Ascomycota, Plectomycetes, Eurotiales,
(family unclassified affiliation based on 18S RNA, sequencing and
homologies)
[0179] Xylaria hypoxylon L. ex Greville
Deposit of Strain, Acc No: CBS 284.96
Classification Ascomycota, Pyrenomycetes, Xylariales,
Xylariaceae
[0180] Poronia punctata (Fr. ex L.) Fr,
Classification: Ascomycota, Pyrenomycetes, Xylariales,
Xylariaceae
[0181] Nodulisporum sp
Isolated from leaf of Camellia reticulata (Theaceae, Guttiferales),
grown in Kunming Botanical Garden, Yunnan Province, China
Classification, Ascomycota, Pyrenomycetes, Xylariales,
Xylariaceae
[0182] Cylindrocarpon sp
Isolated from marine sample, the Bahamas Classification,
Ascomycota, Pyrenomycetes., Hypocreales (unclassified)
[0183] Acremonium sp
Deposit of Strain, Acc. No.: CBS 478.94
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0184] Fusarium anguioides Sherbakoff
Acc No of strain: IFO 4467
Classification Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0185] Fusarium poae (Peck) Wr.
Ex on Acc No of species: ATCC 60883
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0186] Fusarium solani (Mart.)Sacc.emnd.Snyd & Hans.
Acc No of strain: IMI 107.511
Classification, Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0187] Fusarium oxysporum ssp lycopersici (Sacc.)Snyd. &
Hans.
Acc No of strain: CBS 645.78
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0188] Fusarium oxysporum ssp passiflora
Acc No of strain: CBS 744.79
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0189] Gliocladium catenulatum Gilman & Abbott
Acc. No. of strain: CBS 227.48
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Hypocreaceae
[0190] Nectria pinea Dingley
Deposit of Strain. Acc. No. CBS 279.96
Classification: Ascomycota, Pyrenomycetes, Hypocreales,
Nectriaceae
[0191] Volutella colletotrichoides
Acc No of Strain: CBS 400.58
[0192] Classification: Ascomycota, Pyrenomycetes, Hypocreales
(unclassified)
[0193] Sordaria macrospora Auerswald
Ex on Acc No of species: ATCC 60255
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Sordariaceae
[0194] Sordaria fimicola (Roberge) Cesati et De Notaris
Ex on Acc. No. for the species: ATCC 52644 Isolated from dung by H.
Dissing, ISP, KU, Denmark
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Sordariaceae
[0195] Humicola grisea Traeen
ex on Acc No for the species: ATCC 22726
Source: Hatfield Polytechnic
[0196] Classification: Ascomycota, Pyrenomycetes, Sordariales,
(fam. unclassified)
[0197] Humicola nigrescens Omvik
Acc No of strain: CBS 819.73 Classification: Ascomycota,
Pyrenomycetes, Sordariales, (fam. unclassified)
[0198] Scytalidium thermophilum (Cooney et Emerson) Aushtick
Acc No of strain: ATCC 28085 Classification: Ascomycota,
Pyrenomycetes, Sordariales, (fam. unclassified)
[0199] Thielavia thermophila Fergus et Sinden
(syn Corynascus thermophilus) Acc No of strain: CBS 174.70, IMI
145.136
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0200] Isolated from Mushroom compost
[0201] Thielavia terrestris (Appinis) Malloch et Cain
Acc No of strain: NRRL8126
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0202] Cladorrhinum foecundissimum Saccardo et Marchal
Ex on Acc No of species, ATCC 62373
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Lasiosphaeriaceae
[0203] Isolated from leaf of Selandin sp, (Compositaceae,
Asterales), Dallas Mountain, Jamaica
[0204] Syspastospora boninensis
Acc No of strain: NKBC 1515 (Nippon University, profe Tubaki
Collection)
Classification, Ascomycota, Pyrenomycetes, Sordariales,
Cerastomataceae
[0205] Chaetomium cuniculorum Fuckel
Acc. No. of strain: CBS 799.83
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0206] Chaetomium brasiliense Batista et Potual
Acc No of strain: CBS 122.65
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0207] Chaetomium murorum Corda
Acc No of strain: CBS 163.52
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0208] Chaetomium virescens (von Arx) Udagawa
Acc. No. of strain: CBS 547.75
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0209] Myceliophthora thermophila (Apinis) Oorschot
Deposit of Strain, Acc No: CBS 117.65
Classification: Ascomycota, Pyrenomycetes, Sordariales,
Chaetomiaceae
[0210] Nigrospora sp
Deposit of strain, Acc No: CBS 272.96 Isolated from leaf of
Artocarpus altilis Moraceae, Urticales grown in Christiana, Jamaica
Classification: Ascomycota, Pyrenomycetes, Trichosphaeriales,
(family unclassified)
[0211] Nigrospora sp
Isolated from leaf of Pinus yuannanensis, Botanical Garden, Kuning,
Yunnan,
Classification: Ascomycota, Pyrenomycetes, Trichosphaeriales,
Abietaceae, Pinales.
[0212] Diaporthe syngenesia
Deposit of strain, Acc No: CBS 278.96
Classification: Ascomycota, Pyrenomycetes, Diaporthales,
Valsaceae
[0213] Colletotrichum lagenarium (Passerini) Ellis et Halsted
syn Glomerella cingulata var orbiculare Jenkins et Winstead Ex on
acc No of species: ATCC 52609
Classification, Ascomycota, Pyrenomycetes, Phyllachorales
[0214] Exidia glandulosa Fr.
Deposit of Strain. Acc No: CBS 27796
Classification: Basidiomycota, Hymenormycetes, Auriculariales,
Exidiaceae
[0215] Crinipellis scabella (Alb. & Schw.; Fr)Murr
Deposit of strain: Acc No CBS 280.96
Classification: Basidiomycota, Hymenomycetes, Agaricales,
[0216] Panaeolus retirgis (Fr.) Gill.
Acc. No. of strain: CBS 275.47
Classification: Basidiomycota, Hymenomycetes, Agaricales,
Coprinaesae
[0217] Fomes fomentarius (L.) Fr,
Deposit of strain: Acc No. CBS 276.96
Classification: Basidiomycota, Hymenomycetes, Aphyllophorales,
Fomitaceae
[0218] Spongipellis Sp.
Deposit of Strain: Acc No CBS 283.96
Classification: Basidiomycota, Hymenomycetes, Aphyllophorales,
[0219] Bjerkanderaceae (identified and affiliated taxonomically by
18S sequence and homology)
[0220] Trametes sanguinea (Fr.) Lloyd
syn: Polyporus sanguineus; Pycnoporus sanguineus (L.:Fr.) Murril
Acc No of strain: AKU 5062 (Kyoto University Culture
Collection)
Classification, Basidiomycota, Aphyllophorales, Polyporaceae
[0221] Schizophyllum commune Fr
Acc. No. of species: ATCC 38548
Classification: Basidiomycota, Aphyllophorales,
Schizophyllaceae
[0222] Rhizophlyctis rosea (de Bary & Wor) Fischer
Deposit of Strain: Acc No.: CBS 282.96
Classification: Chytridiomycota, Chytridiomycetes,
Spizellomycetales, Spizellomycetaceae
[0223] Rhizomucor pusillus (Lindt) Schipper
syn: Mucor pusillus Acc No of strain: IFO 4578 Ex on Acc No of
species: ATCC 46883
Classification Zygomycota, Zygomycetes, Mucorales, Mucoraceae
[0224] Phycomyces nitens (Kunze) van Tieghem & Le Monnier
Acc No of strain: IFO 4814 Ex on Aco No of species: ATCC 16327
Classification; Zygomycota, Zygomycetes, Mucorales, Mucoraceae
[0225] Chaetostylum fresenii van Tieghem & Le Monnier
syn. Helicostylum fresenii Acc No of strain NRRL 2305
Classification: Zygomycota, Zygomycetes, Mucorales,
Thamnidiaceae
Unclassified:
[0226] Trichothecium roseum
Acc No of strain: IFO 5372
[0227] Coniothecium sp
Endophyte, isolated from leaf of unidentifed higher plant, growing
in Kunming, Yunnan, China
Unclassified and Un-identified:
[0228] Deposit of strain, Acc No.: CBS 271.96
Isolated from leaf of Artocarpus altillis (Moraceae, Urticales),
grown in Christiana, Jamaica
[0229] Deposit of strain, Acc No.; CBS 273.96
Isolated from leaf of Pimenta dioica (Myrtaceae, Myrtales) grown in
Dallas Mountain, Jamaica
[0230] Deposit of strain: CBS 270.96
Isolated from leaf of Pseudocalymma alliaceum (Bignoniaceae,
Solanales) growing in Dallas Mountain, Jamaica Other strains:
[0231] Escherichia coli MC1061 and DH10B.
[0232] Yeast strain: The Saccharomyces cerevisiae strain used was
W3124 (MAT.alpha.; ura 3-52, leu 2-3, 112, h is 3-D200; pep 4-1137;
prc1; HIS3; prb2: LEU2; cir+).
Plasmids:
[0233] The Aspergillus expression vector pH D414 is a derivative of
the plasmid p775 (described in EP 238 023). The construction of
pHD414 is further described in WO 93/11249.
pYES 2.0 (Invitrogen) pA2C477, pA2C193, pA2C357, pA2C371, pA2C385,
pA2C475, pA2C488, pA2C502 (See example 1, 2, 3 and 4),
Isolation of the DNA Sequence of SEQ ID NO: 1, 7, 9, 1113, 15, 21,
or 25, Respectively:
[0234] The full length DNA sequence, comprising the cDNA sequence
of SEC ID NO: 1, 7, 9, 11, 13, 15, 21, or 25, respectively, coding
for the endoglucanase of the invention, can be obtained from the
deposited organism S. cerevisiae, DSM 9770, DSM 10082, DSM 10080,
DSM 10081, E. coli, DSM 10512, DSM 10511, DSM 10571 or DSM 10576,
respectively, by extraction of plasmid DNA by methods known in the
art (Sambrook et al. (1989) Molecular cloning: A laboratory manual,
Cold Spring Harbor lab., Cold Spring Harbor, N.Y.).
PCR primers for molecular screening of cellulases of the present
invention:
[0235] The four degenerate, deoxyinosine-containing oligonucleotide
primers (sense; s and antisense; as1, as2 and as3) corresponding to
four highly conserved amino acid regions found in the deduced amino
acid sequences of Thielavia terrestris cellulase, Myceliophthora
thermophilum cellulase, and two cellulases from Acremonium sp. The
residues are numbered according to the Myceliophthora thermophilum
sequence. The deoxyinosines are depicted by an I in the primer
sequences, and the restriction sites are underlined.
TABLE-US-00021 27 35 NH.sub.2- Thr Arg Tyr Trp Asp Cys Cys Lys
Pro/Thr -COOH (SEQ ID NO: 79) S 5'- CCCCAAGCCTT ACI AGI TAC TGG GAC
TGC TGC AAA AC -3' (SEQ ID NO: 84) HindIII C T T T T G C 106 111
NH.sub.2- Trp Cys Cys Ala Cys Tyr -COOH (SEQ ID NO: 81) as1 3'- CC
ACA ACA CGI ACA AT AGATCTGATC -5' (SEQ ID NO: 85) G G G XbaI 145
152 NH.sub.2- Pro Gly Gly Gly Leu/Val Gly Ile/Leu Phe -COOH (SEQ ID
NO: 82) as2 3'- GGI CCI CCI CCI AAI CCI AAI AA AGATCTGATC -5' (SEQ
ID NO: 86) C G G T 193 198 NH.sub.2- Trp Arg Phe/Tyr Asp Trp Phe
-COOH (SEQ ID NO: 83) as3 3'- CC CGI AAA CTA ACC AAA AGATCTGATC -5'
(SEQ ID NO: 87) T TG G G XbaI
Molecular Screening by Polymerase Chain Reaction (PCR):
[0236] In Vitro Amplification of Genomic DNA and Double-Stranded
cDNA.
[0237] Directional, double-stranded cDNA was synthesized from 5
micrograms of poly(A)+ RNA as described below. Genomic DNA was
isolated according to Yelton et al.
[0238] Approximately 10 to 20 ng of double-stranded,
cellulase-induced cDNA or 100 to 200 ng of genomic DNA from a
selection of fungal strains was PCR amplified in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% (w/v)
gelatin) containing 200 micro-M of each dNTP and 100 pmol of each
degenerate primer in three combinations,
TABLE-US-00022 1) sense, (SEQ ID NO: 84)
5'-CCCCAAGCTTACI.sup.A/.sub.CGITA.sup.C/.sub.TTGGGA.sup.C/.sub.TTG.sup.C/.-
sub.TTG.sup.C/.sub.TAA.sup.A/.sub.G .sup.A/.sub.CC-3' antisense 1,
(SEQ ID NO: 85)
5'-CTAGTCTAGATA.sup.A/.sub.GCAIGC.sup.A/.sub.GCA.sup.A/.sub.GCACC-3';
or 2) sense, (SEQ ID NO: 84)
5'-CCCCAAGCTTACI.sup.A/.sub.CGITA.sup.C/.sub.TTGGGA.sup.C/.sub.TTG.sup.C/.-
sub.TTG.sup.C/.sub.TAA.sup.A/.sub.G .sup.A/.sub.CC-3' antisense 2,
(SEQ ID NO: 86)
CTAGTCTAGAAAIA.sup.A/.sub.G/.sup.TICCIA.sup.A/.sup.C/.sup.GICCICCICCIGG-3'-
; or 3) sense, (SEQ ID NO: 84)
5'-CCCCAAGCTTACI.sup.A/.sub.CGITA.sup.C/.sub.TTGGGA.sup.C/.sub.TTG.sup.C/.-
sub.TTG.sup.C/.sub.TAA.sup.A/.sub.G .sup.A/.sub.CC-3' antisense 3,
(SEQ ID NO: 87)
5'-CTAGTCTAGAIAACCA.sup.A/.sub.GTCA.sup.A/.sub.G.sup.A/.sub.TAIC.sup.G/.su-
b.TCC-3;
a DNA thermal cycler (Landgraf, Germany) and 2.5 units of Taq
polymerase (Perkin-Elmer, Cetus, USA). Thirty cycles of PCR were
performed using a cycle profile of denaturation at 94.degree. C.
for 1 min, annealing at 64.degree. C. for 2 min, and extension at
72.degree. C. for 3 min. Ten microliter aliquots of the
amplification products were analyzed by electrophoresis in 3%
agarose gels (NuSieve, FMC) with HaeIII-digested .phi.174 RF DNA as
a size marker.
Direct Sequencing of the PCR Products.
[0239] Eighty microliter aliquots of the PCR products were purified
using the QIAquick PCR purification kit (Qiagen, USA) according to
the manufacturers instructions. The nucleotide sequences of the
amplified PCR fragments were determined directly on the purified
PCR products by the dideoxy chain-termination method, using 50-150
ng template, the Taq deoxy-terminal cycle sequencing kit
(Perkin-Elmer, USA), fluorescent labeled terminators and 5 pmol of
the sense primer:
5'-CCCCAAGCTTACl.sup.A/.sub.CGITA.sup.C/.sub.TTGGGA.sup.C/.sub.TT-G.sup.C-
/.sub.TTG.sup.C/.sub.TAA.sup.A/.sub.G.sup.A/.sub.CC-3' (SEQ ID NO:
84). Analysis of the sequence data was performed according to
Devereux at alt
Cloning by Polymerase Chain Reaction (PCR):
Subcloning of PCR Fragments.
[0240] Twenty five microliter aliquots of the PCR products
generated as described above were electrophoresed in 0.8% low
gelling temperature agarose (SeaPlaque GTG, FMC) gels, the relevant
fragments were excised from the gels, and recovered by agarase
treatment by adding 0.1 vol of 10.times. agarase buffer (New
England Biolabs) and 2 units per 100 microliters molten agarose to
the sample, followed by incubation at 45.degree. C. for 1.5 h. The
sample was phenol and chloroform extracted, and precipitated by
addition of 2 vols of 96% EtOH and 0.1 of 3 M NaAc, pH 5.2. The PCP
fragments were recovered by centrifugation, washed in 70% EtOH,
dried and resuspended in 20 microliters of restriction enzyme
buffer (10 mM Tris-HCl, 10 mM MgCl.sub.2, 50 mM NaCl, 1 mM DTT).
The fragments were digested with HindIII and XbaI, phenol and
chloroform extracted, recovered by precipitation with 2 vols of 96%
EtOH and 0.1 of 3 M NaAc, pH 5.2, and subcloned into
HindIII/XbaI-cleaved pYES 2.0 vector.
Screening of cDNA Libraries and Characterization of the Positive
Clones
[0241] cDNA libraries in S. cerevisiae or E. coli, constructed as
described below, were screened by colony hybridization (Sambrook,
1989) using the corresponding random-primed (Feinberg and
Vogelstein) .sup.32P-labeled (>1.times.10.sup.9 cpm/microgram)
PCR products as probes. The hybridizations were carried out in
2.times.SSC (Sambrook, 1989), 5.times.Denhardt's solution
(Sambrook, 1989), 0.5% (w/v) SDS, 100 micrograms/ml denatured
salmon sperm DNA for 20 h at 65.degree. C. followed by washes in
5.times.SSC at 25.degree. C. (2.times.15 min), 2.times.SSC 0.5% SDS
at 65.degree. C. (30 min), 0.2.times.SSC 0.5% SDS at 65.degree. C.
