Production And Engineering Thermostability Of Bovine Enterokinase Light Chain(BEK_L) In Pichia Pastoris

Enterokinase(EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum.The enzyme allows any downstream fusion target protein to retain its native N-terminus,without leaving any unwanted amino acid residues on their amino termini.Therefore,the high specificity of enterokinase makes it an ideal tool for cleaving fusion proteins in the field of genetic research.In this research,a recombinant plasmid pPIC9K-BEKL containing the bovine enterokinase light chain(BEKL)sequence was constructed and 5 positive GS115 transformants were selected on gradient YPD-G418 plates with different resistance level of G418.Moreover,the target gene copy number of selected strains were quantified by real-time quantitative polymerase chain reaction(QPCR)and the analytical results showed that both the resistance level of G418 and the target gene copy number of selected strains were positive related with amount of target protein.Then,the expression conditions of selected strains were optimized by muliple-factor optimization method and the recombinant protein was purified by Ni-NTA agarose affinity chromatography.Finally,the yield of purified,active BEKL from this method was about 5.7 mg per liter of fermentation culture,and the specific activity of purified BEKL was 1.25×103 U/mg.The purified BEKL cleaved the synthetic peptide substrate GD4K-NA with kinetic parameters Km=0.59 mM and kcat=45.1 S-1.Besides,the protein molecule weight of BEKL was approximately 43 kDa and the glycosylation was about 20.9%.Furthermore,rational design method was applied to increase the thermostability of bovine enterokinase ligth chain.At first,molecular dynamics software Gromacs v4.5.5,Flex Service,B-FITTER and other softwares were used to predict the flexibility profile of the bovine enterokinase structures.Fragment 63–69,Fragment 85-93 and Fragment 135-139 were confirmed to represent the flexible region.Subsequently,?-turn sequence statistics and stereoscopic criteria of introducing proline were combined to pinpoint appropriate substitution sites by prolines.Besides,cysteine mutants of bovine enterokinase were designed by the software Disulfide by Design.Finally,site-directed mutations(S67P,R87 P,Y136P,C5-C146,C85-C88 and C73-C98)were introduced within the fexible region using Quik ChangeTM method and the thermostability of wide-type and the enterokinase mutants were investigated.The result demonstrated that the half-life(t1/2)and half inactivation(T5010)temperature of the cysteine mutants and R87 P were significantly increased when compared to that of the wild-type enzyme.Meanwhile,the catalytic efficiency(Km/kcat)of R87 P and C85-C88 mutant enzymes remained nearly unchanged.In this study,we obtained high-yeild strains for bovine enterokinase light chain expression and successfully enhanced its thermostability using rational design method without changing its activity.This strategy has potential to be applied to engineer thermostability of other enzyme,which is beneficial for the wider application of biocatalysts in industry.