Screening And Indentification Of A Strain Degradation Of Phytosterol Into Androstenone And Optimizing The Fermentation Conditions Of Transfomation

Androst-4-end-3,17-dione(AD) and androst -1,4-end-3,17-dione (ADD) were commonly used for the synthesis of many steroid drugs. and these steroid compounds play important roles in cell's biological functions. The objective of this work was to select phytosterol biotransforming strains which had the ability of transformation of phytosterol into AD and ADD, and capable of growing in high substrate concentrations from Soil sample. The strain identified for its morphologic, physiological and biochemical characteristics. the optimal conditions for transformation of phytosterol and the methods of analysis substrate and products also investigated. The mainly researching contents are blow:The methods for rapid analysis and determination the cleavage of soybean sterol were studied, in which the substrate and products were produced.With thin 1ayer chromatography, the soybean sterol, AD and ADD were succeed to divide. So the consumption of substrate and the produce of AD and ADD can be monitored rapidly and conveniently. And according to the absorption of substrate and products in ultraviolet, the amount of ADD and AD can be assayed in two wavelength. Liebermann-Burchard adopted to calculate the quantitative of substrate soybean sterol. The high performance liquid chromatography could precise determination the products and HPLC/M S could determine the molecular weight of ADD and AD. The aforesaid methods were convenient to manufactory to assay produce simultaneously.In this work, an efficient strain was screened from 200 conserved strains, which had the ability of transformation of phytosterol into androst-4-end-3,17-dione and androst -1,4-end-3,17-dione. The strain identified for its morphologic, physiological and biochemical characteristics, and 16S ribosomal DNA sequence analysis. The results indicated that the utilization of carbohydrates by the strain ST06-95 include glucose, sucrose, glycerin, etc., and determination was made after cultivation at 30℃for 7 days. It could hydolysate starch and not grow on cellulose. 16S ribosomal DNA analysis showed that the strain ST06-95 belonged to Bacillus sp. Its 16S ribosomal DNA gene sequence was similar to Bacillus Amyloiquefaciens, with identity 99.9%, Therefore, the strain was nominated as Bacillus Amyloiquefaciens ST06-95. The fermentation conditions which affect the products of AD and ADD were investigated.The biotransformation conditions such as the concentration of substrate, the method of substrate added to medium and the time when substrate added to medium have been studied. Furthermore, the fermentation conditions such as the composition of medium, original pH, temperature, inoculation quantity, transformation time were also investigated. The optimal conditions for transformation of phytosterol were as follows: substrate concentration 0.5%, transformation time 160h, original pH 7.0, inoculation quantity 12%, fermentation temperature 30℃, agitation speed 220 r·min-1, use ultrasonic irradiation and surfactant Tween 80 (0.4%) to enhance dissolution of substrate, the ratio of the glycerol and the glucose in the fermentation cultivation medium was 1:4. Under these optimal conditions, the maximum productivity of phytosterol to ADD and AD were increased from 29.5 % to 42.5%, the ratio of ADD and AD was about 10:1. Which provides a good perspective to androstenone industry.The optimization of transformation process in 5L fermentation reactor based on the experiments of flask, the operation parameters of 5L fermentation reactor were established. During the course of culitvation, the temperaturew as controlled in 30℃, and the abundant oxygen could be acquired through adjusting the air flux and the stir rotate speed.The transformation effects (transformation 144h) of 5L fermentation reactor were below: When the substrate adding rate was 0.5%, the substrate transformation rate was 99%, and total production rate was above 45%. When the substrate adding rate was 0.8%, the substrate transformation rate was about 95%, and total production rate was 40%. When the substrate adding rate was 1.0%, the substrate transformation rate was 50%-60%, and total production rate was about 30%.