嗜热β-葡萄糖苷酶基因挖掘、酶学性质表征及转化大豆苷的研究OA

To explore the β-glucosidase gene with high efficiency in converting daidzin to daidzein,using the metagenome DNA of microorganisms from 94 ℃ hot spring in Yuanjiang,Yunnan Province predicted and synthesized the β-glucosidase genes.These genes was transformed into Escherichia coli BL21(DE3)for heterologous expression,its enzymatic properties of the recombinant enzyme were investigated,and its application in the conversion of daidzin to daidzein was explored.The results showed that a thermophilic β-glucosidase gene BglYJ446 from the GH1 family was mined from the metagenome of microorganisms in 94℃ hot spring.The recombinant enzyme encoded by the gene was named BglYJ446.The optimal reaction tem-perature and pH was 85 ℃ and 7.5,respectively.After incubation at 80℃ for 8 h,the relative enzyme activity reached 57.77％.The enzyme exhib-ited good thermal stability within the pH range of 7.5 to 9.0(relative enzyme activity＞70％).The K+and Na+had little effect on BglYJ446 activity,while high concentrations(10 mmol/L)Ni+and Cu2+and other metal ions,10％urea,Tween 20,and β-mercaptoethanol inhibited its enzymatic activity.Under the conditions of methanol and ethanol at a volume fraction of 20％,and 2 000 mmol/L glucose,the relative enzymatic activities of BglYJ446 were 168.21％,93.07％,and 36.83％,respectively.When using β-D-glucopyranoside(p-NPGlu)as the substrate,the Michaelis constant(Km value)of BglYJ446 was 1.72 mmol/L,and the maximum reaction rate(Vmax)was 1 164.56 μmol/(ml·min).At 80 ℃,98.87％of daidzin could be converted using BglYJ446(0.15 U)in 60 min.