A Study On The Properties Of C-terminal And Thermostability Of Thermophilic Protease WF146

Thermophilic protease WF146 is a serine protease and belongs to the subtilisin family.It comes from thermophile Brevibacillus sp.WF146.Early experiental results show that adding,deleting or changing amino acid residues to the C-terminal of zymogen will make a bad impact on the thermostability of its maturase.In order to investigate the C-terminal's function on its biochemical properties,we have constructed a series of mutants which have different "tails".It turns out that regardless of the length and type,the "tails" will reduce the thermostability.So,we can know that the C-terminal of protease WF146 has a irreplaceable effect on its properties of resisting the high temperture.The change and destroy on the structure of C-terminal may affect the whole structure and make it relatively loose which impacts the properties.Meanwhile,we also find that the C-terminal "tails" can lead to a speed down when the zymogen processes into maturase.We suspect the reason of the speed down is that zymogen needs to excise not only the N-terminal but also the C-terminal that has a competition relationship with N-terminal.Moreover,we have used WF146-X-H(WF146's C-terminal with a Leu-Glu-His6)as a temple to build many mutants and added some mutation sites which were screened by our lab.In the construct of 39 mutants,there are lots of mutants have better catalytic activity or(and)thermostability than the wild type and parental type,such as SH47,SH48,SH50,SH51 and SH55.This shows that we can surely get recombinase which are better than natual enzyme using culnulative effect.We still have preliminarily explored the ability of thermophilic protease WF146 and its mutants N63P/A66P and PBL5,which were constructed early in our lab,to degrade keratin and compared with proteinase K.At 50 0C,proteinase K has the best degradation effect,while WF146 and its mutants' degradation effect doesn't have a big difference between each other.At 80 ?,proteinase K inactivates soon,while N63P/A66P and PBL5 can still degrade keratin,and N63P/A66P can basically degrade keratin completely after 8 h.Besides,we find that although the mutant N63P/A66P is inferior to PBL5,its degradation ability to keratin is better than the latter.We hypothesize that the mutant site may have changed the substrate specificity.Whereupon,we add this mutant site to PBL5,and have constructed the mutant protease PBL5N.The thermostability at 80 ? of PBL5N is nearly the same as PBL5,but the degradation ability to keratin of PBL5N is better than PBL5,quitely the same as N63P/A66P.After dealing with th keratin after 8 h,PBL5N can basically degrade keratin completely.In this study,we have investigated and analyzed the properties of C-terminal and thermostability of thermophilic protease WF146,which provide a new way for the directed evolution and industrial application of thermophilic enzymes.