ELECTROPHORETIC STUDY OF SUBSTRATE AND pH DEPENDENCE OF ENDOPROTEOLYTIC ENZYMES IN GREEN MALT*

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Abstract
The endoproteolytic enzymes of malt influence several different aspects of malt and beer quality. For this reason, we are extracting and characterizing these enzymes from green malt. The proteolytic activity of a Morex green malt extract was highest at pH 3.8 with haemoglobin substrate but gelatin hydrolysis was maximal from pH 4.7 to 6.0. Endoproteolytic hydrolysis of a 55% isopropanol-soluble reduced hordein fraction was about three times slower than gelatin hydrolysis but was relatively constant over the pH range from 3.8 to 6.5, although the activity did decrease at more acidic (3.0) or basic (7.0) pH values. To study the green malt proteinases in detail, a non-denaturing electrophoretic system was developed in which substrate proteins—either gelatin, edestin or hordein—were incorporated into an electrophoretic gel. After electrophoresis and incubation of the gels at pH 3.8, 4.7, 5.5, or 6.5 to allow enzymatic hydrolysis, the separated activities were determined by using protein staining to determine where the incorporated substrate had been hydrolysed. Using this system, seven proteolytic activity bands were detected. Five of the bands were maximally active at pH 3.8 and their activities dropped quickly as the pH increased. The other two bands, which migrated more slowly, hydrolysed gelatin more rapidly than they did the other substrates tested. Their gelatinolytic activities increased as the pH was raised (by 3- to 6-fold in the pH range tested). The electrophoretic system described has proven very useful for studying the proteinases of germinating barley seeds. The results indicate that much past research on malt proteinases may not be particularly relevant to the malting and brewing industries because it was conducted under pH conditions and with substrates that are likely quite different from those in the seed during the barley germination process. By using electrophoresis to separate proteinases before analysis, we can now study their individual characteristics and thus can conduct studies more relevant to malting and brewing.