Cellulase enzymes

Details Cellulase enzymes Cellulase enzymes

1999-10-27

2002-08-06

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S200000, C435S252300, C435S320100, C435S410000, C536S023200

Reexamination Certificate

active

06428996

ABSTRACT:

BACKGROUND OF THE INVENTION
Cellulases are enzymes that can hydrolyze the glycosidic linkages in polysaccharides such as cellulose. These enzymes are used in a number of industrial applications where breaking down biomass is beneficial. For example, cellulases can be used as a supplement in animal feed to decrease the production of fecal waste by increasing the digestibility of the feed. Cellulases can also be used to increase the efficiency of alcoholic fermentations (e.g., in beer brewing) by converting undigestible biomass into fermentable sugars. In addition, the “softening” of blue jeans to produce a “stone-washed” look can be facilitated by treating the jeans with cellulases.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a new cellulase isolated from the fungus
Piromyces rhizinflata
. The gene encoding this cellulase is designated cbhA. A portion of an cbhA cDNA is described below.
Accordingly, the invention features a substantially pure polypeptide having an amino acid sequence at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to an amino acid sequence representing the catalytic domain of CBHA (SEQ ID NO:4; described below), the polypeptide encoded by cbhA. The polypeptide is capable of hydrolyzing a polysaccharide such as oat spelt xylan. Such a polysaccharide can also be cellulose (e.g., carboxymethyl cellulose), polysaccharides containing &bgr;-1,3′ or &bgr;-1,4′ glycosidic linkage (e.g., barley &bgr;-glycan), or lechinan.
The invention also includes an isolated nucleic acid encoding a polypeptide of the invention. For example, the invention includes an isolated nucleic acid having a sequence encoding a polypeptide that hydrolyzes a polysaccharide, provided that the nucleic acid hybridizes under stringent conditions to SEQ ID NO:1.
In addition, the invention features any vectors or transformed cells which contain a nucleic acid of the invention. Vectors include nucleic acid vectors, such as expression plasmids, or viral vectors. Transformed cells include eukaryotic and prokaryotic cells.
A “nucleic acid” encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized or modified) DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. An “isolated nucleic acid” refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the 5′ non-coding (e.g., promoter) sequences which are immediately contiguous to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. The term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
By “hybridizes under stringent conditions” is meant specific and non-covalent binding to an immobilized reference nucleic acids in the presence of 0.2×SSC (1.75 g/l NaCl, 0.88 g/l Na
3
citrate.2H
2
O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68° C.
The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) by weight pure. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity or conservation to a reference polypeptide or nucleic acid, the percent identity or conservation is determined by the algorithm of Myers and Miller, CABIOS (1989), which is embodied in the ALIGN program (version 2.0), or its equivalent, using a gap length penalty of 12 and a gap penalty of 4 where such parameters are required. All other parameters are set to their default positions. Access to ALIGN is readily available. See, e.g., http://www2.igh.cnrs.fr\/bin/align-guess.cgi on the Internet.
Other features or advantages of the present invention will be apparent from the following detailed description, the drawings, and also from the claims.


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Denman et al. Appl. Environ. Microbiol. 62 : 1889-1896 (1996).

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