Purification and Some Properties of Maltose Phosphorylase from Enterococcus hirae IFO 3181

Abstract

Maltose phosphorylase was isolated to homogeneity from a cell-free extract of Enterococcus hirae IFO 3181 by chromatographies with phenyl-Sepharose CL-4 B, QAE-Sephadex A-50, hydroxylapatite, and Sephadex G-200. The enzyme was purified about 40-fold with a yield of 32%, and its specific activity was 29.4 units/mg protein. The molecular weight was estimated to be 220, 000 and 90, 000 by HPLC gel filtration on T.SKgel G 3000 SWXL and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme showed optimum activity around pH 6.5-7.5 and its optimum temperature was about 45°C. The enzyme was stable over the range of pH 5.5-8.0 and retained the activity up to about 55°C. Maltose and nigerose were effective substrates for the enzyme reaction, but it could not act on the other disaccharides tested. The Km for maltose, nigerose, phosphate, and arsenate were 1.90, 1.93, 3.37, and 8.33 mM, respectively. The enzyme activity was almost completely inhibited by AgNO₃ and HgCl₂, and also strongly inhibited by CuSO₄, ZnSO₄, and p-chloromercuribenzoic acid.