Improvement of enzymatic stability and catalytic efficiency of recombinant Fusarium oxysporum trypsin with different N-terminal residues produced by Pichiapastoris .

Fusarium oxysporum trypsin (FOT) is afungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichia pastoris by engineering the natural propeptideAPQEIPN.In this study, we constructed two recombinant enzymeswith engineeredamino acid sequences added to the N-terminus of FOT and expressed in P.pastoris . The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature (40 °C) and pH (8.0). However, therecombinants TE and TS showed significantly increased thermal stability at 40°C and 50°C. Moreover, the recombinants TE and TS also showed enhanced of tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation π-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, kcat /Km ),1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198 and G208) and catalytic triad (His42, Asp102 and Ser180), which wouldimprove the electron transfer rate and catalytic efficiency. In addition, N-terminalresidues modification described here may be an useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.