Study On The Enzymatic Resolution Of Biotin Intermediate And Conditions Of Catalysis

In this experiment, 171 strains, which could produce hydrolase of splitting biotin intermediate lactone were screened from the soil samples and strains that lab conserved. Among them, a strain of mildew designated as ZGW12 exhibiting high activity and enantioselectivity was obtained through multiple screening steps and the strain was identified primarily as Fusariwn sp. based on its external appearance properties. The optimum fermentation conditions determined by test were as follows: inoculum size 6%, the initial pH 5.5-6.0, temperature 30癈 and the optimum medium consisted of 20g/L of glucose, 8g/L of peptone, 5g/L of yeast cream, 4g/L corm syrup and 0.10g/L of K₂HPO₄. Optimizing cultural conditions in flask enhanced the production of esterase 8 times approximatively, and a esterase activity of 1846U/g was obtained.The fermentation of enzyme was related to growth partly and the biosynthesis of enzyme was continuous after analysis on growth process and metabolism of ZGW12 Fusarium sp. The producing velocity of enzyme was in direct proportion to cell concentration in exponential growth period and to maximum biomass in stable period. So developing cells in large quantity and prolonging stable period got a high enzyme production.The rest cells of Fusarium sp. ZGW12 were stable at pH 7.2 and under 40℃. Different media affected its activity badly and it got highest activity in water by test. However, it expressed its own highest enzyme activity both in water and in toluol at pH 7.5 and 45℃. Its apparent Vm in water is higher than in toluol, so is its apparent Km after investigation on the reaction dynamics of the rest cell of Fusarium sp. ZGW12 in detail.The conditions for enzyme enantioselective hydrolysis of biotin intermediate were studied using toluol as a compatible medium. The optimum reaction condition was pH7.5 , 45℃, a substrate concentration of 1% and rest cell quality of 6g,, and in this condition a good result of resolution achieved with an ee value of 91.5% obtained after 24 hours' reaction.