title: Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.
abstract: Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC(2)) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5-10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.
authors.affiliation: Department of Chemistry, Science Faculty, İzmir Institute of Technology, Gülbahçe, Urla, İzmir, Turkey.
authors.forename: Hasan Cihad
authors.initials: HC
authors.lastname: Tekedar
authors.forename: Gülşah
authors.initials: G
authors.lastname: Sanlı-Mohamed
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004950:Esterases / Q000737:chemistry / Q000235:genetics*; D004952:Esters / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D056506:Geobacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004952:Esters; D009532:Nickel; D004950:Esterases