Author
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FONTANINI, DEBORA - UNIVERSITY OF WISCONSIN |
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Jones, Berne |
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Submitted to: Brewers Digest
Publication Type: Abstract Only Publication Acceptance Date: 6/19/1999 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Germinated barley contains many proteolytic activities. We are isolating and identifying some of these enzymes so that we can better understand the biochemical events that occur during brewing. Using a two-dimensional (2- D) native PAGE system containing incorporated gelatin substrate, we have identified a major serine protease in green malt. It has an optimum pH of 6 and maintains its activity throughout the kilning process. It may, therefore, hydrolyze proteins during brewing. We extracted this enzyme from green malt and subjected it to chromatographic separation, after which the enzyme yielded a single activity spot on 2-D PAGE analysis. It was further purified on an FPLC Mono-Q column. Characterization with class- specific protease inhibitors revealed that the enzyme was a serine class endoprotease. It is the first serine endoprotease that has been purified from germinated cereal seeds. It had an isoelectric point of about 4.7 and, when tested on PAGE with gelatin as substrate, it was active over a wide range of pH values (neutral to basic). The enzyme did not appear to have tryptic or chymotryptic activity, but it readily digested gelatin immobilized in PAGE gels and beta-purothionin in solution at pH 6. From its hydrolytic specificity, it seems unlikely that the enzyme hydrolyzes storage proteins during brewing. |