Characterization of a Cold-Active Lipase from Psychrobacter cryohalolentis K5^T and Its Deletion Mutants

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K. A. Novototskaya-Vlasova^1*, L. E. Petrovskaya^2*, E. M. Rivkina¹, D. A. Dolgikh^2,3, and M. P. Kirpichnikov^2,3

¹Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya ul. 2, 142290 Pushchino, Moscow Region, Russia; fax: (496) 731-8155; E-mail: nksusha@gmail.com

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-0812; E-mail: lpetr65@yahoo.com

³Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia; fax: (495) 939-4309

^* To whom correspondence should be addressed.

Received December 7, 2012; Revision received December 23, 2012

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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.

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KEY WORDS: cryopeg, permafrost, Psychrobacter cryohalolentis, cold-active lipase, thermostability, deletion mutants

DOI: 10.1134/S000629791304007X

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