Lipase-Catalyzed Regioselective Esterification Of Pyridoxine And The Enzyme Characterization

A lipase-catalyzed reaction system for the preparation of high-purity 5-O-acetylpyridoxine (5-AcPN) was designed and the products were identified. Three kinds of lipases were screened and Novozym 435 lipase (Candida antarctica lipase B) was found to be the best one to catalyze the esterification of pyridoxine (PN) in nonaqueous media. In order to improve the solubility of PN and to elevate the production of 5-AcPN, three reaction media, including AOT/n-hexane reversed micelles, acetonitrile and [bmim]PF6, were used in the reaction. It was found that the site-selectivity in [bmim]PF6 was different from that of the other two, i.e., the 4-OH of PN had the highest activity in [bmim]PF6, however, the 5-OH had the highest activity in acetonitrile and reversed micelles. Finally, acetonitrile and AOT reversed micelles were chosen as the potential media to perform the reaction. Different acyl donors affected significantly not only the degree of conversion but also the regioselectivity.By employing vinyl acetate as acyl donor, Novozym 435-catalyzed esterification of PN was conducted in acetonitrile and AOT reversed micelles. The influence of several parameters such as water content, temperature, substrate mole ratio and enzyme loading on the reaction were analyzed systematically. The analysis confirmed that water content was the most significant factor affecting the esterification of PN. In addition, the response surface methodology (RSM) was used to optimize the reaction conditions for the enzymatic synthesis of 5-AcPN, and the best fitting models were established. The efficiency of the model was experimentally verified. Three sets of optimum reaction conditions were established. As a result, Novozym 435-catalyzed esterification of PN gave a maximum conversion of 99% and the regioselectivity of 93%. Besides, a scale-up experiment was successfully performed based on the optimized condition, indicating the industrial potentials of the technology.The CB-Span 85/Span 85/ hexane reversed micellar system was characterized and evaluated by employing Candida rugosa lipase (CRL)-catalyzed hydrolysis of olive oil as a model reaction. The change of micellar hydrodynamic radius reflected the redistribution of Span 85 and water after enzyme addition. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited different activity behavior vs W0. The optimal pH of the encapsulated lipase kept unchanged, but the apparent activity was significantly higher than that of the enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant decreased with increasing the micelles concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.Magnetic immobilized enzyme, MIE, was prepared by immobilizing CRL onto magnetic nanoparticles. The protection of MIE by disaccharides was investigated. It was shown the stability of MIE increased during drying and storage in the presence of disaccharides. Furthermore, the preservation action of trehalose was the most efficient, secondly was lactose, and sucrose was the worst one, which supported the'glassy state theory'.'Two-step deactivation'model was employed to fit the deactivation kinetics of MIE. Also, the deactivation rate constant and the half-life were obtained. In the presence of trehalose and lactose, the MIE half-lives increased 31- and 23-fold, respectively.