Biochemical Characterization Of Genetically Recombinant Thermophilic Beta-glucosidase And Its Applications

beta-glucosidase is one of the important components of cellulase,but the content and activity are low.Therefore,a heat-tolerant beta-glucosidase producing strain was designed using genetic engineering techniques to discover the enzymatic characterization of a novel glucosidase.Ginseng is rich in ginsenosides,which are glycosides,but the biological activity of common ginsenosides is not as strong as that of rare ginsenosides,which are difficult to isolate and have low yields,and their pharmacological effects are limited,so enzymatic conversion of ginsenosides has become a research hotspot,and the main studies in this paper include the following.（1）The target gene for beta-glucosidase was cloned from the genomic library,obtained by polymerase chain reaction,and the recombinant plasmid was obtained using pET-as a vector and successfully expressed in E.coli.The target protein of RsBgl with a molecular weight of 58 kDa was obtained by heat treatment,Ni-NTA and anion exchange purification.The enzymatic property analysis revealed that RsBgl exerted the best activity at pH 5.5 and 105℃with p-nitrophenyl-β-glucopyranoside as the substrate,and had good thermal stability at 85℃,increasing glucose concentration inhibited the enzyme activity,still had about 60%enzyme activity at the concentration of 0.2 M,good glucose tolerance,Ba²⁺,Mg²⁺,Mn²⁺and Fe²⁺had an activating effect on the enzyme activity and Fe³⁺,K⁺and NH⁴⁺inhibited the enzyme activity.（2）The conversion of ginsenosides by RsBgl was investigated at different times using ginsenosides as substrate at the same optimum pH and the reaction temperature was set at 95°C.Using TLC analysis,it was found that the enzyme could hydrolyze the diol ginsenosides Rb1,Rb2,Rb3,Rc and the triol ginsenoside Rf,with the following conversion pathways:Rb1→Rd→F2→C-K;Rb2→Rd→F2→C-K and Rb2→C-O;Rb3→Rd→F2→C-K and Rb3→C-Mx1;Rc→C-Mc1→C-Mc and Rc→Rd→Rh2;and Rf→Rh1.The conversion rates of RsBgl into ginsenosides were also investigated and the conversion rates obtained after 8 h of reaction were 98.2%,97%,57%,47%and 60%,respectively.The enzymatic characterization of RsBgl obtained by gene recombination can provide theoretical value for industrial production,and its application to the transformation of ginsenosides can transform to produce rare ginsenosides,which plays a scientific value for the preparation of rare ginsenosides.