Terminal truncation improves the activity and soybean protein hydrolysis efficiency of dibasic endopeptidase

Abstract:

[Objective] To realize the heterologous expression of dibasic endopeptidase genes sckex2 and pkkex2 from Saccharomyces cerevisiae and Pichia kudriavzevii in Escherichia coli BL21 and then study the properties of the recombinant KEX2 enzymes and their efficiency of hydrolyzing soybean protein in collaboration with alkaline protease. [Methods] The kex2 from S. cerevisiae and P. kudriavzevii was optimized according to the codon bias of E. coli. After analysis of non-functional region, we redesigned their C-terminal and N-terminal residues by truncation. Four truncated genes, sckex2∆3, sckex2∆4, pkkex2∆3, and pkkex2∆4, were respectively cloned into the vector pGEX-6P-1, and the recombinant plasmids were transformed into E. coli BL21 competent cells. After confirmed by DNA sequencing, the four recombinant strains, E. coli BL21/pGEX-ScKEX2∆3, E. coli BL21/pGEX-ScKEX2∆4, E. coli BL21/pGEX-PkKEX2∆3, and E. coli BL21/pGEX-PkKEX2∆4, were constructed. The recombinant enzymes were purified by GST affinity chromatography and PreScission protease and then their pH stability and thermal stability were determined. With the soybean protein hydrolysis by alkaline protease alone as the control, the yield of small-molecule peptides from the hydrolysis with the recombinant KEX2 in collaboration with alkaline protease was determined. [Results] The recombinant KEX2 and the wild type showed soluble expression in E. coli BL21. SDS-PAGE showed that the purified recombinant enzymes produced single bands. Under the optimal conditions, the wild type showed no enzyme activity, while the mutants showed the maximum specific activity of 47.32 U/g, K_m of 23.61 μmol/L, k_cat of 50.18 s⁻¹, and k_cat/K_m of 2 125.06 L/(mmol·s). After incubation at pH 5.0 for 2 d, the maximum relative activity of the recombinant enzymes was more than 40%. After incubation at 35℃ for 1 h, the relative activity was still over 60%. When the recombinant KEX2 and alkaline protease were used together to hydrolyze soybean protein, more than 39% of the hydrolysates were active peptides with molecular weight less than 500 Da, and the content of active peptides with the molecular weight less than 100 Da was 50% higher than that from the hydrolysis with the alkaline protease alone. [Conclusion] Through terminal truncation, the soluble expression of truncated KEX2 mutants was realized in E. coli. The recombinant enzymes demonstrate high catalytic efficiency and can efficiently hydrolyze soybean protein to release active peptides. This work lays a solid foundation for the value addition and deep hydrolysis of protein-rich biological resources.

Key words:dibasic endopeptidase (KEX2);Escherichia coli;enzymatic properties;soybean protein;active peptides