title: Purification and characterization of beta-glucosidase from Weissella cibaria 37.
abstract: A gene encoding beta-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 beta-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 beta-glucosidase was purified by using a his-tag affinity column. The purified beta-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 micromol/min, respectively, when assayed using pNPG (p-nitrophenyl-beta- D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).
authors.affiliation: Division of Applied Life Science (BK21), Graduate school, Gyeongsang National University, Jinju, Korea.
authors.forename: Kang Wook
authors.initials: KW
authors.lastname: Lee
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Han
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D058836:Weissella / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase