Molecular Characterization of a β-1,4-Endoglucanase Gene from Bacillus subtilis H12

다국어 초록 (Multilingual Abstract)

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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