Studies On Biosynthesis Of Gastrodin And Other Bioactive Natural Products By Microbial Transformation

Gastrodin and salidroside, the major ingredient of the well-known Chinese medicine,were biosynthesized through microbial transformation, which is a new method different from chemical synthesis and traditional plant extraction. In this experiment, mutagenesis of the strain, optimazition of transforming condition, extraction and purification of the enzyme were discussed in order to find the more efficient and economical method for gastrodin and salidroside synthesis and offer a new channel for microbial transformation of other bioactive natural products.Using the method of batch culture and resting culture, Rhizopus LN-1 and pseudomonas Z-1, which have the ability to biosynthesize gastrodin and salidroside were sreened sequently from many fungi, yeast and bacteria according to their TLC and HPLC results. The optimal transforming condition was investigated: 12g resting cell (wet weight) incubated at 28℃for 24 hours, in a 500mL flask containing 120mL of medium, produced a good yield of gastrodin(16%) by adding 2.3mg/mL substrate(final concentration).The optimal initial pH of the medium was 5.5. The maximum yield of salidroside(10%) was acquired under the condition: substrate concentration 1.5mg/mL, initial pH 7.0, incubated at 33℃for 20 hours on a rotary shaker using glucose as carbon source and yeast extract or (NH₄)₂SO₄ as nitrogen source.The preparative scale experiment was carried out under the ideal transforming condition, the products were obtained by extraction, chromatography, and identified on the basis of its ¹H, ¹³CNMR and EI—MS spectral data. On the basis of this, a mutant LN-B with high yield of gastrodin was selected after irradiation of UV and He-Ne laser on the spores and protoplast of the original strain. The isozymegram proved the existence of the mutation.After (NH₄)₂SO₄ precipitation, DEAE-cellulose column chromatography and sepharose CL-6B column chromatography, the enzyme for biosynthesis of gastrodin was purified to one band in SDS-polyacrylamide gel electrophoresis, and its molecular weight was about 63.2kDa. The optimum temperature of the enzyme was 50℃, and the pH was 6.0. The enzyme was stable at less than 50℃, and pH 6.0-9.0.Microorganism performs great roles in biotransformation of bioactive natural product. The fact that gastrodin and salidroside could be synthesized by microbial transformation implies a new approach to gastrodin and salidroside production. As a result, this research will be of a great economic and social value.