产β-葡萄糖苷酶微生物的筛选鉴定及其在人参皂苷Compound K转化中的应用

Screening and Identification of Microorganisms Producing β-glucosidase and Their Application in Transformation of Ginsenoside Compound-K

本试验用七叶苷培养基从西洋参(Panax quinquefolius)的根际土壤中筛选产β-葡萄糖苷酶的菌种,并通过转化试验发现一株可以高效转化人参皂苷Rb1为人参皂苷Compound K(C-K)的菌株。基于16S rDNA基因序列的系统进化树分析确定该菌株为耐热科恩氏菌(Cohnella thermotolerans)。根据单因素试验结果,选取菌液浓度、反应pH值、反应时间3个因素,以C-K的含量为响应值,采用Box-Behnken中心组合试验设计3因素3水平的响应面试验,建立回归模型。确定优化的皂苷转化条件为反应pH 6.4、菌液浓度9.0×10^(8)cfu/mL、反应时间7.5 d,在该条件下人参皂苷转化率为78.36%。通过高效液相色谱分析该菌株转化人参皂苷Rb1的途径为Rb1→Rd→F2→C-K。本研究为稀有皂苷C-K的微生物转化提供了新的菌种来源,也为C-K的工业化生产提供了理论依据。

In this study, microorganisms capable of producing β-glucosidase were isolated from the rhizosphere soil of Panax quinquefolius, and one strain which could transform ginsenoside Rb1 to ginsenoside Compound K(C-K) was discovered with esculin agar. The strain was identified as Cohnella thermotolerans based on the phylogenetic tree constructed from 16 S rDNA sequences. According to single factor experiment results, bacterial concentration, reaction pH value and reaction time were selected as main factors. Using the content of C-K as the response value, the response surface experiment with 3 factors and 3 levels was conducted according to Box-Behnken central composite design. The optimized reaction conditions were as follows: bacterial concentration of 9×10^(8)cfu/mL, pH of 6.4, reaction time of 7.5 d. Under the optimal conditions, the transformation rate was 78.36%. The HPLC analysis showed that the pathway of transformation of Rb1 by the strain was Rb1→Rd→F2→C-K. The results provided a new strain for biotransformation of ginsenoside C-K, and provided theoretical bases for industrial production of C-K.