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 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 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 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 alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan. The enzyme had an apparent Km of 7.92 mg/ml for pullulan, a Km of 1.63 mg/ml for amylopectin, and a Km of 3.1 mg/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50 degrees C. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was not inhibited by alpha, beta, or gamma-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.
Kim
C H
CH
Laboratory of Molecular Biology, KIST, Taejon, Korea.
Choi
H I
HI
Lee
D S
DS
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D002414:Cations, Monovalent; D003470:Culture Media; D005936:Glucans; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase