Expression, Purification And Functional Analysis Ofβ-Glucosidase From The Alkalophilic Bacterium Bacillus Halodurans C-125

β-Glucosidases are one of hydrolases which hydrolyze the (3-glucosidc bonds between carbohydrates. Its unique property determines their broad application in biological industries. Although β-glucosidases serve various functions, our interests are focused on their roles in cellulose hydrolysis, the production of oligomeric sugar gentiana, being as flavor enzyme, cancer diagnosis and remediation. However, for a long time, unstability, low production and specific activity of enzyme are the major bottlenecks to obstruct their application. It is well known that β-Glucosidases have been widely found in microorganisms, but the enzyme production is too low in individual level to obtain enough products. More recently, β-glucosidases from microbes living in extreme environments have attracted research interest due to the potential applications associated with their unusual biochemical properties.The alkalophilic bacterium Bacillus halodurans C-125was isolated in1977and the complete genomic sequence has been available on the NCBI website since2000. According to the gene Bhbgl sequence, the primers were designed and the coding gene was synthesized via the PTDS method. Synthetic Bhbgl was cloned into the prokaryotic expression vector pYM4087and transformed into the competent expression host, E. coli DH5a. As far as we are aware, there are no published reports of a α-glucosidase from B. halodurans C-125, and we now describe the purification and partial characterization of a novel β-glucosidase (Bhbgl) from this bacterium. The enzyme exhibited a high specific activity with oNPGlu and an apparent Km value of0.32mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of～7.0and50℃, respectively. The enzyme was relatively stable under alkaline conditions and>50%activity was retained after incubation at pH9.5for24h at4℃. Recombinant Bhbgl activity was inhibited by5mM Zn2+, F3+e or Cd2+, but was enhanced by1mM Mg and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, D-galactose, xylose, glucose) at concentrations ranging from50-800mM.