Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

Abstract

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.

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

“…Heat stability was increased up to 120 min at 45°C for the loss of 50 % of the CmAP initial activity in the presence of stabilizer Mg 2+ (data not shown). Although CmAPase had lower activity than CmAP that could be the result of the recombinant production method (Nasu et al 2012). CmAP is more close to AP from moderate halophile Halomonas sp.…”

“…For comparison, the K m of E. coli AP was 0.06 mM and a k cat of 62 s with the catalytic efficiency of 1.1×10 6 , whereas the K m of bovine intestinal AP, including its catalytically improved mutants, ranged from 1.3 to 3.9 mM and the k cat ranged from 1,800 to 6,100 s with the catalytic efficiency of 1.4-1.6 × 10 6 s/M (Hauksson et al 2000;Kozlenkov et al 2002). Surprisingly, the native C. marina AP signal peptide was found to be appropriate for E. coli system to produce periplasmic recombinant protein (Nasu et al 2012). Alternatively, the use of E. coli signal peptide included in the expression plasmid pET40b(+) resulted in overproduction of the soluble highly active CmAP (Table 1).…”

“…Currently, recombinant C. marina AP (CmAPase) was expressed by Nasu et al (2012) using the synthetic gene adapted for E. coli and inserted in pET-16b(+) vector and had a specific activity of 12,700 U/mg, which is the highest activity reported for any bacterial or mammalian AP. The specific activity and catalytic efficiency of our recombinant enzyme (CmAP) produced in E. coli on the base of the nature gene of C. marina and pET-40b(+) vector were also the highest among any of the APs studied to date (Tables 1 and 2).…”

“…However, the gel permeation of a recombinant C. marina AP (CmAPase) carried out by Nasu et al (2012) at pH 10.0 resulted in the formation of the enzyme dimer. It seems probable that more flexible links at the dimer interface of the cold-adapted enzymes may ease the structural rearrangement and thereby facilitating more rapid release of phosphate and thus catalytic turnover (Asgeirsson et al 2003).…”

“…The AP from C. marina KMM 296 (CmAP) was found to be relatively heatstable for a psychrophile and functioned independent of extraneous bivalent metal ions and high inorganic phosphorus (P i ) or EDTA concentrations in the incubation medium (Plisova et al 2005). Only a limited number of marine bacterial APs have been reported to be similar to CmAP in that they have unusual structures and cold-adapted properties such as a low thermostability and high catalytic efficiency (Plisova et al 2005;Nasu et al 2012). …”