Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor

Abstract

Objectives To heterologously express peanut ( Arachis ipaensis ) Bowman-Birk inhibitor (BBI) and characterize its properties. Results A putative BBI gene from peanut was overexpressed in Pichia pastoris with the maximal yield of 11.1 mg/L in a 250-mL shaking flask fermentation. The recombinant peanut BBI (rPBBI) was purified and its molecular weight was estimated to be 9 kDa. Purified rPBBI showed 5223.6 TIA/mg inhibitory activity toward trypsin. It retained more than 95% of its inhibitory activity over wide range of temperatures (40 to 90 o C) and pH (2.0 to 10.0) after incubation for 60 min. When presence of 100 mM dithiothreitol, rPBBI lost more than 80% inhibitory activity in 30 min. Conclusion The current investigation expressed a peanut BBI with stable inhibitory activity against trypsin and exposed the potential of heterologous overexpression approach in large scale production of BBI.

Microbial proteases and their applications

“…Endopeptidases can hydrolyze the hydrophobic amino acids forming bonds of oligopeptides and reduce bitterness (Capiralla et al, 2002;Edens et al, 2005;Zhang M. et al, 2021). Aminopeptidases (Lin et al, 2020;Nandan and Nampoothiri, 2020;Song et al, 2020a;Nakamura et al, 2023;Wang et al, 2023) and carboxypeptidase (Ding et al, 2022) from many different sources can continue to hydrolyze end hydrophobic amino acids and then reduce bitterness.…”