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

• Published in: Marine biotechnology (New York, N.Y.) Vol. 17; no. 2; pp. 130 - 143
• Main Authors: Golotin, Vasily, Balabanova, Larissa, Likhatskaya, Galina, Rasskazov, Valery
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01-04-2015; Springer Nature B.V
• Subjects: Alkalies; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Amino Acid Sequence; Animals; Bacteria; Base Sequence; Biomedical and Life Sciences; Biotechnology; Biotechnology - methods; Bivalvia - microbiology; Cations; Cold; Crenomytilus grayanus; DNA Primers - genetics; E coli; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Engineering; Enzymes; Escherichia coli; Freshwater & Marine Ecology; Gene amplification; Gene expression; Half-Life; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Microbiology; Microorganisms; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis; Mutagenesis, Site-Directed; Oceanospirillaceae - enzymology; Original Article; Phosphatase; Plasmids; Protein Conformation; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Studies; Temperature; Vibrio; Zoology; Russia; Biochemical properties; Catalytic efficiency; Purification; Expression; Site-directed mutagenesis; Homology modeling
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-014-9601-0

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 (
Cm
AP) 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.
Cm
AP 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
Cm
AP. The enzyme was stable in a wide range of pH (6.0–11.0).
Cm
AP exhibited its highest activity at the reaction temperature of 40–50 °C and pH 9.5–10.3. The structural features of
Cm
AP could be the reason for the increase in its stability and catalytic turnover. We have modeled the
Cm
AP 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 Mg
2+
bonds in
Cm
AP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in
Cm
AP could be attributed to the adapted mutualistic lifestyle in oceanic waters.