# Characterization of Two Hydrogen Peroxide Resistant Peroxidases from _Rhodococcus apacus_ 1 Cp
## 4 Discussion  
This paper establishes that _Rhodococcus opacus_ 1 CP has similar dye-decolorizing peroxididases (DypA and DypB) described for _Rhodococcus jostii_ RHA1 (Figure A1). Dye-decolorizing peroxididases are heme-containing enzymes [50]. However, the heterologous expression in _E. coli_ limits the production of soluble, active heme-containing enzymes since it doesn't incorporate heme efficiently. Incomplete or inefficient heme incorporation into recombinant proteins have been frequently described [51; 52; 53] especially in the induction of recombinant protein expression systems from highly active vectors such as _E. coli_ expression hosts with T7 promoters. This usually leads to high protein production of enzyme without the heme cofactor. Various techniques have been employed such as the co-expression of \(\delta\)-aminolevulinate to enhance internal heme production and the reconstitution of apoenzyme with hemin chloride [30; 54; 55; 56]. In this study, the supplementation of hemin in growth medium showed good results [57]. A total of 3.72 mg of DypA were purified from 10.8 g of _E. coli_ cells (wet weight) and 16.56 mg of DypB were purified from 7.8 g of _E. coli_ cells (wet weight). Both DyPs from _R. opacus_ 1 CP contain highly conserved residues that are essential for its enzymatic activity: GoxDG with the conserved Asp, distal Arg, and proximal His. Several studies [58; 59; 60] have been done to show that these motifs play a major role on the stability, heme-binding, and biocatalysis of dye peroxididases. The preference of Dyp-type peroxididases to utilize Asp as an acid-base catalyst explains the high activity of these enzymes at low pH since the pKa of Asp is at 3.9 [61; 62]. Meanwhile, the thermostability of DypA and DypB vary from each other. DypB showed more stability at 25 and 30 \({}^{\circ}\)C while DypA already exhibited decrease in activity even at room temperature. Though DypB showed subsequent decrease in activity at 40 \({}^{\circ}\)C after 15 min, DypA was already rendered inactive at this temperature. Suicide inactivation via excessive H\({}_{2}\)O\({}_{2}\) exposure is one of the biggest hurdles when working with peroxididases. The formation of Compound III leads to cofactor destruction, release of heme iron or oxidation of amino acid side chains [63; 64; 65]. For this reason, the standard ABTS assay use only up to 0.2 mM H\({}_{2}\)O\({}_{2}\). However, the Dyp enzymes of _R. opacus_ 1 CP exhibited enzymatic activities at higher concentrations of H\({}_{2}\)O\({}_{2}\). The activity of DypA was retained from 3.9 mM up to about 10 mM before showing decrease in activity while the activity of DypB retained its activity from 2.9 mM up to 5 mM of H\({}_{2}\)O\({}_{2}\) (Table A5) without any detrimental effects. It is still unclear why both enzymes are not inactivated by high concentrations of H\({}_{2}\)O\({}_{2}\). Most DyPs operate only between 0.1 mM and 0.4 mM H\({}_{2}\)O\({}_{2}\)[22; 66]. It has not been investigated yet whether DypA and DypB function as catalase-peroxidase. Most enzymes which can tolerate high concentrations of H\({}_{2}\)O\({}_{2}\) belong to other types of peroxididases. For example, manganese peroxididases (MnP) from _Lenzites betulinus_ retained more than 60% of the initial activity after exposure to 10 mM H\({}_{2}\)O\({}_{2}\) for 5 min at 37 \({}^{\circ}\)C [67]. The results obtained from the kinetic parameters show that DypA was the more efficient catalyst for RB5. Interestingly, both enzymes showed very high Km towards H2O2. A result that is very unusual for peroxididases, but this could be an explanation why it can tolerate high concentrations of H\({}_{2}\)O\({}_{2}\). Despite the lower Km of DypB for RB5, DypA still exhibited better Kcat and higher Kcat/Km. The poor performance of DypB for RB5 can be because that DypB might play a bigger role on lignin degradation than dye degradation. A study [30] showed that the deletion of _dypB_ in _R. jostii_ RHA1 led to impaired activities for lignin breakdown and the deletion of _dypA_ did not show the same effect.