Endo Enzyme-catalyzed Reaction And The Non-aqueous Micellar Nature

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.