Screening For Nitrilase-producing Microbes And Biotransformation Of P-hydroxybenzyl Cyanide By Bacillus Subtilis E9

P-hydroxyphenylacetic acid is widely used in organic synthesis to prepare some valuable medicine, pesticide and liquid crystal material etc. This dissertation is aim to screen for nitrilases which can hydrolyze p-hydroxybenzylcyanide to p-hydroxyphenylacetic acid, and then establish the p-hydroxybenzylcyanide biotransformation process.One high-throughput screen method was established based on p-hydroxyphenylaceti acid detection using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Eight microorganisms were isolated with high nitrilase activity against p-hydroxybenzylcyanide using the enrichment technique. One of these strains numbered E9 was identified as Bacillus subtilis.The culture and nitrilase production conditions of Bacillus subtilis E9 were studied in detail employing P-hydroxybenzylcyanide as substrate. When B.subtilis E9 was cultured in optimized medium consisting of 13.5 g/L glucose, 6.5g/L yeast powder, 5.5 g/L (NH₄)₂SO₄ 6.6 g/L K₂HPO₄, 0.5 g/L KH₂PO₄, 0.5 g/L MgSO₄ and 0.01 g/L FeCl₂, the enzyme activity increased evidently. And it showed that B. subtilis E9 nitrilase was a constitutive enzyme and the nitrilase production was correlated with the cell growth.Biotransformation process of p-hydroxybenzylcyanide by B. subtilis E9, and the nitrilase properties with whole cells were studied. B. subtilis E9 nitrilase could catalyze many substrates to corresponding acid and it exhibited high activity towards p-hydroxybenzylcyanide among the nitriles tested. In the course of biocatalysis of p-hydroxybenzylcyanide to p-hydroxyphenylacetic acid, the optimum transformation temperature and pH were 32.5℃and 8.0-9.0 respectively. The cell activity was inhibited by Cu²⁺, Co²⁺, Hg²⁺, Ni⁺ and activated by Ba²⁺ and Mg²⁺. At the concentration above 1g/L, both substrate and product showed inhibitive effect. Reaction dynamics of resting cell biocatalysis showed that the apparent Michaelis-Menten constant K_m and substrate inhibitive constant K_s were 1.254 mmol/L, 9.218 mmol/L respectively. In addition, the p-hydroxyphenylacetic acid formation rate reached the maximum at p-hydroxybenzylcyanide concentration of 0.446g/L.