High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris

Abstract An alkaline protease (Ap) was cloned from Aspergillus sojae GIM3.33 via RT-PCR technique. A truncated Ap without the signal peptide was successfully expressed in the Pichia pastoris KM71 strain. The following describes the optimal process conditions for the recombinant engineering of a strain expressing a recombinant Ap (rAp) in a triangular flask: inoculum concentration OD 600 value 20.0 in 40 mL working volume (in 500 mL flasks), methanol addition (1.0%; volume ratio), 0.02% biotin solution (60 μL), and YNB primary concentration (13.0 g/L). Under these conditions, the protease activity of rAp in the fermentation broth reached 400.4 ± 40.5 U/mL after induction for three days. The rAp was isolated and purified, and its enzymatic characteristics were tested. Its optimal pH was 10.0, and it remained stable in a pH range of 7.0–10.0. Its optimal temperature was 45 °C and it retained >50% activity at 40 °C for 60 min. The rAp activity was significantly inhibited by PMSF, Zn 2+ and Fe 2+ and the rAp had a broad substrate specificity for natural proteins and synthetic peptide substrates, and preferred substrates at P1 position with large hydrophobic side-chain groups. Compared to Papain (8.7%) and Alcalase (12.2%), the degree of hydrolysis of rAp to soy protein isolate was 16.5%; therefore, rAp was a good candidate for the processing of food industry byproducts.