Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 degrees C and retained >90% activity at 50 degrees C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu2+ and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from beta-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H-429, H-433, and E-453 in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.