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

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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.

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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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Authors and Affiliations

  1. Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya ul. 2, 142290, Pushchino, Moscow Region, Russia

    K. A. Novototskaya-Vlasova & E. M. Rivkina

  2. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    L. E. Petrovskaya, D. A. Dolgikh & M. P. Kirpichnikov

  3. Faculty of Biology, Lomonosov Moscow State University, 119234, Moscow, Russia

    D. A. Dolgikh & M. P. Kirpichnikov

Authors
  1. K. A. Novototskaya-Vlasova
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  2. L. E. Petrovskaya
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  3. E. M. Rivkina
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  4. D. A. Dolgikh
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  5. M. P. Kirpichnikov
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Corresponding authors

Correspondence to K. A. Novototskaya-Vlasova or L. E. Petrovskaya.

Additional information

Published in Russian in Biokhimiya, 2013, Vol. 78, No. 4, pp. 501–512. p ]Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM12-320, February 3, 2013.

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Novototskaya-Vlasova, K.A., Petrovskaya, L.E., Rivkina, E.M. et al. Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5T and its deletion mutants. Biochemistry Moscow 78, 385–394 (2013). https://doi.org/10.1134/S000629791304007X

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  • DOI: https://doi.org/10.1134/S000629791304007X

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