Characterization of Two Hydrogen Peroxide Resistant Peroxidases from Rhodococcus opacus 1CP

: The dye-decolorizing peroxidases (DyP) are a family of heme-dependent enzymes present on a broad spectrum of microorganisms. While the natural function of these enzymes is not fully un-derstood, their capacity to degrade highly contaminant pigments such as azo dyes or anthraquinones make them excellent candidates for applications in bioremediation and organic synthesis. In this work, two novel DyP peroxidases from the organism Rhodococcus opacus 1CP (DypA and DypB) were cloned and expressed in Escherichia coli . The enzymes were puriﬁed and biochemically characterized. The activities of the two DyPs via 2,2 (cid:48) -azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) assay and against Reactive Blue 5 were assessed and optimized. Results showed varying trends for DypA and DypB. Remarkably, these enzymes presented a particularly high tolerance towards H 2 O 2 , retaining its activities at about 10 mM H 2 O 2 for DypA and about 4.9 mM H 2 O 2 for DypB.


Introduction
Different industries are now gearing towards greener solutions-phasing out traditional chemical processes that are often found to be harmful to human and the environment. In search of finding other alternatives, enzymes have been used as a potential agent to drive these sustainable processes as they are more environmental-friendly, non-toxic, and more cost-efficient in the long run [1][2][3]. A particularly interesting family of enzymes that are being used for industrial applications are the dye-decolorizing peroxidases (DyPs) (EC 1.11.1.19), which can oxidize recalcitrant organic compounds using hydrogen peroxide (H 2 O 2 ) as co-substrate [4][5][6][7][8]. This enzyme can degrade different types of dyes, which can make them extremely useful in the treatment of wastewater produced by the textile industry or as decolorizing agents in the cosmetics or food industry [9,10]. Another potential use for these proteins is the valorization of lignin since they are one of the few enzymes able to degrade this natural polymer [11]. Due to the wide availability of lignin, its chemical variety, and its resistance to treatment, the DyPs offer a great potential alternative for the field of synthetic green chemistry [12][13][14][15].
Nevertheless, in the search for an application, two main hurdles have been identified for this type of enzyme: their sensitivity to the H 2 O 2 that they require to operate and the difficulty of heterologous expression [16].
Since the peroxide sensitivity is an issue presented by many different heme-containing proteins [17,18], strategies like the combination with oxidases for in situ production of H 2 O 2 have been attempted [19]. Another option has been the modification of the enzyme to dephosphorylated HincII (for dypA) and SfoI (for dypB) restriction site of pUC19 and then propagated into E. coli DH10B. Plasmids were isolated from white colonies and subjected to DNA sequencing (data not shown).
Confirmed dypA and dypB genes in pUC19 were cleaved at the NdeI and NotI restriction site and ligated into the pET16bP vector. Correct clones were selected by colony PCR and expression plasmids were sent for DNA sequencing using T7-and T7 term-primers. Plasmids with the confirmed dyp genes constructs were then transformed to E. coli Rosetta (DE3) pLysS.
For gene expression, precultures of the expression clones were made by inoculating the E. coli harboring dypA and dypB in an LB medium and then subsequently placed the cultures at 37 • C at 150 rpm overnight. Precultures were then transferred into a 500 mL fresh LB medium containing the antibiotics, ampicillin and chloramphenicol, with the aforementioned concentration and 16 µM hemin. Expression cultures were cultivated at 37 • C at 150 rpm. Once the cultures reach an OD 600 of about 0.5, the temperature was decreased to 20 • C and subsequently induced with 0.1 mM IPTG (isopropyl-ß-D-1thiogalactopyranoside). Cells were harvested at 4 • C by centrifugation (5000 rpm, 20 min) and cell pellets were frozen at −20 • C.

