Improvement Of Thermal Stability Of Lipase LipY2 By Rational Transformation

Lipase can catalyze the hydrolysis of lipids and transesterification in different systems,and is widely used in food,washing and feed industries.The LipY2 derived from Yarrowia lipolytica has the advantages of high catalytic activity,stable production process,and strong acid resistance,so it can adapt to the acidic environment in the human digestive system and is suitable for applications in the food and medical industries.However,the heat-resistance of wild-type LipY2 is not strong,resulting in high production and transportation costs,and the scope of use is also limited.Therefore,this study first used molecular dynamics simulations to study the effect of temperature on the structure of LipY2,and to resolve the thermal instability of the molecular mechanism.Then further rational design of heat-resistant lipases and the use of systematic experiments to prove the thermal stability of these mutants,in order to obtain LipY2 variants with a high thermal stability.In this study,molecular dynamics simulation method was used to analyze the structural changes of LipY2 at different temperatures.It was found that the two regions Alalll-His126 and Gly207-Lys221 located on the surface of the protein were greatly influenced by temperature.Then calculate the theoretical and fold entropy of the region,and on this basis,determine five heat-resistant mutants,named S115P,T117I,G207P,V213P,and K215G.Then,molecular dynamics simulations were performed to analyze the conformational transitions of these mutants at different temperatures.The results showed that the stability of the five mutants was improved compared to wild-type LipY2.The target genes of the mutants were obtained by site-directed mutagenesis.After the expression and protein purification in Pichia pastoris GS115,the enzymatic properties of each mutant were systematically studied.The results showed that the thermostability of the three mutants G207P,V213P and K215G in the Gly207-Lys221 region was increased.After incubation at 50? for 100 min,the remaining enzyme activities were 1.25,1.6 and 1.26 times that of the wild type,respectively.The optimum temperature of V213P mutant was increased from 35? to 40?.The temperature stability of the two mutants in the Ala111-Hisl26 region did not increase,but the specific enzyme activity of the S115P mutant increased from 1047.51 U/mg to 1279.50 U/mg compared to the wild-type LipY2.These findings further demonstrate that the use of molecular dynamics simulations to design mutants has a guiding role in improving enzymatic properties.