title: Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1.
abstract: The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 °C), and it had an optimum pH of 8.0 and an optimum temperature of 60 °C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 °C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 ± 0.030 mM, 3725.8 U/mg, and 1500 ± 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity.
authors.affiliation: Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey.
authors.forename: Fulya
authors.initials: F
authors.lastname: Ay
authors.forename: Hakan
authors.initials: H
authors.lastname: Karaoglu
authors.forename: Kadriye
authors.initials: K
authors.lastname: Inan
authors.forename: Sabriye
authors.initials: S
authors.lastname: Canakci
authors.forename: Ali Osman
authors.initials: AO
authors.lastname: Belduz
mesh_terms: D058677:Anoxybacillus / Q000201:enzymology* / Q000235:genetics; D002087:Butyrates / Q000378:metabolism; D043182:Carboxylesterase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002087:Butyrates; D011994:Recombinant Proteins; C033592:4-nitrophenyl butyrate; D043182:Carboxylesterase