Purification and Characterization of a Glutenin Hydrolysing Proteinase from Wheat Damaged by the New Zealand Wheat Bug, Nysius huttoni

Abstract

Abstract A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni , was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent M r of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of M r 30k and six minor bands of M r 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.