Isolation and some molecular properties of a trypsin-like enzyme from larvae of European corn borer Ostrinia nubilalis Hubner (Lepidoptera:Pyralidae)

A one-step high-yielding procedure is presented for the purification of a trypsin-like proteinase from Ostrinia nubilalis larvae, consisting of benzamidine-sepharose affinity chromatography. The purified enzyme was homogeneous as judged by SDS-PAGE. The enzyme presents a molecular mass of 24 650 Da, a maximum pH activity profile of 9.5, a remarkable thermal stability and an optimum temperature of about 53 degrees C. K-m values determined using N alpha-benzoyl-DL-arginine-ethylester and N alpha-benzoyl-DL-arginine-p-nitro-anilide were 3.2x10(-5) M and 4.1x10(-4) M respectively, The proteinase was inhibited by some typical serine proteinase inhibitors such as N alpha-tosyl-L;lysine chloromethyl ketone, soybean trypsin inhibitors, benzamidine and phenylmethylsulfonyl fluoride. In particular, it,vas competitively inhibited by benzamidine with a K, of 1.2x10(-5) M, whereas it was not affected by cysteine proteinases inhibitors, Comparative analysis of the amino acid composition and N-terminal sequence of O. nubilalis proteinase confirmed that this enzyme is very similar to other serine proteinases from lepidopteran larvae. Copyright (C) 1996 Elsevier Science Ltd