Expression, Characterization And Mutagenesis Study Of A Thermophilic Lipase From Deep-sea Bacterium Geobacillus Stearothermophilus TH10

Thermophilic enzyme is more attractive not only because it is related with the origin of life,but also due to their potential in industry application.Lipase is one of the enzyme that best studied.It has been found in animals,plants and microorganisms. Most of the lipases used in industry are microbial enzymes,which have extensively characterized.Thermophilic lipase is a research hotspot because their excellent thermostability is adorable in industry processing.So far,they have been widely used in food,pharmaceutical,leather process.etc.Hydrothermal vent,as an extreme environment,is characterized by the high pressure,high temperature and lots of reducing substance.Supposedly,there are some special microorganisms.So far,Over 5000 species have been isolated from this enviroment,the order "Nautiliaceae" is found only in this area.These novel microorganisms were considered the best material for thermophilic enzyme study.We isolated a strain,named as TH10,from deep-sea hydrothermal vent sulfide samples 4143-1.16s rRNA result suggests it is a member of Geobacillus.TH10 has optimum growth temperature at 65℃.Geobacillus TH10 lipase gene was obtained by amplifying with designed primer that based on conserved sequence,Then cloned and expressed in E.coli.After purification,we got pure recombinant protein.Enzyme assay shows that TH10 lipase is a thermophilic enzyme with optimum temperature at 80℃,PH at 8.0-9.0.It is inhibitied by all the test metal ions.While,detergent and enzyme inhibitors has little influence on TH10 lipase.Tween20 and chaps can even improve the enzyme activity.Geobacillus TH10 lipase hasα/βthree-tier structure which is shared by all lipase.In addition,it has an extra domain,which is composed of two helix,two sheet and some loops.This domain will cover catalytic region and make protection when at low temperature.Substrate assay shows that TH10 lipase prefer short-chain fatty acid chain especially PNP-C4,when PNP as substrate.While,Staphylococcus lipase,which show most homology with Geobacillus,it is good at degradation of C2-C18.Based on comparison of GSL and SHL,Q142M,I347V,Q142M/I347V three mutants were constructed.Characterization study shows that all the mutants show higher activity toward PNP-C2.Besides,the thermostability of I347V and Q142M / I347V are improved.Due to the lack of Geobacillus lipase structure background,directed evolution is a good alternative strategy.We consructed a mutant library based on error-prone PCR. Preliminary results show that 5mM of MgCl₂ can ensure proper mutation rate.We mesured the activity of mixed clones with PNP-C10 as substrate,it suggested that library contains at least one useful mutation that have increased activity toward long-chain fatty acid.Selection is still in process.Geobacillus TH10 lipase is a good material for studying enzyme stability.We cloned,expressed and then purified the recombinant protein.Characteristic study show that it has higher thermostability than any other Geobacillus.Site directed mutation and directed evolution may facilitate the understanding of structure.