title: Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27.
abstract: A gene encoding a special type of pullulanase from Thermus thermophilus HB27 (TTHpu) was cloned. It has an open reading frame of 1428bp encoding a mature protein with a molecular mass of 52kDa. The gene was expressed in Escherichia coli using pHsh and pET28a vectors. The pHsh expression system produced a 3.6-fold higher recombinant pullulanase than pET28a. The recombinant TTHpu was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The purified TTHpu exhibited highest activity at pH 6.5 and 70°C. More than 90% activity was retained after incubation at 60-70°C for 2h and the half-life was 2h at 80°C. The stability of the enzyme was in a pH range from 6.0 to 8.0. Manganese at 5mM enhanced its activity up to 298%. The Km and Vmax for the enzyme activity on pullulan were 0.0031mgmL(-1) and 23.8μmolmin(-1), respectively. Unlike the most of pullulan-hydrolyzing enzymes described to date, this enzyme can attack α-1,6- and α-1,4-glycosidic linkages in pullulan, and produce a mixture of maltotriose, maltose and glucose. The enzyme could be further employed for industrial saccharification of starch.
authors: College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China. Electronic address: wuhuawei-2000@163.com.
authors: Huawei
authors: H
authors: Wu
authors: College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China.
authors: Xinxin
authors: X
authors: Yu
authors: College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China.
authors: Libing
authors: L
authors: Chen
authors: College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China. Electronic address: guxuwu@yangtzeu.edu.cn.
authors: Guangxu
authors: G
authors: Wu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: Pullulanase; Purification; Soluble expression; Thermus thermophilus HB27; pHsh expression vector