Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Abstract

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus townsendii) liver

“…The current study reported optimum pH value of ALP liver extract of L. townsendii to be about 9.2. These findings are consistent with prevoius studies by Zappa et al (2001), in which they observed that mammalian ALPs possess higher pH optima than procaryotes (E. coli) enzyme, and proposed that substitution of two amino acid residues with corresponding histidine molecules in the mammalian enzymes are responsible for this increase in optimum pH (Holtz and Kantrowitz, 1999;Murphy et al, 1995). These variations in ALP activity which are consequences of adjustments in K m or V max values or both, suggest that pH/activity relationship of ALP may be a factor in the intracellular regulation of its activity.…”

Novel Organophosphate Pesticide Utilizing Alkaline Phosphatase Producing Polyextremophile Bacillus Flexus from Lake Ecosystem of North Gujarat, India

“…100 µl cell free supernatant was added to 1000 µl of p-nitrophenyl phosphate disodium salt (p-NPP) solution (1.35 mM in 50 mM Tris-HCl buffer at pH 9.0) and the mixture was incubated at 35 ˚C for 10 min. ALP activity was measured spectrophotometrically by determining the release of p-nitrophenol (p-NP) at 400 nm [7,8,9]. The amount of orthophosphate released from the organophosphate Pesticide was estimated by Ascorbic acid reduction method at 880 nm [10].…”

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“…The protein content of fractions was determined by measuring O. D. at 280 nm. The protein containing fractions were assayed for ALP activity by p-nitrophenyl phosphate disodium salt (p-NPP) method at 400 nm 5 . The maximum ALP activity containing eluate fraction was further purified by affinity chromatography.…”