Cloning and characterization of a β-glucosidase from marine metagenome
新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质

In the present study, through a functional strategy, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for β-glucosidase and six positive clones were obtained. One of these clones, pSB47B2, was subcloned and further analysed in sequence. The result showed that there was an open reading frame for a novel P-glucosidase, which was nominated as bgl1B. Using pET22b(+) as vector and Escherichia coli BL21(DE3) as host, Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein (rBgl 1B) was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl1B were about 6.5 and 40°C respectively. Under the optimum conditions, rBgl1B hydrolyzed pNPG with an activity up to 39.7 U/mg, K_m and V_(max) being 0.288 mmol/L and 36.9 μmol/min respectively. In addition, rBgl1B could also hydrolyze cellobiose, with a K_m of 0.173 mmol/L and a V_(max) of 35 μmol/min. However, we did not detect evident hydrolytic activity of rBgl1B to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgl1B to pNPG was stimulated to certain degrees by low concentration of Ca~(2+) or Mn~(2+), whereas it exhibited significant tolerance against high Na~+. Distinguished from most of the β-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgl1B exhibited relatively higher activity and stability at pH between 7.0 and 9.0.