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Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

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Abstract

The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10- to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45 °C and 27 min at 40 °C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0–11.0). CmAP exhibited its highest activity at the reaction temperature of 40–50 °C and pH 9.5–10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg2+ bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.

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Acknowledgments

The work was supported by grant from RFBR 12-04-00825-a and the projects 13 НТПI-12, 12-I-П6-10.

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Authors and Affiliations

  1. G.B. Elyakova Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of Russian Academy of Sciences, Prospect 100-letya Vladivostoka, 159, Vladivostok, Russian Federation

    Vasily Golotin, Larissa Balabanova, Galina Likhatskaya & Valery Rasskazov

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  1. Vasily Golotin
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  2. Larissa Balabanova
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  3. Galina Likhatskaya
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  4. Valery Rasskazov
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Correspondence to Larissa Balabanova.

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Golotin, V., Balabanova, L., Likhatskaya, G. et al. Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina . Mar Biotechnol 17, 130–143 (2015). https://doi.org/10.1007/s10126-014-9601-0

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