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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Sequencing of Two Highly Homologous Cellulase Genes, Celh and Celi, from the Anaerobic Fungus Orpinomyces Strain Pc-2

Authors
item Li, Xin Liang
item Ximenes, Eduardo - UNIV BRAZIL
item Chen, Huizhong - NATL CNTR TOX RES
item Cotta, Michael
item Ljungdahl, Lars - UNIV GEORGIA

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: October 1, 2003
Publication Date: January 31, 2004
Citation: Li, X., Ximenes, E.A., Chen, H., Cotta, M.A., Ljungdahl, L.G. 2004. Cloning and sequencing of two highly homologous cellulase genes, celH and celI from the anaerobic fungi Orpinomyces strain PC-2. Proceedings MIE BIOFORUM 2003: Biotechnology of Lignocellulose Degradation and Biomass Utilization, pp. 638-641.

Technical Abstract: Anaerobic rumen fungi produce high levels of cellulases and hemicellulases. These enzymes hydrolyze lignocellulosic polysaccharides using cellulosomal and free-enzyme strategies. As many as 10 cellulase genes, including the 2 new reported here from the polycentric anaerobic fungus Orpinomyces sp. strain PC-2, have been cloned and sequenced. The catalytic domains of these cellulases belong to glycoside hydrolase families 5 and 6. The two new cDNAs, celH and celI, coding for cellulases CelH and CelI, were isolated using fungal dockerin specific PCR amplification products as hybridization probes. CelH and CelI have 491 and 464 amino acid residues, respectively, and share 91% identity on the amino acid sequence level. The two mature enzymes have the same modular structure consisting of an amino terminal fungal dockerin, a linker, and a carboxy terminal family 6 catalytic domain (CD). The CDs of CelA, CelC, CelD, and CelF from the fungus also belong to family 6 and have over 55% amino acid sequence identities between each other. In contrast, the CDs of CelH and CelI have only 34-40% amino acid identities with the CDs of CelA, CelC, CelD, or CelF. These data reveal that multiple homologous cellulase genes are present in the genome of Orpinomyces and that the family 6 glycoside hydrolases of the fungus should be placed into two sub-families. This view is supported by the fact that neither celH nor celI could be expressed as active cellulases in Escherichia coli while the gene products of the other cellulases of the same family when expressed in this bacterium are fully functional.

Last Modified: 7/25/2014
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