Thermoacidophilic Alicyclobacillus Superoxide Dismutase: Good Candidate as Additives in Food and Medicine

Abstract

Thermoacidophilic Alicyclobacillus strains attract great interests as the resource of thermostable or acidic enzymes. In this study, a putative gene encoding superoxide dismutase (AaSOD) was identified in a thermoacidophilic Alicyclobacillus strain. With a 16-fold activity observed, the AaSOD activity expressing in the medium of manganese enrichment was much higher than that in the iron medium. In addition, the purified AaSOD can be reconstituted exclusively with either Fe²⁺ or Mn²⁺, with its Mn-bound protein showing 25-fold activity than that of Fe-bound form. The optimal temperature for AaSOD reaction was 35°C, and was highly stable at any certain temperature up to 80°C. Of particular interest, the enzyme is found to be very stable across a wide pH range spanning from 2.0 to 10.0, which confers its robust stability in the acidic stomach environment and implies striking potentials as food additive and for medical use.

Keywords: acid tolerant; cambialistic Fe/Mn type; superoxide dismutase; thermoacidophilic Alicyclobacillus strain; thermostability.

FIGURE 1

Neighbor-joining phylogenetic tree based on the amino sequences of AaSOD and its closest relatives retrieved from NCBI database. The tree was built by the program MEGA version 7.0. Bootstrap percentages >50% (based on 1000 replications) are shown at branch points.

FIGURE 2

Multiple alignment of AaSOD with other identified Fe, Mn, or Fe/Mn type SODs that retrieved from NCBI database. GsSOD: Geobacillus stearothermophilus, AaSOD: this study, PaSOD: Pseudomonas aeruginosa, TfSOD: Thermus filiformis, EcSOD: Escherichia coli, CaSOD: Chloroflexus aurantiacus, ApSOD: Aeropyrum pernix, AaeSOD: Aquifex aeolicus.

FIGURE 3

Superimposition of the predicted structure of AaSOD (Green) over its template of Geobacillus stearothermophilus Gs-MnSOD (gray) (A) and four predicted residues for manganese binding (B).

FIGURE 4

SDS-PAGE spectrum of AaSOD. Lane M, Marker; lane 1, purified protein after His-Trap affinity column; lane 2, cell debris of the E. coli recombinant harboring AaSOD; lane 3, supernatant of the E. coli recombinant harboring AaSOD induced by IPTG for 12 h at 18°C.

FIGURE 5

Thermostability (A) and optimal temperature (B) of AaSOD. For thermostability measurement, the purified AaSOD was incubated at different temperatures (RT, 60, 70, 80, 90, and 100°C) for 1 h, and subsequently measured the residual activities. The relative activity was calculated as the percentage of the maximum activity at 35°C. To achieve the optimal catalytic temperature, the activity of purified AaSOD was tested at different temperatures (25–80°C) and the highest activity was set as 100% for relative activity calculation.

FIGURE 6

DSC spectrum of AaSOD. The purified AaSOD was scanned from a temperature range of 70–110°C at a rate of 1.5°C min^–1. The transition curve was fitted by non-2-state model and the T_m value was calculated (MN2state) in the Origin software.

FIGURE 7

Stabilities of AaSOD in buffers with different pH values (A) and effect of divalent ions on the AaSOD activity (B). The purified enzyme was incubated in the buffer systems of 200 mM KCl–HCl buffer (pH 1.0–2.0), acetate buffer (pH 2.0–5.0), sodium phosphate buffer (pH 5.0–7.0), Tris–HCl buffer (pH 7.0–9.0), and glycine–NaOH buffer (pH 9.0–10.0) for 1 h, 25°C. The maximal activity was taken as 100% for relative activity calculation. For metal ions effects, the purified enzyme was incubated with 1 mM of divalent metal ions in 50 mM Tris–HCl buffer (pH 8.0), at 25°C for 30 min. The mixture with no ions supplementation was used as a control for relative activity calculation.