The Study Of Soluble Expression,Immobilization And Application Of Lipase Modified By Poly-amino Acids

The lipase has a wide application in enzymatic hydrolysis,esterification,transesterification and acylation.Lipase such as Candida antarctica lipase B(CalB),exhibits high degree of activity,stereoselectivity and operational stability in nonaqueous phase.CalB is one of the most extensively used biocatalysts in organic synthesis for kinetic resolution of a wide-rangeing of racemic substrates to produce optically pure compounds that can be used as chiral versatile scaffolds for pharmaceutical syntheses.At present,eukaryotic organisms,Aspergillus oryzae and Pichia pastoris have been used as hosts for heterologous expression of CalB.However,the operation of eukaryotic expression system is tedious,a long time will be taken to produce the enzyme.Escherichia coli is one of the first options for the recombinant expression of various enzymes due to its advantages,such as high-level expression,short incubation time,the metabolic pathway and fully understood gene regulation mechanism of E.coli.However,CalB is generally expressed in recombinant E.coli as an insoluble inclusion body.In addition,the application of free CalB at the industrial scale is limited because of the instability of free CalB at high temperatures and difficulty of recovering free CalB.Here,CalB modified with poly-amino acids at the N terminal and C terminal was constructed and ligated to a plasmid pET-21 a,and then transformed into E.coliBL21(DE3).After optimizing the culture conditions,nH-CalB-10K(n=2,4,6,8)and 6H-CalB-10 R with high solubility and specific activity were obtained.It is unnecessary to remove the poly-amino acids from CalB,because they are simple and short,and it will not affect the activity of lipase.This method will provide an effective way to achieve soluble expression of enzyme,which enable them to be studied and applied.In nature,silicon-producing tissues of diatom cell wall are mainly formed by silaffin.Silaffin was characterized by polyamine chains with positive charges and high phosphorylation.The formation of diatom cell wall not only controls the structure of silica but also maintains the activity of biological macromolecules.A strategy of biomimetic silicon formation induced by polycationic amino acids,which modified on pCalB,and immobilization of pCalB was established in this paper.The immobilization process is simple and rapid.Without additional coupling agents,pCalB can be efficiently immobilized by the charge interaction between polycationic amino acids modified on pCalB and silicon particles.After optimization experiments,the lipase CalB modified by 6-histidine at the N terminal and 10-lysine at the C terminal(6H-CalB-10K)is easily cross-linked in silica particles with a high immobilization efficiency of 96.8% and activity recovery of 81.5%.Covalent immobilization of lipase pCalB was developed by using aspen powder as carrier,because its great biocompatibility,wide source and low price.In this research,6H-CalB-10 K was covalently immobilized with oxidized aspen powder OAP and grafted aspen powder GAP,respectively.After comparative analysis the stability and catalytic performance of immobilized lipase 6H-CalB-10K-OAP and 6HCalB-10K-GAP,we found that the oxidized aspen powder could effectively combine with the poly-Lys modified CalB and the immobilization effect was not inferior to the grafted aspen powder.The carrier treatment was easier and the immobilization results were better than that reported.It provides a new idea for the development of immobilization technology,by modifying enzyme instead of carriers,the directional immobilization of enzyme can be realized.pCalB modified by polybasic amino acids were also applied to catalyze MBH reaction.MBH reaction is an important C-C addition reaction in chemical synthesis,the product,allyl alcohol is an important intermediate and can be used to synthesize pharmaceutical compounds and complex natural products.The traditional chemical catalysis methods have their disadvantages,such as environmental pollution and harsh condition.Although,there are few reports on enzymatic MBH reaction,the yield was about 7% after 4-6 days reaction.In this work,lipase pCalB catalyzed MBH reaction in phosphoric acid buffer and shortened the reaction time,the yield can reach nearly 33% after 12 hours of reaction.The pCalB inclusion body can also catalyze MBH reaction,especially the poly-His modified pCalB inclusion body(6H-CalB-10H)exhibits superior catalytic performance and 28% yield can be obtained in 18 hours.