Isolation and characterization of a new trypsin-like enzyme from Tenebrio molitor L. larvae.

Abstract

A trypsin‐like enzyme (TLE) was separated and purified from Tenebrio molitor larval midgut enzyme solution by ion‐exchange chromatography on a DEAE‐cellulose column. The purified enzyme was found to be a homogeneous protein by electrophoresis in polyacrylamide gels and on cellulose acetate strips, by electrofocusing in polyacrylamide gels and by SDS‐polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 18300 by ultracentrifugal analysis and 24300 on SDS‐polyacrylamide gel electrophoresis. It has an isoelectric point 8.0, it contains only four half‐cystine residues per molecule and the NH2‐terminal amino acid is isoleucine. TLE possesses a high degree of specificity towards trypsin synthetic substrates such as N‐α‐benzoyl‐DL‐arginine p‐nitroanilide (BAPNA), p‐tosyl‐L‐arginine methyl ester (TAME) and poly‐L‐lysine hydrobromide. The optimal pH for TLE activity was found to be 8.0 and the optimal temperature 50° C. Its Km value when assayed on BAPNA was 0.93mM and on TAME 0.08mM. TLE is stable at neutral pHs and its activity is not affected by Ca2+ and by 0.01 M 1, 4‐dithiothreitol (DTT). It is inactivated by DFP and tosyl‐L‐lysine chloromethylketone (TLCK) and is fully inhibited by the naturally occurring trypsin inhibitors such as trypsin‐and α‐chymotrypsin inhibitor (AA) from soybeans, basic pancreatic trypsin inhibitor (BPTI), chick peas trypsin and chymotrypsin inhibitor (CI) and crystalline soybean trypsin inhibitor (CSBTI), forming with them complexes in a molar ratio of 1:1. The Ki value for AA with BAPNA as substrate is 5.87 10‐7 M and for BPTI 7.92 10‐7M.No common antigenic determinants were noted between TLE and bovine trypsin. This finding together with the relatively low number of ‐S‐S‐ bonds in the TLE molecule indicate that TLE differs in conformation from bovine trypsin.