摘要
本文介绍一种测定固定化酶催化反应动力学参数的新方法。在纤维间扩散阻力和外扩散阻力存在下,改变底物输送流速,测定纤维状固定化酶的一系列对应反应初速度;用双倒数图和Dixon图分别求各种流速下的表观米氏常数(K_m^(?))和表观产物抑制常数,再用所求得的各种表观动力学参数与对应的底物输入酶柱的线速度倒数的线性关系式,两次图解求得本征动力学参数。采用这一方法求得纤维状固定化青霉素酰化酶催化重排酸水解的本征米氏常数(K_m)、产物7-ADCA和苯乙酸抑制常数的本征值(K_p,K_q)分别为6.9,16.8和94.4mmol/L。
This paper presents a method and results of intrinsic kinetic parameters for hydrolysis of 7-phenylacetamide-aminodesacetoxycephalosporanic acid catalyzed by fibrous immobilized penicillin amidase. The initial velocities were determined under intrafibrous and external diffusion limitations. The apparent kinetic parameters were obtained either by Lineweaver-Burk plot or by Dixon plots. The intrinsic parameters were obtained by reploting the corresponding apparent values against the superficial substrate feeding velocities. The intrinsic K_m, K_p and K_q were found to be 6.9, 16.8 and 94.4 mmol/L, respectively. By comparing with the data reported for the catalytic hydrolysis of benzylpenicillin catalyzed by the same immobilized enzyme, we suggested that the method just reported can be used for kinetic parameter determination in the presence of intrafibrous and external diffusion limitations.
关键词
青霉素
酰化酶
扩散
动力学
固相酶
penicillin
amidase
diffusion
kinetic
immobilized enzyme
external diffusion
