海因酶法制备D-苯丙氨酸的酶催化过程动力学

ENZYMATIC CATALYSIS DYNAMICS OF PREPARATION OF D-PHENYLALANINE

采用自行筛选的兼有海因酶和N 氨甲酰氨基酸水解酶活性的Burkholderiacepecia 10 0 3菌种 ,利用海因酶法大规模制备D 苯丙氨酸 ,对其中涉及的各过程的动力学参数进行了测定 .结果表明 :L 苄基海因的消旋速率常数为 3 975× 10 -3 min-1;海因酶的米氏常数为 16 7894mmol·L-1,最大反应速率为 0 6 12 7mmol·L-1·min-1;N 氨甲酰氨基酸水解酶的米氏常数为 0 82 6 88mmol·L-1,最大反应速率为 4 82 8× 10 -4mmol·L-1·min-1.对DL 5 苄基海因的溶解、L 苄基海因的消旋、D 海因的水解开环及其中间产物 (N 氨甲酰苯丙氨酸 )的水解脱酰氨过程建立了动力学模型 ,并在此基础上进行了动力学参数显著性分析和优化 .结果表明 :对于这一级联酶转化反应 ,D 海因酶的水解反应是快速反应 ,而N 氨甲酰氨基酸脱氨甲酰的反应速率极小 ,是该过程的控制步骤 .提高氨甲酰水解酶的活力将有助于提高总体的转化速率 ,而L 海因的消旋速率则是影响外消旋苄基海因转化率的主要因素 .

The Burkholderia cepecia 1003 screened by the authors' laboratory, which contained hydantoinase and N-carbamoylase activities, was used to prepare D-phenylalanine on a large scale. The dynamic parameters of the whole bioconversion process were measured, and the results showed that kr was 3.975×10-3 min-1, Km and rm of hydantoinase and N-carbamoylase were 16.7894 mmol &middotL-1, 0.82688 mmol&middotL-1, 0.6127 mmol&middotL-1&middotmin-1 and 4.828×10-4 mmol&middotL-1&middotmin-1, respectively. Simulation was made including processes of dissolution of DL-BH, racemization of L-BH, hydrolysis of D-BH and hydrolysis of N-carbamyl phenylalanine. The significance of parameters in this model was investigated and these parameters were optimized. The results showed that the reaction rate of D-BH hydrolysis was higher than that of N-carbamyl phenylalanine hydrolysis, and the latter was the limiting step of the whole process. Promotion of N-carbamoylase activity was helpful to D-phenylalanine production. The rate of L-BH racemization was the main factor, which influenced the conversion of racemic BH.