title: Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24.
abstract: A thermostable pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus caldophilus GK-24 by chromatographic methods, including gel-filtration and ion-exchange chromatography. The specific activity of the enzyme was increased 431-fold with a recovery of 13.2%. The purified enzyme was a monomer, M(r) = 65 kDa as estimated by SDS-PAGE and gel filtration. The pI was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75 degrees C and stable up to 95 degrees C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4 degrees C for 24 h. The activity of the enzyme was stimulated by Mn2+ and Mg2+ ions. Ni2+, Ca2+, Co2+ 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 caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by alpha-, beta-, or gamma-cyclodextrins. The N-terminal sequence [(AIa-Pro-Gln-(Asp or Tyr)- Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] showed some similarity to those of bacterial pullulanases.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dong-Guk University, Kyung-Ju City, Kyung-Pook, South Korea.
authors.forename: C H
authors.initials: CH
authors.lastname: Kim
authors.forename: O
authors.initials: O
authors.lastname: Nashiru
authors.forename: J H
authors.initials: JH
authors.lastname: Ko
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
chemical_list: D006026:Glycoside Hydrolases; C100181:pullulanase