title: Screening and characterization of a novel cellulase gene from the gut microflora of Hermetia illucens using metagenomic library.
abstract: 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.
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea.
authors.forename: Chang-Muk
authors.initials: CM
authors.lastname: Lee
authors.forename: Young-Seok
authors.initials: YS
authors.lastname: Lee
authors.forename: So-Hyeon
authors.initials: SH
authors.lastname: Seo
authors.forename: Sang-Hong
authors.initials: SH
authors.lastname: Yoon
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Bum-Soo
authors.initials: BS
authors.lastname: Hahn
authors.forename: Joon-Soo
authors.initials: JS
authors.lastname: Sim
authors.forename: Bon-Sung
authors.initials: BS
authors.lastname: Koo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004175:Diptera / Q000235:genetics*; D004795:Enzyme Stability; D041981:Gastrointestinal Tract / Q000382:microbiology*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D002480:Cellulase