副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究

Expression,Purification and Enzymatic Properties of β-glucosidase from Lactobacillus paracasei

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei) TK1501β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86. 63kDa和675. 56U/mg。最适作用温度和pH分别为30℃和6. 5。Mg^2+和Ca^2+对β-葡糖苷酶酶活抑制作用最小,Cu^2+几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的Km和Vmax分别为1. 44mmol/L和58. 32mmol/(L·s),催化系数kcat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。

To improve the conversion efficiency of glucoside to small molecule compounds,the gene encoded β-glucosidase from Lactobacillus paracasei TK1501 was inserted into pET28 a(+), and further transformed into E. coli BL21( DE3) for heterologous expression. The recombinant enzyme,which purified by nickel affinity chromatography,is conferred with the high specific activity of 675. 56 U/mg,and the molecular weight of 86. 63 kDa. The biochemical characterization of this recombinant enzyme shows that it exhibit the highest bio-activity in 30℃ and pH of 6. 5,and the β-glucosidase activity was barely inhibited by Mg^2+ and Ca^2+,but largely by Cu^2+ with even no catalytic activity. It was also found that this enzyme possess a broad substrates specificity toward genistin,daidzin,daidzein,geniposide,salicin,heptosporin,polydatin and arbutin. Finally,The kinetics characteristics shows that Km and Vmax of this enzyme are 1. 44 mmol/L and 58. 32 mmol/( L·s),respectively,and the catalytic coefficient( kcat) is 3 982/s using β-pNPG as the substrate. The all results above show that the β-Glucosidases from Lactobacillus paracasei TK1501 play important roles in the process of hydrolysis of soybean isoflavone and synthesis of glycosides.