Enzyme Purification, Quantification, and Verification
Cell pellets were thawed and resuspended in 4-fold volume of TED buffer (25 mM Tris-HCl, 2 mM EDTA, 2 mM DTT, pH 8.0) plus the lysozyme solution (25 mM Tris-HCl, 1% lysozyme, pH 8.0) and incubated for 30 min on ice before disruption via ultrasonication. The soluble proteins were separated from the cell debris by centrifugation at 4 • C (18,500 rpm, 40 min).
His-tagged DyPs were purified on a Ni-sepharose colume (45 mL Ni 2+ loaded Chelating Sepharose Fast Flow (Amersham Bioscience), using ECONO system (BIO-RAD) laboratories equipped with a UV monitor and tempered at 4 • C. DypA-containing crude extracts were spiked with 500 mM NaCl prior to loading. The column was pre-equilibrated with a filtered and degassed mixture of Buffer A (DypA: 25 mM Tris-HCl, 500 mM NaCl, pH 8.0; DypB: 25 mM Tris-HCl, pH 8.0) and Buffer B (Buffer A plus 500 mM (DypA) or 400 mM (DypB) imidazole) which was 90%:10% for Dyp A and 95%:5% for DypB, respectively. Proteins were eluted by a linear gradient of increasing imidazole concentration from 10% to 100% Buffer B over 12 column volumes. Eight ml fractions were collected and assayed for peroxidase activity, protein content, and purity via SDS-PAGE. Pure and most active fractions were pooled, dialyzed against 200-fold volume 10mM Tris-HCl buffer (pH 8.0) and stored in aliquots at −20 • C.
Bradford assay was used to determine protein concentration, using bovine serum albumin (BSA) as a standard. Meanwhile, sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used to verify the purity of the proteins and to determine the sizes.

Enzyme Characterization
Two assays were used to characterize DypA and DypB. First, peroxidase activity was determined spectrophotometrically using the 2,2 -azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS)-assay. Standard ABTS assay contained 1 mM ABTS, 0.1 M KPi buffer at pH 5.0, 0.25 mM H 2 O 2 and the corresponding enzyme. After mixing, the increase in extinction at 420 nm was recorded for 1 min at room temperature. ABTS assay was further optimized to determine the best conditions for the DyPs from Rhodococcus opacus 1CP. The pH profile was determined with the standard ABTS assay using Britton-Robinson buffer ( Meanwhile, dye-decolorizing peroxidase activity was determined spectrophotometrically using the Reactive Blue 5 (RB5)-assay [42]. Typically, 1 mL RB5-assay contained 500 µL 288 µM RB5 solution, (480-x) µL 25 mM citrate buffer (pH 3.6), 20 µL 0.03% H 2 O 2 and an appropriate amount of enzyme (2.5 nM DypA and 22.6 nM DypB). After mixing, the decrease in extinction at 600 nm was detected immediately and run for 1 min at 20 • C for DypA and 30 • C for DypB.
The temperature optimum was determined for both the optimized ABTS and RB5 assay for each enzyme varying the temperature from 4-60 • C. The H 2 O 2 tolerance in the presence of ABTS was tested using the optimized ABTS assay. The H 2 O 2 concentration was varied from 0.02-200 mM.
Control reactions were performed without enzyme, H 2 O 2 or both. Conversion of substrate was only observed when both enzyme and H 2 O 2 were present. For reference, 1 U is defined as the conversion of 1 µmol ABTS or 1 µmoL RB5 within 1 min. Measurements were carried out at least in triplicates.

Dye-Decolorization Capability
Additional dyes were utilized to check if the DyPs can decolorize other dyes. The concentration of each dye was adjusted so that the initial absorbance of each was around 1 in 25 mM citrate buffer (pH 3.6). The respective final concentration in a 1 mL assay was as follows: Reactive Blue 5 (137 µM, ε 600-20 . The structures of the dyes used in this study can be seen on Figure 1. Control reactions were included as described earlier. The extinction coefficient for each dye was determined in triplicates from a 1 mg mL −1 stock solution of the dye in 25 mM citrate buffer (pH 3.6) at 20 • C and 30 • C using the LAMBERT-BEER law and taking the dilution factor of the dye in buffer into account. λ max was ascertained for each dye performing a wave scan from 300 to 800 nm.
The extinction coefficient for each dye was determined in triplicates from a 1 mg mL −1 stock solution of the dye in 25 mM citrate buffer (pH 3.6) at 20 °C and 30 °C using the LAMBERT-BEER law and taking the dilution factor of the dye in buffer into account. λmax was ascertained for each dye performing a wave scan from 300 to 800 nm.

