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Poster Presentation 6-39
A Cellulase Gene (CelF) from Orpinomyces PC-2 Has an Intron and Encodes a Cellulase (CelF), Containing a Cellulose Binding Domain
Huizhong Chen, Xin-Liang Li, David L. Blum, Eduardo A. Ximenes and Lars G. Ljungdahl
Center for Biological Resource Recovery & Department of Biochemistry and Molecular Biology University of Georgia A214 Life Sciences Building Athens, GA 30602-7229
Telephone: (706) 542-7640; Fax: (706) 542-2222; E-mail: larsljd@arches.uga.edu
Anaerobic fungi found in remuns and cecums of herbivores produce a broad spectrum of highly active hydrolytic enzymes for plant cell wall degradation. The enzymes exist free or bound in cellulosomal complexes. A cDNA (1,520 bp), designated CelF, isolated from a cDNA library of the anaerobic rumen fungus Orpinomyces PC-2 constructed in Escherichia coli, encodes a polypeptide (CelF) of 432 amino acids. Starting from the N-terminus celF contains a signal peptide, a carbohydrate binding domain (CBD), an Asn-rich linker region, and a catalytic domain (CD). The CD of CelF is highly homologous to the CDs of CelA and CelC of the same fungus, and other family 6 glycosyl hydrolases. The CelF lacks a docking domain present in CelA and CelC and is a free enzyme. The recombinant CelF with a molecular mass of 45.5 kDa lacks the N-terminal signal peptide of 20 amino acid residues. Km and Vmax with carboxymethyl cellulose (CMC) as the substrate at pH 6.0 and 40ºC were 4.37 mg/mL and 173, respectively. Optimal activity was between pH 5.8 to 6.2 and 40 to 50ºC. The enzyme hydrolyzes celooligosaccharides, microcrystalline cellulose (Avicel), amorphous-swollen cellulose, and CMC, indicating it has both cellobiohydrolase and endoglucanase activities. The genomic DNA of CelF contains an 111 bp intron, in the region coding for the CBD with features common to introns of genes from aerobic fungi.
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