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KMID : 0545120140240091196
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 9 p.1196 ~ p.1206
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Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic Library
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Lee Chang-Muk
Lee Young-Seok
Yoon Sang-Hong
Kim Soo-Jin
Hahn Bum-Soo
Sim Joon-Soo
Koo Bon-Sung
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Abstract
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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 cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase 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 thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.
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KEYWORD
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Cellulase, Hermetia illucens, Metagenomic library, Purification, Characterization, Carboxymethyl cellulose
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