(30 min) and finally in 5.times.SSC (2.times.15 min) at 25.degree.
C. The positive cDNA clones were characterized by sequencing the
ends of the cDNA inserts with pYES 2.0 polylinker primers
(Invitrogen, USA), and by determining the nucleotide sequence of
the longest cDNA from both strands by the dideoxy chain termination
method (Sanger et al.) using fluorescent labeled terminators.
Qiagen purified plasmid DNA (Qiagen, USA) was sequenced with the
Taq deoxy terminal cycle sequencing kit (Perkin Elmer, USA) and
either pYES 2.0 polylinker primers (Invitrogen, USA) or synthetic
oligonucleotide primers using an Applied Biosystems 373A automated
sequencer according to the manufacturer's instructions. Analysis of
the sequence data was performed according to Devereux et al.
[0242] Extraction of total RNA was performed with guanidinium
thiocyanate followed by ultracentrifugation through a 5.7 M CsCl
cushion, and isolation of poly(A).sup.+ RNA was carried out by
oligo(dT)-cellulose affinity chromatography using the procedures
described in WO 94/14953.
cDNA Synthesis
[0243] Double-stranded cDNA was synthesized from 5 micrograms
poly(A).sup.+ RNA by the RNase H method (Gubler and Hoffman Gene
25:263-269 (1983), Sambrook et al. (1939) Molecular cloning: A
laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor,
N.Y.) using the hair-pin modification developed by F. S. Hagen
(pers. comm.). The poly(A).sup.+ RNA (5 micrograms in 5 microliters
of DEPC-treated water) was heated at 70.degree. C. for 8 min. in a
pre-siliconized, RNase-free Eppendorph tube, quenched on ice and
combined in a final volume of 50 microliters with reverse
transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM
MgCl.sub.2, 10 mM DTT, Bethesda Research Laboratories) containing 1
mM of dATP, dGTP and dTTP and 0.5 mM 5-methyl-dCTP (Pharmacia), 40
units human placental ribonuclease inhibitor (RNasin, Promega),
1.45 micrograms of oligo(dT).sub.18-Not I primer (Pharmacia) and
1000 units SuperScript II RNase H reverse transcriptase (Bethesda
Research Laboratories). First-strand cDNA was synthesized by
incubating the reaction mixture at 45.degree. C. for 1 hour. After
synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a
MicroSpin S-400 HR (Pharmadia) spin column according to the
manufacturer's instructions.
[0244] After the gelfiltration, the hybrids were diluted in 250
microliters second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl,
4.6 mM MgCl.sub.2, 10 mM (NH.sub.4).sub.2SO.sub.4, 0.16 mM
.beta.NAD+) containing 200 micro-M of each dNTP, 60 units E. coli
DNA polymerase I (Pharmacia), 5.25 units RNase H (Promega) and 15
units E. coli DNA ligase (Boehringer Mannheim). Second strand cDNA
synthesis was performed by incubating the reaction tube at
16.degree. C. for 2 hours and additional 15 min. at 25.degree. C.
The reaction was stopped by addition of EDTA to a final
concentration of 20 mM followed by phenol x's and chloroform
extractions.
Mung Bean Nuclease Treatment
[0245] The double-stranded cDNA was precipitated at -20.degree. C.
for 12 hours by addition of 2 vols 96% EtOH, 0.2 vol 10 M
NH.sub.4Ac, recovered by centrifugation, washed in 70% EtOH, dried
and resuspended in 30 microliters Mung bean nuclease buffer (30 mM
NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO.sub.4, 0.35 mM DTT, 2%
glycerol) containing 25 units Mung bean nuclease (Pharmacia). The
single-stranded hair-pin DNA was clipped by incubating the reaction
at 30.degree. C. for 30 min., followed by addition of 70
microliters 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction and
precipitation with 2 vols of 96% EtOH and 0.1 vol 3 M NaAc, pH 5.2
on ice for 30 min.
Blunt-Ending with T4 DNA Polymerase
[0246] The double-stranded cDNAs were recovered by centrifugation
and blunt-ended in 30 microliters T4 DNA polymerase buffer (20 mM
Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing
0.5 mM of each dNTP and 5 units T4 DNA polymerase (New England
Biolabs) by incubating the reaction mixture at 16.degree. C. for 1
hour. The reaction was stopped by addition of EDTA to a final
concentration of 20 mM, followed by phenol and chloroform
extractions, and precipitation for 12 hours at -20.degree. C. by
adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.
Adaptor Ligation, Not I Digestion and Size Selection:
[0247] After the fill-in reaction the cDNAs were recovered by
centrifugation, washed in 70% EtOH and dried. The cDNA pellet was
resuspended in 25 microliters ligation buffer (30 mM Tris-Cl, pH
7.8, 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM ATP) containing 2.5
micrograms non-palindromic BstXI adaptors (invitrogen) and 30 units
T4 ligase (Promega) and incubated at 15.degree. C. for 12 hours.
The reaction was stopped by heating at 65.degree. C. for 20 min.
and then cooling on ice for 5 min. The adapted cDNA was digested
with Not I restriction enzyme by addition of 20 microliters water,
5 microliters 10.times. Not I restriction enzyme buffer (New
England Biolabs) and 50 units Not I (New England Biolabs), followed
by incubation for 2.5 hours at 37.degree. C. The reaction was
stopped by heating at 65.degree. C. for 10 min. The cDNAs were
size-fractionated by gel electrophoresis on a 0.8% SeaPlaque GTG
low melting temperature agarose gel (FMC) in 1.times.TBE to
separate unligated adaptors and small cDNAs. The cDNA was
size-selected with a cut-off at 0.7 kb and rescued from the gel by
use of beta-Agarase (New England Biolabs) according to the
manufacturer's instructions and precipitated for 12 hours at
-20.degree. C. by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH
5.2.
Construction of Libraries
[0248] The directional, size-selected cDNA was recovered by
centrifugation, washed in 70% EtOH, dried and resuspended in 30
microliters 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. The cDNAs were
desalted by gelfiltration through a MicroSpin S-300 HR (Pharmacia)
spin column according to the manufacturers instructions. Three test
ligations were carried out in 10 microliters ligation buffer (30 mM
Tris-Cl, pH 7.8, 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM ATP)
containing 5 microliters double-stranded cDNA (reaction tubes #1
and #2), 15 units T4 ligase (Promega) and 30 ng (tube #1), 40 ng
(tube #2) and 40 ng (tube #3, the vector background control) of
BstXI-NotI cleaved pYES 2.0 vector. The ligation reactions were
performed by incubation at 16.degree. C. for 12 hours, heating at
70.degree. C. for 20 min. and addition of 10 microliters water to
each tube. One microliter of each ligation mixture was
electroporated into 40 microliters electrocompetent E. coli DH10B
cells (Bethesda research Laboratories) as described (Sambrook et
al. (1989) Molecular cloning: A laboratory manual, Cold Spring
Harbor lab., Cold Spring Harbor, N.Y.). Using the optimal
conditions a library was established in E. coli consisting of
pools. Each pool was made by spreading transformed E. coli on
LB+ampicillin agar plates giving 15.000-30.000 colonies/plate after
incubation at 37.degree. C. for 24 hours. 20 ml LB+ampicillin was
added to the plate and the cells were suspended herein. The cell
suspension was shaked in a 50 ml tube for 1 hour at 37.degree. C.
Plasmid DNA was isolated from the cells according to the
manufacturer's instructions using QIAGEN plasmid kit and stored at
-20.degree. C.
[0249] One microliter aliquots of purified plasmid DNA (100
ng/microliter) from individual pools were transformed into S.
cerevisiae W3124 by electroporation (Becker and Guarante, Methods
Enzymol. 194: 182-187 (1991)) and the transformants were plated on
SC agar containing 2% glucose and incubated at 30.degree. C.
Identification of Positive Colonies
[0250] After 3-5 days of growth, the agar plates were replica
plated onto a set of SC+galactose-uracil agar plates containing
0.1% AZCL HE cellulose. These plates were incubated for 3-7 days at
30.degree. C. Endoglucanase positive colonies were identified as
colonies surrounded by a blue halo.
[0251] Cells from enzyme-positive colonies were spread for single
colony isolation on agar, and an enzyme-producing single colony was
selected for each of the endoglucanase-producing colonies
identified.
Characterization of Positive Clones
[0252] The positive clones were obtained as single colonies, the
cDNA inserts were amplified directly from the yeast colony using
biotinylated polylinker primers, purified by magnetic beads
(Dynabead M-280, Dynal) system and characterized individually by
sequencing the 5'-end of each cDNA clone using the
chain-termination method (Sanger et al. (1977) Proc. Natl. Acad.
Sci. U.S.A. 74: 5463-5467) and the Sequence system (United States
Biochemical).
[0253] The nucleotide sequence was determined of the longest cDNA
from both strands by the dideoxy chain termination method (Sanger
et al.) using fluorescent labeled terminators. Plasmid DNA was
rescued by transformation into E. coli as described below. Qiagen
purified plasmid DNA (Qiagen, USA) was sequenced with the Taq deoxy
terminal cycle sequencing kit (Perkin Elmer, USA) and either pYES
2.0 polylinker primers (Invitrogen, USA) or synthetic
oligonucleotide primers using an Applied Biosystems 373A automated
sequencer according to the manufacturer's instructions. Analysis of
the sequence data was performed according to Devereux et at
Isolation of a cDNA Gene for Expression in Aspergillus
[0254] An endoglucanase-producing yeast colony was inoculated into
20 ml YPD broth in a 50 ml grass test tube. The tube was shaken for
2 days at 30.degree. C. The cells were harvested by centrifugation
for 10 min. at 3000 rpm.
[0255] DNA was isolated according to WO 94/14953 and dissolved in
50 microliters water. The DNA was transformed into E. coli by
standard procedures. Plasmid DNA was isolated from E. coli using
standard procedures, and analyzed by restriction enzyme analysis.
The cDNA insert was excised using appropriate restriction enzymes
and ligated into an Aspergillus expression vector.
Transformation of Aspergillus oryzae or Aspergillus Niger
[0256] Protoplasts may be prepared as described in WO 95/02043, p.
16, line 21-page 17, line 12, which is hereby incorporated by
reference.
[0257] 100 microliters of protoplast suspension is mixed with 5-25
micrograms of the appropriate DNA in 10 microliters of STC (1.2 M
sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl.sub.2. Protoplasts are
mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid). The
mixture is left at room temperature for 25 minutes. 0.2 ml of 60%
PEG 4000 (BDH 29576): 10 mM CaCl.sub.2 and 10 mM Tris-HCl, pH 7.5
is added and carefully mixed (twice) and finally 0.85 ml of the
same solution is added and carefully mixed. The mixture is left at
room temperature for 25 minutes, spun at 2500 g for 15 minutes and
the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more
sedimentation the protoplasts are spread on minimal plates (Cove,
Biochem. Biophys. Acta 113: 51-56 (1966)) containing 1.0 M sucrose,
pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to
inhibit background growth. After incubation for 4-7 days at
37.degree. C. spores are picked and spread for single colonies.
This procedure is repeated and spores of a single colony after the
second resolution is stored as a defined transformant.
Test of A. oryzae Transformants
[0258] Each of the transformants were inoculated in 10 ml YPM and
propagated. After 2-5 days of incubation at 37.degree. C., 10 ml
supernatant was removed. The endoglucanase activity was identified
by AZCL HE cellulose as described above.
Hybridization conditions (to be used in evaluating property ii) of
the DNA construct of the invention): Suitable conditions for
determining hybridization between a nucleotide probe and a
homologous DNA or RNA sequence involves presoaking of the filter
containing the DNA fragments or RNA to hybridize in 5.times.SSC
(standard saline citrate) for 10 min, and prehybridization of the
filter in a solution of 5.times.SSC (Sambrook et al. 1989),
5.times.Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and
100 micrograms/ml of denatured sonicated salmon sperm DNA (Sambrook
et al. 1989), followed by hybridization in the same solution
containing a random-primed (Feinberg, A. P. and Vogelstein, B.,
Anal, Biochem. 132:6-13 (1983)), .sup.32P-dCTP-labeled (specific
activity >1.times.10.sup.9 cpm/microgram) probe for 12 hours at
ca. 45.degree. C. The filter is then washed two times for 30
minutes in 2.times.SSC, 0.5% SDS at preferably not higher than
50.degree. C., more preferably not higher than 55.degree. C., more
preferably not higher than 60.degree. C., more preferably not
higher than 65.degree. C., even more preferably not higher than
70.degree. C., especially not higher than 75.degree. C.
[0259] The nucleotide probe to be used in the hybridization is the
DNA sequence corresponding to the endoglucanase encoding part of
the DNA sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 21 or 25,
respectively, and/or the DNA sequence obtainable from the plasmid
in S. cerevisiae, DSM 9770, DSM 10082, DSM 10080, DSM 10081, E.
coli, DSM 10512, DSM 10511: DSM 10571 or DSM 10576,
respectively.
Immunological Cross-Reactivity
[0260] Antibodies to be used in determining immunological
cross-reactivity may be prepared by use of a purified cellulase.
More specifically, antiserum against the cellulase of the invention
may be raised by immunizing rabbits (or other rodents) according to
the procedure described by N. Axelsen et al. in: A Manual of
Quantitative Immunoelectrophoresis, Blackwell Scientific
Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe,
Immunochemistry in Practice, Blackwell Scientific Publications,
1982 (more specifically pp. 27-31). Purified immunoglobulins may be
obtained from the antisera, for example by salt precipitation
((NH.sub.4).sub.2 SO.sub.4), followed by dialysis and ion exchange
chromatography, e.g., on DEAE-Sephadex. Immunochemical
characterization of proteins may be 2 done either by Qutcherlony
double-diffusion analysis (O. Ouchterlony in: Handbook of
Experimental Immunology (D. M., Weir, Ed.), Blackwell Scientific
Publications, 1967, pp. 655-706), by crossed immunoelectrophoresis
(N. Axelsen et al., supra, Chapters 3 and 4), or by rocket
immunoelectrophoresis (N. Axelsen et al., Chapter 2).
Media
[0261] YPD: 10 g yeast extract, 20 g peptone, H.sub.2O to 900 ml.
Autoclaved, 100 ml 20% glucose (sterile filtered) added.
[0262] YPM: 10 g yeast extract, 20 g peptone, H.sub.2O to 900 ml.
Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.
[0263] 10.times. Basal salt: 75 g yeast nitrogen base, 113 g
succinic acid, 68 g NaOH, H.sub.2O ad 1000 ml, sterile
filtered.
[0264] SC-URA: 100 ml 10.times. Basal salt, 28 ml 20% casamino
acids without vitamins, 10 ml 1% tryptophan, H.sub.2O ad 900 ml,
autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20%
galactose added.
[0265] SC-URA agar: SC-URA, 20 g/l agar added.
[0266] PD agar: 39 g potato dextrose agar, DIFCO 0013: add
deionized water up to 1000 ml; autoclave (121.degree. C. for 15-20
min).
[0267] PC agar: Potatoes and carrots (grinded, 20 g of each) and
water, added up to 1000 ml, are boiled for 1 hr; agar (20 g/l of
Merck 1614); autoclave (121.degree. C. for 20 min)
[0268] PC liquid broth: as PC agar but without the Agar
[0269] PD liquid broth: 24 g potato dextrose broth, Difco 0549,
deionized water up to 1000 ml; autoclave (121.degree. C. for 15-20
min)
[0270] PC and PD liquid broth with cellulose: add 30 g Solcafloc
(Dicacel available from Dicalite-Europe-Nord, 9000 Gent, Belgium)
per 1000 ml
[0271] PB-9 liquid broth: 12 g Rofec (Roquette 101-0441) and 24 g
glucose are added to 1000 ml water; pH is adjusted to 5.5; 5 ml
mineral oil and 5 g CaCO.sub.3 are added per 1000 ml. Autoclave
(121.degree. C. for 40 min)
[0272] YPG liquid broth: 4 g yeast extract (Difco 0127), 1 g
KH.sub.2PO.sub.4 (Merck4873), 0.5 g MgSO.sub.4.7H.sub.2O Merck
5886, 15 g Dextrose, Roquette 101-0441, 0.1 ml Pluronic (101-3088);
deionized water up to 1000 ml; autoclave (20 min at 121.degree.
C.)
[0273] Dilute salt solution (DS): Make up two stock solutions:
P-stock: 13.61 g KH.sub.2PO.sub.4; 13.21 g
(NH.sub.4).sub.2PO.sub.4, 17.42 g KH.sub.2PO.sub.4; deionized water
up to 100 ml Ca/Mg stock: 7.35 g CaCl.sub.2, 2H.sub.2O, 10.7 g
MgCl.sub.2, 6H.sub.2O, deionized water up to 100 ml; pH adjusted to
7.0; autoclaving (121.degree. C.; 20 min)
[0274] Mix 0.5 ml P-stock with 0.1 ml Ca/Mg stock
[0275] add deionized water up to 1000 ml
[0276] AZCL HE cellulose (Megazyme, Australia).
