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KMID : 0545120120220121705
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 12 p.1705 ~ p.1713
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Purification and Characterization of Beta-Glucosidase from Weissella cibaria 37
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Lee Kang-Wook
Han Nam-Soo
Kim Jeong-Hwan
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Abstract
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A gene encoding β-glucosidase was cloned from Weissella cibaria 37, an isolate from human feces. Sequence analysis showed that the gene could encode a protein of 415 amino acids in length, and the translated amino acid sequence showed homology (34-31%) with glycosyl hydrolase family 1 β-glucosidases. The gene was overexpressed in E. coli BL21(DE3) using pET26b(+) and a 50 kDa protein was overproduced, which matched well with the calculated size of the enzyme, 49,950.87 Da. Recombinant β-glucosidase was purified by using a his-tag affinity column. The purified β-glucosidase had an optimum pH and a temperature of 5.5 and 45oC, respectively. Among the metal ions (5mM concentration), Ca2+ slightly increased the activity (108.2%) whereas Cu2+ (46.1%) and Zn2+ (56.7%) reduced the activity. Among the enzyme inhibitors (1 mM concentration), SDS was the strongest inhibitor (16.9%), followed by pepstatin A (45.2%). The Km and Vmax values of purified enzyme were 4.04 mM and 0.92 μmol/min, respectively, when assayed using pNPG (p-nitrophenyl-β- D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).
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KEYWORD
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Weissella cibaria, beta-glucosidase, overexpression
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