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

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 incorpor ated 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 germinat ing barley seeds. The results indicate that much past research on malt proteinases may not be particu larly 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.

Appearance of Endoproteolytic Enzymes during the Germination of Barley

“…Typically, 100 g of material was homogenized in 0.05 M sodium acetate buffer, pH 4.7, containing 2 mm cysteine and 1 mM EDTA and centrifuged, and the supernatant was dialyzed against 5 mm sodium acetate buffer at pH 5.0 as described previously (29). This 'crude extract' was frozen in 0.5-mL portions and stored at -200C.…”

“…We recently concluded a study using electrophoresis with nondenaturing gels containing incorporated substrate proteins that revealed that there were seven proteolytic activity bands in a crude green malt (4-d-germinated barley) extract (29). Five of the endoproteinase bands were maximally active at pH 3.8, and the other two were maximally active at pH 5.5 to 6.5.…”

“…Enzymes active at pH 6.5 are designated neutral proteinases. Most of acidic enzymes hydrolyzed both gelatin and edestin, neutral enzymes degraded only gelatin, and the proteinase present in steeped barley could only be detected using edestin (29). For this reason, both of these substrates were utilized.…”

Barley and Malt Proteins and Proteinases: II. The Purification and Characterisation of Five Malt Endoproteases, Using the Highly Degradable Barley Protein Fraction (HDBPF) Substrate

“…They were shown to hydrolyse hordein and their formation was shown to be induced by gibberellin during germination. All these purifications were based on the use of non-barley protein substrates, such as casein, haemoglobin and gelatin for the detection and assay of the purified enzymes 2,3,9,18,19,22 .…”

“…However, the latter could not be confirmed at lower concentrations of the inhibitor. This flies in the face of the most widely reported conclusion, which stipulates that the most active, at acidic pH, green malt endoproteases are cysteine class endoproteases [2][3][4][5][6][7]9,10,17,21,22 . It is very important to note that our enzymes were purified from malt rather than green malt, which is very commonly used.…”