title: Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01.
abstract: Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS-PAGE. The values of Km and Vmax, calculated by the Michaelis-Menten equation, were 1077μM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70°C and pH 8.5. Activities at 50, 55 and 60°C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5-10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% β-sheet, 22.7% β-turn and 27% random coil.
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Bahram Pooreydy
authors.initials: BP
authors.lastname: Golaki
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran. Electronic address: aminzade@nigeb.ac.ir.
authors.forename: Saeed
authors.initials: S
authors.lastname: Aminzadeh
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran. Electronic address: karkhane@nigeb.ac.ir.
authors.forename: Ali Asghar
authors.initials: AA
authors.lastname: Karkhane
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Bagher
authors.initials: B
authors.lastname: Yakhchali
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Parisa
authors.initials: P
authors.lastname: Farrokh
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Seyed Hossein
authors.initials: SH
authors.lastname: Khaleghinejad
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Asal Akhavian
authors.initials: AA
authors.lastname: Tehrani
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran.
authors.forename: Sina
authors.initials: S
authors.lastname: Mehrpooyan
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008049:Lipase / Q000302:isolation & purification* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D009930:Organic Chemicals / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D003902:Detergents; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase
keywords: Cloning; Cohnella sp. A01; Expression; Purification; Thermostable lipase