内切酶催化反应及非水溶液中胶束性质的研究

【作者】 孔哲；

【导师】 张洪林；

【作者基本信息】 曲阜师范大学 ， 物理化学， 2007， 硕士

【摘要】 随着各种蛋白质纯化技术的发展，纤维素酶的分离纯化研究也取得了很大进步。纤维素酶来源广泛，组成复杂，具有高度的底物专一性，其对纤维素的降解需要三种主要成份的协同作用才能有效地进行。对纤维素酶进一步的分离提纯并系统研究它的酶催化反应可为研究纤维素酶的协同作用提供理论基础，从而提高酶解效率，使纤维素资源得到有效利用，缓解日益严重的环境和能源问题。非水溶液中表面活性剂性质的研究可以为进一步研究纤维素酶的胶束酶催化奠定理论基础。一些纤维素不溶于水而溶于某些有机溶剂，在非水环境中研究纤维素酶对纤维素的降解是酶催化反应新的研究方向。具体研究内容如下：1．内切葡聚糖酶的分离纯化利用凝胶层析的方法对纤维素酶的C_x酶组分进行了分离提纯，用SDS-聚丙烯酰胺凝胶电泳法和DNS显色法对分离所得的C_x酶组分的纯度和酶活进行了检验得到活性较高的C_x酶组分。2．内切葡聚糖酶最佳酶解条件的研究用微量量热仪测出C_x酶降解纤维素在不同酸度和不同温度下的热功率—时间曲线，按照热动力学理论和对比进度法解析出反应的米氏常数（K_m）和表观最大速率(V_max)，建立最大速率与酸度和温度的关系式，从而得到C_x酶催化反应的最佳酸度（pH=5.20）和最佳温度（322.9K）。3．常见离子对C_x酶催化反应的影响测定了在不同浓度的离子存在时的热功率—时间曲线并进行解析，通过比较得出相同浓度的不同离子以及同一离子在不同浓度时对C_x酶催化反应的抑制与激活作用的规律。得到了Na⁺，Li⁺，Cd²⁺，K⁺，Ca²⁺对C_x酶催化反应有激活作用，并且激活作用由Na⁺到Ca²⁺逐渐减弱；Co²⁺，Cu²⁺，Mg²⁺，Ba²⁺对C_x酶催化反应有抑制作用，其抑制作用由Co²⁺到Ba²⁺逐渐增强：Cl^-，Si₂O₈^2-，NO₃^-对C_x酶催化反应有激活作用，其激活作用由Cl^-到NO₃^-逐渐减弱；SO₄^2-和SO₃^2-对C_x酶催化反应有抑制作用，其抑制作用为SO₄^2-小于SO₃^2-。在所选浓度范围内，激活或抑制作用均随离子浓度的增加而增强。5．SLA和SDS在DMF体系中胶束类型的确定十二烷基羧酸钠（SLA）和十二烷基硫酸钠（SDS）在N，N-二甲基甲酰胺（DMF）体系中的胶束均属于O／W型胶束。6．CMC及热力学函数的确定及规律性用微量量热仪测定了SLA和SDS在DMF体系中，在不同温度下和不同浓度的各种醇存在时胶束形成过程的热功率-时间曲线。根据曲线转折点所对应的体积，获得临界胶束浓度（CMC）；从曲线上的面积，求得热效应，从而得到标准形成焓（△H_m^θ）；根据△H_m^θ和CMC的数据，运用表面活性剂作用原理及热力学理论计算出△G_m^θ和△S_m^θ。结果表明：SLA和SDS在DMF体系中，在含有相同浓度、相同碳原子数的醇时，CMC、△H_m^θ和△S_m^θ的值随着温度的升高而增加，△G_m^θ的值随着温度的升高而降低；在相同温度及相同浓度的醇存在时，CMC、△H_m^θ、△G_m^θ和△S_m^θ的值都随着醇中碳原子数目的增加而降低；在相同温度下、相同碳原子数的醇存在时，CMC和△G_m^θ的值随着醇的浓度的增加而增大，而△H_m^θ和△S_m^θ的值随着醇的浓度的增加而减小。本文的创新之处1．将纤维素酶的内切葡聚糖酶组分进行分离提纯并单独研究其酶催化反应为系统研究纤维素酶的协同作用及寻找三种主要成分之间的最佳酶配比奠定理论基础。利用微量量热法，根据热动力学理论获得热动力学参数并用来表征C_x酶催化反应的特征及规律，确定酶催化降解纤维素的最佳条件的研究是酶催化反应研究的新课题。2．利用微量量热法研究非水溶液中表面活性剂，助表面活性剂及溶剂的相互作用，用测得的CMC及胶束形成过程的热效应，得到胶束形成焓△H_m^θ，进而计算出△G_m^θ和△S_m^θ。这是获得CMC和热力学函数的新方法。更多还原

【Abstract】 With the development of purification technology of protein, the separation and purification of cellulase has been advanced greatly. Cellulase’s source is widespread, its composition is complex and its effect on substrate is highly specificity. Cellulose’s degradation by cellulase is effectively done only when the three main constituents of cellulase cooperate with each other. Further separation and purification of cellulase and systematic study of its enzyme catalysis can offer theoretical basis to the research of cellulase’s cooperation. So, the efficiency of enzyme catalysis can be improved, the cellulose resource can be effectively used and the more and more severe problems of environment and energy can be relaxed.The property research of surfactant in non-aqueous solution can provide theoretical basis for the research of micelle enzyme catalysis of cellulase. Some cellulose is not water miscible, but it can be dissolved in certain organic solvent. The study of cellulose’s degradation by cellulase in non-aqueous environment is a new research subject.The main contents are as follows:1. the separation and purification of endoglucanaseThe endoglucanase of cellulase was separated and purified by gel chromatography. The purity and enzyme activity of endoglucanase were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and DNS development process. The endoglucanase with high activity was got.2. the study of optimum condition of endoglucanase’s enzyme catalysisThe power-time curves of cellulose’s degradation by endoglucanase at different acidities and different temperatures were determined by microcalorimetry. Using thermodynamic theory and reduced extent method, the Michaelis constant （K_m） and the maximum velocity (V_max) of the reaction were obtained. The relationships between maximum velocity and acidity or temperature were established and the optimal acidity （pH=5.20） and the optimal temperature （322.9K） of enzyme catalysis were got.3. the influence of common ions on enzyme catalysis of endoglucanaseThe power - time curves of enzyme catalysis without ions and with the existence of ions of the same or different concentrations were determined and analyzed. By comparing, the regularities of activation or inhibition of different ions of the same concentration or of the same ion of different concentrations to enzyme catalysis were seen. It was found that Na⁺, Li⁺, Cd²⁺, K⁺ and Ca²⁺ were active to the enzyme catalysis and the effect weaken from Na⁺ to Ca²⁺; Co²⁺, Cu²⁺, Mg²⁺ and Ba²⁺ were inhibitory to the reaction and the effect reinforced from Co²⁺ to Ba²⁺; Cl^-, Si₂O₈^2- and NO₃^- were active to the enzyme catalysis and the effect weaken from Cl^- to NCV; SO₄^2- and SO₃^2- were inhibitory to the reaction and the effect of SO₄^2- was weaker than that of SO₃^2-. The activation or inhibition increased with the increment of ion’s concentration in the selected concentration range.5. the determination of micelle’s type of SLA and SDS in DMF systemThe micelles of Sodium laurate （SLA） and Sodium dodecyl sulfate （SDS） in DMF system all belonged to O/W type.6. the determination and regularity of CMC and thermodynamic functionsThe power-time curves of the micelle formation process were determined for SLA and SDS in DMF system under different temperatures and different alcohols of different concentrations by titration microcalorimetry. From the corresponding volume of the lowest point of the curves, the critical micelle concentration （CMC） was determined; from the area up the curves, the thermal effect and ΔH^θ_m were measured; according to the action principle of surfactant and thermodynamic theory, the ΔG^θ_m and ΔS^θ_m were calculated. The conclusions were as follows: For SLA and SDS in DMF system, the CMC, ΔH^θ_m and ΔS^θ_m increased while the ΔG^θ_m decreased with the increment of temperature in the presence of same alcohol’s concentration and carbon number; the CMC, ΔH^θ_m, ΔG^θ_m and ΔS^θ_m all decreased with the increment of alcohol’s carbon number in the presence of same temperature and same alcohol’s concentration; the CMC and ΔG^θ_m increased while the ΔH^θ_m and ΔS^θ_m decreased with the increment of alcohol’s concentration in the presence of same temperature and alcohol’s carbon number.The innovation of this paper:1. The separation and purification of endoglucanase of cellulase and exclusive study of its enzyme catalysis can provide theoretical basis for the research of cellulase’s cooperation and for the optimum condition seeking of the three main constituents. Thermodynamic parameters which obtained by microcalorimetric method and thermodynamic theory are used to characterize endoglucanase’s enzyme catalysis and to determine the. optimum condition of the enzyme catalysis. This is a new subject for enzyme catalysis research.2. microcalorimetric method is used to study the interactivity of surfactant, cosurfactants and solvents in non-aqueous solution. The CMC and thermal effect are determined and the ΔH^θ_m is obtained, then the ΔG^θ_m and ΔS^θ_m are calculated. This is a new method to gain CMC and thermodynamic function.更多还原