Microbial Transformation Of Cucurbitacin B-glucoside From Pedicellus Melo

Cucurbitacins B with a broad range of bioactivity, such as anti-tumor, anti-hepatic lesion, anti-chemocarcinogenesis, immunity enhancing, has been used for adjuvant therapy of chronic hepatitis and primary liver cancer for many years. At present, the main production method of cucurbitacin B is by extraction from traditional Chinese medicine Pedicellus Melo., but the yield is very low. Cucurbitacins B from Pedicellus Melo can be found in glycosylated forms, Cucurbitacin B-2-O-glucoside, and its content is almost close to cucurbitacin B. Because of lacking glycosidase for hydrolyzing glycoside compounds, the human body can not absorb them efficiently, only if they are hydrolyzed to aglycon. Therefore cucurbitacin B-2-O-glucoside from Pedicellus Melo can not be applied as a drug. Microorganisms possess a large amount of enzymes and a great power of decomposition and biotransformation. Cucurbitacin B-2-O-glucoside can be hydrolyzed to cucurbitacin B by utilizing enzymes from microbes, and the productivity of cucurbitacin B from Pedicellus Melo could be improved obviously if the biotransformation is applied to cucurbitacin B-2-O-glucoside. The advantagements of the biotransformation are distinguished of mild reaction conditions, high specificity and environmental benefits compared with chemical processes. In the paper, the biotransformation of cucurbitacin B-2-O-glucoside to cucurbitacin B by the enzyme from Streptomyces sp is established for the first time, and also biotransformation process was optimized for improving the productivity.Firstly,109strains of fungi and actinomyces were isolated from soil and sewage, and the crude enzymes from these strains were used as catalysts for biotransformation cucurbitacin B-2-O-glucoside to cucurbitacin in the extracts from Pedicellus Melo. By comparing the yield of cucurbitacin B, a strain, designated as RW-2, could give a high yield, was selected as the electee for the further study. The strain RW-2was identified as a strain of Streptomyces sp. based on morphological characteristics, comparision of16S rDNA nucleotide sequence and phylogenetic analysis.Secondly, for improving the molar yield of cucurbitacins B from cucurbitacin B-2-O-glucoside, the single factor experiment and response surface methodology were applied to optimize the enzyme production process and biotransformation methods. The results showed that the optimal process was as follows:The medium composition for enzyme production are potato200g/L, sucrose30g/L and soybean powder15g/L with the initial pH of medium7.2; the fermentation temperature is28℃; the shaking speed is180r/min and the fermentation time is4d. The biotransformation was conducted in the system containing8mL twice diluted crude enzyme broth,1mL substrate solution and1mL MnSO4solution (50mm), and was incubated at32℃and180r/min for16h. Under the optimized condition, the molar yield of cucurbitacin B could reach99.5%with the concentration of3.23g/L. Compared to the initial17.2%and1.80g/L, they were increased by82.3%and79.4%, respectively.Finally, cucurbitacin B was separated with chromatograph by using the column packed with MCI CHP20P. The different concentrations of ethanol were used as elution for washing out the product. The elution curve showed that cucurbitacin B was washed out when60%ethanol was used as elutions. After the elutions were collected together and the solvent was evaporated, the cucurbitacin B was harvested with a yield of78.5%and purity of98.4%. The molecular weight and structure of cucurbitacin B was confirmed by LC-MS and1H-NMR.