修饰脂肪酶催化水解反应的动力学和热力学研究  

Study on the Hydrolysis Catalyzed by Modified Pocine Pancreas Lipase by Kinetics and Thermodynamics

作　　者：熊亚红[1] 苏健鸿[1] 刘小平[1] 

机构地区：[1]华南农业大学理学院生物材料研究所,广东广州510642

出　　处：《分子催化》2010年第5期435-442,共8页Journal of Molecular Catalysis(China)

基　　金：华南农业大学"211工程"重点学科建设项目(2009B010100001);国家自然科学基金(青年科学基金项目)(20906034B0604);广东省科技计划项目(2008B080701011)

摘　　要：采用邻苯二甲酸酐(PA)对猪胰脂肪酶(PPL)进行了化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰度,并比较了酶修饰前后的紫外光谱和荧光光谱;采用动力学方法考察了酸碱和热对修饰酶水解活性和稳定性的影响,并计算了一系列动力学和热力学参数.实验结果表明:PA对PPL的修饰度为30%,脂肪酶PPL经PA修饰后,其紫外特征吸收峰发生红移,吸收强度降低,而且最大荧光发射峰发生蓝移,荧光强度增大;PPL经PA修饰前后,催化水解反应的最适pH和最适温度未发生变化,仍为pH7.5和40℃,但修饰后酶催化水解的效率和酶与底物的亲和力得到有效提高,反应的活化能Ea、自由能ΔG≠、活化焓ΔH≠及活化熵ΔS≠均明显降低,如最适条件下修饰酶PA-PPL和脂肪酶PPL的转化数kcat和反应的活化能Ea分别为39.8 s-1、25.8 kJ/mol和34.4s-1、43.8kJ/mol;PPL经PA修饰后,酶分子热变性的热力学参数(变性活化能Ea,d、变性自由能ΔGd≠、变性活化焓ΔHd≠及变性活化熵ΔSd≠)均增大,半衰期t1/2延长,酶分子的热稳定性也得到有效提高.Pocine pancreas lipase（PPL） was modified with phthalic anhydride（PA） and the average amino modification yield was determined by trinitrobenzene sulfonic acid.Ultra violet spectrum and fluroscence spectrum of modified lipase PA-PPL were compared with those of unmodified lipase PPL.Effects of pH and temperature on hydrolysis activities and stabilities of PA-PPL and PPL were studied by kinetic method and series of kinetic and thermodynamic parameters were calculated.The experimental results showed that the average amino modification yield was 30% under the experimental conditions.After PPL was modified by PA,red shift of PPL＇s characteristic ultraviolet peak and absorbance decrease occurred and blue shift of fluroscence emission peak and fluroscence intensity incrase were taken place.After PA modification,PPL＇s optimal pH and temperature for catalysis weren＇t changed and these values were pH 7.5 and 40 ℃ respectively.Chemical modification of PPL with PA caused that catalysis efficence and substrate affinity were enhanced and all of activation energy（Ea） and activation free energy（ΔG≠） and activation enthalpy（ΔH≠） and activation entropy（ΔS≠） for catalysis decreased obviously.For example,turnover number（kcat） and activation energy（Ea） ＋under the optimal conditions for hydrolysis of PA-PPL and PPL were 39.8 s-1,25.8 kJ/mol and 34.4 s-1,43.8 kJ/mol respectively.Chemical modification of PPL with PA also resulted that the values of thermodynamic parameters for thermal denaturation,such as activation energy（Ea,d）,activation free energy（ΔG≠d）,activation enthalpy（ΔH≠d） and activation entropy（ΔS≠d） for denaturation,increased markedly and the half-life time（t1/2） became longer.So PA modification also enhanced thermal stability of PPL.

关 键 词：脂肪酶 化学修饰 稳定性 动力学 热力学参数 

分 类 号：Q55[生物学—生物化学] O643.32[理学—物理化学]