title: Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5(T) and its deletion mutants.
abstract: A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30-35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.
authors.affiliation: Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia. nksusha@gmail.com
authors.forename: K A
authors.initials: KA
authors.lastname: Novototskaya-Vlasova
authors.forename: L E
authors.initials: LE
authors.lastname: Petrovskaya
authors.forename: E M
authors.initials: EM
authors.lastname: Rivkina
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D003080:Cold Temperature; D004789:Enzyme Activation / Q000187:drug effects; D008049:Lipase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism*; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D008049:Lipase