title: Characterization of recombinant amylopullulanase (gt-apu) and truncated amylopullulanase (gt-apuT) of the extreme thermophile Geobacillus thermoleovorans NP33 and their action in starch saccharification.
abstract: A gene encoding amylopullulanase (gt-apu) of the extremely thermophilic Geobacillus thermoleovorans NP33 was cloned and expressed in Escherichia coli. The gene has an open reading frame of 4,965 bp that encodes a protein of 1,655 amino acids with molecular mass of 182 kDa. The six conserved regions, characteristic of GH13 family, have been detected in gt-apu. The recombinant enzyme has only one active site for α-amylase and pullulanase activities based on the enzyme kinetic analyses in a system that contains starch as well as pullulan as competing substrates and response to inhibitors. The end-product analysis confirmed that this is an endoacting enzyme. The specific enzyme activities for α-amylase and pullulanase of the truncated amylopullulanase (gt-apuT) are higher than gt-apu. Both enzymes exhibited similar temperature (60 °C) and pH (7.0) optima, although gt-apuT possessed a higher thermostability than gt-apu. The overall catalytic efficiency (K(cat)/K(m)) of gt-apuT is greater than that of gt-apu, with almost similar substrate specificities. The C-terminal region of gt-apu appeared to be non-essential, and furthermore, it negatively affects the substrate binding and stability of the enzyme.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Jaurez Road, New Delhi 110021, India.
authors.forename: M
authors.initials: M
authors.lastname: Nisha
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase