title: Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg.
abstract: A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5(T) was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by P. cryohalolentis K5(T) , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0-5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca(2+) , Mn(2+) , and EDTA whereas Zn(+2) , Cu(+2) , Co(+2) , Ni(+2) , and Mg(+2) inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Puschino, Moscow Region, Russia. nksusha@gmail.com
authors.forename: Ksenia
authors.initials: K
authors.lastname: Novototskaya-Vlasova
authors.forename: Lada
authors.initials: L
authors.lastname: Petrovskaya
authors.forename: Sergey
authors.initials: S
authors.lastname: Yakimov
authors.forename: David
authors.initials: D
authors.lastname: Gilichinsky
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000235:genetics; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D054712:Salinity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012800:Siberia; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D003902:Detergents; D008670:Metals; D011994:Recombinant Proteins; D012997:Solvents; D017830:Octoxynol; D004950:Esterases