A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

Abstract

Background: Alkaline 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.

Results: The 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 Ca(2+) 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.

Conclusions: The presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.

Figure 1

Identification of putative alkaline phosphatase from metagenome. Sequencing of subclone (pSTV28-AP6) expressing alkaline phosphatase activity resulted in the assembly of a 5,428-bp contig. Three intact ORFs (ORF1 - ORF3) and two partial ORFs (PORF1 and PORF2) with conserved domains were identified through BLAST search.

Figure 2

Multiple alignment of the deduced amino acid sequence of the mAP gene derived from the metagenome with other alkaline phosphatases (APs). APs are identified by their GenBank or PDB accession numbers: Sphingomonas sp. strain BSAR-1 AP (SPAP, ABL96598), Escherichia coli AP (ECAP, BAE76164), Vibrio sp. G15-21 AP (VAP, AAK94204), Antarctic bacterium TAB5 AP (AAP, CAB82508), shrimp AP (SAP, PDB 1SHQ), human placenta AP (HPAP, PDB 1ZEB), Shewanella sp. AP1 AP (SCAP, BAB85685), and Sinorhizobium meliloti 1021 AP (SMAP, NP 385195). Highly conserved amino acid residues are highlighted in black, and gaps are denoted by dots. Gray-shaded amino acids are conserved in at least six of the nine APs shown. The amino acid residues forming the metal binding site and catalytic residues of mAP are denoted by asterisks (★) and closed circle (●), respectively. Numbers along the sequences indicate the positions of the amino acid residues starting from the initial Met for each AP.

Figure 3

Phylogenetic analysis of mAP. A phylogenetic tree based on the similarities of full-length deduced amino acid sequences was constructed with MEGA 5.2 software using the neighbor-joining method. GenBank or PDB accession numbers are given after the species designation. Numbers at nodes are bootstrap values based on 1,000 samplings.

Figure 4

Signal peptide sequence, heterologous expression, and translocation of mAP. (A) N-terminal amino acid sequence of mAP showing the predicted Tat motif and signal peptide cleavage site at Ala⁸¹. (B) Expression of mAP in the E. coli BL21(DE3) transformant harboring pET-mAP at various temperatures from 15°C to 37°C. Lane M, protein standards; lane EV, total proteins expressed in BL21(DE3) cells with pET-21a at 15°C; lane T, IPTG-induced total proteins of BL21(DE3) with pET-mAP at various temperatures. (C) Secretion of mAP. mAP-expressing cells grown with 0.5 mM IPTG for 24 h were fractionated into total (lane T), periplasmic (lane P), and cytoplasmic/membrane (lane C) fractions and subjected to SDS-PAGE. Lanes M and EV are the same as in (B). (D) Purification of mAP. mAP expressed at 15°C was purified using the periplasmic fraction. Lane M, protein standards; lane P, IPTG-induced periplasmic fraction; lane mAP; purified mAP protein after dialysis and concentration. Arrows indicate precursor and mature mAP.

Figure 5

Molecular mass determination of mAP by size exclusion chromatography. mAP and six molecular mass standard proteins (peak a, ferritin; peak b, adolase; peak c, albumin; peak d, ovalbumin; peak e, chymotrypsinogen A; and peak f, RNase A) were each subjected to size exclusion chromatography on a Superose 6 HR column that had been pre-equilibrated with 25 mM Tris–HCl buffer containing 150 mM NaCl at pH 7.6. The molecular mass of mAP was estimated by comparing the retention time of mAP with the standard curve (plot of retention time versus logMW) obtained for the molecular mass standard proteins (inset).

Figure 6

Effects of pH and temperature on mAP activity. AP activity was measured by a spectrophotometric method using p-nitrophenyl phosphate (pNPP). (A) Optimum pH. Enzymatic activity was assayed at 37°C in the presence of 0.5 mM pNPP as a substrate using 50 mM Tris–HCl buffer (pH 7.0–8.5) or 50 mM DEA buffer (pH 8.0 to 10.5). (B) Optimum temperature. Enzymatic activity was assayed at 20–80°C in the presence of 0.5 mM pNPP as a substrate using 50 mM DEA buffer (pH 9.0). (C) Thermal stability. Residual activity of mAP (●) was determined at 37°C after 15 min incubation at temperature ranges from 20°C to 100°C. Bacterial AP (▲) was also assayed under the same conditions. (D) Kinetic studies of mAP. Enzymatic activity was assayed under standard condition with 0.5 mM CaCl₂ using a range of pNPP.