Molecular Modification On Catalytic Activity Of Thermomyces Lanuginosus Lipase

Thermomyces lanuginosus lipases(TLL)are widely used in industry fields such as biodiesel,detergents,food,cosmetics,leather,and medicine industry for good thermostability,excellent catalytic activity and tolerance in alkaline environment.However,lipases for industry use have strict requirements such as activity and tolerant ability,optimization on lipase properties can efficiently minimize production cost.In order to improve the catalytic activity of TLL and so forth,we performed computer-aided design,point-saturation mutagenesis,and site-specific mutagenesis to improve TLL catalytic,and the resultant TLL mutants were identified with enhanced catalytic activity and were suited for biodiesel preparation.1.Semi-rational protein engineering using point-saturation mutagenesis technique was performed on the three amino acid of TLL(G23,D62,and D96).There are totally 5760 TLL mutants screened,and 12 TLL mutants with improved catalytic activity were identified.(1)By analyzing the nature of the mutants,we found that the 96 th amino acid of TLL is closely related to its catalytic activity and enzymatic properties,and the compound mutations at the three positions of G23,D62,and D96 can obtain more abundant enzymatic properties.mutant.(2)And the optimal pH of mutants G23A/D62K/D96 H,D62R/D96 E,D96Q,D96 M,D96K,D62T/D96 R becomes 10.0;the optimal temperature of D62T/D96 R,G23V/D96 R,G23L/D62 V increases 60?;G23T/D96 R was treated at 80? for45 min,the relative enzyme activity was 47.3%,the thermal stability was improved compared with TLL;the pH stability range of D96 M and D96 K was wider than that of TLL;D96M,D96 K,G23L/D62 V The affinity and catalytic efficiency of G23V/D96 R for substrate are higher than TLL.2.Rosetta ddg_monomer program was applied to calculate the free energy changes in order to predict TLL mutant with improved stability.We discovered that charged amino acids of TLL may influence TLL stability based on analysis of Rosettaresults.Therefore,mutagenesis on 8 charged residues D111,D158,D165,R205,R209,R232,E239,and D254 were performed.3.We use the ddg_monomer module of Rosetta software to perform unit point saturation mutation calculations and predict the stability of TLL mutants with improved stability.After summarizing and analyzing the prediction results,we found that the charged amino acids on the surface of TLL have the potential to affect the stability of TLL.Therefore,we performed site-directed mutations on the TLL charged amino acids D111,D158,D165,R205,R209,R232,E239,and D254.(1)As a result,we found that mutants on four negatively charged amino acids D111,D158,D165 and E239 had improved catalytic activity.Moreover,improvement on the transesterification rate of biodiesel production were observed in the above TLL mutants.Structural analysis suggested that the negatively charged amino acids D111,D158,D165,and E239 are far from the active sites of TLL,and surface electrostatic potential of the TLL mutants were different from the wild type TLL which induced property alteration.Specifically,one TLL mutant,D165 E,possessed the best catalytic activity and thermal stability,and the transesterification rate of biodiesel production is as high as 93.56%.(2)The optimal temperature of mutants D111 E,D158F,D158 H,and D165 E was increased to 60?;D165A and R209 D were treated at 80? for 45 min,and the relative residual enzyme activity was reduced to about 50%.The pH stability of D158 H,D164E,R232 E,E239R and D254 R is improved;the affinity and catalytic efficiency of R232 F,D165A and E239 R for substrates are higher than those of TLL.In conclusion,TLL mutants with improved catalytic activity,thermostability,and biodiesel production were obtained based on the semi-rational and rational design.The resultant TLL mutants were excellent candidate for biodiesel industry,and our protein engineering methods would provide new insight for similar studies.