This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

Direct electronic coupling of peroxidases with bio-compatible interfaces allows for investigation of enzyme's electro-catalytic properties that are essential in the design of bio-electronic devices. Here, a novel dye decolourising-type peroxidase from Pseudomonas putida MET94 (PpDyP) is immobilised on Ag electrodes coated with an alkanethiol self-assembled monolayer. Structural features of the active site, heterogeneous electron transfer and electro-catalytic properties of immobilised PpDyP are addressed by combination of surface enhanced spectroscopic and electrochemical approaches. They reveal that the structural integrity of the heme pocket of PpDyP is preserved upon immobilisation, the enzyme is electronically coupled to the electrode, and it exhibits efficient catalytic activity. Importantly, no significant modulation of the midpoint redox potential (E m ) of the immobilised protein (E m À300 mV) is observed with respect to that in solution (E m,sol À260 mV). This study provides important structural and mechanistic insights into immobilised DyP-type peroxidase, capable of efficient decolourisation of numerous dyes, revealing PpDyP as a promising candidate for biotechnological applications.

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ARTICLE This journal isBsDyP from Bacillus subtilis belongs to the new dye-decolourising peroxidase (DyP) family. Here we use transient kinetics to provide details on the catalytic cycle of BsDyP. The reaction of BsDyP with H 2 O 2 exhibits saturation behaviour consistent with a two-step mechanism involving the formation of an E-H 2 O 2 intermediate (K 1 = (12 ± 1) × 10 -6 M) followed by formation of Compound I (k 1 = 22 ± 1 s -1 ). We demonstrate that the k 1obs is pH-dependent and controlled by an ionisable group with a pK a of 4.3 suggesting the involvement of distal Asp. The reaction of Compound I with guaiacol obeys second order kinetics (k 3 ' = (0.21 ± 0.01) × 10 6 M -1 s -1 ) while the reaction of Compound II with guaiacol shows saturation kinetics (K 4 = 22 ± 5) × 10 -6 M and k 4 = 0.13 ± 0.01 s -1 ) and is the rate-limiting step in the BsDyP catalytic cycle. We furthermore use transient and steady-state kinetics, spectroscopic and electrochemical approaches to investigate the role of distal Asp240, Arg339 and Asn244 and proximal Asp383 residues in BsDyP. All mutations of distal residues affect particularly the K 1 (and K m ) for H 2 O 2 leading to catalytic efficiencies (k cat /K m ) of only one to two orders of magnitude lower than in the wild type. Notably, a significant improvement in the catalytic efficiency for reducing substrates is observed in variants. We conclude that the Asp and Arg residues are important for the proper binding of H 2 O 2 to the haem but none is individually indispensable for promoting H 2 O 2 (de)protonation and O-O bond cleavage. The obtained kinetic data suggest an important role of the distal Asn in modulating the acidbase catalysis of BsDyP. Our findings contribute to the establishment of structural determinants of DyPs that underlie their mechanistic properties; this has implications for their potential in biotechnological applications and sheds more light on subfamily-dependent features of these enzymes.