Sequence Analysis
A BLASTp search [47] of DypA and DypB sequence from R. opacus 1CP was per formed against the NCBI database. Sequence-based alignment of Dyps and other peroxi dases (out-group) was done using ClustalW [48]. The maximum likelihood tree was gen erated from 1000 bootstraps using Mega X [49]. NCBI accession numbers used are listed on Table A4.

Identification of Dyp Genes
The dyp genes from Rhodococcus opacus 1CP were compared to the DypA and DypB from Rhodococcus jostii RHA1 via a BLAST search. Results showed (data not shown) th DypA of R. opacus 1CP is 98% similar to that of R. jostii DypA and shared only 33% simi larity to the DyP enzyme of Geobacillus geotrichum. Meanwhile, DypB of R. opacus 1CP have 97% similarity to the DypB of R. jostii RHA1 and only 29% to DyP of G. geotrichum.
Dye-decolorizing peroxidases are often classified into four distinct classes based on their homologies [3]. Class A, B, and C generally include Dyps of bacteria belonging t the phyla Proteobacteria, Actinobacteria, Firmicutes, and Bacteroides, while Class D in cludes fungal Dyps. Thereby, Class A and B form a cluster distinguishable from Class C

Identification of Dyp Genes
The dyp genes from Rhodococcus opacus 1CP were compared to the DypA and DypB from Rhodococcus jostii RHA1 via a BLAST search. Results showed (data not shown) the DypA of R. opacus 1CP is 98% similar to that of R. jostii DypA and shared only 33% similarity to the DyP enzyme of Geobacillus geotrichum. Meanwhile, DypB of R. opacus 1CP have 97% similarity to the DypB of R. jostii RHA1 and only 29% to DyP of G. geotrichum.
Dye-decolorizing peroxidases are often classified into four distinct classes based on their homologies [3]. Class A, B, and C generally include Dyps of bacteria belonging to the phyla Proteobacteria, Actinobacteria, Firmicutes, and Bacteroides, while Class D includes fungal Dyps. Thereby, Class A and B form a cluster distinguishable from Class C and D indicating that they share more sequence similarities than to the other two groups. As expected, DypA and DypB from R. opacus 1CP are assigned to Class A and B, respectively, and are the closest related to Dyps of other rhodococci like R. jostii RHA1, R. opacus PD630 or R. opacus B4 ( Figure A1).

Biochemical Properties of DyPA and DyPB
pET16bp_dypA and pET16bp_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 • C ( Figure 2). DypA showed already a decrease of 15% in its activity at 25 • C after about 2 h of incubation while more than 20% decrease after 1 h at 30 • C. The subsequent loss of activity at 30 • C was further observed after 2 h. At 40 • 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 • C. Though a decrease in activity can already be observed for DypB after 30 min at 40 • 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 • C and 60 • 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 1CP 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).
at 25 °C after about 2 h of incubation while more than 20% decrease after 1 h at 30 °C. The subsequent loss of activity at 30 °C was further observed after 2 h. At 40 °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 °C. Though a decrease in activity can already be observed for DypB after 30 min at 40 °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 °C and 60 °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 1CP 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).     0. Both enzymes were tested with different buffers using Britton-Robinson buffer as a standard for comparison with respect to activity. Potassium phosphate buffer was used only for DypB since most of the buffers tested showed a decrease in activity when tested to citrate, citrate phosphate, and sodium acetate buffers.
Tolerance to H2O2 was tested by checking different concentrations and monitored via ABTS assay. DypA obtained the highest activity with about 4 mM H2O2 and sustained relatively high activity until 10 mM of H2O2 ( Figure 5). Meanwhile, the optimum concentration for DypB was about 3 mM H2O2 then showed subsequent loss of activity. Interestingly, both Dyps still exhibited some activity at 195mM of H2O2 (Table A5).  (Table A5).

