The Study Of The Immobilization Of Lipase Based On Biomimetic Mineralization

Biomimetic mineralization, which employs biological molecules to induce and drive the formation of inorganic oxides at room temperature, ambient pressure and neutral pH, and can control the morphology of the inorganic particles by the conformation of biological template, provides a facile new method for enzyme immobilization. Lipases have wide applications in pharmaceutic industry and biotechnology, because they can catalyze not only the hydrolysis reaction in aqueous solution but also esterification and trans-esterification reactions with high region- or enantioselectivity in organic solvent. In the present study, we investigate the immobilization of lipase based on biomimetic mineralization, and its application in the resolution of (RS)-1-phenylethanol. The details were as follows:We studied the biomimetic synthesis of tiania and zirconia induced by different proteins, particularly, cytochrome c was employed for the first time in vitro to template and catalyze the formation of titania and zirconia. Both the synthetic titania and zirconia induced by cytochrome c were amorphous spherical particles. The morphology and size of the inorganic materials were controlled by changing the pH and temperature in the reaction condition.The inducing and templating function of cytochrome c during the formation of the titania and zirconia particles was investigated and the mechanism of mineralization was proposed.The approach of the biomimetic synthesis of titania was employed for the encapsulation of lipase. The optimized immobilization conditions were:5mg/mL protamine and 6mg/mL lipase in phosphate buffer (pH7.5,50mmol/L) and 0.25mol/L water-stable Ti-BALDH, and the encapsulation efficiency and recovery of lipase were 70.1% and 20.3%, respectively. It was found that the optimum pH of free and encapsulated lipase in titania were pH 7.0 and 8.0, respectively, while the optimum temperature were 37℃and 45℃. Additionally, the encapsulated lipase in titania exhibited significantly enhanced pH, thermal, storage and recycling stability.The biomimetic zirconia approach was utilized for the immobilization of lipase, and under the optimum encapsulation conditions:20mg/mL protamine and 5mg/mL lipase in phosphate buffer (pH 6.5,70mmol/L) and 0.01mol/L water-stabile K2ZrF6, the encapsulation efficiency and the activity of the encapsulated lipase were 70% and 0.15U/mg, respectively. Compared to the free lipase, the immobilized lipase in zirconia showed significantly enhanced pH, temperature and storage stability, and the encapsulated lipase in zirconia retained 60% of its initial activity after 6 cycles.Finally, the chiral resolution of (RS)-1-phenylethanol by the encapsulated lipase in zirconia was studied in organic solvent, and it was found that the encapsulated lipase could catalyze the resolution of (RS)-1-phenylethanol with a high conversion (49.9%) and an excellent enantioselectivity (ees,99%) in octane, at 50℃,180rpm for 48 h.