PURIFICATION AND BIOCHEMICAL-PROPERTIES OF AN ALKALINE PULLULANASE FROM ALKALOPHILIC BACILLUS SP S-1

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 140 kDa on denatur ated and natural conditions. The pI was 5.5. The pullulanase, when res olved by SDS-PAGE, was negative for Schiff staining, suggesting that t he 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-degrees-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-degrees-C for 24 h. The presence of pullulan protected the enzyme fr om heat inactivation, the extent depending upon the substrate concentr ation. The activity of the enzyme was stimulated by Mn2+ ions. Ca2+ io ns and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan. The enzyme had an apparent K(m) of 7.92 mg/ml f or pullulan, a K(m) of 1.63 mg/ml for amylopectin, and a K(m) of 3.1 m g/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50-de grees-C. The enzyme caused the complete hydrolysis of pullulan to malt otriose. The activity was not inhibited by alpha, beta, or gamma-cyclo dextrins. The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bac terial cultivation.