Electrostatic Networks Of Penicillium Expansum Lipase Affect Enzymatic Properties

To explore the relationship between the electrostatic networks and catalytic properties of penicillium expansum lipase(PEL),site-directed mutagenesis was used,some charged residues in PEL were chosen and the enzymatic properties of variants were analyzed.The main contents include:1.Sites selectingAsp44?Asp70?Asp74?Asp76?Glu83?Glu113?Glu127?Glu207?Arg63?Arg97?Arg101 Arg182?Lys16?Lys202?Lys219 and Lys226 were selected.These sites are significant members of electrostatic networks in PEL,8 of them with negative charge(Asp or Glu),others with positive charge(Arg or Lys).Besides,all these sites are close to the center of lid domain(sites 67-75)or catalytic triad(Ser132-His241-Asp188)in PEL.2.Construction of recombinant plasmid PET-47b(+)-PEL-X and pre-screeningTo uncover the influence on catalytic properties of these charged residues,site-directed mutagenesis was used to reverse the charge of residues,namely,the negative charged residues(Asp or Glu)were changed to Lys which with positive charge,and vice versa(Arg or Lys?Asp).Then,the variants were expressed in E.coli.BL21(DE3)with recombinant plasmids.The results suggest,Asp44Lys?Asp76Lys?Glu83Lys?Arg97Asp?Arg101Asp?Lys202Asp and Lys219Asp show different activity in tributyrin identification plate,the other 9 variants without activity?Subsequently,the 9 non-active sites were changed between the same charged residues(which we called "Gay replacement"),namely,Asp(?)HGlu or Arg(?)Lys.And we got another three active variants,they are Lys 16Arg?Glu113Asp and Lys226Arg.3.Eeukaryotic expression and enzymatic properties analysis of active variantsThe plasmid pPIC3.5K-PEL-X with single mutant Asp44Lys?Asp76Lys?Glu83Lys?Arg97Asp?Arg101Asp?Lys202Asp?Lys219Asp?Lys16Arg?Glu113Asp and Lys226Arg were constructed by site-directed mutagenesis respectively.By electroporation,the recombinant plasmid pPIC3.5k-PEL-X was integrated to Pichia pastoris GS115 after linearization with SalI endonuclease.Multi-copy strains were selected with MD plate and tributyrin identification plate.Finally,five multi-copy strains were obtained after year's selecting.Pure PELs were collected respectively after fermentation with multi-copy strains.Enzymatic properties of variants were analyzed with pure PELs.The results are as follows:(1)Asp44LysThe concentration of PEL-Asp44Lys in crude enzyme extract is 0.06 mg/mL,about 30%of WT;The specific activity towards 4-nitrophenyl pamitate is 96 U/mg,about 2.67 times of WT;The optimal reaction temperature is 32?;The optimal pH is 8.0,the same with WT;After dealing with different pH buffers,the relative residual activity up to 100%in pH 7.0 buffer.It prefers short chain fatty acid;After dealing with 20%acetonitrile,20%acetone and 20%ethanol,the residual activities are 0%,29%and 58%respectively,while WT's are 1%,31%,26%respectively,suggests that PEL-Asp44Lys has better tolerance towards ethanol.(2)Glu83LysThe concentration of PEL Glu83Lys in crude enzyme extract is 0.11 mg/mL,about 55%of WT;The specific activity towards 4-nitrophenyl pamitate is 2928 U/mg,81.33 times of WT;The optimal reaction temperature is 32?;After dealing in 45 ? water-bath for 10 minute,the residual activity remains 39%,13 times of WT's;The optimal pH is 8.0;It tolerates pH5.0-11.0,after dealing with dififerent pH buffers,the residual activities are remain more than 70%,and dealing with water(as control,pH5.0-6.0),the residual activity up to 100%;It prefers long chain fatty acid,especially 4-nitrophenyl laurate(C12)and 4-nitrophenyl palmitate(C16);After dealing with 20%acetonitrile,20%acetone and 20%ethanol,the residual activities are 0.10%,100%and 73%respectively,suggests that PEL-Glu83Lys has excellent tolerance towards acetone and ethanol.(3)Arg97AspThe concentration of PEL-Arg97Asp in crude enzyme extract is 0.09 mg/mL,about 45%of WT;The specific activity towards 4-nitrophenyl pamitate is 63 U/mg,1.75 times of WT;The optimal reaction temperature is 32?;The optimal pH is 7.0;After dealing with dififerent pH buffers,the relative residual activity up to 100%in pH 7.0 buffer.It prefers short chain fatty acid;After dealing with 20%acetonitrile,20%acetone and 20%ethanol,the residual activities are 0%,67%and 33%respectively,suggests that PEL-Arg97Asp has better tolerance towards acetone compared with WT.(4)Arg101AspThe concentration of PEL-ArglOlAsp in crude enzyme extract is 0.24 mg/mL,1.2 times of WT.The specific activity towards 4-nitrophenyl pamitate is 27 U/mg,about 75%of WT;The optimal reaction temperature is 36?;The optimal pH is 7.0;After dealing with different pH buffers,the relative residual activity up to 100%in pH 7.0 buffer.It prefers It prefers short chain fatty acid;After dealing with 20%acetonitrile,20%acetone and 20%ethanol,the residual activities are 40%,60%and 20%respectively,suggests that PEL-Arg101 Asp has better tolerance towards acetone.(5)Lys226ArgThe concentration of PEL-Lys226Arg in crude enzyme extract is 0.32 mg/mL,about 1.6 times of WT;The specific activity towards 4-nitrophenyl pamitate is 21 U/mg,only 58.33%of WT;The optimal reaction temperature is 32?;The optimal pH is 8.0,the same with WT;pH-stability,tolerate pH5.0-11.0,and dealing with water(as control,pH5.0-6.0),the residual activity up to 100%;It prefers short chain fatty acid,and it is a little interested in 4-nitrophenyl laurate(C12);After dealing with 20%acetonitrile,20%acetone and 20%ethanol,the residual activities are 6%,21%and 31%respectively,suggests that PEL-Lys226Arg has huger tolerance towards ethanol.