【作者】 黄伟；

【导师】 廖丹葵；

【作者基本信息】 广西大学 ， 生物化工， 2012， 硕士

【摘要】 酶解过程中动力学研究不仅在理论意义上加深对酶解过程的理解,而且能有效地指导生产实践,通过模型推算出获得目标水解产物所需的酶解条件。目前对于酶解蓝圆鲹蛋白的研究主要停留在工艺条件的优化以及酶的选择等方面,其酶解反应过程规律研究则相对较少。常规米氏方程以恒定动力学参量米氏常数来建模的传统方法很难真实地表征整个反应历程。本文分别用宏观动力学模型、3D图形以及集总动力学模型等三种方法对碱性蛋白酶-蓝圆鲹蛋白酶解体系表征。基于米氏方程的反应机理,加入酶失活、底物抑制和产物抑制等影响因素,并引入水解度定义,建立了宏观动力学表征碱性蛋白酶-蓝圆鲹蛋白酶解体系的动力学模型。结果发现,在pH8.5,温度t为47.5℃的条件下,酶解体系中底物抑制和产物抑制的作用可以忽略。同时为了提高效率和减小工作量,提出用D-最优设计法对模型参数估算过程进行优化。通过验证比较得,D-最优设计能应用到酶解体系水解度动力学模型参数的估算,构建的水解度模型为DH=5.781n[(-0.107+55.584[E0]/[S0])×t+1],平均相对误差为6.67%,能较好的从宏观上表征体系的酶解过程,对其工艺条件优化有一定的指导意思和理论依据。在表征过程中引入酶解产物中多肽片段族的分子量和质量百分数两个因素,采用3D图形表征碱性蛋白酶-蓝圆鲹蛋白酶解体系,使其具备更明确的目的性。通过体积排阻色谱测定酶解体系中不同水解度下酶解产物的多肽片段族的分子量分布,对多肽片段族的质量百分含量与水解度、分子量的进行3D曲面函数拟合,得到表征酶解体系的数学表达式为：m=10.4194+1.1209DH-0.0329(DH)2-0.0006(DH)3+0.0999(DH)2·Mw+23.7492Mw-16A377(Mw)2+11.6233(Mw)3-0.8423DH·(Mw)2-2.2178DH·Mw,验证得其平均相对误差为12.75%,表明该3D模型能比较准确的反映酶解体系中质量百分数与水解度和分子量之间的关系,能较好表征酶解体系的反应历程。采用集总动力学表征碱性蛋白酶-蓝圆鲹蛋白酶解体系。依据蓝圆鲹蛋白溶解特性以及组分的动力学特性将酶解体系划为四个集总,并设计其集总动力学反应网络。分别测定了集总组分的米氏常数、产物抑制常数和底物抑制常数等本征动力学参数,并采用Marquardt法估算集总动力学反应网络的动力学参数,对集总动力学模型进行实验验证表明：集总动力学模型能较好的描述碱性蛋白酶-蓝圆鲹蛋白酶解体系中组分的变化规律以及动力学反应机理,对定向获取多肽片段族提供理论支持。更多还原

【Abstract】 Recently, the researchs of enzymatic hydrolysis Decapterus Maruadsi protein are the optimization of process conditions and the choice of enzyme, which was more than the law of enzymatic hydrolysis the process. The study of the dynamics in the enzymatic hydrolysis process is not only to deepen the understanding of enzymatic hydrolysis process, but also to obtain the goal of hydrolysis products of the hydrolysis conditions and effectively guide the production practice. The traditional methods modeled by the conventional Michaelis-Menten equation with constant kinetic parameters Michaelis constant is difficult to truly characterize the entire reaction course. In this paper, alkaline protease-Decapterus Maruadsi protein hydrolyzate system was characterized by the three methods of the macro-dynamic model,3D graphics, and the lumped kinetic model.Macro-dynamics characterization of alkaline protease-Decapterus Maruadsi protein hydrolyzate system was based on the reaction mechanism of the Michaelis-Menten equation, added influencing factors of the enzyme inactivation, substrate inhibition and product inhibition. Then the reaction ratestudy was substituted by the degree of hydrolysis, in order to establish the degree of hydrolysis kinetic model. The results of the degree of hydrolysis kinetic showed that the conditions of pH8.5, temperature47.5℃, the enzymatic system substrate inhibition and product inhibition could be ignored. Meanwhile, in order to improve efficiency and reduce the workload, estimating the model parameters was optimized by the D-optimal design method. The D-optimal design can be well applied to the estimation of the degree of hydrolysis of the enzymatic system dynamic model parameters. The macro enzymatic hydrolysis process was better characterized by the degree of hydrolysis of model, DH=5.781n [(-0.107+55.584[Eo]/[So])×t+1], the average relative error of6.67%, with certain guidance meaning and theoretical basis for optimizing process conditions.3D graphic characterization of alkaline protease-Decapterus Maruadsi protein hydrolyzate system was with a more clear purpose by introducing the molecular weight and the mass percentage of peptide segments. The molecular weight distribution of peptide fragments in the hydrolysates under different degree of hydrolysis was determined by size exclusion chromatography, the results was showed:the component of molecular weight greater than10kDa was constantly reduction. The variation other of virtual components were not obvious. In order to more systematic and intuitive characterization of the enzymatic system as a function of the degree of hydrolysis and molecular weight, weight percent based on the peptide fragments, the3D surface map had been drawn. Virtual component of more than lOkDa continued to decline, the following5kDa gradually increased, while5～10kDa firstly increased and then decreased. Which can be inferred that the main reaction course of the enzymatic system high molecular weight protein was gradually degraded into small molecular weight.3D surface fitted function to characterize the enzymatic system of mathematical expressions:m=10.4194+1.1209DH-0.0329(DH)2-0.0006(DH)3+0.0999(DH)2-Mw+23.7492Mw-16.4377(Mw)2+11.6233(Mw)3-0.8423DH·(Mw)2-2.2178DH·Mw, the average relative error12.75%. The reaction mechanism of the enzymatic system was characterized by the3D model,via the relationship the mass percentage, molecular weight and the degree of hydrolysis.Lumped kinetic characterization of alkaline protease-Decapterus Maruadsi protein hydrolyzate system was based on the solubility characteristics of Decapterus Maruadsi protein and the dynamic characteristics of the components to be designated as the four lumped. Then lumped components were measured Michaelis constant, product inhibition constants and substrate inhibition constant, with the Marquardt method to estimate the kinetic parameters of lumped kinetic reaction network. The lumped kinetic model was showed: components and kinetic reaction mechanism were described by the lumped kinetic model, which provided theoretical support to obtain peptide fragments.更多还原