Uses
[0277] During washing and wearing, dyestuff from dyed fabrics or
garment will conventionally bleed from the fabric which then looks
faded and worn. Removal of surface fibers from the fabric will
partly restore the original colours and looks of the fabric. By the
term "colour clarification", as used herein, is meant the partly
restoration of the initial colours of fabric or garment throughout
multiple washing cycles.
[0278] The term "de-pilling" denotes removing of pills from the
fabric surface.
[0279] The term "soaking liquor" denotes an aqueous liquor in which
laundry may be immersed prior to being subjected to a conventional
washing process. The soaking liquor may contain one or more
ingredients conventionally used in a washing or laundering
process.
[0280] The term "washing liquor" denotes an aqueous liquor in which
laundry is subjected to a washing process, i.e., usually a combined
chemical and mechanical action either manually or in a washing
machine. Conventionally, the washing liquor is an aqueous solution
of a powder or liquid detergent composition.
[0281] The term "rinsing liquor" denotes an aqueous liquor in which
laundry is immersed and treated, conventionally immediately after
being subjected to a washing process, in order to rinse the
laundry, i.e., essentially remove the detergent solution from the
laundry. The rinsing liquor may contain a fabric conditioning or
softening composition.
[0282] The laundry subjected to the method of the present invention
may be conventional washable laundry. Preferably, the major part of
the laundry is sewn or unsewn fabrics, including knits, wovens,
denims, yarns, and toweling, made from cotton, cotton blends or
natural or manmade cellulosics (e.g., originating from
xylan-containing cellulose fibers such as from wood pulp) or blends
thereof. Examples of blends are blends of cotton or rayon/viscose
with one or more companion material such as wool, synthetic fibers
(e.g., polyamide fibers, acrylic fibers, polyester fibers,
polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene
chloride fibers, polyurethane fibers, polyurea fibers, aramid
fibers), and cellulose-containing fibers (e.g., rayon/viscose,
ramie, flax/linen, jute, cellulose acetate fibers, lyocell).
Detergent Compositions
[0283] According to one aspect of the present invention, the
present endoglucanases may typically be components of a detergent
composition. As such, they may be included in the detergent
composition in the form of a non-dusting granulate, a stabilized
liquid, or protected enzymes. Non-dusting granulates may be
produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and
4,661,452 (both to to Novo Industri A/S) and may optionally be
coated by methods known in the art, Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molecular weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono and di- and triglycerides of fatty
acids, Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in patent GB 1483591.
Liquid enzyme preparations may, for instance, be stabilized by
adding a polyol such as propylene glycol, a sugar or sugar alcohol,
lactic acid or boric acid according to established methods. Other
enzyme stabilizers are welt known in the art. Protected enzymes may
be prepared according to the method disclosed in EP 238,216.
[0284] The detergent composition of the invention may be in any
convenient form, e.g., as powder, granules, paste or liquid. A
liquid detergent may be aqueous, typically containing up to 70%
water and 0-30% organic solvent, or nonaqueous.
[0285] The detergent composition comprises one or more surfactants,
each of which may be anionic, nonionic, cationic, or zwitterionic.
The detergent will usually contain 0-50% of anionic surfactant such
as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS),
alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate
(AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty
acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may
also contain 0-40% of nonionic surfactant such as alcohol
ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,
nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine
oxide, ethoxylated fatty acid monoethanolamide, fatty acid
monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as
described in WO 92/06154).
[0286] The detergent composition may additionally comprise one or
more other enzymes such as amylase, lipase, cutinase, protease,
peroxidase, and oxidase, e.g., laccase.
[0287] The detergent may contain 1-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, citrate, nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTMPA), alkyl- or
alkenylsuccinic acid, soluble silicates or layered silicates (eg.,
SKS-6 from Hoechst). The detergent may also be inbuilt, i.e.,
essentially free of detergent builder.
[0288] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),
polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA),
polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers and lauryl methacrylate/acrylic acid copolymers.
[0289] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine (TAED) or Nonanoyloxybenzenesulfonate
(NOBS). Alternatively, the bleaching system may comprise
peroxyacids of, erg., the amide, imide, or sulfone type.
[0290] The enzymes of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative such
as, e.g., an aromatic borate ester, and the composition may be
formulated as described in, e.g., WO 92/19709 and WO 92/19708.
[0291] The detergent may also contain other conventional detergent
ingredients such as, e.g., fabric conditioners including clays,
foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil-redeposition agents, dyes,
bactericides, optical brighteners, or perfume.
[0292] The pH (measured in aqueous solution at use concentration)
will usually be neutral or alkaline, e.g., in the range of
7-11.
[0293] Particular forms of detergent compositions within the scope
of the invention include:
1) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00023 Linear alkylbenzenesulfonate (calculated as acid)
7-12% Alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 EO)
1-4% or alkyl sulfate (e.g., C.sub.16-18) Alcohol ethoxylate (e.g.,
C.sub.14-15 alcohol, 7 EO) 5-9% Sodium carbonate (as
Na.sub.2CO.sub.3) 14-20% Soluble silicate (as Na.sub.2O,
2SiO.sub.2) 2-6% Zeolite (as NaA1SiO.sub.4) 15-22% Sodium sulfate
(as Na.sub.2SO.sub.4) 0-6% Sodium citrate/citric acid (as
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) 0-15% Sodium
perborate (as NaBO.sub.3.cndot.H.sub.2O) 11-18% TAED 2-6%
Carboxymethylcellulose 0-2% Polymers (e.g., maleic/acrylic acid
copolymer, PVP, 0-3% PEG) Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., suds suppressors,
perfume, 0-5% optical brightener, photobleach)
2) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00024 Linear alkylbenzenesulfonate (calculated as acid)
6-11% Alcohol ethoxysulfate (e.g., C.sub.12-18 alcohol, 1-2 EO or
1-3% alkyl sulfate (e.g., C.sub.16-18) Alcohol ethoxylate (e.g.,
C.sub.14-15 alcohol, 7 EO) 5-9% Sodium carbonate (as
Na.sub.2CO.sub.3) 15-21% Soluble silicate (as Na.sub.2O,
2SiO.sub.2) 1-4% Zeolite (as NaA1SiO.sub.4) 24-34% Sodium sulfate
(as Na.sub.2SO.sub.4) 4-10% Sodium citrate/citric acid (as
C.sub.6H.sub.5Na.sub.3O.sub.7/C.sub.6H.sub.8O.sub.7) 0-15%
Carboxymethylcellulose 0-2% Polymers (e.g., maleic/acrylic acid
copolymer, PVP, 1-6% PEG) Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., suds suppressors,
perfume) 0-5%
3) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00025 Linear alkylbenzenesulfonate (calculated as acid)
5-9% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) 7-14%
Soap as fatty acid (e.g., C.sub.16-22 fatty acid) 1-3% Sodium
carbonate (as Na.sub.2CO.sub.3) 10-17% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 3-9% Zeolite (as NaA1SiO.sub.4) 23-33%
Sodium sulfate (as Na.sub.2SO4) 0-4% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 8-16% TAED 2-8% Phosphonate (e.g.,
EDTMPA) 0-1% Carboxymethylcellulose 0-2% Polymers (e.g.,
maleic/acrylic acid copolymer, PVP, 0-3% PEG) Enzymes (calculated
as pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., suds
suppressors, perfume, 0-5% optical brightener)
4) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00026 Linear alkylbenzenesulfonate (calculated as acid)
8-12% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO) 10-25%
Sodium carbonate (as Na.sub.2CO.sub.3) 14-22% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 1-5% Zeolite (as NaA1SiO.sub.4) 25-35%
Sodium sulfate (as Na.sub.2SO.sub.4) 0-10% Carboxymethylcellulose
0-2% Polymers (e.g., maleic/acrylic acid copolymer, PVP, 1-3% PEG)
Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., suds suppressors, perfume) 0-5%
5) An aqueous liquid detergent composition comprising
TABLE-US-00027 Linear alkylbenzenesulfonate (calculated as acid)
15-21% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO or
12-18% C.sub.12-15 alcohol, 5 EO) Soap as fatty acid (e.g., oleic
acid) 3-13% Alkenylsuccinic acid (C.sub.12-14) 0-13% Aminoethanol
8-18% Citric acid 2-8% Phosphonate 0-3% Polymers (e.g., PVP, PEG)
0-3% Borate (as B.sub.4O.sub.7) 0-2% Ethanol 0-3% Propylene glycol
8-14% Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., dispersants, suds suppressors, 0-5% perfume,
optical brightener)
6) An aqueous structured liquid detergent composition
comprising
TABLE-US-00028 Linear alkylbenzenesulfonate (calculated as acid)
15-21% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or 3-9%
C.sub.12-15alcohol, 5 EO) Soap as fatty acid (e.g., oleic acid)
3-10% Zeolite (as NaA1SiO.sub.4) 14-22% Potassium citrate 9-18%
Borate (as B.sub.4O.sub.7) 0-2% Carboxymethylcellulose 0-2%
Polymers (e.g., PEG, PVP) 0-3% Anchoring polymers such as, e.g.,
lauryl 0-3% methacrylate/acrylic acid copolymer; molar ratio 25:1;
MW 3800 Glycerol 0-5% Enzymes (calculated as pure enzyme protein)
0.0001-0.1% Minor ingredients (e.g., dispersants, suds suppressors,
0-5% perfume, optical brighteners)
7) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00029 Fatty alcohol sulfate 5-10% Ethoxylated fatty acid
monoethanolamide 3-9% Soap as fatty acid 0-3% Sodium carbonate (as
Na.sub.2CO.sub.3) 5-10% Soluble silicate (as Na.sub.2O, 2SiO.sub.2)
1-4% Zeolite (as NaA1SiO.sub.4) 20-40% Sodium sulfate (as
Na.sub.2SO.sub.4) 2-8% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 12-18% TAED 2-7% Polymers (e.g.,
maleic/acrylic acid copolymer, PEG) 1-5% Enzymes (calculated as
pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., optical
brightener, suds 0-5% suppressors, perfume)
8) A detergent composition formulated as a granulate comprising
TABLE-US-00030 Linear alkylbenzenesulfonate (calculated as acid)
8-14% Ethoxylated fatty acid monoethanolamide 5-11% Soap as fatty
acid 0-3% Sodium carbonate (as Na.sub.2CO.sub.3) 4-10% Soluble
silicate (as Na.sub.2O, 2SiO.sub.2) 1-4% Zeolite (as NaA1SiO.sub.4)
30-50% Sodium sulfate (as Na.sub.2SO.sub.4) 3-11% Sodium citrate
(as C.sub.6H.sub.5Na.sub.3O.sub.7) 5-12% Polymers (e.g., PVP,
maleic/acrylic acid copolymer, 1-5% PEG) Enzymes (calculated as
pure enzyme protein) 0.0001-0.1% Minor ingredients (e.g., suds
suppressors, perfume) 0-5%
9) A detergent composition formulated as a granulate comprising
TABLE-US-00031 Linear alkylbenzenesulfonate (calculated as acid)
6-12% Nonionic surfactant 1-4% Soap as fatty acid 2-6% Sodium
carbonate (as Na.sub.2CO.sub.3) 14-22% Zeolite (as NaA1SiO.sub.4)
18-32% Sodium sulfate (as Na.sub.2SO.sub.4) 5-20% Sodium citrate
(as C.sub.6H.sub.5Na.sub.3O.sub.7) 3-8% Sodium perborate (as
NaBO.sub.3.cndot.H.sub.2O) 4-9% Bleach activator (e.g., NOBS or
TAED) 1-5% Carboxymethylcellulose 0-2% Polymers (e.g.,
polycarboxylate or PEG) 1-5% Enzymes (calculated as pure enzyme
protein) 0.0001-0.1% Minor ingredients (e.g., optical brightener,
perfume) 0-5%
10) An aqueous liquid detergent composition comprising
TABLE-US-00032 Linear alkylbenzenesulfonate (calculated as acid)
15-23% Alcohol ethoxysulfate (e.g., C.sub.12-15 alcohol, 2-3 EO)
8-15% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or 3-9%
C.sub.12-15alcohol, 5 EO) Soap as fatty acid (e.g., lauric acid)
0-3% Aminoethanol 1-5% Sodium citrate 5-10% Hydrotrope (e.g.,
sodium toluensulfonate) 2-6% Borate (as B.sub.4O.sub.7) 0-2%
Carboxymethylcellulose 0-1% Ethanol 1-3% Propylene glycol 2-5%
Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minor
ingredients (e.g., polymers, dispersants, 0-5% perfume, optical
brighteners)
11) An aqueous liquid detergent composition comprising
TABLE-US-00033 Linear alkylbenzenesulfonate (calculated as acid)
20-32% Alcohol ethoxylate (e.g., C.sub.12-15 alcohol, 7 EO, or
6-12% C.sub.12-15 alcohol, 5 EO) Aminoethanol 2-6% Citric acid
8-14% Borate (as B.sub.4O.sub.7) 1-3% Polymer (e.g., maleic/acrylic
acid copolymer, 0-3% anchoring polymer such as, e.g., lauryl
methacrylate-/acrylic acid copolymer) Glycerol 3-8% Enzymes
(calculated as pure enzyme protein) 0.0001-0.1% Minor ingredients
(e.g., hydrotropes, dispersants, 0-5% perfume, optical
brighteners)
12) A detergent composition formulated as a granulate having a bulk
density of at least 600 gal comprising
TABLE-US-00034 Anionic surfactant (linear alkylbenzenesulfonate,
25-40% alkyl sulfate, alpha-olefinsulfonate, alpha-sulfo fatty acid
methyl esters, alkanesulfonates, soap) Nonionic surfactant (e.g.,
alcohol ethoxylate) 1-10% Sodium carbonate (as Na.sub.2CO.sub.3)
8-25% Soluble silicates (as Na.sub.2O, 2SiO.sub.2) 5-15% Sodium
sulfate (as Na.sub.2SO.sub.4) 0-5% Zeolite (as NaA1SiO.sub.4)
15-28% Sodium perborate (as NaBO.sub.3.cndot.4H.sub.2O) 0-20%
Bleach activator (TAED or NOBS) 0-5% Enzymes (calculated as pure
enzyme protein) 0.0001-0.1% Minor ingredients (e.g., perfume,
optical brighteners) 0-3%
13) Detergent formulations as described in 1)-12) wherein all or
part of the linear alkylbenzenesulfonate is replaced by
(C.sub.12-C.sub.18) alkyl sulfate. 14) A detergent composition
formulated as a granulate having a bulk density of at least 600 g/l
comprising
TABLE-US-00035 (C.sub.12-C.sub.18) alkyl sulfate 9-15% Alcohol
ethoxylate 3-6% Polyhydroxy alkyl fatty acid amide 1-5% Zeolite (as
NaA1SiO.sub.4) 10-20% Layered disilicate (e.g., SK56 from Hoechst)
10-20% Sodium carbonate (as Na.sub.2CO.sub.3) 3-12% Soluble
silicate (as Na.sub.2O, 2SiO.sub.2) 0-6% Sodium citrate 4-8% Sodium
percarbonate 13-22% TAED 3-8% Polymers (e.g., polycarboxylates and
PVP = 0-5% Enzymes (calculated as pure enzyme protein) 0.0001-0.1%
Minor ingredients (e.g., optical brightener, photo 0-5% bleach,
perfume, suds suppressors)
15) A detergent composition formulated as a granulate having a bulk
density of at least 600 g/l comprising
TABLE-US-00036 (C.sub.12-C.sub.18) alkyl sulfate 4-8% Alcohol
ethoxylate 11-15% Soap 1-4% Zeolite MAP or zeolite A 35-45% Sodium
carbonate (as Na.sub.2CO.sub.3) 2-8% Soluble silicate (as
Na.sub.2O, 2SiO.sub.2) 0-4% Sodium percarbonate 13-22% TAED 1-8%
Carboxymethyl cellulose 0-3% Polymers (e.g., polycarboxylates and
PVP) 0-3% Enzymes (calculated as pure enzyme protein) 0.0001-0.1%
Minor ingredients (e.g., optical brightener, 0-3% phosphonate,
perfume)
16) Detergent formulations as described in 1)-15) which contain a
stabilized or encapsulated peracid, either as an additional
component or as a substitute for already specified bleach systems.
17) Detergent compositions as described in 1), 3), 7), 9) and 12)
wherein perborate is replaced by percarbonate. 18) Detergent
compositions as described in 1), 3), 7), 9), 12), 14) and 15) which
additionally contain a manganese catalyst. The manganese catalyst
may, e., be one of the compounds described in "Efficient manganese
catalysts for low-temperature bleaching", Nature 369, 1994, pp.
637-639. 19) Detergent composition formulated as a nonaqueous
detergent liquid comprising a liquid nonionic surfactant such as,
e.g., linear alkoxylated primary alcohol, a builder system (e.g.,
phosphate), enzyme and alkali. The detergent may also comprise
anionic surfactant and/or a bleach system.
