Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164).
Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.
Fahmy
Afaf S
AS
Department of Molecular Biology, National Research Centre, Tahrir St, Dokki, 12311 Cairo, Egypt.
Ali
Ahmed A
AA
Mohamed
Saleh A
SA
D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004534:Egypt; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D014908:Triticum / Q000201:enzymology*
D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; C026018:azocasein; D003546:Cysteine Endopeptidases