Purification and Properties of Bacillus acidopullulyticus Pullulanase

Abstract

Two active forms (F1 and F2) of Bacillus acidopullulyticus pullulanase (EC 3.2.1.41 pullulan 6-glucanohydrolase) from Promozyme 200L (Novo Industri A/S) were purified using 50% (v/v) acetone precipitation and chromatographies on CM-Toyopearl 650S, Toyopearl HW-55S, and Butyl-Toyopearl 650S columns. The FI and F2 forms showed single bands in polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The optimum pHs and specific activities of F1 and F2 were about 5.0 and 200 ∼ 220 units/mg, respectively. The molecular weights of F1 and F2 were estimated as 115,000 and 116,000, and their isoelectric points were 5.0 and 5.2. The values of F1 and F2 for pullulan were the same, 2.5 mm glucose equivalents, and their k_o values were calculated as 2.9 × 10³ and 3.6 × 10³ s⁻¹, respectively, from the Lineweaver-Burk plots. These K_o values of F and F2 for pullulan are larger than that (2.9 × 10² sec⁻¹ of Klebsiella pneumoniae pullulanase. The activities of F1 and F2 on glycogen β-limit dextrins were 26∼46% of those on pullulan, but the activity of K. pneumoniae pullulanase on glycogen β-limit dextrins was 3∼6% of that on pullulan.