| Object:Bacillus subtilis strain, produce lipase were fixed in artificial zeolite, and inspect the feasibility of the immobilize methods, estimate the catalysis ability and catalytic conditions of immobilized enzyme; discusse the process of immobilized enzyme catalysis biodiesel, exzamination each factor of influence for esterification rate, and through orthogonal test determine the best esterification reaction conditions To increase the utilization ratio and catalytic effect of lipase, For industrial use immobilized enzyme to catalysis of bio-diesel provide experimental theory.Methods:This study use the bacillus subtilis's lipase for materials, through separation of lipase and immobilized in artificial zeolite. The lipase was immobilized by using the method of Silang-adsorpsi. enzymatic activity was determinated by p-NPP, designed the single factor experiments and orthogonal experimental to finded out the best reaction conditions. And then made it to be the Biodiesel, Olive oil as a catalytic substrate, dimethyl acyl donor for the synthesis of biodiesel was investigated enzyme dosage, molar ratio of methanol to oil, moisture content, catalytic temperature, catalyst time, and different catalytic substrate of the catalytic process; establishment of orthogonal experiment design, high performance liquid chromatography were used to detect the rate changes of conversion, analysis the best conditions for catalysis bio-diesel by the method of qualitative and quantitative.Results:1. Bacillus subtilis lipase was fixed by using cross-linking-absorption. Extracted lipase by using ammonium sulfate precipitation, the results of the experiment confirmed that the best immobilization effect was when the carrier is Zeolite and the concentration of the glutaraldehyde is 3%. The measured kinetic constants was Km= 12.5μmol/L, Vmax= 1.896μmol/ml/min.2. Determination of enzymatic properties of immobilized enzyme. Immobilized lipase and free lipase temperature, pH, heat stability, stability operations, the results show that the immobilized enzyme reaction temperature is 40℃, relative to free enzyme increased 5℃, the optimum reaction pH Value of 8.0, an increase of 0.5 than the free enzyme; thermal stability was enhanced. After continuous reaction activity of the remaining five batches of 78%.3. By orthogonal design, the results show that the temperature of 50℃, pH8.0,45min reaction time conditions, the immobilized enzyme to achieve maximum catalytic ability.4. Catalysis using immobilized enzyme preparation for bio-diesel. HPLC determination of the esterification rate as a detection method to determine the optimization of the immobilized enzyme transesterification conditions:methanol to oil ratio of 4:1, enzyme amount was 0.1g/g·oil, reaction temperature 50℃, Reaction time 24h, then this condition, the conversion rate of esterification of 41.5%.Conclusion:1. Use the artificial zeolite as the carrier, glutaraldehyde as crosslinking agent, through crosslinking-adsorption method to immobilized bacillus subtilis lipase is effective and feasible immobilized method, successfully, and determined its basic enzymology constants.2. Through the determination of enzymology characteristics, the results show that bacillus subtilis lipase thermal stability and pH stability are improved, and can be duplicated, showing the advantages of the immobilized enzyme, accord with the purpose of immobilized enzymes.3. Using enzymes method to catalyze synthesis biodiesel, and using thin layer chromatography and high-performance liquid chromatography analysis bio-diesel by the method of qualitative and quantitative. Confirmed that this method can be used to determine the production of biodiesel.4. Use single factor experiments and orthogonal test, can systemic investigation the best affects of immobilized enzyme's main factors of biodiesel production, is a rigorous and completely analysis method. |
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