Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-15
2001-04-24
Achutamurthy, Ponnathapu (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100
Reexamination Certificate
active
06222028
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 eglA. A portion of an eglA 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 EGLA (SEQ ID NO:4; described below), the polypeptide encoded by eglA. The polypeptide is capable of hydrolyzing a polysaccharide containing a &bgr;-1,3′ or &bgr;-1,4′ glycosidic linkage. Such a polysaccharide can be cellulose (e.g., carboxymethyl cellulose), polysaccharides containing both &bgr;-1,3′ and &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 containing a &bgr;1,3′ or &bgr;1,4′ glycosidic linkage, 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.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a cellulase enzyme, nucleic acids encoding it, and vectors and cells containing such nucleic acids. Contemplated within the scope of this invention are recombinant nucleic acids or viruses which allow production of EGLA in a transformed cell or transgenic organism or allow ease of producing specific or non-specific mutations within the EGLA reading frame. These recombinant nucleic acids or viruses may further include any one of a variety of sequences flanking or within the EGLA coding sequences, such as strong constitutive promoters within the EGLA coding sequence, as introns containing cis-elements that allow high level expression, or efficient polyadenylation signals.
Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the isolation of EGLA polypeptides and nucleic acids described below, utilize the present invention to its fullest extent. The following examples are to be construed as merely illustrative of how one skilled in the art can isolate and use EGLA polypeptides and nucleic acids from biological sources, and are not limitative of the remainder of the disclosure in any way. For example, once the sequence of the egla CDNA is known, any egla sequence can be obtained by PCR amplification of mRNA or genomic DNA. Any publications cited in this disclosure are hereby incorporated by reference.
The anaerobic fungus
Piromyces rhizinflata
, strain 2301, was cultivated anaerobically at 39° C. in a modified semi-defined medium as described in Lowe et al., J. Gen. Microbiol. 131:2225-2229, 1985. The mycelia were harvested from the culture media, lyophilized, frozen in liquid nitrogen, and ground into a powder. The powder was homogenized in extraction buffer containing 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl, 2% SDS, and 1% &bgr;-mercaptoethanol. An equal volume of a 1:1 mixture of phenol/chloroform was added, and the resulting mixture vortexed for 60 seconds and then centrifuged. The aqueous phase was extracted with the phenol/chloroform again. A one-third volume of 8 M LiCl was then added to the extracted mixture. The mixture was centrifuged sufficiently to pellet the RNA, which was washed with 2 M LiCl, followed by 80% ethanol. The washed RNA was then resuspended in diethyl pyrocarbonate (DEPC)-treated water.
Polyadenylated RNA was isolated from total RNA using a standard oligo-(dT)-cellulose chromatography column. The construction of a cDNA expression library was carried out using a Stratagene kit. The library was screened for cellulase activity by overlaying plaques with 0.7% (w/v) agarose containing 0.2% (w/v) carboxymethyl cellulose (CMC). The plates were incubated at 39° C. overnight, then stained with a 0.1% (w/v) aqueous solution of Congo red and destained with 1 M NaCl as described in Teather et al., App. Environ. Microbiol. 43:777-780, 1982. Cellulase-producing plaques were surrounded by a clear halo visible against a red background. The positiv
Chang Chia-Chieh
Cheng Kuo-Joan
Liu Jin-Hao
Tsai Cheng-Fang
Academia Sinica
Achutamurthy Ponnathapu
Fish & Richardson P.C.
Kerr Kathleen
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