A Simple Reducing Sugar Assay for Measuring β-Glucanase Activity in Malt, and Various Microbial Enzyme Preparations

Abstract

The reducing sugar method of Dygert, Florida, and Thoma (11) employing neocuproine hydrochloride as color reagent and glycine as the copper chelating agent was adapted to measure reducing sugars liberated from barley β-glucan, lichenin, and laminarin by the action of β-glucanases from malt and Bacillus amyloliquefaciens. Studies showed that bacterial β-glucanase gave the same results on barley β-glucan and lichenin, but did not attack laminarin. Malt contained β-glucanases which attacked both lichenin and laminarin. Lichenin and laminarin had high reducing sugar backgrounds which restricted the useful linear range. Reduction with sodium-borohydride virtually eliminated this background. The optimum pH and temperature for bacterial β-glucanase activity on lichenin were 6.5 and 40–45° C, respectively, while malt β-glucanases displayed optima of pH 4.0 and 40°C on lichenin and pH 4.5 and 45°C on laminarin. Km values were 0.053% for bacterial β-glucanase on lichenin, 0.67% for malt β-glucanase on lichenin, and 0.05% for malt β-glucanase on laminarin.