Effects Of Enzymatic Properties Of Fusarium Oxysporum Trypsin With N-terminal Residual Amino Acides In P.pastoris

Fungal Fusarium oxysporum trypsin?FOT?is a serine protease similar to mammal trypsin.,which is widely used in various fields such as industry,food and medicine.FOT could be successfully expressed in Pichia pastoris.In this study,the recombinant TE?with peptide YVEF?and TS?with peptide YV?expressed in Pichia pastoris were compared to the wildtype FOT of the enzyme properties.The results showed that the FOT,TE and TS displayed the same optimum temperature?40°C?and pH 8.0.However,the recombinant TE and TS respectively with the peptide YVEF and YV showed significantly increased thermostability at 40°C and 50°C.Moreover,the recombinant TE and TS also showed the pH tolerance under alkaline conditions.Compared with the wildtype FOT,the intramolecular Hydrogen bonds and the cation?-interactions of the recombinant TE and TS were significantly increased.The recombinant TE and TS has also a significantly increased the catalytic efficiency(referring to the specificity constant,k_cat/K_m)1.75-fold and 1.23-fold to the wild FOT.In silico modeling analys is uncovered that introduction of the peptide YVEF and YV resulted in a shorter distance between substrate binding pocket?D174,G198 and G208?and catalytic triad?His42,Asp102 and Ser180?which will improve the electron transfer rate and catalytic efficiency.The results suggested that engineering of the N-terminus with residual peptides might be an effective approach for improve the properties of the trypsin.