Comparitive enzyme kinetics of two allelic forms of barley (Hordeum vulgare L.) β-amylase
Ma, YF and Stewart, DC and Eglinton, JK and Logue, SJ and Langridge, P and Evans, E, Comparitive enzyme kinetics of two allelic forms of barley (Hordeum vulgare L.) β-amylase, Journal of Cereal Science, 31 pp. 335-344. ISSN 0733-5210 (2000) [Refereed Article]
The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta-amylase (EC 18.104.22.168) encoded on chromosome 4H by the Bmy1-Sd1 and Bmy1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2L beta-amylases after germination. The K(m) value for green malt beta-amylase was less than that of mature grain beta-amylase for both varieties when potato starch was used as a substrate, although V(max) was similar. This indicated that proteolysis after germination increased the affinity of beta-amylase for potato starch. No significant kinetic differences were observed between beta-amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta-amylase. Sd1 beta-amylase from green malt exhibited a lower K(m) value for potato starch than Sd2L beta-amylase, demonstrating that at non-saturating starch concentrations Sd1 beta-amylase is better able to hydrolyse starch than Sd2L beta-amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K(m) values for beta-amylase increased, whereas V(max) values decreased. Maltose, the hydrolytic product of beta-amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta-amylases with respect to potato starch and amylose. Taken together the kinetic observations for beta-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process. (C) 2000 Academic Press.