title: Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.
abstract: As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.
authors.affiliation: Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997, Moscow, Russia lpetr65@yahoo.com.
authors.forename: Lada E
authors.initials: LE
authors.lastname: Petrovskaya
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.
authors.forename: Ksenia A
authors.initials: KA
authors.lastname: Novototskaya-Vlasova
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Spirina
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Durdenko
authors.affiliation: Department of Chemistry, Lomonosov Moscow State University, Leninskiye Gory, 1/3, 119991, Moscow, Russia.
authors.forename: Galina Yu
authors.initials: GY
authors.lastname: Lomakina
authors.affiliation: Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, 119121, Moscow, Russia.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Zavialova
authors.affiliation: Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, 119121, Moscow, Russia Institute of Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38 k.2, 119334, Moscow, Russia.
authors.forename: Evgeny N
authors.initials: EN
authors.lastname: Nikolaev
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.
authors.forename: Elizaveta M
authors.initials: EM
authors.lastname: Rivkina
mesh_terms: D020816:Amino Acid Motifs; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D000069463:Olive Oil / Q000378:metabolism; D065946:Permafrost / Q000382:microbiology*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
chemical_list: D000069463:Olive Oil; D004950:Esterases
keywords: GCSAG motif; HSL family; esterase; metagenome; microcosm; permafrost