Improving Thermostability Of Yarrowia Lipolyticalipase 2 And Ehancing Expression Levelsof The Mutants

In this study,wecombined disulfide bonds and used molecular dynamic simulationsto investigate the thermostable effect of multiple disulfide bonds on Yarrowia lipolytica lipase 2?Lip2?,andco-expressed chaperones PDI?Protein disulfide isomerase?and transcription factor HAC1?The homologous to the activating transcript factor/cAMP-response bingding protein?with mutants of Lip2 to enhance the expression levels.It would improve the value of Lip2 used in the feed industry.The researchconsisted of four sections.In experiment?,we combined strongly effective disulfide bond mutation sites?S8-214,S60-69,S191-241,S122-196 and S118-177?that our laboratory identified previously,to investigate the thermostable effect of multiple disulfide bonds in Lip2.In experiment?,we used the multi-template approach to modeling engineered disulfide bonds with the Bridge D program in a larger conformational space,to screen other disulfide bond mutation sites,and new mutation sites were joinedinthe mutations library;subsequently,we investigated the combined regional effect by combining disulfide bonds from same and diffirent regions;then iterative mutation werecontinued on the base in experiment I.In experiment?,we investigated the thermostable effect of multiple disulfide bonds on Lip2 by using molecular dynamic simulations from microscopic view.In experiment?,we co-expressed chaperones PDI and signaling molecules HAC1 with mutants of Lip2 to ehance expression levels.The results showed as follows:1.The mutant could not express normally while disulfide bond mutation sites S191-241 wascombined with S8-214;and catalytic activity of the mutant sharply declined while disulfide bond mutation sites S191-241 wascombined with S122-196.While mutation sites S8-214,S60-69,S122-196 and S118-177 were combined mutually,their own bondcould form properly.We obtained mutantsby introducing two,three and four disulfide bonds?2S:S214+69,S214+196,S214+177,S69+196,S69+177 and S196+177;3S:S214+69+196,S214+69+177,S69+196+177 and S214+196+177;4S:4s?with iterative combination.The thermal stability of themutant4s increased substantially,Td?Dnaturation temperature?and T₅₀¹⁵?Half lost temperaturefor 15 min?was 64.77and 68.35?,increased by16.45 and 27.49?compared with Native?wild-type?,respectively.2.We screened three strongly effective disulfide bond muta nts,S1-242,S2-210 and S14-216 by using the multi-template approach of Bridge D program.Their T_d were 63.69,58.80 and 54.02?,increasedby 15.37,10.48 and 5.7?compared with Native,respectively.3.The thermal stability of mutants that combining weaker disulfide bonds from diffirent regions,S214+69(t⁵⁵_1/2=94.95 min)and S214+69+196(t⁶⁰_1/2=72.96 min),were superior to that combining stronger disulfide bonds from same regions,S214+210(t_1/2⁵⁵=44.43 min)and S214+210+216(t_1/2⁶⁰=25.57 min).4.Compared with Native,the thermal stability of muta nts,5s and 6s,increased more substantially.Their T_d were 68.55 and 70.85?,increased 20.23 and 22.53?,respectively.Their T_opt?Optimum temperature?were 55?,p H_opt?Optimum pH?were between 4.5⁵.0,and their reaction temperature and pH range were enlarged.5.The results ofhyperthermia molecular dynamics simulation showed that the RMSD?Root mean square deviation?,Rg?Radius of gyration?and the ability of maintaining secondary structure of mutants 4s,5s and 6s were improved compared with Native,but could not identify thermostable tendency between ofthem.The cluster analysis showed theunfoldingintermediate state timesof4s,5s and 6s wereprolonged,and the thermostable tendencycould beestimated.6.We found that PDI could significantly improve theexpression level of 5s and 6s,increased by 60%?P<0.05?and 80%?P<0.05?respectively,byco-expressing mutants 5s and6s with chaperones recombinant vector p AO815-PDI.While co-expressingmutants 5sand6s withtranscription factor recombinant vector p AO815-HAC1 and combined recombinant vector p AO815-PDI/HAC1,we found that HAC1 had no significant effect of theexpression level of 5sand6s?P>0.05?.