蛋白质酶促水解反应机理与动力学模型

Mechanism and Kinetic Model of Enzymatic Hydrolysis of Protein

研究蛋白质酶促水解制备生物活性多肽的反应机理与动力学行为,并建模表征不同反应机制的酶解曲线.基于对不同实验条件下反应时程特性的分析,以经典的米氏方程为理论基础,提出了同时考虑蛋白质单底物水解、蛋白酶失活以及底物或产物抑制因素的机理模型,并构建了指数形式动力学方程dh/dt=aexp(-bh),其中参数a和b对于不同反应机理具有不同的表达形式和确定的数值结果.对于牛血清白蛋白(BSA)-胰蛋白酶(trypsin)模型体系,经实验拟合平均相对误差为4.73%,并求得该体系相应热力学和动力学常数:Km=0.074 8 g/L,Ks=7.961 g/L,Kd=9.358min-1,k2=38.439 min-1,Ea=64.826 kJ/mol,Ed=80.031 kJ/mol.所建整套模型可用于蛋白质酶解反应过程的模拟、热动力学常数的计算和生物反应器的优化设计.

The bioreaction mechanism and kinetic behavior of protein enzymatic hydrolysis in active peptides preparation were investigated to model and characterize the enzymatic hydrolysis curves. Taking into account single substrate hydrolysis, enzyme inactivation and substrate or product inhibition, the reaction mechanism could be deduced from a series of experimental results carried out in a batch stirred tank reactor at different substrate concentrations, enzyme concentrations and temperatures based on M-M equation, and an exponential equation dh/dt = aexp（-bh） was also established,where parameters a and b have different expressions according to different reaction mechanisms, and have different values for different reaction systems. For BSA-trypsin model system, the regressive results agree well with the experimental data, i, e. the average relative error was only 4.73%, and the reaction constants were determined as Km =0. 074 8 g/L, Ks = 7. 961 g/L, k,j = 9. 358 min^-1, k2 =38. 439 min^-1, Ea =64. 826 kJ/mol, Ed= 80.031 kJ/mol in accordance with the proposed kinetic model, The whole set of exponential kinetic equations can be used to model the bioreaction process of protein enzymatic hydrolysis, to calculate the thermodynamic and kinetic constants, and to optimize the operation parameters for bioreactor design.