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Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5T

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Abstract

We have cloned the gene coding for AT877—a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5T. Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8–C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15–25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.

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Acknowledgments

The work was supported by Russian Academy of Sciences programs”The origin of the biosphere and the evolution of geo-biological systems. The microbial biosphere”, “Molecular and Cellular Biology”, grants SS-1766.2014.4, RFBR 13-04-12405 ofi_m, 12-05-01085 and 14-04-31573 mol_a.

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Authors and Affiliations

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

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

  2. Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Moscow, 142290, Russian Federation

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

  3. Department of Biology, M. V. Lomonosov Moscow State University, Leninskie gory, 1, Moscow, 119234, Russian Federation

    D. A. Dolgikh & M. P. Kirpichnikov

Authors
  1. L. E. Petrovskaya
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  2. K. A. Novototskaya-Vlasova
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  3. E. A. Kryukova
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  4. E. M. Rivkina
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  5. D. A. Dolgikh
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  6. M. P. Kirpichnikov
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Correspondence to L. E. Petrovskaya.

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Communicated by S. Albers.

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Petrovskaya, L.E., Novototskaya-Vlasova, K.A., Kryukova, E.A. et al. Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5T . Extremophiles 19, 161–170 (2015). https://doi.org/10.1007/s00792-014-0695-0

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  • DOI: https://doi.org/10.1007/s00792-014-0695-0

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