Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium Meiothermus ruber and characterization of the recombinant enzyme

Abstract

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg2+ ions was required. Zn2+ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62 degrees C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60 degrees C, whereas at 80 degrees C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

“…4. Surprisingly, negligible activity was detected in the absence of exogenous metal ions in the reaction mixture, and similar results have been observed for the recombinant AP from Meiothermus ruber [41]. Howeve, the crude enzyme extract, supernatant, without exogenous metal ions supplemented, was activated.…”

“…Under the optimal conditions, the recombinant Gtd AP hydrolyzed an artificial substrate pNPP with a K m of 31.5 μM and V max of 430 μM/min. A similar K m was found in APs from mesophilic E. coli [43] and thermophilic Meiothermus ruber [41], which ranges from 21 to 55 μM.…”

“…Further analysis showed that Gtd AP for total thermal inactivation was only 15 min by a heat treatment of 80°C and the half-life value at 70°C was 8 min (Fig. 3c), which is less stable than its counterparts in various thermophiles [9,22,29,30,[39][40][41][42]. For example, Thermus caldophilus retained 80% activity after incubation at 80°C for 12 h [39] and even E. coli AP exhibited a half-life value at 80°C more than 6 h [8].…”

Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237

“…Consequently, thermostable enzymes have attracted more attention. To date, numerous thermostable APases have been isolated and characterized from various thermophiles, including Thermus species (Kim et al, 1997;Pantazaki et al, 1998;Yuan et al, 1998), Thermotoga neapolitana (Dong & Zeikus, 1997), Meiothermus ruber (Yurchenko et al, 2003), Bacillus stearothermophilus (Mori et al, 1999), Scytalidium thermophilum (Guimaraes et al, 2001), Pyrococcus abyssi (Zappa et al, 2001;Minamihata et al, 2012), and Bacillus licheniformis (Banik & Pandey, 2009;Divya et al, 2016). However, these thermostable APases exhibit drawbacks such as low activity, low thermostability, or low tolerance to alkaline pH and detergents.…”