title: Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.
abstract: Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Zebardast Roodi
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Saeed
authors.initials: S
authors.lastname: Aminzadeh
authors.affiliation: Department of Biotechnology Engineering, Faculty of New Technologies Engineering, Shahid Beheshti University G.C., Tehran, Iran.
authors.forename: Naser
authors.initials: N
authors.lastname: Farrokhi
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: AliAsghar
authors.initials: A
authors.lastname: Karkhane
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Kamahldin
authors.initials: K
authors.lastname: Haghbeen
mesh_terms: D056449:Bacillales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000737:chemistry / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D000071825:Extremophiles / Q000201:enzymology*; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D005936:Glucans; D008670:Metals; D011994:Recombinant Proteins; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase