Abstract
BackgroundAlkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.ResultsThe gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.ConclusionsThe presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.
Highlights
Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications
The novel AP genes phoX and phoK were identified in Vibrio cholerae [7] and Sphingomonas sp. strain BSAR-1 [8], respectively; these genes were shown to be activated by Ca2+ and share no homology with phoA
For functional screening of AP, the DmpR of Genetic Enzyme Screening System (GESS) can be activated upon binding to p-nitrophenol, which is released from p-nitrophenyl phosphate hydrolysis by AP, and turn on the egfp expression
Summary
Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Alkaline phosphatase (AP; EC 3.1.3.1) is a ubiquitous enzyme widely distributed from microorganisms to humans [1] and functions to catalyze the hydrolytic cleavage of phosphate monoesters under alkaline conditions, releasing inorganic phosphate from many phosphate-containing compounds. This enzyme plays important roles in microbial ecology through its involvement in phosphate metabolism [2], signal transduction [3], and bacterial virulence [4]. PhoA-like APs are found in many other bacteria and have conserved domains with those in ECAP [5]. The novel AP genes phoX and phoK were identified in Vibrio cholerae [7] and Sphingomonas sp. strain BSAR-1 [8], respectively; these genes were shown to be activated by Ca2+ and share no homology with phoA
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