A glutathione peroxidase from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506: Cloning and heterologous expression of the gene and characterization of recombinant enzyme

Abstract

A glutathione peroxidase (GPx) gene, designated as PsGPx, was cloned from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506 and expressed in Escherichia coli. The full-length PsGPx contained a 585-bp encoding 194 amino acids with predicted molecular masses of approx. 21.7 kDa. Multiple sequence alignments revealed that PsGPx belonged to the thioredoxin-like superfamily. PsGPx was heterologously overexpressed in E. coli, purified and characterized. The maximum catalytic temperature and pH value for recombinant PsGPx (rPsGPx) were 30°C and pH 9.0, respectively. rPsGPx retained 45% of the maximum activity at 0°C and exhibited high thermolability with a half-life of approx. 40 min at 40°C. In addition, the enzymatic activity of rPsGPx was still manifested under 3 M NaCl. The K_m and V_max values of the recombinant enzyme using GSH and H₂O₂ as substrates were 1.73 mM and 16.28 nmol/mL/min versus 2.46 mM and 21.50 nmol/mL/min, respectively.

Keywords: Antarctic sea ice; expression; gene; glutathione peroxidase; psychrotrophic.

Figure 1.

The methodology flowchart for the cloning and heterologous expression of the PsGPx gene.

Figure 2.

Multiple sequence alignment between the deduced PsGPx and other GPxs. The species names and GenBank accession numbers are as follows: Pseudoalteromonas haloplanktis TAC125 (YP_340977), Pseudoalteromonas agarivorans (WP_004589433), Pseudoalteromonas luteoviolacea (WP_005493401), Shewanella baltica OS155 (YP_001555602), Shewanella frigidimarina (YP_751531). ‘▾’ and ‘▽’ indicate the amino acids defining the active site and the polypeptide binding, respectively.

Figure 3.

SDS-PAGE (12.5%) analysis of the purified rPsGPx. (a) Lane 1, protein molecular weight marker; Lane 2, the purified rPsGPx under reducing condition (with 2-mercapto ethanol) ; Lane 3, total extracts of the BL21/pETGPx cells with IPTG induction; Lane 4, total extracts of the BL21/pET-28a(+). (b) Lane 1, the purified rPsGPx under nonreducing condition (without 2-mercapto ethanol); Lane 2, protein molecular weight marker.

Figure 4.

(a) Effect of temperatures on the purified rPsGPx activity. (b) Effect of temperatures on the stability of the purified rPsGPx. •30°C; (○) 40°C; (▾) 50°C. The residual catalytic activity after pre-incubation was assayed at 30°C, 40°C and 50°C for 10, 20, 30, 40, 50, and 60 min.

Figure 5.

Effect of pH on enzymatic activity (a) and stability (b) of the purified rPsGPx.

Figure 6.

Effect of NaCl on the purified rPsGPx activity.