title: Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis K5(T).
abstract: We describe cloning and expression of genes coding for lipase Lip2Pc and lipase-specific foldase LifPc from a psychrotrophic microorganism Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg (the lense of overcooled brine within permafrost). Upon expression in Escherichiacoli Lip2Pc accumulated in inclusion bodies while chaperone was synthesized in a soluble form. An efficient protocol for solubilization and subsequent refolding of the recombinant lipase in the presence of the truncated chaperone was developed. Using this procedure Lip2Pc with specific activity of 6900U/mg was obtained. Contrary to published data on other lipase-chaperone complexes, refolded Lip2Pc was mostly recovered from the complex with chaperone by metal-affinity chromatography. Recombinant Lip2Pc displayed maximum lipolytic activity at 25°C and pH 8.0 with p-nitrophenyl palmitate (C16) as a substrate. Activity assays conducted at different temperatures revealed that the recombinant Lip2Pc is a cold-adapted lipase with ability to utilize substrates with long (C10-C16) hydrocarbon chains in the temperature range from +5 to +65°C. It demonstrated relatively high stability at temperatures above 60°C and in the presence of various metal ions or organic solvents (ethanol, methanol, etc.). Non-ionic detergents, such as Triton X-100 and Tween 20 decreased Lip2Pc activity and SDS completely inhibited it.
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290 Pushchino, Moscow Region, Russian Federation. nksusha@gmail.com
authors.forename: Ksenia
authors.initials: K
authors.lastname: Novototskaya-Vlasova
authors.forename: Lada
authors.initials: L
authors.lastname: Petrovskaya
authors.forename: Elena
authors.initials: E
authors.lastname: Kryukova
authors.forename: Elizaveta
authors.initials: E
authors.lastname: Rivkina
authors.forename: Dmitry
authors.initials: D
authors.lastname: Dolgikh
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D001110:Arctic Regions; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D058849:Protein Refolding; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D008049:Lipase; C504169:lipase foldase
keywords: Cryopeg; Lipase; Lipase-specific foldase; Permafrost; Psychrobacter cryohalolentis; Refolding