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KMID : 0903520080510040299
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
2008 Volume.51 No. 4 p.299 ~ p.304
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Molecular Characterization of a β-1,4-Endoglucanase Gene from Bacillus subtilis H12
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Oh Jin-Hwan
Cha Jeong-Ah
Yoon Min-Ho
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Abstract
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A β-1,4-endoglucanase gene from Bacillus subtilis H12 was cloned into Escherichia coli JM109 (pBC8) and sequenced. The endoglucanase gene with an insert DNA of 2.5 kb possessed an open reading frame of 1,500 bp encoding a mature protein of 499 amino acids with a calculated molecular mass of 55 kDa. The deduced amino acid sequence showed similarity to those of the known neutral cellulase genes of B. subtilis PAP115 (99.2%) and BSE616 (97.8%), as well as the alkaline gene of Bacillus sp. N4 (55.1%). The endoglucanase activity expressed by E. coli (pBC8) was localized in the periplasmic fraction (80%) and the cytoplasmic fraction (20%). An endoglucanase was purified from the periplasmic fraction by performing gel filtration and anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 31 kDa by SDS-PAGE, and the maximum activity occurred at pH 7 and 40oC. The enzyme easily hydrolyzed soluble substrates such as carboxymethyl cellulose and barely β-glucan, whereas the sigmacell and xylan, the known insoluble substrates, were not entirely hydrolyzed.
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KEYWORD
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Bacillus subtilis H12, cloning, β-1, 4-endoglucanase, purification
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