[0294] The endoglucanase may be incorporated in concentrations
conventionally employed in detergents. It is at present
contemplated that, in the laundry composition of the invention, the
cellulase may be added in an amount corresponding to 0.0001-10 mg
(calculated as pure enzyme protein) of cellulase per liter of wash
liquor.
[0295] According to yet another aspect of the present invention,
endoglucanase may typically be a component of a fabric conditioning
or softener composition. Examples of conventional softener
compositions are disclosed in, e.g., EP 0 233 910.
Textile Applications
[0296] In another embodiment, the present invention relates to use
of the endoglucanase of the invention in the biopolishing process.
Bio-Polishing is a specific treatment of the yarn surface which
improves fabric quality with respect to handle and appearance
without loss of fabric wettability. The most important effects of
Bio-Polishing can be characterized by less fuzz and pilling,
increased gloss/luster, improved fabric handle, increased durable
softness and altered water absorbency. Bio-Polishing usually takes
place in the wet processing of the manufacture of knitted and woven
fabrics. Wet processing comprises such steps as, e.g., desizing,
scouring, bleaching, washing, dying/printing and finishing. During
each of these steps, the fabric is more or less subjected to
mechanical action. In general, after the textiles have been knitted
or woven, the fabric proceeds to a desizing stage, followed by a
scouring stage, etc. Desizing is the act of removing size from
textiles. Prior to weaving on mechanical looms, warp yarns are
often coated with size starch or starch derivatives in order to
increase their tensile strength. After weaving, the size coating
must be removed before further processing the fabric in order to
ensure a homogeneous and wash-proof result. It is known that in
order to achieve the effects of Bio-Polishing, a combination of
cellulytic and mechanical action is required. It is also known that
"super-softness" is achievable when the treatment with a cellulase
is combined with a conventional treatment with softening agents. It
is contemplated that use of the endoglucanase of the invention for
bio-polishing of cellulosic fabrics is advantageous, e.g., a more
thorough polishing can be achieved. Bio-polishing may be obtained
by applying the method described, e.g., in WO 93/20278.
Stone-washing
[0297] It is known to provide a "stone-washed" look (localized
abrasion of the colour) in dyed fabric, to especially in denim
fabric or jeans, either by washing the denim or jeans made from
such fabric in the presence of pumice stones to provide the desired
localized lightening of the colour of the fabric or by treating the
fabric enzymatically, in particular with cellulytic enzymes. The
treatment with an endoglucanase of the present invention may be
carried out either alone such as disclosed in U.S. Pat. No.
4,832,864, together with a smaller amount of pumice than required
in the traditional process, or together with perlite such as
disclosed in WO 95/09225.
Pulp and Paper Applications
[0298] In the papermaking pulp industry, the endoglucanase of the
present invention may be applied advantageously, e.g., as follows:
[0299] For debarking; pretreatment with the endoglucanase may
degrade the cambium layer prior to debarking in mechanical drums
resulting in advantageous energy savings. [0300] For
defibrillation: treatment of a material containing cellulosic
fibers with the endoglucanase prior to refining or beating may
result in reduction of the energy consumption due to the
hydrolyzing effect of the cellulase on the interfiber surfaces. Use
of the endoglucanase may result in improved energy savings as
compared to the use of known enzymes, since it is believed that the
enzyme composition of the invention may possess a higher ability to
penetrate fiber waits. [0301] For fiber modification, i.e.,
improvement of fiber properties where partial hydrolysis across the
fiber watt is needed which requires deeper penetrating enzymes
(eg., in order to make coarse fibers more flexible). Deep treatment
of fibers has so far not been possible for high yield pulps, e.g.,
mechanical pulps or mixtures of recycled pulps. This has been
ascribed to the nature of the fiber watt structure that prevents
the passage of enzyme molecules due to physical restriction of the
pore matrix of the fiber wall, it is contemplated that the present
endoglucanase is capable of penetrating into the fiber wall, [0302]
For drainage improvement. The drainability of papermaking pulps may
be improved by treatment of the pulp with hydrolyzing enzymes,
e.g., cellulases. Use of the present endoglucanase may be more
effective, e.g., result in a higher degree of loosening bundles of
strongly hydrated micro-fibrils in the fines fraction (consisting
of fiber debris) that limits the rate of drainage by blocking
hollow spaces between fibers and in the wore mesh of the paper
machine. The Canadian standard freeness (CSF) increases and the
Schopper-Riegler drainage index decreases when pulp in subjected to
cellulase treatment, see, e.g., U.S. Pat. No. 4,923,565; TAPPI
T227, SCANC 19:65 ence. [0303] For inter fiber bonding. Hydrolytic
enzymes are applied in the manufacture of papermaking to pulps for
improving the interfiber bonding. The enzymes rinse the fiber
surfaces for impurities, eg., cellulosic debris, thus enhancing the
area of exposed cellulose with attachment to the fiber wall, thus
improving the fiber-to-fiber hydrogen binding capacity. This
process is also referred to as dehornification. Paper and board
produced with a cellulase containing enzyme preparation may have an
improved strength or a reduced grammage, a smoother surface and an
improved printability. [0304] For enzymatic deinking. Partial
hydrolysis of recycled paper during or upon pulping by use of
hydrolyzing enzymes such as cellulases are known to facilitate the
removal and agglomeration of ink particles. Use of the present
endoglucanase may give a more effective loosening of ink from the
surface structure due to a better penetration of the enzyme
molecules into the fibrillar matrix of the fiber wall, thus
softening the surface whereby ink particles are effectively
loosened. The agglomeration of loosened ink particles are also
improved, due to a more efficient hydrolysis of cellulosic
fragments found attached to ink particles originating from the
fibers.
[0305] The treatment of lignocellulosic pulp may, e.g., be
performed as described in WO 91/14819, WO 91/14822, WO 92/17573 and
WO 92/18688.
Degradation of Plant Material
[0306] In yet another embodiment, the present invention relates to
use of the endoglucanase and/or enzyme preparation according to the
invention for degradation of plant material, eg., cell walls.
[0307] It is contemplated that the novel endoglucanase and/or
enzyme preparation of the invention is useful in the preparation of
wine, fruit or vegetable juice in order to increase yield,
Endoglucanases according to the invention may also be applied for
enzymatic hydrolysis of various plant cell-wall derived materials
or waste materials, e.g., agricultural residues such as
wheat-straw, corn cobs, whole corn plants, nut shells, grass,
vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the
like. The plant material may be degraded in order to improve
different kinds of processing, facilitate purification or
extraction of other components tike purification of beta-glucan or
beta-glucan oligomers from cereals, improve the feed value,
decrease the water binding capacity, improve the degradability in
waste water plants, improve the conversion of, eg., grass and corn
to ensilage, etc.
[0308] The following examples illustrate the invention.
EXAMPLE 1
[0309] Cellulytic enzymes from 4 fungi, belonging to 3 families
under two orders within the Ascomycetes were detected by expression
cloning; corresponding DNA sequences were determined; the enzymes
heterologously expressed, and produced by liquid fermentation,
characterized and demonstrated to give good performance in colour
clarification assays.
[0310] Isolate CBS 117.65, CBS 478.94, NRRL 8126, and ATCC 10523
were grown in shake flask cultures on cellulose enriched potato
dextrose broth, incubated for 5 days at 26.degree. C. (shaking
conditions, 150 rpm).
A. Cloning and Expression of an Endoglucanase from Myceliophthora
thermophila, Acremonium sp., and Thielavia terrestris and Volutella
colletotrichoides
[0311] mRNA was isolated from Myceliophthora thermophila,
Acremonium sp., Thielavia terrestris and Volutella
colletotrichoides, respectively, grown in a cellulose-containing
fermentation medium with agitation to ensure sufficient aeration.
Mycelia were harvested after 3-5 days' growth, immediately frozen
in liquid nitrogen and stored at -80.degree. C. Libraries from
Myceliophthora thermophila, Acremonium sp., Thielavia terrestris
and Volutella colletotrichoides, respectively, each consisting of
approx. 106 individual clones were constructed in E. coli as
described with a vector background of 1%.
[0312] Plasmid DNA from some of the pools from each library was
transformed into yeast, and 50-100 plates containing 250-400 yeast
colonies were obtained from each pool.
[0313] Endoglucanase-positive colonies were identified and isolated
on SC-agar plates with the AZCL HE cellulose assay. cDNA inserts
were amplified directly from the yeast colonies and characterized
as described in the Materials and Methods section above.
[0314] The DNA sequence of the cDNA encoding the endoglucanase from
Myceliophthora thermophila is SEQ ID NO: 1 and the corresponding
amino acid sequence is SEQ ID NO: 2. The cDNA is obtainable from
the plasmid in DSM 9770.
[0315] The DNA sequence of the cDNA encoding the endoglucanase from
Acremonium sp. is SEQ ID NO: 7 and the corresponding amino acid
sequence is SEQ ID NO: 8. The cDNA is obtainable from the plasmid
in DSM 10032.
[0316] The DNA sequence of the cDNA encoding the endoglucanase from
Thielavia terrestris is SEQ ID NO: 11 and the corresponding amino
acid sequence is SEQ ID NO: 12. The cDNA is obtainable from the
plasmid in DSM 10081.
[0317] The DNA sequence of the cDNA encoding the endoglucanase from
Volutella colletotrichoides is SEQ ID NO: 21 and the corresponding
amino acid sequence is SEQ ID NO: 22. The cDNA is obtainable from
the plasmid in DSM 10571.
[0318] Total DNA was isolated from a yeast colony and plasmid DNA
was rescued by transformation of E. coli as described above. In
order to express the endoglucanases in Aspergillus, the DNA was
digested with appropriate restriction enzymes, size fractionated on
gel, and a fragment corresponding to the endoglucanase gene from
Myceliophthora thermophila, Acremonium sp., Thielavia terrestris
and Volutella colletotrichoides, respectively, was purified. The
genes were subsequently ligated to pHD414, digested with
appropriate restriction enzymes, resulting in the plasmids pA2C193,
pA2C357, pA2C385 and pA2C488, respectively.
[0319] After amplification of the DNA in E. coli the plasmids were
transformed into Aspergillus oryzae as described above,
Test of A. oryzae Transformants
[0320] Each of the transformants were tested for endoglucanase
activity as described above. Some of the transformants had
endoglucanase activity which was significantly larger than the
Aspergillus oryzae background. This demonstrates efficient
expression of the endoglucanases in Aspergillus oryzae. The
transformants with the highest endoglucanase activity were selected
and inoculated in a 500 ml shake flask with YPM media. After 3-5
days of fermentation with sufficient agitation to ensure good
aeration, the culture broth was centrifuged for 10 minutes at 2000
g and the supernatant recovered.
B. Determination of Endoglucanase Activity The cellulytic activity
of the endoglucanase may be determined relative to an analytical
standard and expressed in the unit S-CEVU.
[0321] Cellulytic enzymes hydrolyze OMC, thereby decreasing the
viscosity of the incubation mixture. The resulting reduction in
viscosity may be determined by a vibration viscometer (e.g., MIVI
3000 from Sofraser, France).
[0322] Determination of the cellulytic activity, measured in terms
of S-CEVU, may be determined according to the analysis method AF
301.1 which is available from the Applicant upon request.
[0323] The S-CEVU assay quantifies the amount of catalytic activity
present in the sample by measuring the ability of the sample to
reduce the viscosity of a solution of carboxymethylcellulose (CMC).
The assay is carried out at 40.degree. C., pH 7.5 using a relative
enzyme standard for reducing the viscosity of the CMC
substrate.
[0324] Assay for determination of endoglucanase activity in terms
of SAVI units using phosphoric-acid swollen cellulose (PASC):
Definition: 1 SAVI-U is the amount of enzyme which forms an amount
of reducing carbohydrates equivalent to 1 micro-mol of glucose per
minute.
Assay Condition:
[0325] Enzyme solution, 0.5 ml
[0326] 4 g/l PASC in 0.1 M Buffer: 2.0 ml
[0327] 20 min, 40.degree. C.
Sensitivity:
[0328] Max 0.1 SAVIU/ml=approx. 1 S-CEVU/ml (CMC viscosity)
[0329] Min 0.01 SAVIU/ml=approx. 0.1 S-CEVU/ml
Determination of Formation of Reducing Sugars:
[0330] The reducing groups assay was performed according to Lever,
M. A new reaction for colormetric determination of carbohydrates.
Anal. Biochem. 1972. Vol 47 (273-279). Reagent mixture was prepared
by mixing 1.5 gram p-hydroxybenzoic-acid hydrazide (PHBAH) with 5
gram sodium tartrate in 100 ml 2% sodium hydroxide.
Substrate:
[0331] PASC stock solution was prepared the following way using ice
cold acetone and phosphoric acid. 5 gram of cellulose (Avicel.RTM.)
was moistened with water, and 150 ml ice cold 85% ortho-phosphoric
acid was added. The mixture was placed in ice bath under slow
stirring for 1 hr. Then 100 ml ice cold acetone was added with
stirring. The slurry was transferred to a Buchner filter with pyrex
sintered disc number 3 and then washed three times with 100 ml ice
cold acetone, and sucked as dry as possible after each wash.
Finally, the filter cake was washed twice with 500 ml water, sucked
as dry as possible after each wash The PASC was mixed with
deionized water to a total volume of 300 ml, blended to homogeneity
(using the Ultra Turrax Homogenizer) and stored in refrigerator (up
to one month).
[0332] Substrate equilibration with buffer: 20 gram phosphoric acid
swollen cellulose PASC stock solution was centrifuged for 20 min at
5000 rpm., the supernatant was poured of; the sediment was
resuspended in 30 ml of buffer and centrifuged for 20 min. at 5000
rpm., the supernatant was poured of, and the sediment was
resuspended in buffer to a total of 60 g corresponding to a
substrate concentration of 5 g cellulose/liter.
Buffer for pH 8.5 determination: 0.1 M Barbital, Buffer for pH 10
determination: 0.1 M Glycine.
Procedure:
A. Dilution of Enzyme Samples
[0333] The enzyme solution is diluted in the same buffer as the
substrate.
2. Enzyme Reaction
[0334] The substrate in buffer solution is preheated for 5 min. at
40.degree. C. (2 ml). Then the enzyme solution (diluted to between
0.2 and 1 S-CEVU/ml) 0.5 ml is added and mixed for 5 sec. Enzymes
blanks are obtained by adding the stop reagent before enzyme
solution. Incubate for 20 min. at 40.degree. C., The reaction is
stopped by adding 0.5 ml 2% NaOH solution and mixing for 5 sec.
[0335] The samples are centrifuged for 20 min. at 5000 rpm. 1 ml
supernatant is mixed with 0,5 ml PHBAH reagent and boiled for 10
min. The test tubes are cooled in an ice water bath.
3. Determination of Reducing End Groups:
[0336] The absorbency at 410 nm is measured using a
spectrophotometer. Blanks are prepared by adding sodium hydroxide
before adding enzyme solution.
[0337] A standard glucose curve was obtained by using glucose
concentrations of 5, 10, 15 and 25 mg/l in the same buffer and
adding PHBAH reagent before boiling. The release of reducing
glucose equivalent is calculated using this standard curve.
4. Calculation of Catalytic Activity:
[0338] Measure absorbance at 410 nm
1) Standard Curve
[0339] (Glucose)-(H.sub.2O) vs concentration of glucose
2) Enzyme Sample
[0340] (Sample)-(Blank)
Calculate Glucose Concentration According to a Standard Curve
Activity (SAVIU/ml):
[0341] X ( mg glucose / l ) * Dilution 180.16 ( MW of glucose ) *
20 ( min ) ##EQU00001##
C. Purification and Characterization of the Endoglucanase from M.
Thermophila
[0342] Aspergillus oryzae transformed with pA2C193 was grown on YPM
medium for 4 days. The liquid was then centrifuged and sterile
filtered.
[0343] The sample was concentrated by ultrafiltration on AMICON
cells using a DOW membrane GR61PP with cut-off 20 kD. The
Uf-concentrate was analyzed for S-CEVU/ml and SaviU/ml with the
following result:
TABLE-US-00037 UF-concentrate S-CEVU/ml SaviU/ml 9.25 ml 570 41
Purification:
[0344] 2 ml of the UF-concentrate was diluted 5 times to tower the
ionic strength and filtered through 0.22 micro-m disk fitter. This
sample was applied to a Mono Q.RTM. HR5/5 Pharmacia column,
equilibrated with 50 mM Tris/HCl buffer, pH 7.5, (buffer A) and a
flow of 1 ml/min. After wash to baseline, with buffer A, the column
was eluted with a Tris/HCl buffer, pH 7.5, containing 1 M NaCl
(buffer B), the elution gradient was 0-50% buffer B in 1 hour.
[0345] After 36 min. a peak complex showed up, 1 ml fractions were
picked up and the first 10 fractions showed cellulase activity on
COM/Agarose/congo-red plates.
[0346] These fractions were pooled and concentrated, by
ultrafiltration on AMICON cells using a DOW membrane GR61PP with
cut-off 20 kD, to 3 ml.
