Purification and Biochemical Properties of an Alkaline Pullulanase from Alkalophilic Bacillus sp. S-1

Abstract

A novel extracellular pullulanase (PUL-E, pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified from the alkalophilic Bacillus sp. S-1. The purified enzyme had a molecular mass of about 140kDa on denaturated and natural conditions. The pI was 5.5. The pullulanase, when resolved by SDS-PAGE, was negative for Schiff staining, suggesting that the enzyme is not a glycoprotein. The N-terminal amino acid sequence of the enzyme was Phe-Leu-Asn-Met-Ser-(Trp-Phe). The enzyme displayed a temperature optimum of around 60°C and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at 4°C for 24 h. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn²⁺ ions. Ca²⁺ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the α-1, 6-linkages of amylopectin, glycogens, α, β-limited dextrin, and pullulan. The enzyme had an apparent K_m of 7.92 mg/ml for pullulan, a K_m of 1.63 mg/ml for amylopectin, and a K_m of 3.1mg/ml for α, β-limited dextrin, when measured at pH 9.0 and 50°C. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was not inhibited by α, β, or γ-cyclodextrins. The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bacterial cultivation.