生物转化法制备葫芦素B产酶菌株的筛选与鉴定

Isolation and Identification of a Microbial Strain for Production of Cucurbitacin B by Biotransformation

葫芦素B(CuB)是临床用于治疗肝炎和肝癌等疾病的药物,开发以葫芦素B葡萄糖苷(CuBg)为原料生产CuB的生物转化工艺,具有潜在的工业应用价值,获得一株特异性产酶菌株是转化工艺开发的首要工作。首先利用甜瓜蒂和污水富集培养产酶菌株,平板分离法获得纯培养;之后以含CuBg的甜瓜蒂提取物为底物,筛选出能将CuBg转化为CuB高产菌株,并对筛选获得的菌株进行分类鉴定;最后对转化工艺进行优化,提高转化率。结果分离到一株编号为RW-2放线菌菌株,其滤除菌丝的发酵液处理甜瓜蒂提取物,其中的CuBg被转化为CuB,CuB的含量显著提高;经形态学鉴定和16S r DNA序列分析,确定该菌株是一株链霉菌(Streptomyces sp.);经过转化工艺优化后,该菌株发酵制备的粗酶液转化甜瓜蒂提取物,其中CuB的含量从1.50 g/L提高到3.27 g/L,提高了1.18倍,CuB的转化得率达99.5%。利用生物转化法将低活性的CuBg转化高活性的CuB,具有反应条件温和、工艺简单,转化得率高的优点,在药物CuB生产中有一定的应用价值。

Cucurbitacin B （CuB） is a drug used in the treatment of hepatitis and liver cancer. Developing a biotransformationprocess for production of CuB from its glycoside form-cucurbitacin B glucoside （CuBg） has some potential applications in the phar-maceutical industry. The first work of the biotransformation is to obtain a special microbial strain for producing enzyme. In the paper,the pedicels of Cucumis melo and sewage mixture were incubated for enriching enzyme producing strains. And then, the culture super-natant of strains was used as crude enzyme to treat the pedicels of C. melo for selecting a strain which can convert CuBg to CuBspecifically. After the selected strain was identified, the biotransformation process was optimized to improve the conversion yield. As aresult, a strain, designated as RW-2, was isolated from the enrichment. The culture broth of strain RW-2 without cells was used totreat the extracts of pedicels of C. melo, CuBg can be converted to CuB without degradation, and the concentration of CuB wasimproved significantly. Based on morphological characteristics and analysis of 16S rDNA, strain RW-2 was identified as a species ofStreptomyces sp.. After optimization of biotransformation process ,the concentration of CuB in the extracts of pedicels of C. melo couldbe increased to 3.27 g/L from 1.50 g/L by biotransformation （ 1. 18 times improvement）. The molar yield of CuB from CuBgreached 99.5%. The biotransformation process developed in the paper has the advantages of mild reaction conditions, simple opera-tions, and high productivity, which may greatly increase CuB production from C. melo pedicels in the pharmaceutical industry.