Production and Partial Characterisation of Bifunctional Cellulase Genes of Anaerobic Gut Fungus Orpinomyces sp. GMLF19

Fiber-degrading properties of anaerobic gut fungus, Orpinomyces sp. GMLF19, were investigated when it was grown on various carbon sources, namely carboxymethylcellulose, barley, oat, corncob and wheat straw. Fungi were developed under anaerobic conditions, and carboxymethylcellulase (CMCase), lichenase, and xylanase activities were determined by using DNS method. Meanwhile, the decreasing order of the enzyme activity of Orpinomyces sp. GMLF19 was observed to be lichenase > CMCase > xylanase. Relative activities of these enzymes in the isolate GMLF19 were found to be the highest when the culture was grown for 5 days. Cellulase gene domain was reproduced from chromosomal DNA and transferred into Escherichia coli with the aid of pCT plasmid vector and CMCase, lichenase and xylanase activities were determined using DNS method. The gene showed relatively higher CMCase activity and moderate lichenase activity while no xylanase activity was observed suggesting that the gene is bi-functional. The cell-associated activity of overexpressed Cel19 enzyme (E. coli cells containing this transferred gene were designated as C19) was relatively higher than extracellular activity. It was also recorded that this gene has 2 dockerin domains and 1 catalytic domain with the length of 1.585 bp, and it coded 50.59 kDa size of the protein which belongs to glycosyl hydrolase family 6.