期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 64, 期 11, 页码 2281-2289出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.64.2281
关键词
Bacillus pseudofirmus; alkaliphile; cellulase; thermostability; site-directed mutagenesis
Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta -glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-amino-acid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg(111), His(151), Glu(190), His(262), Tyr(264), and Glu(305) in catalysis and/or substrate binding of the enzyme.
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