Biochemistry (Moscow)

, Volume 78, Issue 4, pp 385–394

Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants

  • K. A. Novototskaya-Vlasova
  • L. E. Petrovskaya
  • E. M. Rivkina
  • D. A. Dolgikh
  • M. P. Kirpichnikov
Article

Abstract

A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5T 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.

Key words

cryopeg permafrost Psychrobacter cryohalolentis cold-active lipase thermostability deletion mutants 

Abbreviations

CHAPS

3[(3-cholamidopropyl)dimethylam-monio]-propanesulfonic acid

EDTA

ethylenediamine tetra-acetic acid

HSL

hormone sensitive lipase

IPTG

isopropyl β-D-1-thiogalactopyranoside

PMSF

phenylmethylsulfonyl fluoride

p-NPB

p-nitrophenylbutyrate

SDS

sodium dodecyl sulfate

SOE-PCR

splicing by overlapping extension PCR

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Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • K. A. Novototskaya-Vlasova
    • 1
  • L. E. Petrovskaya
    • 2
  • E. M. Rivkina
    • 1
  • D. A. Dolgikh
    • 2
    • 3
  • M. P. Kirpichnikov
    • 2
    • 3
  1. 1.Institute of Physicochemical and Biological Problems in Soil ScienceRussian Academy of SciencesPushchino, Moscow RegionRussia
  2. 2.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  3. 3.Faculty of BiologyLomonosov Moscow State UniversityMoscowRussia

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