&bgr;-glucosidase coding sequences and protein from...

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase

Reexamination Certificate

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C435S183000, C435S252300, C435S320100, C435S069100, C435S254210, C435S254300, C435S254500, C435S254600, C435S254110, C435S252350, C435S252310, C435S255500, C435S254230, C536S023100, C536S023200

Reexamination Certificate

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06184018

ABSTRACT:

BACKGROUND OF THE INVENTION
The field of the present invention is the area of cellulolytic enzymes, nucleotide sequences encoding them and recombinant host cells and methods for producing them.
Cellulosic biomass, photosynthesized by solar energy with CO
2
and H
2
O, is one of the most important renewable energy resources on earth. Its effective utilization through biological processes is one approach to overcoming the shortage of foods, feeds and fuels, expected as a consequence of the explosive increase in human population [Ohmiya et al. (1997)
Biotechnol. Gen. Engineer. Rev
. 14:365-414]. Several types of enzymes are required for complete hydrolysis of cellulose to glucose, including endoglucanase, exoglucanase or cellobiohydrolase and &bgr;-glucosidase [Filho (1996)
Can. J. Microbiol
. 42:1-5].
&bgr;-Glucosidase (&bgr;-D-glucoside glucohydrolase; EC 3.2.1.21) is common among plants, fungi and bacteria. &bgr;-Glucosidase has aroused considerable interest primarily because of its involvement in the biological conversion of cellulosic material. The enzymatic saccharification of cellulosic materials to D-glucose is known to require the synergistic action of three classes of enzymes: endo-1,4-&bgr;-D-glucanohydrolase (EC 3.2.1.74), 1,4-&bgr;-D-cellobiohydrolase (EC 3.22.1.91), and 1,4-&bgr;-D-glucosidase (&bgr;-glucosidase; EC 3.2.1.21). Endo-1,4-&bgr;-D-glucanases act randomly on cellulose chains, whereas 1,4-&bgr;-D-cellobiohydrolases cleave cellobiosyl residues from the ends of cellulose chains, generating cellobiose as the main product. &bgr;-Glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides [Freer (1993)
J. Biol. Chem
. 268:9337-9342].
Anaerobic fungi have been isolated from the alimentary tracts of herbivores and other environments [Li et al. (1997)
Appl. Environ. Microbiol
. 63:628-635; Wubah and Kim (1994) Abstracts of the 94
th
Gen. Meet. of the American Society for Microbiology. Las Vegas, Nev., USA]. They produce highly active hydrolytic enzymes [Borneman et al. (1989)
Appl. Environ. Microbiol
. 55:1066-1073]. Genes encoding several cellulases and xylanases have been cloned and sequenced from anaerobic fungi
Neocallimastix patriciarum
[Black et al. (1994)
Biochem. J
. 299:381-387; Denman et al. (1996)
Appl. Environ. Microbiol
. 62:1889-1896; Gilbert et al. (1992)
Mol. Microbiol
. 6:2065-2072; Zhou et al. (1994)
Biochem. J
. 297:359-364], Piromyces sp. [Fanuti et al. (1995)
J. Biol. Chem
. 270:29314-29322] and Orpinomyces sp. [Chen et al. (1998)
FEMS Microbiol. Letts
. 159:63-68; Li et al. (1997)
Appl. Environ. Microbiol
. 63:628-635]. In addition, genes coding for three mannanases from Piromyces sp. [Fanutti et al. (1995)
J. Biol. Chem
. 270:29314-29322; Millward-Sadler et al. (1996)
FEMS Microbiol. Lett
. 141:183-188] and one 1,3-1,4-&bgr;-D-glucanase from Orpinomyces sp. [Chen et al. (1997)
J. Bacteriol
. 179:6028-6034] have been cloned and sequenced. However, genes coding for &bgr;-glucosidases of anaerobic fungi have not been reported even though several such enzymes from Neocallimastix [Herbaud and Fevre (1990)
Appl. Environ. Microbiol
. 56:3164-3169; Li and Calza (1991 A)
Enzyme Microb. Technol
. 13:622-628; Li and Calza (1991B)
Biochem. Biophys. Acta
1080:148-154], Orpinomyces [Chen et al. (1994)
Appl. Environ. Microbiol
. 60:64-70], and Piromyces [Teunissen et al. (1992)
Arch. Microbiol
. 158:276-281] have been purified and characterized.
There is a longfelt need in the art for &bgr;-glucosidase enzymes with catalytic properties which allow for improved saccharification of cellulosic materials and partial breakdown products thereof
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a &bgr;-glucosidase and the nucleotide sequences encoding it from Orpinomyces PC-2. The coding sequence for the protein, including its signal peptide and the stop codon, is given in SEQ ID NO:1, nucleotides 39-2012. The mature &bgr;-glucosidase is encoded at nucleotides 87-2009, exclusive of the stop codon. The deduced amino acid sequences of the signal peptide and of the mature protein is given in SEQ ID NO:2. Alternative &bgr;-glucosidase sequences are SEQ ID NO:2, amino acids 24-641 or SEQ ID NO:2, amino acids 33-641.
Also within the scope of the present invention are nonnaturally occurring recombinant DNA molecules comprising all synonymous sequences encoding the &bgr;-glucosidase of the present invention, recombinant host cells comprising the aforementioned DNA molecules, and methods for the synthesis of recombinant &bgr;-glucosidase of the present invention. Preferably, the coding sequence for the &bgr;-glucosidase is operably linked to transcription and translation control sequences functional in the desired host cell. A desired recombinant host cell is a yeast, as specifically exemplified, a
Saccharomyces cerevisiae
cell genetically engineered to contain and express the &bgr;-glucosidase coding sequences of the present invention. Other recombinant host cells of the present invention include, without limitation, fungi such as Aspergillus spp., Trichoderma spp., Pichia spp., Aureobasidium spp. and bacteria, including but not limited to Bacillus spp.