[0347] This sample was applied to a HiLoad 26/60 Superdex 75.TM.
prep grade Pharmacia column, equilibrated with 100 mM Na-Acetate
buffer, pH 6.35, and a 1 ml/min flow.
[0348] After 82 min. a peak showed up, 1 ml fractions were picked
up and the first 10 fractions showed cellulase activity on
CMC/Agarose/congo-red plates.
[0349] These fractions were pooled and the following results were
obtained;
A.sub.280=0.15
A.sub.280/A.sub.260=1.62
Mw(SDS)=22 kD
[0350] pl=3.5-5
Purity on SDS-PAGE=100%
S-CEVU/ml=28.5
S-CEVU/A.sub.280=188
S-CEVU/mg-436
[0351] Extinction coefficient=54880 (calculated)
Mw(calculated)=22 kD
[0352] The Extinction coefficient is based on the content of
tyrosine, tryptophane and cysteine calculated from the sequence of
SEQ ID NO: 2 (the amino acid sequence). SOS-Page was performed on
NOVEX Pre-Cast Gels 4-20% Tris-Glycine Gel 1.0 mm.times.10
Well.
[0353] IEF was performed on Pharmacia PAGplate pH 3.5-9.5, the
activity was visualized by CMC-Congored overlaying.
[0354] Determination of K.sub.M & k.sub.cat:
k.sub.m and k.sub.cat was determined in the same manner as the
determination of SAVI Units at pH 8.5 with a substrate
concentration up to 8 g/l.
[0355] The following results were obtained:
k.sub.cat 38 per sec. k.sub.m 5 g/l, phosphoric acid swollen
cellulose, pH 8.5. Specific activity on CMC at pH 7.5; 436 S-CEVU
per mg protein. D. Determination of pH and Temperature Profile of
the Endoglucanase from M. thermophila
[0356] The pH profile was determined at the following
conditions:
[0357] Buffers of pH values between 2.5 and 10.0 were made by
mixing 0.1 M Tri-sodium phosphate with 0.1 M citric acid. Purified
endoglucanase was diluted to ensure the assay response to be within
the linear range of the assay. The substrate was a 0.4% suspension
of AZCL-HE-cellulose (MegaZyme) mixed 1:1 with the
citrate/phosphate buffer to a final substrate concentration of 0.2%
AZCL-H E-cellulose. 1 ml substrate in Eppendorf.RTM. 1.5 ml
polypropylene tubes were added 10 microliters of enzyme solution
and incubated for 15 minutes in Eppendorf.RTM. temperature
controlled Thermomixers before heat-inactivation of enzymes for 20
minutes at 95.degree. C. in a separate Thermomixer. The tubes were
centrifuged and 200 microliters of each supernatant was transferred
to a well in a 96 well microtiter plate and OD was measured at 620
nm in an ELISA reader (Labsystems Multiskan.RTM. MCC/1340).
[0358] For the pH optimum incubations took place at 30.degree. C.
For each pH value, three tubes were added enzyme and incubated
before heat-inactivation, whereas one tube (the blank) was added
enzyme and heat-inactivated immediately. The mean value of the
three incubated samples was calculated and the blank value was
subtracted.
[0359] The following pH profile was determined:
TABLE-US-00038 pH Relative Activity 2.5 <10% 3 <10% 3.5 22% 4
87% 4.5 89% 5 100% 6 94% 6.5 86% 7 78% 7.5 73% 8 68% 8.5 54% 9 31%
10 18%
[0360] It is seen that the endoglucanase has more than 60% activity
between pH 4.0 and 8.0 and optima activity at pH 5.0-6.0.
Temperature Profile:
[0361] The temperature optimum was determined in the same manner at
pH 5.5. The temperatures ranged from 30.degree. C. to 80.degree. C.
For each temperature three incubations were carried out and the
mean calculated. Three blanks were produced by immediate
heat-inactivation of enzyme and the mean was subtracted from the
incubated sample values.
[0362] Is seen that the endoglucanase has optimal activity at
50-70.degree. C.
TABLE-US-00039 Temp. (.degree. C.) 30 40 50 60 70 80 Relative
Activity 74% 77% 99% 100% 93% 62%
[0363] The temperature stability was determined in the same manner
at pH 5.5 and 30.degree. C., and, further, the enzyme solutions
were preheated for 1 hour at the actual temperature and cooled on
ice. The residual activity is shown below in % of the activity of a
non-preheated enzyme sample:
TABLE-US-00040 Temp. (.degree. C.) 40 50 60 70 80 Relative Activity
95% 84% 92% 86% 24%
E. Color Clarification of Myceliophthora Cellulase (SEQ ID NO: 2)
Measured as Removal of Surface Fibrils and Fibers Protruding from
the Yarn of a Textile Containing Cellulosic Fibers
TABLE-US-00041 Apparatus Terg-o-tometer Liquid volume 100 ml
Agitation 150 movements/min with vertical stirrer Rinse time 5 min
in tapwater Washing temp 40.degree. C. Washing liqour 0.05 M
phosphate buffer pH 7.0 Washing time 30 min Repetitions 2 Enzymes
Myceliophthora cellulase (SEQ ID NO: 2) Dosage 500 and 2500
S-CEVU/l Textile 2 swatches of aged black 100% cotton 5 .times. 6
cm (0.9 gram) Drying Tumble dry Evaluation The light remission is
measured by a Datacolor Elrepho Remission spectrophotometer.
Remission is calculated as delta L (Hunter Lab-values). When the
surface fibrils and fibers protruding from the yarn are removed by
the cellulase, the surface of the black fabric appears darker, and
lower L values are obtained.
[0364] The sample is compared with a blind sample, i.e., washed
without enzyme:
TABLE-US-00042 No cellulase 500 ECU/l 2500 ECU/l 0.00 -1.41
-1.91
Delta L-Values Compared to Blind Sample.
[0365] The data shows that Myceliophthora cellulase without CBD
gives good color clarification under the conditions tested.
F. Construction of the Gene Fusions Between the Endoglucanase from
Myceliophthora thermophila and the 43 kD Endoglucanase from
Humicola insolens
[0366] The purpose of the two constructions was to make derivatives
of the endoglucanase from M. thermophila with the linker and CBD
from the 43 kD endoglucanase from H. insolens (disclosed in WO
91/17243). The native endoglucanase from M. thermophila do not have
a linker and/or a cellulose binding domain, CBD.
[0367] CM1: Construction 1 consists of the endoglucanase from M.
thermophila (225 amino acids) and the 72 C-terminal amino acids
from the H. insolens 43 kD endoglucanase.
[0368] CM2: Construction 2 consists of the endoglucanase from M.
thermophila (225 amino acids) and the 83 C-terminal amino acids
from the H. insolens 43 kD endoglucanase.
[0369] The 43 kD endoglucanase cDNA from H. insolens was cloned
into pHD414 in such a way that the endoglucanase gene was
transcribed from the Taka-promoter. The resulting plasmid was named
pCaHj418.
[0370] In a similar way the cDNA encoding the endoglucanase from M.
thermophila was cloned into in pHD414 and the resulting plasmid was
named pA2C193. Primers;
TABLE-US-00043 primer 1: (SEQ ID NO: 88)
5'-CGGAGCTCACGTCCAAGAGCGGCTGCTCCCGTCCCTCCAGCAGCACC AGCTCTCCGG-3'
primer 2: (SEQ ID NO: 89)
5'-CCGGAGAGCTGGTGCTGCTGGAGGGACGGGAGCAGCCGCTCTTGGAC GTGAGCTCCG-3'
primer 3: (SEQ ID NO: 90)
5'-CGGAGCTCACGTCCAAGAGCGGCTGCTCCCGTAACGACGACGGCAAC TTCCCTGCCG-3'
primer 4 (SEQ ID NO: 91)
5'-CGGCAGGGAAGTTGCCGTCGTCGTTACGGGAGCAGCCGCTCTTGGAC GTGAGCTCCG-3'
Take-pro primer: (SEQ ID NO: 92) 5'-CAACATCACATCAAGCTCTCC-3'
AMS-term primer: (SEQ ID NO: 93) 5'-CCCCATCCTTTAACTATAGCG-3'
[0371] The endoglucanase fusions were constructed by the PCR
overlap-extension method as described by Higuchi et al. 1988.
Construction 1:
[0372] Reaction A: The Polymerase Chain Reaction (PCR) was used to
amplify the fragment of pCaHj418 between primer 1 and AMG-term.
primer (the linker and CBD from the 43 kD endoglucanase from H.
insolens). Reaction B. PCR amplification of the fragment between
Taka-pro, primer and primer 2 in pA2C193, the endoglucanase gene
from M. thermophila. Reaction C: The two purified fragments were
used in a third PCR in the presence of the primers flanking the
total region, i.e., Taka-pro. primer and AMG-term. primer.
Construction 2:
[0373] The same procedure was used where primer 3 and primer 4 had
replaced respectively primer 1 and primer 2.
[0374] The fragment amplified in reaction C was purified, digested
with restriction enzymes Xba I and BsstE II. The purified digested
fragment was ligated into pA2C193 digested with restriction enzymes
Xba I and BsstE II.
[0375] Competent cells from E. coli strain DH5.alpha.F' (New
England Biolabs.) were transformed with the ligated plasmid and
colonies containing the gene fusion were isolated. The sequence of
the cloned part was verified by DNA sequencing.
[0376] The sequences of the genes in the two constructs are SEQ ID
NO: 3 and SEQ ID NO: 5.
[0377] Polymerase Chain Reactions were carried out under standard
conditions, as recommended by Perkin-Elmer.
[0378] Reactions A and B started with 2 min. at 94.degree. C.
followed by 20 cycles of (30 sec. at 94.degree. C., 30 sec. at
50.degree. C. and 1 min. at 72.degree. C.) and end with 4 min. at
72.degree. C.
[0379] Reaction C started with (2 min. at 94.degree. C., 1 min. at
52.degree. C. and 2 min. at 72.degree. C.), followed by 15 cycles
of (30 sec. at 94.degree. C., 30 sec. at 52.degree. C. and 90 sec.
at 72.degree. C.) and end with 4 min. at 72.degree. C.
[0380] The two constructs were transformed into Aspergillus oryzae
as described above.
G. Purification and Characterization of Cloned Cellulases with
Cellulose Binding Domains:
[0381] The cloned product is recovered after fermentation by
separation of the extracellular fluid to from the production
organism.
[0382] About one gram of cellulase is then highly purified by
affinity chromatography using 150 gram of Avicel in a slurry with
20 mm Sodium-phosphate pH 7.5.
[0383] The Avicel is mixed with the crude fermentation broth, which
contains total about 1 gram of cellulase. After mixing at 4.degree.
C. for 20 min the Avicel enzyme is packed into a column with a
dimension of 50 times 200 mm about 400 ml total.
[0384] The column is washed with the 200 ml buffer, then washed
with 0.5 M NaCl in the same buffer until no more protein elutes,
Then washed with 500 ml 20 mm Tris pH 8.5. Finally the pure full
length enzyme is eluted with 1% triethylamine pH 11.8.
[0385] The eluted enzyme solution is adjusted to pH 8 and
concentrated using a Amicon cell unit with a membrane DOW GR61PP
(polypropylene with a cut off of 20 KD) to above 5 mg protein per
ml.
[0386] The purified cellulases were characterized as follows;
TABLE-US-00044 Mw S-CEVU SDS-PAGE pI Molar E.280 per A.280
Myceliophthora 43 kD 4 74.950 135 (SEQ ID NO: 4) Acremonium 40 kD 5
68.020 185 (SEQ ID NO: 8) Thielavia 35 kD 4.3 52.470 75 (SEQ ID NO:
12) pH Activity Thermostability above 50% N-terminal DSC
Myceliophthora 5.0-9.0 Blocked. 80.degree. C. (SEQ ID NO: 4)
Acremonium 6.0-9.5 Blocked. 61.degree. C. (SEQ ID NO: 8) Thielavia
5.0-9.0 ASGSG--- 83.degree. C. (SEQ ID NO: 12)
[0387] The purified cellulases was analysed for MW by SDS-PAGE and
using standard LMW protein marker kit from Pharmacia the MW was
calculated for the cellulases. The MW is apparently higher than the
MW of the composition of the coding amino acids and is due to the
fact the linker region is O-glycosylated resulting in this higher
MW. The pt was determined using a Pharmacia Ampholine PAG plate pH
3.5 to 9.5 and again using a Pharmacia kit with known pl
proteins.
[0388] The molar extinction coefficient was calculated based on the
amin acids composition using the known absorbance of Tryptophan,
Tyrosine and Cystein.
[0389] pH activity profile was obtained using CMC substrate,
incubation for 20 min at 40.degree. C. at a 0.5 pH interval and
measuring the formation of reducing sugars. The relative activity
at the different pH was calculated and the table contains the
intervat with more than 50% relative activity has been
measured.
[0390] The N-terminal was determined for the purified cellulase
using a Applied Biosystems model 473A sequencer. The protein
sequencer was run according to the manufacturer's instructions.
[0391] Two of the cellulases were blocked, this is due to the
N-terminal glutamine which forms a MC pyroglutamate which can not
be detected and which blocks further sequencing.
[0392] Differential scanning calometry ("DSC") was done at neutral
pH (7.0) using a MicroCalc Inc. MC calorimeter with a constant scan
rate and raising the temperature from 20 to 90.degree. C. at a rate
of 900 per hour.
[0393] Raising antibody. The cellulases from Myceliophthora,
Acremonium and Thielavia were used for raising antibody in rabbits.
0.1 mg of the purified cellulases in 0.9% NaCl solution mixed with
Freunds adjuvant immediately prior to injection. The rabbits were
immunized 10 times with one week interval. The immunoglobulin G
fraction (IgG) was purified by ammonium sulfate precipitation (25%
saturation). The precipitate was solubilized in water and then
dialyzed extensively against sodium acetate buffer (pH 5.0, 50 mM)
altering with deionized water. After filtration, the IgG fraction
was stabilized with sodium azide (0.01%).
[0394] Using immunodiffusion in agar plates all three cellulases
form a single immunoprecipitate with its homologous antiserum and
no precipitate was seen between the 3 cloned cellulases and the
sera raised against the other two cellulases.
H-I. Performance of Endoglucanase of Construction 1 (SEQ ID NO: 3)
Measured in Buffer as Removal of Surface Fibrils and Fibers
Protruding from the Yarn of a Textile Containing Cellulosic
Fibers
TABLE-US-00045 Apparatus Terg-o-tometer Liquid volume 100 ml
Agitation 150 movements/min (rpm) Rinse time 5 min in tap water
Washing temp 40.degree. C. Water Hardness 1 mM CaCl.sub.2 Washing
liquor 0.05 M phosphate buffer pH 7.0 Washing time 30 min
Repetitions 2 Textile 2 swatches of aged black, 100% cotton 5
.times. 6 cm Drying Tumble dry
Evaluation:
[0395] The light remission was measured by a Macbeth Color Eye 7000
Remission spectrophotometer. Remission is calculated as delta L
(Hunter Lab-values). When the surface fibrils and fibers protruding
from the yarn were removed by the cellulase, the surface appeared
more bright and lower L values were obtained.
Results:
TABLE-US-00046 [0396] S-CEVU/l 0 250 1000 Inventive enzyme 0 -1.4
-1.6
[0397] The data show that the enzyme of the invention gives very
good color clarification under the conditions tested.
H-I. Performance of Cloned Endoglucanase from Thielavia terrestris
(SEQ ID NO: 12) in Buffer Measured as Removal of Surface Fibrils
and Fibers Protruding from the Yarn of a Textile Containing
Cellulosic Fibers
TABLE-US-00047 7Apparatus Terg-o-tometer Liquid volume 100 ml
Agitation 150 movements/min with vertical stirrer Rinse time 10 min
in tapwater Washing temp 40.degree. Washing liquor 0.05 M phosphate
buffer. pH 7.0 Washing time 30 min Repetitions 2 Textile 2 swatches
of aged black cotton 5 .times. 6 cm (app. 150 g/m.sup.2) Drying
Tumble dry
Evaluation:
[0398] The light remission was measured by a Datacolor Elrepho
Remission spectrophotometer. Remission is calculated as delta L
(Hunter Lab-values). When the surface fibrils and fibers protruding
from the yarn are removed by the cellulase, the surface of the
black fabric appears darker and nicer, and lower L values are
obtained.
Results:
TABLE-US-00048 [0399] S-CEVU/l 0 50 200 Inventive enzyme 0 -0.66
.+-. 0.10 -1.32 .+-. 0.06
[0400] The data show that the cellulose gives good color
clarification under the conditions tested.
H-III Performance Of Endoglucanase Of Volutella colletrichoides
(SEQ ID NO: 9) Measured in Buffer as Removal of Surface Fibrils and
Fibers Protruding from the Yarn of a Textile Containing Cellulosic
Fibers
TABLE-US-00049 Apparatus Terg-o-tometer Liquid volume 100 ml
Agitation 150 movements/min with vertical stirrer Rinse time 5 min
in tapwater Washing temp 40.degree. Washing liqour 0.05 M phosphate
buffer pH 7.0 Washing time 30 min Repetitions 2 Dosage 2.5
S-CEVU/ml Textile 2 swatches of aged black 100% cotton 5 .times. 6
cm (0.9 gram) Drying Tumble dry
Evaluation,
[0401] The light remission is measured by a Datacolor Elrepho
Remission spectrophotometer. Remission is calculated as delta L
(Hunter Lab-values). When the surface fibrils and fibers protruding
from the yarn are removed by the cellulase, the surface of the
black fabric appears darker, and lower L values are obtained.
[0402] The sample is compared with a blind sample, i.e., washed
without enzyme:
TABLE-US-00050 No cellulase With cellulase 0.00 -0.57
Delta L remission values compared to blind sample.
[0403] The data shows that the Volutella colletrichoides cellulase
gives good color clarification under the conditions tested.
H-IV. Performance of Cloned Cellulases from Thielavia terrestris
and Acremonium sp. CBS 478.94 in high pH Heavy Duty Detergent
Measured as Removal of Surface Fibrils and Fibers Protruding from
the Yarn of a Textile Containing Cellulosic Fibers
TABLE-US-00051 Apparatus Terg-o-tometer Liquid volume 150 ml
Agitation 150 movements/min with vertical stirrer Rinse time 10 min
in tapwater Washing temp 35.degree. C. Washing liqour 1.0 g/l US
type HDG (zeolite/soda built, anionic/nonionic weight ratio
>2.5) pH 10.0 Hardness 1.0 mM CaCl.sub.2 0.34 mM MgCl.sub.2
Washing time 12 min Repetitions 6 Textile 2 swatches of aged black
cotton 5 .times. 6 cm (app. 150 g/m.sup.2) 2 swatches of heavy
knitted cotton 5 .times. 6 cm (app. 600 g/m.sup.2) Drying Tumble
dry
Evaluation:
[0404] The light remission is measured by a Datacolor Elrepho
Remission spectrophotometer. Remission is calculated as delta L
(Hunter Lab-values). When the surface fibrils and fibers protruding
from the yarn are removed by the cellulase, the surface of the
black fabric appears darker and nicer, and lower L values are
obtained. Different dosages of cloned cellulases from Thielavia
terrestris (SEQ ID NO: 12) and Acremonium sp. CBS 478.94 (SEQ ID
NO: 8), respectively, (denoted A and B, respectively) were
tested.
Results:
TABLE-US-00052 [0405] S-CEVU/l 0 500 2000 A 0 -2.09 .+-. 0.22 -2.86
.+-. 0.19 B 0 -0.60 .+-. 0.36 -1.96 .+-. 0.23
[0406] The data show that both cellulases gives good color
clarification under the conditions tested.
H-V. Performance of Cellulases Cloned from Thielavia terrestris and
Acremonium sp. CBS 478.94, and Construction 1 (SEQ ID NO: 3)
Measured as Removal of Surface Fibrils and Fibers Protruding from
the Yarn of a Textile Containing Cellulosic Fibers
TABLE-US-00053 Apparatus Terg-o-tometer Liquid volume 150 ml
Agitation 150 movements/min with vertical stirrer Rinse time 10 min
in tapwater Washing temp 35.degree. C. Hardness 1.0 mM CaCl.sub.2
0.34 mM MgCl.sub.2 Washing liqour 2.0 g/l HDL (neutral, citrate
built HDL, with nonionic/anionic weight ration >0.5) pH 7.5
Washing time 30 min Repetitions 2 Textile 2 swatches of aged black
cotton 5 .times. 6 cm (app. 150 g/m.sup.2) 2 swatches of heavy
knitted cotton 4 .times. 7 cm (app. 600 g/m.sup.2) Drying Tumble
dry
Evaluation:
[0407] The light remission is measured by a Datacolor Elrepho
Remission spectrophotometer. Remission is calculated as delta L
(CIE Lab-values). When the surface fibrils and fibers protruding
from the yarn are removed by the cellulase, the surface of the
black fabric appears darker and nicer, and lower L values are
obtained. Three different dosages of cloned cellulases from
Thielavia terrestris (SEQ ID NO: 12) and Acremonium sp. CBS 478.94
(SEQ ID NO: 8) and the construction 1 (SEQ ID NO: 3), respectively,
(denoted A and B and C, respectively) were tested.
Results:
TABLE-US-00054 [0408] S-CEVU/l 0 100 200 400 A 0 -3.06 .+-. 0.24
-3.15 .+-. 0.27 -3.92 .+-. 0.26 B 0 -1.75 .+-. 0.27 -3.08 .+-. 0.32
-3.51 .+-. 0.44 C 0 -1.84 .+-. 0.39 -1.70 .+-. 0.47 -2.30 .+-.
0.61
[0409] The data show that all cellulases give very good color
clarification under the conditions tested.
I. Application of Endoglucanases from Thielavia terrestris,
Acremonium sp. and Construction 1 (SEQ ID NO: 3) in Denim
Finishing
Experimental
TABLE-US-00055 [0410] Apparatus: Washing machine Wascator FL 120
Liquid volume: 20 L Fabric: 1.1 kg denim fabric, 141/2 oz 100%
cotton Desizing: 10 min, 55.degree. C., pH 7 50 ml Aquazyme 120 L
2.5 g/l Phosphate buffer Abrasion: 2 hours;
[0411] pH and temperature varied according to the following
table:
TABLE-US-00056 Enzyme SEQ ID Activity pH/temp Buffer system NO: 3
1400 S-CEVU/g 6/55.degree. C. 2.5 g/l phosphate buffer NO: 12 292
S-CEVU/g 5/65.degree. C. 1 g/l citrate buffer NO: 8 782 S-CEVU/g
7/45.degree. C. 2.5 g/l phosphate buffer
Inactivation: 15 min, 80.degree. C.
[0412] 1 g/l sodium carbonate
Rinses: Three rinse cycles of 5 min in cold tap water
Evaluation:
[0413] Abrasion: The remission from the fabric was determined at
420 nm using a Texflash 2000 as a measure of the abrasion
level.
[0414] The results from the treatment of the denim fabric with
different endoglucanases of the invention is shown in the following
table:
TABLE-US-00057 Abrasion Enzyme Dosage Trial conditions 420 nm Blank
0 S-CEVU/g textile pH 6, 55.degree. C. 9.96 SEQ ID NO: 3 10
S-CEVU/g textile pH 6, 55.degree. C. 14.37 Blank 0 S-CEVU/g textile
pH 5, 65.degree. C. 9.26 SEQ ID NO: 12 10 S-CEVU/g textile pH 5,
65.degree. C. 16.86 Blank 0 S-CEVU/g textile pH 7, 45.degree. C.
9.47 SEQ ID NO: 8 10 S-CEVU/g textile pH 7, 45.degree. C. 14.08
[0415] All tested cellulases show excellent performance in denim
finishing, although each enzyme is unique in its own way, When
applying the enzyme corresponding to SEQ ID NO: 3 for denim
finishing it is possible to reach a high abrasion level with a
minimum of strength loss. When treating denim with the enzyme
corresponding to SEQ ID NO: 12, a very high wash down can be
reached which leaves the fabric with an almost bleached appearance.
Denim finishing with the enzyme corresponding to SEQ ID NO: 8 gives
a high abrasion level at a tow temperature optimum which makes it
possible to reduce the processing temperature and save energy.
J. Use of Cloned Cellulases from Acremonium sp. and Thielavia
Terrestris for Biopolishing of Lyocell Fibers
[0416] Lyocell fibers which are sold under the trade name Tencel
are spun from wood pulp its cellulose in a more environmentally
friendly waterbased solvent than is the case for normal viscose
production). How ever, the fibers have a tendency to fibrillate
when they are processed into textiles which is seen on the surface
and denoted "fuzz". By using cellulases it is possible to
permanently remove the exposed and Fuzzy fibers and significantly
improve the look of the finished fabric, the treatment generally
known as Biopolishing. The endoglucanases of the present invention
are especially suited for the removal of Lyocell surface
fibers,
Materials and Methods
[0417] The textile substrate was either 100% woven or different
kinds of jersey knitted dark blue almost. The dark colour and
jersey knit was preferred in order to enhance the visual effects
which simplified the evaluation. A woven 70/30 Tencel/Rayon blend
was also used to a lesser extent.
[0418] The assays were either performed in 200 ml scale using a
Launder-o-meter or in the 20 l scale using a Wascator. The
treatment time was 60 min at 55.degree. C. in Wascator and 60-90
min in LOM. The buffer was 2 g/l sodium acetate adjusted to pH 5
with acetic acid, The fabric to liquid ratio was 1:10 but in the
Launder-o-meter 20 steel balls with a diameter of 14 mm (11 g each)
was used to obtain sufficient mechanical abrasion. The biopolishing
was immediately followed by inactivation using 2 g/l sodium
carbonate at 80.degree. C. for 15 min followed by rinsing in cold
water.
[0419] The results were evaluated using a fuzz note scale from 1-5
were 1 is the fibrillated look of the starting material and 5 is a
high quality look with no visible fibers on the surface. Since the
performance of endocellulases is specific towards a surface
treatment the weightloss is below 2% and is therefore not included
in the evaluation. Two cellulases were evaluated: the cellulases
cloned from Acremonium sp. (SEQ ID NO: 8) and from Thielavia
terrestris (SEQ ID NO: 12).
[0420] The two cellulases are able to defibrillate both Tencel and
Tencel blended fabrics. By using an endoglucanase of the invention,
only small fibrils are removed rather than whole fibers such as is
the case when using acid cellulase mixtures from Trichoderma. The
strength loss of the treated fabric is therefore kept at a minimum
when using endoglucanases of the present invention.
[0421] The following dosages gave a superior defibrillation, i.e.,
fuzz note 4 or above:
15 S-CEVU/g fabric of cellulase from Acremonium sp (SEQ ID NO: 8);
and 10 S-CEVU/g fabric of cellulase from Thielavia terrestris (SEQ
ID NO: 12).
EXAMPLE 2
A New Cellulytic Enzyme was by Expression Cloning as Well as by PCR
Cloning Detected to be Produced by a Plant Pathogen, Isolated from
Soy Bean Seeds and Identified as Macrophomina Phaseolina
Production of Biomass for PCR and Expression Cloning
Procedures:
[0422] Isolate CBS 281.96 was grown in shake flask cultures on
cellulose enriched potato dextrose broth, incubated for 5 days at
260 C (shaking conditions: 150 rpm).
A. Cloning and Expression of an Endoglucanase from Macrophomina
Phaseolina
[0423] mRNA was isolated from Macrophomina phaseolina, grown in a
cellulose-containing fermentation medium with agitation to ensure
sufficient aeration. Mycelia were harvested after 3-5 days' growth,
immediately frozen in liquid nitrogen and stored at -80.degree. C.
A library from Macrophomina phaseolina, consisting of approx.
10.sup.6 individual clones was constructed in E. coli as described
with a vector background of 1%.
[0424] Plasmid DNA from some of the pools was transformed into
yeast, and 50-100 plates containing 250-400 yeast colonies were
obtained from each pool.
[0425] Endoglucanase-positive colonies were identified and isolated
on SC-agar plates with the AZCL HE cellulose assay, cDNA inserts
were amplified directly from the yeast colonies and characterized
as described in the Materials and Methods section above. The DNA
sequence of the cDNA encoding the endoglucanase is SEQ ID NO: 13
and the corresponding amino acid sequence is SEQ ID NO: 14.
[0426] The cDNA is obtainable from the plasmid in DSM 10512.
[0427] Total DNA was isolated from a yeast colony and plasmid DNA
was rescued by transformation of E. coli as described above. In
order to express the endoglucanase in Aspergillus, the DNA was
digested with appropriate restriction enzymes, size fractionated on
gel, and a fragment corresponding to the endoglucanase gene was
purified, The gene was subsequently ligated to pHD414, digested
with appropriate restriction enzymes, resulting in the plasmid
pA2C477.
[0428] After amplification of the DNA in E. coli the plasmid was
transformed into Aspergillus oryzae as described above.
Screening of the cDNA Library by Hybridization and Characterization
of the Positive Clones.
[0429] Approximately 6000 colony forming units (c.f.u.) from the
Macrophomina phaseolina cDNA library in E. coli was screened by
colony hybridization using a random-primed .sup.32P-labeled PCR to
product from M. phaseolina as probe, The PCR product was generated
as described in the Materials and Methods section. The positive
cDNA clones were characterized by sequencing the ends of the cDNA
inserts, and by determining the nucleotide sequence of the longest
cDNA from both strands. The DNA sequence of the cDNA encoding the
endoglucanase is SEQ ID NO: 13 and the corresponding amino acid
sequence is SEQ ID NO: 14.
B. Construction of Gene Fusion Between the Endoglucanase from
Macrophomina Phaseolina and the 43 kD Endoglucanase from Humicola
insolens
[0430] One construction was prepared in order to make a derivative
of the endoglucanase from M. phaseolina with the linker and CBD
from the 43 kD endoglucanase from H. insolens (disclosed in WO
91/17243) The native endoglucanase from M. phaseolina does not have
a linker and/or a cellulose binding domain, CBD.
[0431] The construction consists of the endoglucanase from M.
phaseolina (222 amino acids) and the 72 C-terminal amino acids from
the H. insolens 43 kD endoglucanase (SEQ ID NO: 24).
[0432] The 43 kD endoglucanase cDNA from H. insolens is cloned into
pHD414 in such a way that the endoglucanase gene is transcribed
from the Taka-promoter. The resulting plasmid is named
pCaHj418.
[0433] The cDNA encoding the endoglucanase from M. phaseolina (SEQ
ID NO: 13) is cloned into pYES2.0 as a BstX I/Not I fragment and
the resulting plasmid is named pC1C477.
[0434] Primers:
TABLE-US-00058 primer 1: (SEQ ID NO: 94)
5'-GGTCGCCCGGACTGGCTGTTCCCGTACCCCCTCCAGCAGCACCAGCT CTCCGG-3' primer
2: (SEQ ID NO: 95)
5'-CCGGAGAGCTGGTGCTGCTGGAGGGGGTACGGGAACAGCCAGTCCGG GCGACC-3'
pYES2.0 F.HT primer: (SEQ ID NO: 96) 5'-CGGACTACTAGCAGCTGTAATACG-3'
AMO-term Primer: (SEQ ID NO: 93) 5'-CCCCATCCTTTAACTATAGCG-3'
[0435] The endoglucanase fusion is constructed by the PCR
overlap-extension method as described by Higuchi et al. 1988.
[0436] Reaction A: The Polymerase Chain Reaction (PCR) is used to
amplify the fragment of pCaHj418 between primer 1 and AMG-term.
primer (the linker and CBD from the 43 kD 2 endoglucanase from H.
insolens).
[0437] Reaction B: PCR amplification of the fragment between
pYES2.0 F.HT primer and primer 2 in pC1C477, the endoglucanase gene
from M. phaseolina.
[0438] Reaction C. The two purified fragments are used in a third
PCR in the presence of the primers flanking the total region, i.e.,
pYES2.0 F.HT primer and AMG-term. primer.
[0439] The fragment amplified in reaction C is purified, digested
with restriction enzymes, e.g. Xba I and BamHI. The purified
digested fragment is ligated into pHD414 digested with restriction
enzymes, e.g., Xba I and BamH I.
[0440] Competent cells from E. coli strain DH5.alpha.F' (New
England Biolabs) are transformed with the ligated plasmid and
colonies containing the gene fusion are isolated. The sequence of
the cloned to part was verified by DNA sequencing.
[0441] Polymerase Chain Reactions are carried out under standard
conditions, as recommended by Perkin-Elmer.
[0442] Reaction A and B start with 2 min. at 94.degree. C. followed
by 20 cycles of (30 sec. at 94.degree. C., 30 sec. at 52.degree. C.
and 1 min. at 72.degree. C.) and ends with 4 min. at 72.degree.
C.
[0443] Reaction C starts with (2 min. at 94.degree. C., 1 min. at
52.degree. C. and 2 ml. at 72.degree. C.), followed by 20 cycles of
(30 sec. at 94.degree. C., 30 sec. at 52.degree. C. and 90 sec. at
72.degree. C.) and ends with 4 min. at 72.degree. C.
[0444] The construct may be transformed into Aspergillus oryzae as
described above.
EXAMPLE 3
Cloning and Expression of an Endoglucanase from Acremonium sp. and
Sordaria fimicola Production of Biomass for Expression Cloning
Procedures
[0445] Isolates CBS 478.94 and ATCC 52644, respectively, were grown
in shake flask cultures on cellulose enriched potato dextrose
broth, incubated for 5 days at 260 C (shaking conditions: 150
rpm).
[0446] mRNA was isolated from Acremonium sp., CBS 478.94, and
Sordaria fimicola, ATCC 52644, respectively, grown in a
cellulose-containing fermentation medium with agitation to ensure
sufficient aeration. Mycelia were harvested after 3-5 days' growth,
immediately frozen in liquid nitrogen and stored at -80.degree. C.
Libraries from Acremonium sp., and Sordaria fimicola, respectively,
each consisting of approx. 106 individual clones were constructed
in E. coli as described with a vector background of 1%.
