소 반추위 메타게놈에서 새로운 섬유소분해효소 유전자(cel5C) 클로닝 및 유전산물의 특성

Abstract

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in E. coli EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with Sau3AI, ligated into the BamHI site of the pBluescript II SK+ vector, and transformed into E. coli DH5 α. We obtained a 1.5 kb insert DNA, designated cel5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from E. coli DH5α, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and 50℃. We examined whether the cel5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the cel5C gene came from the unidentified cow rumen bacteria.