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KMID : 0545120180280091482
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 9 p.1482 ~ p.1492
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Improvement of Enzymatic Stability and Catalytic Efficiency of Recombinant Fusariumoxysporum Trypsin with Different N-Terminal Residues Produced by Pichiapastoris
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Yang Ning
Ling Zhenmin
Peng Liang
Liu Yanlai
Liu Pu
Zhang Kai
Aman
Shi Juanjuan
Li Xiangkai
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Abstract
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Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino 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, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature (40°C) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at 40°C and 50°C. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wildtype 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 would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.
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KEYWORD
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Trypsin, N-terminal peptide, catalytic efficiency, Pichia pastoris
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