[0447] Plasmid DNA from some of the pools from each library was
transformed into yeast, and 50-100 plates containing 250-400 yeast
colonies were obtained from each pool.
[0448] Endoglucanase-positive colonies were identified and isolated
on SC-agar plates with the AZCL HE cellulose assay. cDNA inserts
were amplified directly from the yeast colonies and characterized
as described in the Materials and Methods section above.
[0449] The DNA sequence of the cDNA encoding the endoglucanase from
Acremonium sp. is SEQ ID NO: 9 and the corresponding amino acid
sequence is SEQ ID NO: 10. The cDNA is obtainable from the plasmid
in DSM 10080.
[0450] The partial DNA sequence of the cDNA encoding the
endoglucanase from Sordaria fimicola is SEQ ID NO-25 (Nucleotide
sequence of the 5'-end of the cDNA) and the corresponding amino
acid sequence is SEQ ID NO: 26. The cDNA is obtainable from the
plasmid in DSM 10576.
[0451] Total DNA was isolated from a yeast colony and plasmid DNA
was rescued by transformation of E. coli as described above. In
order to express the endoglucanase in Aspergillus, the DNA was
digested with appropriate restriction enzymes, size fractionated on
gel, and a fragment corresponding to the endoglucanase gene from
Acremonium sp. and Sordaria fimicola, respectively, was purified.
The genes were subsequently ligated to pHD414, digested with
appropriate restriction enzymes, resulting in the plasmids pA2C371
and pA2C502, respectively.
[0452] After amplification of the DNA in E. coli the plasmids were
transformed into Aspergillus oryzae as described above.
EXAMPLE 4
A. Cloning by PCR an Endoglucanase from Crinipellis scabella, CBS
280.96
[0453] Isolate CBS 280.96 was grown in static flask cultures,
holding wheat bran medium (per flask, 300 g wheat bran added 450 ml
salt solution), incubated for 6 days at 26.degree. C. After
incubation the wheat bran was extracted with distilled water (300
ml per flask) and the extract tested for endoglucanase activity
(0.1% AZCL-HE-Cellulose (megazyme) in 1% agarose (Litex agarose,
Medinova). Activity was observed on the plates holding pH of 3.0,
7.0 and 9.5.
[0454] mRNA was isolated from Crinipellis scabella grown as
describe above. Mycelia were harvested after 3-5 days' growth,
immediately frozen in liquid nitrogen and stored at -80.degree. C.
A library from Crinipellis scabella, consisting of approx. 10.sup.6
individual clones was constructed in E. coli as described with a
vector background of 1%.
[0455] Approximately 10 000 colony forming units (c.f.u.) from the
Crinipellis scabella cDNA library in E. coli was screened by colony
hybridization using a random-primed .sup.32P-labeled PCR product
from C. scabella as probe. The PCR product was generated as
described in the Materials and methods section. The positive cDNA
clones were characterized by sequencing the ends of the cDNA
inserts, and by determining the nucleotide sequence of the longest
cDNA from both strands.
[0456] The DNA sequence of the cDNA encoding the endoglucanase is
SEQ ID NO: 15 and the corresponding amino acid sequence is SEQ ID
NO: 16.
[0457] The cDNA is obtainable from the plasmid in DSM 10511.
[0458] Total DNA was isolated from a yeast colony and plasmid DNA
was rescued by transformation of E. coli as described above. In
order to express the endoglucanase in Aspergillus, the DNA was
digested with appropriate restriction enzymes, size fractionated on
gel, and a fragment corresponding to the endoglucanase gene was
purified. The gene was subsequently ligated to pHD414, digested
with appropriate restriction enzymes, resulting in the plasmid
pA2C475.
[0459] After amplification of the DNA in E. coli the plasmid was
transformed into Aspergillus oryzae as described above.
Construction of Two Gene Fusions Between the Endoglucanase from
Crinipellis Scabella and the Linker/CBD Region of the 43 kDa
Endoglucanase from Humicola insolens.
[0460] The native endoglucanase from Crinipellis scabella neither
has a linker nor a cellulose binding domain (CBD). In addition, the
full-length cDNA contains an ATG start codon upstream from the
endoglucanase encoding open reading frame (ORF), presumably
resulting in scrambled translation initiation upon heterologous
expression of the cDNA, such as in the yeast Saccharomyces
cerevisiae and the filamentous fungus Aspergillus oryzae. Thus, two
gene fusions between the endoglucanase from Crinipellis scabella
and the linker/CBD region of the 43 kD endoglucanase from Humicola
insolens (disclosed in WO 91/17243) has been constructed using
splicing by overlap extension (SOE) (Horton et al, 1989).
[0461] Construction 1 consists of the cDNA encoding the 226-residue
endoglucanase from C. scabella fused by PCR with the 3'-end cDNA of
H. insolens coding for the linker and CBD region (72 amino acids)
at the COOH-terminus of the H. insolens 43 kD endoglucanase. The
second hybrid construct is identical to the abovementioned gene
fusion, except that the first five residues from the putative
signal peptide have been deleted by PCR resulting in a shorter
signal, which starts with the second in-frame ATG start codon.
Plasmid Constructs
[0462] The plasmid pC1C475 contains the full-length cDNA from C.
scabella, cloned into BstXI/NotI-cut yeast expression vector pYES
2.0, the plasmid pC1C144 contains the full-length cDNA from H.
insolens, cloned into the BstXI site of pYES 2.0.
Splicing by Overlap Extension
[0463] Two PCR fragments encoding the core region of the
endoglucanase from C. scabella were generated in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% gelatin;
containing 200 micro-M each dNTP), using 50-100 ng of pC1C475 as
template, and 250 pmol of the reverse primer
(5'-GACCGGAGAGCTGGTGTGCTGGAGGGTTTACGACACAGCCCGAGATATAGTG-3 (SEQ ID
NO: 97)) in two combinations with 300-350 pmol of each forward
primer (forward no. 1 5'-CCCCAAGTTGACTTGGAACCAATGGTCCATCG-3' (SEQ
ID NO: 98), forward no. 2
5'-CCCCAAGCTTOCATOCAAACATGCTTAAAACOGTOG-3, (SEQ ID NO: 99)), a DNA
thermal cycler (Landgraf, Germany) and 2.5 units of Taq polymerase
(Perkin-Elmer, Cetus, USA). Thirty cycles of PCR were performed
using a cycle profile of denaturation at 94.degree. C. for 1 min,
annealing at 55.degree. C. for 2 min, and extension at 72.degree.
C. for 3 min. The PCR fragment coding for the linker and CBD of the
endoglucanase of H. insolens was generated in PCR buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% gelatin:
containing 200 micro-M each dNTP) using 100 ng of the pC1C144
template, 250 pmol forward primer
(5'-CACTAATATCTCGGCTGTGTTCGTAACCCTCCAGCAGCACCAGCTCTCCGGTC-3'(SEQ ID
NO:100)), 250 pmol of the pYES 2.0 reverse primer
(5'-GGGCGTGAATGTAAGCGTGACATA-3' (SEQ ID NO-101)), a DNA thermal
cycler (Landgraf, Germany) and 2.5 units of Taq polymerase
(Perkin-Elmer, USA). Thirty cycles of PCR were performed as above.
The PCR products were electrophoresed in 0.7% low gelling
temperature agarose gels (SeaPlaque, FMC), the fragments of
interest were excised from the gel and recovered by treatment with
agarase (New England Biolabs, USA) according to the manufacturer's
instructions, followed by phenol extraction and ethanol
precipitation at -20.degree. C. for 12 h by adding 2 vols of 96%
EtOH and 0.1 vol of 3 M NaAc.
[0464] The recombinant hybrid genes between the endoglucanase from
Crinipellis scabella and the linker/CBD region of the 43 kD
endoglucanase from Humicola insolens were generated by combining
the overlapping PCR fragments from above (ca 50 ng of each
template) in two combinations in PCR buffer (10 mM Tris-HCl, pH
8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.01% gelatin; containing 200
micro-M each dNTP). The SOE reaction was carried out using the DNA
thermal cycler (Landgraf, Germany) and 2.5 units of Taq polymerase
(Perkin-Elmer, Cetus, USA). Two cycles of PCR were performed using
a cycle profile of denaturation at 94.degree. C. for 1 min,
annealing at 55.degree. C. for 2 min, and extension at 72.degree.
C. for 3 min, the reaction was stopped, 250 pmol of each end-primer
(forward no. 1 5'-CCCCAAGCTTGACTTGGAACCAATGGTCCATCC-3' (SEQ ID NO:
98), forward no. 2 5'-CCCCAAGCCTCCATCCAAACATGCTTAAAACGCTCG-3' (SEQ
ID NO: 99), reverse primer 5'GGGCGCTGAATGTAAGCGTGACATA-3' (SEQ ID
NO: 101)) was added to the reaction mixture, and an additional 30
cycles of PCR were performed using a cycle profile of denaturation
at 94.degree. C. for 1 min annealing at 55.degree. C. for 2 min,
and extension at 72.degree. C. for 3 min.
Construction of the Expression Cassettes for Heterologous
Expression in Aspergillus oryzae
[0465] The PCR-generated, recombinant fragments were
electrophoresed in a 0.7% low gelling temperature agarose gel
(SeaPlaque, FMC), the fragments of interest were excised from the
gel and recovered by treatment with agarase (New England Biolabs,
USA) according to the manufacturer's instructions, followed by
phenol extraction and ethanol precipitation at -20.degree. C. for
12 h. The DNA fragments were digested to completion with HindIII
and XbaI, and ligated into HindIII/XbaI-cleaved pHD414 vector
followed by electroporation of the constructs into E. coli DH10B
cells according to the manufacturer's instructions (Life
Technologies, USA).
[0466] The nucleotide sequence of the resulting gene fusions was
determined from both strands as described in the Materials and
methods section, SEQ ID NOS: 17 and 19. The constructs may be
transformed into A. oryzae as described.
EXAMPLE 5
PCR Facilitated Detection of Said Type of Cellulytic Enzyme from 46
Filamentous and Monocentric Fungi, Representing 32 Genera, from 23
Families, Belonging to 15 Orders of 7 Classes, Covering all in all
all Four Groups of the True Fungi: Ascomycetous, Basidiomnycetous,
Chytridiomycetous and Zygomycetous fungi
5.1 Materials
[0467] 1. Diplodia gossypina Cooke
Deposit of Strain, Acc No: CBS 274.96
[0468] 2. Ulospora bilgramii (Hawksw et al.) Acc No of strain: NKBC
1444,
3. Microsphaeropsis sp
[0469] 4. Ascobolus stictoideus Speg. Acc No of strain: Q026 (Novo
Nordisk collection) Isolated from goose dung, Svalbard, Norway 5.
Saccobolus dilutellus (Fuck) Sacc. Deposit of strain: Acc No CBS
275.96 6. Penicillium verruculosum Peyronel Ex on Acc No of species
ATCC 62396 7. Penicillium chrysogenum Thom
Acc No of Strain: ATCC 9480
[0470] 8. Thermomyces verrucosus Pugh et al
Deposit of Strain, Acc No.: CBS 285.96
[0471] 9. Xylaria hypoxylon L. ex Greville Deposit of Strain. Acc
No: CBS 284.96 10. Poronia punctata (Fr. ex L.) Fr.
Ref:A.Munk: Danish Pyrenomycetes,
Dansk Botanisk Arkiv, Vol 17, 1 1957
11. Nodulisporam sp
[0472] Isolated from leaf of Camellia reticulata (Theaceae,
Guttiferales),
Kunming Botanical Garden, Yunnan Province, China
12. Cylindrocarpon sp
[0473] Isolated from marine sample, the Bahamas 13. Fusarium
anguioides Sherbakoff Acc No of strain: IFO 4467 14. Fusarium poae
(Peck) WV. Ex on Acc No of species: ATCC 60883 15. Fusarium solani
(Mart.)Sacc.emnd.Snyd & Hans. Acc No of strain: IMI 107.511 16.
Fusarium oxysporum ssp lycopersici (Sacc.)Snyd. & Hans. Acc No
of strain: CBS 645.78 17. Fusarium oxysporum ssp pass/flora Acc No
of strain: CBS 744.79 18. Gliocladium catenulatum Gilman &
Abbott Acc. No of strain: ATCC 10523 19. Nectria pinea Dingley
Deposit of Strain, Acc. No. CBS 279.96 20. Sorda a macrospora
Auerswald ex on Acc No of species. ATCC 60255 21. Humicola grisea
Traeen ex on Acc No for the species: ATCC 22726 22. Humicola
nigrescens Omvik Acc No of strain: CBS 819.73 23. Scytalidium
thermophilum (Cooney et Emerson) Austwick Acc No of strain: ATCC
28085 24. Thielavia thermophila Fergus et Sinden (syn Corynascus
thermophilus) Acc No of strain: CBS 174.70, IMI 145.136 25.
Cladorrhinum foecundissimum Saccardo et Marchal Ex on Acc No of
species: ATCC 62373 26. Syspastospora boninensis Acc No of strain:
NKBC 1515 (Nippon University, profe Tubaki Collection) 27.
Chaetomium cuniculorum Fuckel Acc. No, of strain: CBS 799.83 28.
Chaetomium brasiliense Batista et Potual Acc No of strain: CBS
122.65 29. Chaetomium murorum Corda Acc No of strain: CBS 163.52
30. Chaetomium virescens (von Arx) Udagawa Acc. No. of strain: CBS
547.75
31. Nigrospora sp
[0474] Deposit of strain, Acc No: CBS 272.96
32. Nigrospora sp
[0475] Isolated from: 33. Diaporthe syngenesia Deposit of strain,
Acc No: CBS 278.96 34. Colletotrichum lagenarium (Passerini) Ells
et Halsted syn Glomerella cingulata var orbiculare Jenkins et
Winstead Ex on acc No of species: ATCC 52609 35. Exidia glandulosa
Fr.
Deposit of Strain, Acc No: CBS 277.96
[0476] 36. Fomes fomentarius (L.) Fr. deposit of strain: Ac No. CBS
276.96
37. Spongipellis (?)
Deposit of Strain, Acc No CBS 283.96
[0477] 38. Rhizophlyctis rosea (de Bary & Wor) Fischer
Deposit of Strain: Acc No.: CBS 282.96
[0478] 39. Rhizomucor pusillus (Lindt) Schipper syn: Mucor pusillus
Acc No of strain: IFO 4578 40. Phycomyces nitens (Kunze) van
Tieghem & Le Monnier Acc No of strain: IFO 4814 41.
Chaetostylum fresenii van Tieghem & Le Monnier syn.
Helicostylum fresenii Acc No of strain NRRL 2305 42. Trichothecium
roseum, Acc No of strain: IFO 5372
43. Coniothecium sp,
[0479] Endophyte, isolated from leaf of flowering plant,
Kunming, Yunnan, China
[0480] 44. Deposit of strain, Acc No., CBS 271.96 Coelomycete,
isolated from leaf of Artocarpus altilis
(Moraceae, Urticales), Christiana, Jamaica
[0481] 45, Deposit of strain, Acc No.: CBS 273.96 Coelomycete,
isolated from leaf of Pimenta dioica
(Myrtaceae, Myrtales), Dallas Mountain, Jamaica
[0482] 46. Deposit of strain, CBS 270.96 Coelomycete, isolated from
leaf of Pseudocalymma alliaceum (Bignoniaceae, Solanales) growing
in Dallas Mountain, Jamaica
5.2 Procedure
Maintenance of Strains and Production of Biomass:
[0483] The strains were maintained on agar in petrie dishes (9 cm)
or on slants (see list of Media: PCA and PDA). 44 of the strains
were grown in shake flasks under the following growth conditions:
general fungal media as PC, PD and PB 9 or YPG (see list of media);
incubation time from 3 to 9 days, temperature 26.degree. C.; rpm
between 150 and 175. Strain No 14 (F. poae) was grown on wheat bran
for 15 days (26.degree. C.; static) Strain No 38 was grown in
dilute salt solution (DS/2), added 1 cm.sup.2 pieces of autoclaved
filter paper.
Activity Test:
[0484] Activity was tested on 0.1% AZCL-HE-Cellulose (Megazyme)
plates (14 cm Petrie dishes), made up in 1% agarose (HSB, Litex
Agarose, Medinova). All tests were done in triplicate, viz.
AZCL-HE-Cellulose dissolved in three buffers, adjusted to pH 3, 7
or 9.5 (using various proportions of the following two ingredients
Citric acid monohydrate, Merck art. No 100244 (21.0 g) dissolved in
water, making a total of 1000 ml, 0.1M tri-Sodium dodecabrohydrate,
Merck art. no. 6578 (38 g), dissolved in water, making a total of
1000 ml. The mixing is done immediately before use.
Harvesting of Biomass:
[0485] The biomass was harvested by filtering (mesh adjusted to the
growth of the fungus, the finest used for fungi which have highly
sporulating mycelium as, e.g., Fusarium spp.) The biomass on the
filter was scraped into a sterile plastic bag and immediately
frozen (by submerging into liquid nitrogen).
