Activation Of Esterification Activity Of Recombinant Lipase From Rhizopus Chinensis In Non-aqueous Media

Lipase-catalyzed esterification reaction in non-aqueous media has important industrial applications.However,the esterification activity of natural lipase is generally low.It is of great theoretical and practical value to improve the non-aqueous catalytic activity of lipase.The lipase from the filamentous fungi Rhizopus chinensis has high esterification activity,but the heterologously-expressed lipase does not exhibit obvious esterification activity.In this study,the recombinant Rhizopus chinensis prosequence lipase?r27RCL?and mature lipase?mRCL?were treated with different methods to improve the esterification activity,aiming to lay the foundation for its industrial application.The major contents and results of this research are described as follows:?1?The lipase r27RCL was treated with surfactants and other activating methods in order to improve the esterification activity.The results demonstrate that different surfactants have different influence on esterification activity,among which the surfactant n-dodecyl-?-D-maltoside?DDM?has the best activating effect.The critical factor for DDM-induced activation is the molar ratio of DDM to r27RCL rather than concentration of DDM.Esterification activity cannot be activated until the molar ratio is greater than 30,and then increases with the amount of DDM.Changing the solution pH and contents of inorganic salts can influence the activating effect of DDM on r27RCL.Increasing the concentration of phosphate buffer can strengthen the DDM-induced activation,another 96%increase in specific activity.?2?In terms of mRCL expressed as inclusion bodies in Escherichia coli,effects of in vitro refolding and addition of surfactants in the refolding process on esterification activity were studied.Due to the formation of inclusion bodies in E.coli,refolding process was conducted after the improvement of the expression level of mRCL by the optimization of the expression conditions.Under the condition of initial protein concentration of 100?g·mL^-1,4?and pH 8.0,72%of protein can be recovered by dialysis against solution with gradient reduction in urea concentration,of which the esterification activity is low.Addition of DDM in refolding process increased the esterification activity of m RCL by 32 folds.?3?The strongly activated RCLs were applied to catalyze non-aqueous reactions.The DDM-salts-activated r27RCL was used to catalyze the synthesis of ethyl hexanoate in organic media and ethyl oleate in solvent-free system.The results that higher reaction rate and conversion were obtained suggests the applicability and universality of the activated lipase in esterification reaction.