苯甲酰甲腈水解酶产生菌的筛选及应用分析-screening and application analysis of benzonitrile hydrolase producing bacteria.docxVIP

高可达 98.22%。同时，本研究考察了 CCZU10-1 腈水解酶对其它腈类化 合物的降解情况，结果表明目标菌株具有较宽的底物谱。 最后，利用海藻酸钙包埋法对细胞进行固定化，并考察了其部分催 化特性，确定最适反应条件：底物浓度 150 mmol·L-1，反应温度为 35℃， 反应 pH 为 7.0。对固定化细胞的稳定性研究结果表明，与游离细胞相比， 固定化细胞能够表现出更高的稳定性，可以重复使用多次。并且，利用 固定化细胞进行催化反应，产物易分离，操作简便。进一步对固定化细 胞催化水解苯甲酰甲腈获得的产物苯甲酰甲酸进行制备，最终通过高效 液相色谱法及核磁共振分析确定产物的正确性。 关键词：苯甲酰甲腈; 苯甲酰甲酸; 腈水解酶; 筛选; 优化 ABSTRACT Benzoylformic acid is an important intermediate for organic synthesis because it could be widely used in the synthesis of pesticides and medicine. With the development of science and technology, the application of benzoylformic acid continues to expand and the demand is also increasing year by year. Many experts and scholars take more and more attention to the highly effective synthesis. Many methods can be used for synthesizing benzoylformic acid. The nitrilase-mediated method for biotransforming α-oxophenylacetonitrile into benzoylformic acid gains great interest under mild reaction conditions compared to chemical methods. The quality of product is good and the process is in line with the requirements of the development of green chemistry, so it has a broad prospect for development. In this study, a high-throughput nitrilase screening method and the isolation of nitrilase-producer strain, the optimization of fermentation conditions and catalytic reaction conditions, expanding the substrate spectrum, and immobilizing cells by the calcium-alginate immobilization method and the optimization of some catalytic conditions of immobilized cells were described. Finally, the main product of biotransforming α-oxophenylacetonitrile by CCZU10-1 was analyzed. Firstly, a high-throughput nitrilase screening method was built based on a modified ferric hydroxamate spectrophotometry. The color-generation reactions were optimized as follows: 0.5 mL dicyclohexylcarbodiimide (0.60 mol·L-1) and 1.0 mL hydroxyl-ammonium perchlorate (0.070 mol·L-1) were added into the 50 μL of the testing sample，and then the mixture was incubated for 15 min at room tem