Study On The Soluble Expression Of Lipase In Escherichia Coli

Lipase has a wide range of applications in industrial production fields such as pharmaceuticals,detergents,food additives,and fine chemicals,and is one of the most important enzymes in the industry.Although various lipases are currently expressed in a variety of expression systems,they still have shortcomings.Especially when lipase is overexpressed in Escherichia coli,a host with good universality,it is very easy to produce inclusion bodies due to the incorrectly fold,which lead to hardly meet the requirements of industrial production.Therefore,probing the factors affecting the soluble expression of lipase in Escherichia coli,improving expression efficiency,reducing costs and simplifying production technology are of great significance for promoting the development of its fundmental and applied research.In this study,the amino acid sequence of lipase Sp L from Sphingomonas sp.HXN-200 was modified by directed evolution,and a high-throughput screening system for increased protein soluble expression was constructed based on the fusion of Galactoside hydrolase.The effect of N-terminal nucleotide sequence of lipase on the translation and folding of the whole protein was studied.We expect this work can benefit the elucidation of the general rule of lipase soluble expression in E.coli,and promote the development and utilization of lipase.This research focuses on the following three aspects:1.Optimizing the expression conditions of lipase.To optimize the Sp L expression,expression conditions involved strain,vector,temperature,speed,time,types of flask and concentration of inducer were all investigated.Finally,we found as E.coli BL21(gold)was used as the host,p RSF-Duet-1 as the vector,induce by 0.1 m M IPTG,and expression at 22 ℃,200 rpm in for 18 h in flask with baffle,the best expression results can be achieved.2.Constructing a high-throughput screening system for lipase mutants and improving its solublity by error-prone PCR.We first constructed a a high-throughput screening system based on a fusion galactosidase,then error-prone PCR was employed to construct libraries for the solubility screening.Finally,a mutant with maintained activity and about 50-fold more solubility than wild-type was achieved.3.Investigation of the relationship between nucleotide sequence and protein soluble expression.While the amino acid sequence is same,different nucleotide sequences will also lead to different expression result.Here,we investigated the codes of 15 amino acids located on the N-terminal,by replacing them with the initial nucleotides,better expression result was achieved.