Purification of isoamylases from the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), by high-performance liquid chromatography and their interaction with partially-purified amylase inhibitors from wheat

Abstract

Amylase isozymes from the adult rice weevil, Sitophilus oryzae (L.), were purified by high-performance liquid chromatography (HPLC) on a Synchropak AX-300 analytical ion exchange column. The isozymes, designated RW-1 and RW-2, had similar pH optima between pH 4.5 and 5.0, molecular weights of about 56,000, similar amino acid profiles that included high concentrations of aspartic acid and low levels of cystine and methionine, and identical action patterns for hydrolysis of amylose that were typical of α-amylases. Kinetic constants of RW-1 and RW-2 for hydrolysis of soluble starch were significantly different. K m values for starch were 0.246% for RW-1 and 0.066% for RW-2. V max values for starch were 837 AU/mg protein for RW-1 and 308 AU/mg for RW-2. One amylase unit (AU) was the amount of enzyme that produced 1.0 mg maltose hydrate/min at 30°C. Naturally-occurring amylase inhibitors in ammonium sulfate precipitates of a saline extract of wheat flour were fractionated by HPLC on a Synchropak AX-300 preparative column. Significant differences were found in the sensitivity of RW-1 and RW-2 to several major inhibitor fractions. Inhibition patterns of the HPLC fractions toward RW-1 were similar to that against purified amylase of S. granarius (L.), whereas inhibitory activity against RW-2 paralleled that against amylase from Tenebrio molitor L. A zymogram technique for electrophoretic demonstration of amylase inhibitors is described.