Purification of a Trypsin-Like Enzyme and Cloning of Its Gene from Chinese Ground Beetle (Eupolyphaga sinensis)

An insect trypsin-like enzyme with similar biochemical properties to psychrophilic analogs was purified from Chinese ground beetle (Eupolyphaga sinensis) for the first time. The purified trypsin-like enzyme was designated as E.sinensis trypsin-like enzyme (ESTL) with molecular weight of 22.8kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maximal activity of ESTL was observed at pH9.5, and the temperature optimum of ESTL was observed at 45C by its amidolytic effect on the substrate benzoyl-L-Arg-p-nitroanilide. The analysis of inhibitors showed that specific inhibitor of serine proteases (phenylmethanesulfonyl fluoride) and trypsin inhibitors (tosyl-lysine chloromethyl ketone, bovine pancreatic trypsin inhibitor and benzamidine) inactivated ESTL almost completely. Over the range of tested temperature of 10-30C, the catalytic efficiency (k(cat)/K-m) of the ESTL was about five times than that of bovine trypsin. The N-terminal sequencing of ESTL revealed the following sequence: I(1)VGGSTTTIQ(10)NFPYQVSL, and the complementary DNA (cDNA) of ESTL was cloned by rapid amplification of cDNA ends. Practical Applications In recent years, increasing attention has been drawn to the psychrophilic (cold-adapted) enzymes. Psychrophilic trypsins with high activity may be interesting for several industrial applications of enzymes, such as in certain food-processing operations that require low-processing temperatures. For example, cod trypsin is already used in food production and cosmetics. In this article, a trypsin-like enzyme [Eupolyphaga sinensis trypsin-like enzyme (ESTL)] was purified from Chinese ground beetle by a relatively simple method, and the complementary DNA (cDNA) of this enzyme was cloned by rapid amplification of cDNA ends. This enzyme was characterized for molecular and enzymatic properties, as demonstrated that it had high activity at low and moderate temperature compared with the mammalian trypsins, and maintained good thermal stability compared with psychrophilic trypsins. ESTL has potential interest in food industry.