5.3 Results
[0486] I. Using the PCR screening and amplification techniques
described in Materials and Methods the following partial cDNA
sequences were obtained:
[0487] Saccobolus dilutellus (Fuck) Sacc., CBS 275.96: SEQ ID NO:
27 (and the deduced amino acid sequence in SEQ ID NO: 28);
[0488] Thermomyces verrucosus, CBS 285.96; SEQ ID NO: 29 (and the
deduced amino acid sequence in SEQ ID NO: 30);
[0489] Xylaria hypoxylon, CBS 284.96 SEQ ID NO: 31 (and the deduced
amino acid sequence in SEQ ID NO: 32);
[0490] Fusarium oxysporum ssp lycopersici, CBS 645.78: SEQ ID NO:
33 (and the deduced amino acid sequence in SEQ ID NO: 34);
[0491] Nectria pinea, CBS 279.96: SEQ ID NO: 35 (and the deduced
amino acid sequence in SEQ ID NO:36);
[0492] Humicola grisea, ATCC 22726: SEQ ID NO: 37 (and the deduced
amino acid sequence in SEQ ID NO: 38);
[0493] Humicola nigrescens CBS 819.73 SEQ ID NO: 39 (and the
deduced amino acid sequence in SEQ ID NO: 40);
[0494] Cladorrhinum foecundissimum ATCC 62373: SEQ ID NO: 41 (and
the deduced amino acid sequence in SEQ ID NO: 42);
[0495] Syspastospora boninensis, NKBC 1515: SEQ ID NO: 43 (and the
deduced amino acid sequence in SEQ ID NO: 44):
[0496] Nigrospora sp., CBS 272.96: SEQ ID NO: 45 (and the deduced
amino acid sequence in SEQ ID NO: 46);
[0497] Chaetostylum fresenii: SEQ ID NO: 47 (and the deduced amino
acid sequence in SEQ ID NO: 48);
[0498] Exidia glandulosa, CBS 277.96: SEQ ID NO: 49 (and the
deduced amino acid sequence in SEQ ID NO: 50);
[0499] Coniothecium sp.: SEQ ID NO: 51 (and the deduced amino acid
sequence in SEQ ID NO: 52);
[0500] Deposition No. CBS 271.96: SEQ ID NO: 53 (and the deduced
amino acid sequence in SEQ ID NO: 54);
[0501] Deposition No, CBS 270.96: SEQ ID NO: 55 (and the deduced
amino acid sequence in SEQ ID NO: 56);
[0502] Diplodia gossypina, CBS 274.96: SEQ ID NO: 57 (and the
deduced amino acid sequence in SEQ ID NO: 58);
[0503] Ulospora bilgramii, NKBC 1444: SEQ ID NO: 59 (and the
deduced amino acid sequence in SEQ ID NO: 60);
[0504] Penicillium verruculosum, ATCC 62396: SEQ ID NO: 61 (and the
deduced amino acid sequence in SEQ ID NO: 62);
[0505] Poronia punctata: SEQ ID NO: 63 (and the deduced amino acid
sequence in SEQ ID NO: 64);
[0506] Fusarium anguioides, IFO 4467 SEQ ID NO: 65 (and the deduced
amino acid sequence in SEQ ID NO: 6);
[0507] Thielavia thermophila, CBS 174.70: SEQ ID NO: 67 (and the
deduced amino acid sequence in SEQ ID NO: 68);
[0508] Chaetomium cuniculorum, CBS 799.83: SEQ ID NO: 69 (and the
deduced amino acid sequence in SEQ ID NO: 70);
[0509] Chaetomium virescens: SEQ ID NO: 71 (and the deduced amino
acid sequence in SEQ ID NO: 72);
[0510] Colletotrichum lagenarium: SEQ ID NO: 73 (and the deduced
amino acid sequence in SEQ ID NO: 74);
[0511] Phycomyces nitens: SEQ ID NO: 75 (and the deduced amino acid
sequence in SEQ ID NO: 76); and
[0512] Trichothecium roseum: SEQ ID NO: 77 (and the deduced amino
acid sequence in SEQ ID NO: 78);
[0513] II. Using the PCR screening and amplification techniques
described in Materials and Methods partial cDNA encoding partially
for the enzyme of the invention was obtained and the plasmid was
deposited according to the Budapest Treaty:
Escherichia coli, DSM 10583, deposition date 13 Mar. 1996; cDNA
from Trichothecium roseum; Escherichia coli, DSM 10584, deposition
date 13 Mar. 1996, cDNA from Syspastospora boninensis; Escherichia
coli, DSM 10585, deposition date 13 Mar. 1996; cDNA from Cheatomium
murorum; Escherichia coli, DSM 10587, deposition date 13 Mar. 1996;
cDNA from Sordaria fimicola; Escherichia coli, DSM 10588,
deposition date 13 Mar. 1996; cDNA from the unidentified strain CBS
273.96; Escherichia coli, DSM 10586, deposition date 13 Mar. 1996;
cDNA from Spongipeillis sp.
Color Clarification of Crude Supernatants
[0514] During normal wash the fabric will often fade. However, the
fabric appearance is improved and the original colours are much
better preserved or maintained if the fabric is washed with a
cellulase giving color clarification. Color clarification is
measured as removal of surface fibrils and fibers protruding from
the yarn of a textile containing cellulosic fibers.
TABLE-US-00059 Apparatus Terg-o-tometer Liquid volume 100 ml
Agitation 150 movements/min with vertical stirrer Rinse time 5 min
in tapwater Washing temp 40.degree. Washing liqour 0.05 M phosphate
buffer pH 7.0 Washing time 30 min Repetitions 2 Enzymes Crude
supernatants from the strains shown below. Dosage Two dosages from
200, 500, 1000 or 2500 S-CEVU/l Textile 2 swatches of aged black
100% cotton 5 .times. 6 cm (0.9 gram) Drying Tumble dry
Evaluation:
[0515] The light remission is measured by a Datacolor Elrepho
Remission spectrophotometer. Remission is calculated as delta L
(Hunter Lab-values). When the surface fibrins and fibers protruding
from the yarn are removed by the cellulase, the surface of the
black fabric appears darker, and lower L values are obtained.
[0516] The samples are compared with a blind sample, i.e., washed
without enzyme. Below is shown the delta L remission values
compared to a blind sample.
REFERENCES
Background of the Invention
[0517] 1. GB-A-1368599 [0518] 2. EP-A-0 307 564 [0519] 3. EP-A-0
435 876 [0520] 4. WO 91/17243 [0521] 5. WO 91/10732 [0522] 6. WO
91/17244 [0523] 7. WO 95/24471 [0524] 8. WO 95/26398 [0525] 9.
Methods in Enzymology, 1988, Vol. 160, p. 200-391 (edited by Wood,
W. A. and Kellogg, S. T.). [0526] 10. Beguin. P., "Molecular
Biology of Cellulose Degradation", Annu. Rev. Microbiol. (1990),
Vol. 44, pp. 219-248. [0527] 11. Henrissat, B., "Cellulases and
their interaction with cellulose", Cellulose (1994), Vol. 1, pp.
169-196. [0528] 12. T.-M. Enveri, "Microbial Cellulases" in W. M.
Fogarty, Microbial Enzymes and Biotechnology, Applied Science
Publishers, 183-224 (1983). [0529] 13. Beguin, P. and Aubert, J-P.,
"The biological degradation of cellulose", FEMS Microbiology
Reviews 13 (1994) 2558. [0530] 14. Sheppard, P. O., et al., "The
use of conserved cellulase family-specific sequences to clone
Cellulase homologue cDNAs from Fusarium oxysporum, Gene, (1994),
Vol. 15, pp. 163-167. [0531] 15. Saloheimo, A., et al., "A novel,
small endoglucanase gene, egl5, from Trichoderma reesei, isolated
by expression in yeast", Molecular Microbiology (1994), Vol. 13(2),
pp. 219-228. [0532] 16. van Arsdell, J. N. et at., (1987) Cloning,
characterization, and expression in Saccharomyces cerevisiae of
endoglucanase I from Trichoderma reesei, Bio/Technology 5: 60-64.
[0533] 17. Penttila, M. et at, (1986) Homology between cellulase
genes of Trichoderma reesei complete nucleotide sequence of the
endoglucanase I gene. Gene 45: 253-263. [0534] 18. Saloheimo, M. et
al, (1988) EGIII, a new endoglucanase from Trichoderma reesei the
characterization of both gene and enzyme. Gene 63: 11-21. [0535]
19. Gonzales, R., et at., "Cloning, sequence analysis and yeast
expression of the egl1 gene from Trichoderma longibrachiatum",
Appl. Microbiol. Biotechnol., Vol. 38, pp. 370375 (1992). [0536]
20. Ooi, T. et at. "Cloning and sequence analysis of a cDNA for
cellulase (FI-CMCase) from Aspergillus aculeatus" Curr. Genet.,
Vol. 18, pp. 217-222 (1990), [0537] 21. Ooi, T. et al., "Complete
nucleotide sequence of a gene coding for Aspergillus aculeatus
cellulase (Fl-CMCase)" Nucleic Acids Research, Vol. 18, No. 19, p.
5884 (1990). [0538] 22. Xue, G. et at., "Cloning and expression of
multiple cellulase cDNAs from the anaerobic rumen fungus
Neocallimastix patriciarum in E. coli, J. Gen. Microbiol., Vol.
138, pp. 1413-1420 (1992). [0539] 23. Xue, G. et al., "A novel
polysaccharide hydrolase cDNA (celD)) from Neocallimastix
patriciarum encoding three multifunctional catalytical domains with
high endoglucanase, cellobiohydrolase and xylanase activities", J.
Gen. Microbiol., Vol. 138, pp. 2397-2403 (1992). [0540] 24. Zhou,
L, et al., "Intronless celB from the anaerobic fungus
Neocallimastix patriciarum encodes a modular family A
endoglucanase", Biochem. J., Vol, 297, pp. 359-364 (1994). [0541]
25. Dalboge, H. and Heldt-Hansen, H. P. "A novel method efficient
expression cloning of fungal enzyme genes", Mol. Gen. Genet., Vol.
243, pp. 253-260 (1994). [0542] 26. Ali, B. R. S. et al.,
"Cellulases and hemicellulases of the anaerobic fungus Piromyces
constitute a multiprotein cellulose-binding complex and are encoded
by multigene families", FEMS Microbiol. Lett., Vol. 125, No. 1, pp.
15-21 (1995). [0543] 27. DNA Data Bank of Japan (DDBJ). [0544] 28.
Wang, H. Y. and Jones, R. W.: "Cloning, characterization and
functional expression of an endoglucanase-encoding gene from the
phytopathogenic fungus Macrophomina phaseolina", Gene, 158:125-128,
1995. [0545] 29. Wang, H. Y. and Jones, R. W.: "A unique
endoglucanase-encoding gene cloned from the phytopathogenic fungus
Macrophomina phaseolina", Appl. and Environm. Microbiology,
61:2004-2006, 1995. [0546] 30. B. Henrissat: Biochem. J., 280: 309,
316, 1991. [0547] 31. Schauwecker, F., Wanner, G., Kahmann, R.:
"Filament-specific expression of a cellulase gene in the dimorphic
fungus Ustilago maydis", 1995, Biological Chemistry Hoppe-Seyler,
376:617-625. [0548] 32. WO 93/20193 [0549] 33. WO 94/21801 [0550]
34. WO 94/26880 [0551] 35. WO 95/02043
THE DRAWINGS
[0551] [0552] 1. Feng and Doolittle, 1987, J. Mol. Evol. 25:
351-360. [0553] 2. NIH Data Base (Entrez, version spring 1996)
available on internet. [0554] 3. Eriksson, O. E. & Hawksworth,
D. L.: Systema Ascomycetum vol 12 (1993). [0555] 4, Julich, W.:
Higher Taxa of Basidiomycetes, Bibliotheca Mycologia 85, 485 pp
(1981). [0556] 5. O'Donnell, K., Zygomycetes in culture, University
of Georgia, US, 257 pp (1979). [0557] 6. Hawksworth, D. L., Kirk,
P. M., Sutton, B. C. and Pegler, D. N.: Dictionary of the fungi,
International to Mycological institute, 616 pp (1995). [0558] 7.
Von Arx, J. A.: The genera of fungi sporulating in culture, 424 pp
(1981).
DETAILED DESCRIPTION
[0558] [0559] 1. Ford et al., Protein Expression and Purification
2: 95-107, 1991. [0560] 2. Cunningham and Wells, Science 244,
1081-1085, 1989. [0561] 3. de Vos et at., Science 255: 306-312,
1992. [0562] 4. Smith et al., J. Mol. Biol. 224: 899-904, 1992.
[0563] 5. Wlodaver et al. FEBS Lett 309: 59-64, 1992. [0564] 6.
Tomme, P. et al. "Cellulose-Binding Domains: Classification and
Properties" in "Enzymatic Degradation of Insoluble Carbohydrates",
John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series,
No. 618, 1996. [0565] 7. WO 90/00609 [0566] 8. WO 95/16782 [0567]
9. Needleman, S. B. and Wunsch, C. D., Journal of Molecular
Biology, 48-443-453, 1970. [0568] 10. WO 94/14953 [0569] 11.
Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring
Harbor, N.Y. [0570] 12. Beaucage and Caruthers, Tetrahedron Letters
22: 1859-1869 (1981). [0571] 13. Matthes et at., EMBO Journal 3:
801-805 (1984). [0572] 14. U.S. Pat. No. 4,683,202 [0573] 15. Saiki
et at., Science 239: 487-491 (1988). [0574] 16. Hitzeman et al., J.
Biol. Chem. 255 12073-12080 (1980). [0575] 17. Alber and Kawasaki,
J. Mol. Appl. Gen. 1 419-434 (1982). [0576] 18. Young et at. in
Genetic Engineering of Microorganisms for Chemicals (Hollaender et
at, eds.), Plenum Press, New York, 1982). [0577] 19. U.S. Pat. No.
4,599,311 [0578] 20. Russell et al., Nature 304: 652-654 (1983).
[0579] 21. McKnight et al., The EMBO J. 4: 2093-2099 (1985). [0580]
22. P. R. Russell, Gene 40, pp. 125-130 (1985). [0581] 23. U.S.
Pat. No. 4,870,008 [0582] 24. Hagenbuchle et at., Nature 289, pp.
643-646 (1981), [0583] 25. L. A. Valls et al., Cell 48, 1987, pp.
887-897. [0584] 26. WO 87/02670 [0585] 27. M. Egel-Mitani et at.,
Yeast 6, pp. 127-137 (1990). [0586] 28. U.S. Pat. No. 4,546,082
[0587] 29. EP 16 201 [0588] 30. EP 123 294 [0589] 31. EP 123 544
[0590] 32. EP 163 529 [0591] 33. WO 89/02463 [0592] 34. WO 92/11378
[0593] 35. U.S. Pat. No. 4,599,311 [0594] 36. U.S. Pat. No.
4,931,373 [0595] 37. U.S. Pat. No. 4,870,008 [0596] 38. U.S. Pat.
No. 5,037,743 [0597] 39. U.S. Pat. No. 4,845,075 [0598] 40. U.S.
Pat. No. 4,931,373 [0599] 41. Gleeson et at., J. Gen. Microbiol.
132, pp. 3459-3465 (1986). [0600] 42, U.S. Pat. No. 4,882,279
[0601] 43. EP 272 277 [0602] 44. EP 230023 [0603] 45. Malardier et
at., 1989, Gene 78-147-156. [0604] 46. WO 93/11249. [0605] 47. WO
94/14953. [0606] 48. WO 95/02043. [0607] 49. Horton, R. M., Hunt,
H. D., Ho, S, N., Pullen, J. K., and Pease, L. R., Gene 77, 61-68
(1989) [0608] 50. Dalboge, H., and Heldt-Hansen, H., Mol. Gen.
Genet 243, 253-260 (1994) [0609] 51. Christensen, T., Wodike, H.,
Boel, E., Mortensen, S. B., Hjortshoj, K., Thim, L., and Hansen,
[0610] M. T., Bio/Technology 6, 1419-1422 (1988) [0611] 52. Sanger,
F., Nicklen, S., and Coulson, A. R., Proc. Nat. Acad. Sci. U.S.A.
74, 5463-5467 (1977). [0612] 53. Devereux, J., Haeberli, P., and
Smithies, O., Nucleic Acids Res. 12, 387-395 (1984). [0613] 54.
Becker, D. M. & Guarante, L., Methods Enzymol. 194; 182-187
(1991). [0614] 55. Gubler, U. & Hoffman, B. J., Gene 25:
263-269 (1983). [0615] 56. R. Higuchi, B. Krummel, and R. K. Saiki,
A general method of in vitro preparation and specific mutagenesis
of DNA fragments: study of protein and DNA interactions, Nucl.
Acids Res. 16: 7351-7367 (1988). [0616] 57, Sanger, F., Nicklen, S.
& Coulson, A. R., Proc. Natl. Acad. Sri. U.S.A. 74: 5463-5467
(1977). [0617] 58. N. Axelsen et al., A Manual of Quantitative
Immunoelectrophoresis, Blackwell Scientific Publications, 1973,
Chapters 2, 3, 4 and 23.
Sequence CWU 1
1
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