Activities of DyPA and DypB with RB5 as a Substrate
Aside from ABTS, Reactive Blue 5 (RB5) is also used as a standard substrate for dyedecolorizing peroxidases. The optimal reaction conditions differentiate considerably when using RB5 as a representative substrate for Dyps. Though potassium phosphate buffer showed the best activity for DypB during the ABTS assay, it showed a more signif-   Tolerance to H2O2 was tested by checking different concentrations and monitored via ABTS assay. DypA obtained the highest activity with about 4 mM H2O2 and sustained relatively high activity until 10 mM of H2O2 ( Figure 5). Meanwhile, the optimum concentration for DypB was about 3 mM H2O2 then showed subsequent loss of activity. Interestingly, both Dyps still exhibited some activity at 195mM of H2O2 (Table A5).  (Table A5).

Activities of DyPA and DypB with RB5 as a Substrate
Aside from ABTS, Reactive Blue 5 (RB5) is also used as a standard substrate for dyedecolorizing peroxidases. The optimal reaction conditions differentiate considerably when using RB5 as a representative substrate for Dyps. Though potassium phosphate buffer showed the best activity for DypB during the ABTS assay, it showed a more significant reduction for RB5 (data not shown). Hence, for RB5, citrate buffer was used for all assays. Both Dyps showed the best activity at pH 3.6 ( Figure 6).
Thermal stability for RB5 assay was also determined. For DypA, most reactions done at room temperature for the RB5 were relatively stable ( Figure 6). In contrast to DypB, only at 30 °C that it showed a fairly stable reaction for RB5 and showed a 20% decrease in  (Table A5).

Activities of DyPA and DypB with RB5 as a Substrate
Aside from ABTS, Reactive Blue 5 (RB5) is also used as a standard substrate for dyedecolorizing peroxidases. The optimal reaction conditions differentiate considerably when using RB5 as a representative substrate for Dyps. Though potassium phosphate buffer showed the best activity for DypB during the ABTS assay, it showed a more significant reduction for RB5 (data not shown). Hence, for RB5, citrate buffer was used for all assays. Both Dyps showed the best activity at pH 3.6 ( Figure 6).

Kinetic Parameters for DypA and DypB
Using Reactive Blue 5 to differentiate properties between DypA and DypB of Rhodococcus opacus 1CP, it was shown that DypA showed better preference for the anthraquinone representative (Figures 8 and 9; Table 1). Thermal stability for RB5 assay was also determined. For DypA, most reactions done at room temperature for the RB5 were relatively stable ( Figure 6). In contrast to DypB, only at 30 • C that it showed a fairly stable reaction for RB5 and showed a 20% decrease in activity for other temperatures (Figure 7). This showed a stark contrast to the behavior of the DyPs since DypA exhibited more stability with RB5 than the ABTS assay while DypB had an opposite trend.

Kinetic Parameters for DypA and DypB
Using Reactive Blue 5 to differentiate properties between DypA and DypB of Rhodococcus opacus 1CP, it was shown that DypA showed better preference for the anthraquinone representative (Figures 8 and 9; Table 1).

Kinetic Parameters for DypA and DypB
Using Reactive Blue 5 to differentiate properties between DypA and DypB of Rhodococcus opacus 1CP, it was shown that DypA showed better preference for the anthraquinone representative (Figures 8 and 9; Table 1).

DypA and DypB Activities against Different Dyes
Seven dyes were tested to check the dye-decolorizing capability of Dyps. The dyes were comprised of two anthraquinone dyes (Reactive Blue 5 and Reactive Blue 19), diazo dye (Reactive Black 5), copper complex azo dye (Reactive Red 23), single azo dyes (Reactive Orange 16 and Reactive Yellow 37), and the polyanthraquinone dye with poly(vinylamine) sulfonate backbone (Poly R-478).

