title: Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana.
abstract: The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5-7 and 80°C and had a half-life of 88 min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity.
authors: Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea.
authors: Jinho
authors: J
authors: Kang
authors: Kyung-Min
authors: KM
authors: Park
authors: Kyoung-Hwa
authors: KH
authors: Choi
authors: Cheon-Seok
authors: CS
authors: Park
authors: Go-Eun
authors: GE
authors: Kim
authors: Doman
authors: D
authors: Kim
authors: Jaeho
authors: J
authors: Cha
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D005936:Glucans; D011994:Recombinant Proteins; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase