# Characterization of Two Hydrogen Peroxide Resistant Peroxidases from _Rhodococcus apacus_ 1 Cp
## 3 Results
### Biochemical Properties of DyPA and DyPB  
pET16 bp_dypA and pET16 bp_dypB harboring the respective Dyp-genes were used for expression and allowed the production of recombinant proteins after induction with IPTG and upon addition of hemin. Soluble fractions of cell disruption were strongly reddish colored indicating the presence of heme-containing enzymes. Dyps were isolated on Ni-sepharose columns checking presence and purity via SDS-PAGE (Figures A2 and A3). The theoretical molecular size of 48.48 kDa (DypA) and 39.81 kDa (DypB) corresponded to the observed single band at around 50 kDa and 40 kDa, respectively. Various parameters were checked to determine the optimal conditions for these enzymes to operate on. First, the thermal stability of DyPs were determined by incubating the enzymes up to 60 \({}^{\circ}\)C (Figure 2). DypA showed already a decrease of 15% in its activity at 25 \({}^{\circ}\)C after about 2 h of incubation while more than 20% decrease after 1 h at 30 \({}^{\circ}\)C. The subsequent loss of activity at 30 \({}^{\circ}\)C was further observed after 2 h. At 40 \({}^{\circ}\)C, DypA exhibited only 15% activity on the first reading and did not show further activity after 10 min. In contrast, DypB showed more heat resistance by retaining its activities even after 5 h on 30 \({}^{\circ}\)C. Though a decrease in activity can already be observed for DypB after 30 min at 40 \({}^{\circ}\)C, it was still able to retain 90% of its activities during the first 15 min of incubation. Both enzymes did not show any activity at 50 \({}^{\circ}\)C and 60 \({}^{\circ}\)C, respectively (data not shown). Britton-Robinson buffer was used since it offers a wide range of pH to be tested. As seen on Figure 3, both Dyps from _Rhodococcus opacus_ 1 CP operated better on acidic pH. DypA performed best with pH 4.3 and showed a significant reduction with pH 6 (Figure 3). Meanwhile, DypB performed relatively well with a wider range of pH around pH 4-6, showing close values in its specific activity. Different buffers were checked to determine the most suitable buffer for DypA and DypB assays. Since the best pHs for DypA and DypB were pH 4.3 and pH 5, respectively, the buffers tested were prepared with the same optimal pH relative to Britton-Robinson buffer. At pH 4.3, DypA performed twice as better on citrate buffer and citrate phosphate buffer compared to Britton-Robinson (Figure 4A). With citrate buffer, a 50% increase was observed in its activity. Sodium acetate buffer, on the other hand, showed the same activity to Britton-Robinson buffer. DypB was also tested on the same buffers used with DypA. However, all of the buffers tested for DypA were inhibitory to the activity of DypB. Potassium phosphate buffer was checked and DypB showed an increase on its activity (Figure 4B). Tolerance to H\({}_{2}\)O\({}_{2}\) was tested by checking different concentrations and monitored via ABTS assay. DypA obtained the highest activity with about 4 mM H\({}_{2}\)O\({}_{2}\) and sustained relatively high activity until 10 mM of H\({}_{2}\)O\({}_{2}\) (Figure 5). Meanwhile, the optimum concentration for DypB was about 3 mM H\({}_{2}\)O\({}_{2}\) then showed subsequent loss of activity. Interestingly, both Dyps still exhibited some activity at 195 mM of H\({}_{2}\)O\({}_{2}\) (Table A5). Figure 3: Specific activity of (**A**) DypA and (**B**) DypB with 1 mM ABTS as the substrate and 0.25 mM H\({}_{2}\)O\({}_{2}\) using Britton-Robinson buffer with pH ranges from pH 2 to 6 to check the optimal pH for each enzyme. Figure 2: Thermal stability of (**A**) DypA and (**B**) DypB. The enzymes were incubated at different temperatures and subjected to standard ABTS assay to determine changes in the activity. DypA exhibited about 13 U/mg while DypB showed about 12 U/mg. These values represent the 100% so as to compare the relative activity of the enzyme after subsequent incubation to different temperatures. Results for higher temperatures were not shown because the enzyme was rendered inactive immediately.