Heterologous expression of aminopeptidase (AoAPase) from Aspergillus oryzae RIB40 and the application in debittering cod peptides

Abstract:

[Objective] To explore a specific aminopeptidase that can debitter cod peptides and further increase the value of this fish. [Methods] Aspergillus oryzae RIB40-derived aminopeptidase (AoAPase) was expressed in Pichia pastoris KM71, and its characteristics were investigated. The cod peptides-debittering ability of AoAPase was evaluated by ANS probe and electronic tongue. The content and molecular weight of free amino acids in the cod peptides hydrolysate were detected by high performance liquid chromatography (HPLC), and the surface microstructure of cod peptides was observed under scanning electron microscope (SEM). Moreover, the DPPH-, hydroxyl- and ABTS-scavenging activities were determined to evaluate the antioxidant properties of cod peptides before and after AoAPase treatment. [Results] AoAPase was successfully expressed in KM71, with the molecular weight of about 41 kDa. AoAPase activity reached 2 238 U/mL under optimal conditions of 70℃ and pH 8.0. Its activity was enhanced by Ca²⁺. In addition, AoAPase exhibited substrate specificity, and its K_m and V_max values towards Leu-pNA were 5.95 mmol/L and 43.58 μmol/(mL·min), respectively. AoAPase could completely eliminate the bitterness of cod peptides by removing N-terminal hydrophobic amino acids and reducing the content of bitter peptides (500-1 000 Da). The cod peptide powder after AoAPase treatment became more delicate and loose with no significant change in antioxidant activity. [Conclusion] The recombinant AoAPase can remove the bitter taste of cod peptides without influencing their bioactivity. This study lays a theoretical foundation for the application of AoAPase in protein debittering.

Key words:aminopeptidase;heterologous expression;debittering;cod peptides;antioxidant activity