Effects Of Charged Residues In Lid And C-Terminal Peptide On The Catalytic Activity And Selectivity Of HL1232 Lipase

HL1232 lipase(also known as PLA1),is a preparation obtained from the fusion of the genes of the lipase from Thermomyces lanuginosus and the phospholipase from Fusarium oxysporum.This new enzyme presents not only lipase activity but also phospholipase activity.HL1232 lipase displays interface activation,which is related with the lid domain.The position of lid determines the enzyme in an inactive or active conformation.Based on the previous results of our laboratoty,the effects of three charged residues in lid region on the function of this lipase were further studied by site-directed mutagenesis in the current paper.In addition,lipase HL1232 is widely applied in oil degumming industry because of its ability of hydrolysis of phospholipids.With the analysis and alignment of amino sequence,the role of C-terminal peptide on hydrolysis of phospholipids was studied.This research mainly includes the following contents:1,The effects of charged residues in lid domain on the catalytic activity of HL1232 lipase.On the basis of previous research,mutants R81 E,R84E and R87 R were designed by replacing residues with opposite electronic charge.Furthermore,multi-mutants R81AR84 M,R81AE87Q,R84ME87 Q and R81AR84ME87 Q were conducted to determine the effects of these three residues on the enzymatic characteristics more comprehensive.With the pGAPZaA-HL1232 plasmid,the expression vectors of mutants were successfully constructed by site-directed mutagenesis.After electroporation,expression with Pichia pastoris X-33,and purification with Q-anion exchange column,enzymes with high purity were obtained.Biochemical properties of hydrolysis towards olive oil and soy lecithin of wild-type and mutants were determined.Compared with wild-type,for R84 E mutant,the specific activity of hydrolysis towards both lipids and phospholipids was improved.While it was decreased for mutant E87 R.Besides,the optimal temperature of E87 R mutnats was dramatically changed.The optimal temperature for wild-type to hydolyze olive oil and soy lecithin is 45℃ and 55℃,while it decreased to be 30℃ and 45℃ respectivity.The specific activity of R81 E mutant was similar to wild-type,and the optimal conditions for it to hydrolyze both olive oil and soy lecithin are the same.Besides,even though the specific activity with certain single-mutant was improved,except for R81AE87 Q mutant,the activity was decreased for other three multi-mutants.The change in catalytic activity of properties was not cumulative by plurality mutation of amino acids.2,The effects of C-terminal peptide on the substrate selectivity of HL1232 liapse.Based on the analysis of amino sequence,mutants D263-317,D270-317,S263-269 and S263-269D270-317 were constructed to study the role of C-terminal peptide on the phospholipase activity of HL1232 lipase.Besides,D266 might play an important role in the catalytic characteristics of HL1232 lipase,and mutants D266 A,D266E and D266 E were obtained.After heterologous expression and purification,the enzymatic properties of these mutants were analyzed.The results showed that deletion or substitution of the C-terminal peptide,the enzymatic properties of HL1232 lipase would be changed greatly.The optimal pH for wild-type to hydrolyze olive oil and soy lecithin was 7.0 and 5.0 respectivity.However,the optimal pH for D263-317 and S263-269 mutants to hydrolyze soy lecithin was increased to be 7.0,and the optimal pH for S263-269 mutant to hydrolyze olive oil was increased to be 9.0.Delete the whole C-terminal peptide(mutant D263-269),the(phospho)lipase activity was decreased.Substitution of 263-269 residues would alter the hydrolysis preference towards lipids and phospholipids.The PL/TG was changed from 0.828 of wild-type to 0.802(D263-317),0.601(D270-317),0.328(S263-269),and 0.115(S263-269D270-317),respectively.3,Analysis by bioinformatics,it could be found that the interactions(including Cation-π,hydrogen bonds,salt bridges,hydrophobic interactions,etc.)formed by residues of lid with the surrounding residues and external environment would play an important role in the conformational transition of lid domain.They influence the flexibility and stability of the lid and thus affect the catalytic efficiency of the enzyme.The C-terminal peptide of HL1232 lipase might contribute to the polarity of the catalytic center and then encourage this enzyme to hydrolyze phospholipids.