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

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 Mg 2+ 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.

Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain

“…From Fig.3, where the effect of temperature on the activity of alkaline phosphatase is shown, it appears that maximum activity was obtained at 50 °C, a value similar of that found for marine bacterium Cobetia marina and Neurospora crassa [8,28], but higher than that referred to bos Taurus alkaline phosphatase (25 °C) [29], Pyrococcus furiosus alkaline phosphatase (37 °C) [30] and Pinctada fucata alkaline phosphatase (45 °C) [7]. Enzyme retains a high activity in a wide temperature range from about 30-70 °C if compared with other alkaline phosphatases [8,31,32].…”

“…Enzyme retains a high activity in a wide temperature range from about 30-70 °C if compared with other alkaline phosphatases [8,31,32].…”

“…The enzyme exhibited maximum activity at about pH 11 and a relative high residual activity at pH 12 (80% of the maximum). As known, at the pH values ≥10.0, the substrate-binding residues oriented toward the bound substrate become neutral and negatively charged, respectively [8,23,24]. In the case of some alkaline phosphatases can be inactive at some pH values.…”

“…Alkaline phosphatases are widely distributed in nature and knowledge of the alkaline phosphatases has increased remarkably during the last few years [6]. ALPs in marine organisms play important roles in cell phosphate metabolism, which is related to the absorption of phosphate and calcium from seawater and the biomineralization process in marine organisms [7,8]. Furthermore, alkaline phosphatase is an important enzyme in recycling phosphate in marine environments as well as within living cells.…”

“…Its distribution is dependent on environmental factors such as phosphate availability, dominant Page 5 of 31 A c c e p t e d M a n u s c r i p t 5 habitants, water depth, salinity, temperature, and the internal ratio of nitrogen to phosphorus in higher organisms and microbial communities [8,9].…”