REFERENCES:
Ximenes et al. (1998) “Cloning and Sequencing of a &bgr;-Glucosidase Gene from the Anaerobic Fungus Orpinomyces sp. Strain PC-2 and Its Expression inSaccharomyces cerevisiae” Abstract. General Meeting of the American Society of Microbiology. 98:337.
Archer and Peberdy (1997) “The Molecular Biology of Secreted Enzyme Production by Fungi”Critical Reviews in Biotechnology17:273-306.
Bhat et al. (1993) “Purification and Characterization of an Extracellular &bgr;-Glucosidase from the Thermophilic FungusSporotrichum thermophileand Its Influence on Cellulase Activity”Journal of General Microbiology139:2825-2832.
Borneman et al. (1989) “Fermentation Products and Plant Cell Wall-degrading Enzymes Produced by Monocentric and Polycentric Anaerobic Ruminal Fungi”Applied and Environmental Microbiology55:1066-1073.
Breves et al. (1997) “Genes Encoding Two Different &bgr;-Glucosidases ofThermoanaerobacter brockiiAre Clustered in a Common Operon”Applied and Environmental Microbiology63:3902-3910.
Chen et al. (1998) “Two Genes of Anaerobic Fungus Orpinomyces sp. Strain PC-2 Encoding Cellulases with Endoglucanase Activities May Have Arisen by Gene Duplication”FEMS Microbiology Letters159:63-68.
Chen et al. (1997) “Sequencing of a 1,3-1,4-&bgr;-D-Glucanase (Lichenase) from the Anaerobic Fungus Orpinomyces Strain PC-2: Properties of the Enzyme Expressed inEscherichia coliand Evidence that the Gene Has a Bacterial Origin”Journal of Bacteriology179:6028-6034.
Chen et al. (1995) “A Cyclophilin from the Polycentric Anaerobic Rumen Fungus Orpinomyces sp. Strain PC-2 Is Highly Homologous to Vertebrate Cyclophilin B”Proc. Natl. Acad. Sci. USA92:2587-2591.
Chen et al. (1994) “Isolation and Properties of an Extracellular &bgr;-Glucosidase from the Polycentric Rumen Fungus Orpinomyces sp. Strain PC-2”Applied and Environmental Microbiology60:64-70.
Chen et al. (1992) “Purification and Characterization of Two Extracellular &bgr;-Glucosidases fromTrichoderma reesei” Biochimica et Biophysica Acta1121:54-60.
Cummings and Fowler (1996) “Secretion ofTrichoderma reesei&bgr;-Glucosidase bySaccharomyces cerevisiae” Curr. Genet.29:227-233.
Desrochers et al. (1981) “High Production of &bgr;-Glucosidase inSchizophyllum commune:Isolation of the Enzyme and Effect of the Culture Filtrate on Cellulose Hydrolysis”Applied and Environmental Microbiology41:222-228.
Fanutti et al. (1995) “The Conserved Noncatalytic 40-Residue Sequence in Cellulases and Hemicellulases from Anaerobic Fungi Functions as a Protein Docking Domain”Journal of Biological Chemistry270:29314-29322.
Freer, S.N. (1993) “Kinetic Characterization of a &bgr;-Glucosidase from a Yeast,Candida wickerhamii” The Journal of Biological Chemistry268:9337-9342.
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