Extraction and Assay of Proteolytic Activities in Sorghum Malt

Abstract

A method has been developed to assay the proteinase and carboxypeptidase activities in sorghum malt.The method specifically addresses two problems encountered with some other assays for malt proteolytic activity; that of enzyme inextractability and the ubb of alien substrates. It was found that as with barley malt proteolytic enzymes, a high proportion of sorghum malt proteolytic activity was inextractable with sodium chloride solution. Systematic investigation into the extraction of proteolytic enzymes from sorghum malt led to the development of a novel extractant viz. 0.6 M Lithium iodide Li I plus 3.33 mM dithiothreitol. This extractant appears to solubilize a significantly higher proportion of the malt proteolytic activities with a single extraction than previously used buffers. Assay is carried out Using purified Kafirin, the sorghum prolamin storage protein, as substrate. Proteinase is measured in terms of total nitrogen solubilized and carboxypeptidase as free a-amino nitrogen solubilized.

Modification of the Methods for the Extraction of Carboxypeptidase and Proteinase Activities from Sorghum Malts

“…A proportion of sorghum malt proteolytic enzymes, like those of barley malt, are not extractable with NaCl 27 . Consequently, the EBC endopeptidase assay, for example, frequently measures less than 50% of the activity in the malt 19 . Mazhar et al 29 reported an increase in the extractability of prolamin proteins (kafirins) from sorghum malt using varying concentrations of both t-butanol and 2-mercaptoethanol.…”

“…Mazhar et al 29 reported an increase in the extractability of prolamin proteins (kafirins) from sorghum malt using varying concentrations of both t-butanol and 2-mercaptoethanol. Similarly, Evans and Taylor 19 , extracted both proteinase and carboxypeptidase enzymes from sorghum malts using a novel extractant, containing 0.6M lithium iodide plus 3.33 mM DTT, to enhance the extractability/activity of these enzymes. Recently, Agu and Palmer 1 reported a higher endopeptidase enzyme activity from sorghum malt using 0.2 M sodium acetate buffer containing 5 mM 2-ME as extractant.…”

“…The enzymes were extracted as outlined by Evans and Taylor 19 with some modifications by Okolo and Ezeogu 38 . Sorghum malt grists, 1.0 g, from each of the samples malted for 3 and 5 days was extracted separately with 15 mL of the 0.1 M citrate-phosphate buffer, pH 7.0, containing 2 mM 2-mercaptoethanol (2-ME) or 0.2% (w/v) Cyst.…”

“…Consistent with earlier reports 19 , the terms proteinase and carboxypeptidase are used here to describe the proteolytic activities measured in terms of solubilization of total nitrogen and free ␣-amino nitrogen (FAN), respectively. In the absence of kafirin, the sorghum prolamin storage protein 19,46 , BSA was used as substrate contrary to earlier criticisms against the use of alien substrates 9,17,18,21 since proteolytic activity is influenced by the amino acid sequence and tertiary structure of the substrate 43 .…”

“…In the absence of kafirin, the sorghum prolamin storage protein 19,46 , BSA was used as substrate contrary to earlier criticisms against the use of alien substrates 9,17,18,21 since proteolytic activity is influenced by the amino acid sequence and tertiary structure of the substrate 43 . To avoid this problem, Baxter 5 assayed barley malt proteolytic activities using hordein, the barley prolamin storage protein as substrate.…”