金黄节杆菌CYC705腈水解酶催化亚氨基二乙酸合成的研究

Arthrobacter aurescens CYC705 nitrilase catalyzed synthesis of iminodiacetic acid

摘要:

将金黄节杆菌CYC705(Arthrobacter aurescens CYC705) 腈水解酶用于生物催化合成亚氨基二乙酸(IDA)，从生物催化剂的形式、生物催化反应过程优化和反应体系放大三个方面进行了考察。在氨基载体固定化酶、环氧基载体固定化酶、海藻酸钠固定化细胞、壳聚糖固定化细胞和游离全细胞几种生物催化剂形式中，壳聚糖固定化细胞催化效率最高、稳定性最好。通过反应体系、反应温度、金属离子、底物浓度、固定化细胞投量等因素的优化，确定了最佳的生物催化反应条件：以50 mmol/L pH=6.6的磷酸氢二钠-柠檬酸缓冲液作为反应体系，底物亚氨基二乙腈(IDAN)的浓度为200 mmol/L，添加CoCl2至终浓度为1 mmol/L，反应温度37 ˚C，固定化细胞投量为0.25 g每5 mL反应体积。在此条件下，反应2h可将IDAN完全转化为IDA。进一步将反应体系放大10倍，催化200 mmol/L的IDAN完全转化为IDA仅需1h。

Abstract:

The nitrilase from Arthrobacter aurescens CYC705 was used to catalyze the synthesis of iminodiacetic acid (IDA) in this work. The biocatalyst form, optimization of biocatalysis process and scale-up of the reaction system were investigated in detail. Several biocatalyst forms including the aminated carrier immobilized nitrilase, epoxy carrier immobilized nitrilase, sodium alginate immobilized cells, chitosan immobilized cells and free whole cells were prepared and compared. It was found that the chitosan immobilized cells showed the highest catalytic efficiency and the best stability. After optimizing the reaction system, reaction temperature, metal ion concentration, substrate concentration and addition amount of chitosan immobilized cells, the optimal reaction conditions were obtained as follows: taking 50 mmol/L pH 6.6 Na2HPO4-citric acid buffer as the reaction medium, iminodiacetonitrile (IDAN) concentration 200 mmol/L, final CoCl2 concentration in the reaction medium of 1 mmol/L, reaction temperature 37 ˚C, chitosan immobilized cells addition amount 0.25 g/5 mL reaction volume. Under these conditions, IDAN could be hydrolyzed to IDA completely in 2 h. Further, the reaction system was amplified 10 times. It was found that the time required to catalyze the full conversion of 200 mmol/L IDAN to IDA was only 1h.