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KMID : 0613820120220040437
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Journal of Life Science
2012 Volume.22 No. 4 p.437 ~ p.446
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Cloning and Characterization of Cellulase Gene (cel5C) from Cow Rumen Metagenomic Library
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Kim Min-Keun
Barman Dhirendra Nath
Kang Tae-Ho
Kim Jung-Ho
Kim Hoon
Yun Han-Dae
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Abstract
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A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $EE.celi 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.celi DH5α. 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.celi 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.
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KEYWORD
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Rumen metagenome, cellulase, cel5C, unculturable bacterium, CMC-SDS-PAGE
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