DypA and DypB Activities against Different Dyes
Seven dyes were tested to check the dye-decolorizing capability of Dyps. The dyes were comprised of two anthraquinone dyes (Reactive Blue 5 and Reactive Blue 19), diazo dye (Reactive Black 5), copper complex azo dye (Reactive Red 23), single azo dyes (Reactive Orange 16 and Reactive Yellow 37), and the polyanthraquinone dye with poly(vinylamine) sulfonate backbone (Poly R-478).

DypA and DypB Activities against Different Dyes
Seven dyes were tested to check the dye-decolorizing capability of Dyps. The dyes were comprised of two anthraquinone dyes (Reactive Blue 5 and Reactive Blue 19), diazo dye (Reactive Black 5), copper complex azo dye (Reactive Red 23), single azo dyes (Reactive Orange 16 and Reactive Yellow 37), and the polyanthraquinone dye with poly(vinylamine) sulfonate backbone (Poly R-478).
DypA showed strong preferences for the two anthraquinone representatives as it exhibited about 114 ± 2.94 U/mg and 5.91 ± 0.6 U/mg for Reactive Blue 5 and Reactive Blue 19, respectively. Reactive Black 5 and Reactive Red 23 were also moderately accepted but not as efficient compared to the two anthraquinone dyes. DypB, on the other hand, only showed activity against the two anthraquinone dyes. Interestingly, both dyes did not have any activity on single azo dye structures used in this study. Poly R-478, an anthraquinone-based polymeric dye, was somewhat acted on by DypA. However, it only showed a low activity of 1.15 ± 0.4 mU/mg at 0.4 µM concentration of Poly R-478.
Overall, DypA was a better dye converter than DypB as it acted on about 4 dyes out of the 7 that were tested. However, the substrate scope of the DypA and DypB from Rhodococcus opacus 1CP was still not up to par compared to known dye-decolorizing peroxidases.
Dye-decolorizing peroxidases 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 δ-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 1CP contain highly conserved residues that are essential for its enzymatic activity: GxxDG 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 peroxidases. The preference of DyPtype peroxidases 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 • C while DypA already exhibited decrease in activity even at room temperature. Though DypB showed subsequent decrease in activity at 40 • 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 peroxidases. 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 1CP 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) [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.
As the name suggests, dye-decolorizing peroxidases were believed to play a role in decolorizing a wide array of dyes such as azo dyes, heterocyclic dyes, and anthraquinone dyes [26,68]. These enzymes are known to attack the anthraquinone skeleton. In contrast to other DyPs, DypA from R. opacus 1CP did not show a huge substrate scope. Although it exhibited the best activity against RB5 and Reactive Blue 19, the azo dyes, Reactive Black 5 and Reactive Red 23, were only accepted up to some extent. As for DypB, it only moderately accepted RB5 and Reactive Blue 19 but not comparable to the activity of DypA. This strongly suggests that DypB of R. opacus 1CP has only a minimal role for dye decolorization.

Conclusions
This work offers insights to the enzymatic properties of the recombinant DypA and DypB from Rhodococcus opacus 1CP. Moreover, the addition of hemin to expression cultures led to enhanced heme incorporation when using E. coli as expression hosts. Therefore, it generated high amount of functional DypA and DypB. Although both enzymes have shown activities against ABTS and RB5, DypA performed better than DypB. Both enzymes also showed tolerance to high H 2 O 2 concentrations but poorly degraded dyes, suggesting that dyes are not their physiological substrate. It is also noteworthy to investigate if DypB does play a role on lignin degradation. Moreover, a study [69] showed that the dye-decolorizing peroxidase Pf DyP B2 from Pseudomonas fluorescences Pf0-1 can insert carbene into an N-H bond. Investigation of these non-natural reactions can be a nice addition to the enzymatic reactions that these dye-decolorizing peroxidases can do.     Dictyostelium discoideum AX4 EAL70759 Figure A1. Cladogram of the dye-decolorizing peroxidase superfamily including DypA and DypB from R. opacus 1CP and important representatives of the plant-type peroxidase superfamily. The tree was inferred using the maximum likelihood method and JTT matrix-based model (CITE). Bootstrap values (%) were generated from 1000 replicates. This analysis involved 52 amino acid sequences.