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°C. K(m) values determined using Nα- benzoyl-DL-arginine-ethylester and Nα-benzoyl-DL-arginine-p-nitro-anilide were 3.2 x 10-5 M and 4.1 x 10-4 M respectively. The proteinase was inhibited by some typical serine proteinase inhibitors such as Nα-tosyl-L- lysine chloromethyl ketone, soybean trypsin inhibitors, benzamidine and phenylmethylsulfonyl fluoride. In particular, it was competitively inhibited by benzamidine with a K(i) of 1.2 x 10-5 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.