Recombinant Chymotrypsin-Like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

Abstract

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Tenebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni2+-NTA agarose. The specificity constants (kcat/KM) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA were 7, 4.2 and 0.9 (µM∙min)–1, respectively. Optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable at the alkaline pH range, and in the presence of BSA also in the acidic region. Peptidase was inhibited by synthetic inhibitors such as PMSF, TPCK, chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e. g., stain removers, detergents, etc.).