Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic Library

A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulose activity with molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50 degrees C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20 similar to 50 degrees C and pH 4.0 similar to 10.0. CS10 exhibited little loss of cellulose activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thin-layer chromatography suggested that CS10 is an endo-beta-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.