On the presence and activities of proteolytic enzymes in vital wheat gluten

Two vital wheat gluten samples were submitted to an Osborne type fractionation. The proteolytic activities of the resulting fractions were evaluated. Gluten contained endoproteolytic, exoproteolytic, carboxypeptidase, aminopeptidase and N alpha-benzoylarginine-p-nitroanilide hydrolase activities. After extraction with 0.5 M NaCl, and subsequently with 70% (v/v) ethanol, little activity remained in the extracted gluten. Upon autodigestion of gluten no (microbial) enzymes were released. High specificities of gluten-associated proteolytic enzymes were noted and their effects were clearly visible on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The sum of lysine, leucine, phenylalanine, tyrosine and arginine accounted for ca. 40-44% of the released amino acids, while they only make up ca. 18% of vital gluten proteins. Good correlations were found between the proportions of the amino acids released by the different Osborne fractions as a result of autodigestion, indicating that the gluten hydrolysing enzymes found in the different Osborne fractions are probably the same. Autodigestion of gluten proteins was reduced by ca. 73-76% upon addition of pepstatin A (0.2 mM), an inhibitor of aspartic proteases, and by c. 39-41% by phenylmethylsulphonylfluoride (1.0 mM), a serine protease inhibitor. (C) 1997 Academic Press Limited.