title: Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304.
abstract: A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K m and V max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.
authors.affiliation: Research Center of Bioresource & Bioenergy, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, People's Republic of China.
authors.forename: Youran
authors.initials: Y
authors.lastname: Li
authors.forename: Dandan
authors.initials: D
authors.lastname: Niu
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhengxiang
authors.initials: Z
authors.lastname: Wang
authors.forename: Guiyang
authors.initials: G
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007517:Isoamylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008320:Maltose / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008320:Maltose; D013213:Starch; D007517:Isoamylase