Biochemical Characteristic Analysis And Molecular Modification Of Lipase From Aspergillus Niger

Lipases（triacylglycerol hydrolases;EC 3.1.1.3）can catalyze the hydrolysis and synthesis of triglycerides and other water-insoluble esters at lipid-water interfaces.The current study found that lipase can catalyze a series of reactions such as ester synthesis,amidation,alcoholysis,and transesterification,and they are stable in various organic solvents and do not require cofactors,making them a versatile biocatalyst.In the previous research of the research group,the transcriptome of Aspergillus niger was analyzed,and many genes encoding lipase in Aspergillus niger were excavated,and their enzymatic properties and molecular structure were deeply analyzed,which may help to reveal the molecular mechanism of lipase catalyzing the synthesis of flavor substances.The structure-function relationship of the lipase catalytic pocket was studied by site-directed mutagenesis and molecular docking,which provides a new idea for protein engineering.In this paper,two lipases Lip A and Lip B from Aspergillus niger CBS513.88 were successfully expressed in Escherichia coli,and the properties of the two recombinant lipases were further studied.The results of substrate specificity showed that Lip A had hydrolytic activity on short and medium-chain（C2-C8）p-nitrophenol esters（p NPs）,and had the strongest hydrolytic activity on p NP-C4;Lip B showed both medium and long-chain p-nitrophenol esters.The hydrolysis activity was the highest,and the hydrolysis activity on p NP-C2 substrate was the highest.The optimum temperature and p H of Lip A are 40℃and7.0,and it has good stability at 40-50℃and p H 6.0-8.0.1mmol/L Mg²⁺can promote the activity of Lip A,Fe³⁺,acetonitrile and ethyl acetate have obvious inhibitory effect on the activity of Lip A.The optimum temperature and p H of Lip B are 50℃and 8.0,and it has good stability at 40-60℃and p H 5.0-8.0;Mg²⁺,Ca²⁺,hexane and heptane can promote the hydrolysis activity of Lip B,Fe³⁺and dodecane had the most obvious inhibitory effect on the enzyme activity of Lip B.The study of esterification characteristics showed that lipase Lip B could catalyze the synthesis of ethyl lactate.Under the condition that the molar ratio of lactic acid and ethanol was 1:1 at 50°C for 12 h,the synthesis amount of ethyl lactate was 54 mg/L.A preliminary exploration was made on the application of catalyzed production of flavor substance ethyl lactate in the brewing industry.The secondary structure composition of Lip A was analyzed by bioinformatics,and the binding of substrates with different length carbon chains to the template protein was simulated by combining homology modeling and molecular docking.Six amino acid residues near the catalytic pocket were selected and scanned by alanine.The strategy resulted in 6 mutants（R116A,Y190A,P241A,C243A,M250A,L253A）.The stability results of the mutant enzymes showed that the thermal stability of M250A was improved,and the thermal stability of C243A was significantly decreased;the stability of P241A was improved under acidic conditions,the stability of Y190A was reduced under acidic conditions,and the stability of R116A and C243A under alkaline conditions was reduced.Stability under conditions is reduced.The results of kinetic parameter analysis showed that the substrate specificity of the mutant enzymes was changed,and the mutant enzymes Y190A and C243A enhanced the specificity for medium and long-chain substrates.Substrate specificity has a greater impact,while disulfide bond has a great effect on the thermal stability of the enzyme...