Rational Design Of Lipase For The Long-chain Polyunsaturated Fatty Acids Selectivity

The long-chain polyunsaturated fatty acid eicosapentaenoic acid(EPA)has very important biological activities,but free EPA is easily oxidized and has poor stablility,while EPA glycerides have good stability and good absorption in the human body.Therefore,the use of EPA glycerides has become a research hotspot in functional foods,dietary supplements,and pharmaceuticals.Compared with traditional physical and chemical methods,the use of lipase to generate EPA glycerides has the advantages of mild reaction conditions and low energy consumption.However,lipases with high specificity for EPA are lacking in industrial production.In order to achieve efficient and targeted synthesis of EPA glycerides,this study selected lipases that can catalyze the esterification of EPA and glycerol,through rational design to improve the specificity of the enzyme for EPA and anlyze the selection mechanism of lipases for fatty acids.The main contents of this study are as follows:1.Lipase Lipk107 was expressed and its eight mutant expression strains were successfully constructed.Their hydrolase activity was measured.The crude enzyme activities of mutants clu67,clu53,clu64,clu86,and clu36 were all increased compared with the wild type,and the enzyme activities of the mutants clu183 and clu68 were partially lost.The ability of Lipk107,clu101,clu86,clu67,and clu36 to catalyze the esterification of EPA to glycerides was initially determined by TLC and mass spectrometry.Clu101 and clu36 hardly react with EPA,clu67 converts MAG to DAG faster than DAG to TAG,and clu86 converts MAG and DAG to TAG more efficiently than Lipk107 and clu67.Combined with the analysis of the docking results,it was found that the enzyme activity is affected by the distance between the atoms of key residues.2.Lipase MAS1 was successfully expressed and purified in E.coli.The best induction results were obtained under the conditions of 20℃,220 rpm,0.1 mM IPTG and 12 h of induction.Using p-nitrophenol myristate as the substrate,the specific activity of MAS 1 was determined to be 97.189 U/mg,and its optimum pH,optimum temperature and pH stability were also determined.The ability of lipase MAS1 to catalyze EPA to glycerides was identified.3.MAS1 was successfully expressed in Pichia pastoris.After codon optimization,the average GC content of the MAS1 gene decreased from 67.74%to 40.98%,the CAI increased from 0.51 to 0.97,and the frequency of high frequency codons(91%-100%)also increased from 16%to 86%.The optimal induction condition of lipase MAS1 was 0.5%methanol induction for 96h,and the target protein was of higher purity without complicated purification process.4.There were 34 mutants of lipase MAS1 obtained by rational design.These mutant proteins were successfully expressed in E.coli.Using the hydrolase activity of 4-nitrophenol myristate as a high-throughput screening method,23 active mutant lipases were screened out,and their esterification ability was identified by mass spectrometry.The hot-spot residues of lipase MAS1 were predicted and the residues that played a key role in the esterification process were analyzed by MM/BGSA.