Study On The Thermostability And Catalytic Function Of Thermomyces Lanuginosus Lipase By Rational Design

Thermomyces lanuginosus lipase(TLL)has been widely applied in food,medicine,biodiesel and leather industry due to its high efficacy and specificity.On account of the harsh conditions of biotechnological treatments in industrial process,it is still difficult for TLL to meet the high quantity of industrial needs.Therefore,it is important to optimize the properties of TLL.In this study,the thermostability and catalytic function of TLL were modified by means of rational design,computer-aided design,sequence alignment,homology modeling,and site-directed mutagenesis,TLL mutants with improved thermostability and catalytic ability were finally obtained.TLL mutants with improved thermostability were predicted by computer-aided design with two free energy calculation modules Rosetta ddg?monomer and Cartesian?ddG.First,we conducted all single mutants of TLL,in total 5111.Then the above two calculating modules were applied to calculate the Gibbs free energy changes(??G)of each TLL mutant.Three mutants(T114W,R175W,R195W)were predicted by Rosetta ddg?monomer with improved stability for its??G is less than-5kcal/mol.The results showed that the optimum temperature of the three mutants was the same as the TLL,which was 40?;the relative residual enzyme activities of mutants T114W and R195W treated at 70?for 90 min increased by 13.46%and22.88%,respectively,compared with that of TLL.The thermal tolerant ability of T114W and R195W was improved.On the other hand,Rosetta Cartesian?ddG predicted 35 mutants including all P256 saturation mutants with improved stability.Experimental validation discovered that the optimal reacting temperature of TLL mutants N11V,S79V,S79I,P204I,P256I,and P256M was 45°C,5°C higher than that of wild-type TLL;the optimal reacting temperature of P256A?P256V?P256E?P256 was 50°C,10°C higher than that of TLL;the T_mvalues of G112W?G112Y?G112M?S79V?S79I?N11V?S170V?P204I?P204C?P256Q?P256G?P256E?P256K?P256D?P256I?P256M were all increased compared with that of TLL;besides,the biodiesel yields of N11V?S79V?P256H?P256Q?P256I?P256Y?P256E were higher than that of TLL.Since thermostability of mutants have additive effect,we chose the best TLL mutants on each amino acid(N11V?S79V?P204I?P256E and P256A),and conducted complex mutants.Experimental assays showed that the optimal temperature of N11V/S79V/P256E was increased by 5?compared with the TLL;residual activities of mutants S79V/P256A and N11V/S79V/P256A were increased by 32.82%and43.86%compared with TLL when treated at 80?for 60 min.Our study proved that the predicting accuracy of Rosetta Cartesian?ddG is higher than that of ddg?monomer.Based on the predicting results of Cartesian?ddG,a variety of thermostable lipases suited for industrial production have been obtained.Additionally,we optimized the catalytic function of TLL.It is known that lipase from Aspergillus oryzae(AOL)has the ability to hydrolyze mono-and diacylglycerol.However,TLL can only catalyze triacylglycerol.Therefore,we compared the structure and sequence of AOL and TLL,conducted three complex TLL mutants after superimposing AOL and TLL.Besides,it is known that the lipase's active site consists two binding pockets,and the alcohol-binding pocket was found important for catalytic performance and substrate preference.We substituted the alcohol binding-site residues of TLL to that of the AOL,and conducted eight TLL mutants.Finally,it revealed that I255F,L259V,G266D,and one complex TLL mutant were able to catalyze MADG.In conclusion,the thermostability and catalytic ability of TLL were optimized by rational design,TLL mutants with improved thermostability,enhanced catalytic efficiency,substrate preference and biodiesel yield were obtained.Two Gibbs energy calculating modules of Rosetta were systematically compared,our study proved that the strategy that using computer-aided method to predict stable lipase mutants was effective and reliable;the important amino acids which may influence the substrate preference of TLL were discovered and expanded in this study;the obtained TLL mutants with improved properties could be applied for industrial use,and our study provides a good reference for future study.