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Currently, there is increasing interest in assessing the potential of bacterial laccases for industrial and environmental applications especially in harsh conditions. The environmental impact of the textile industry requires novel and effective technologies to mitigate the presence of dyes in wastewaters before discharging into the environment. Dyes usually remain stable in the presence of a variety of chemicals, light and are recalcitrant to microbial degradation. Among available technologies the biological treatments offer environmentally friendly strategies for decolorizing and detoxifying these compounds. The recent discovery of versatile laccases in streptomycetes opens up new opportunities for their commercial application. The aim of this study is to assess the potential of a novel bacterial laccase SilA produced by Streptomyces ipomoeae CECT 3341 active over wide temperature and pH ranges for use as an eco-friendly, biological treatment for the degradation of textile dyes. Insights into the enhancement of the oxidative action of this enzyme through the use of natural redox mediators are presented together with an assessment of the potential toxicity of the degradation products. Our results confirm that the combination of the laccase and natural mediators such as acetosyringone and methyl syringate enhanced the decolorization and detoxification of a variety of textile dyes up to sixfold and 20-fold, respectively. Mediator concentration was found to have a significant effect (p 55 °C) and salinity and neutral pH, conditions which are unfavourable for conventional fungal laccases. Keywords: Laccase, Dyes decolorization, Streptomyces, Natural mediators, Detoxification

Chitosan-genipin films present a bluish-green color due to the conjugated double bonds formed when monomeric or dimeric genipin residues bridge chitosan. This phenomenon limits their use when colorless materials are required. In this work, a two-step oxidation strategy was developed aiming to remove color from chitosan-genipin films while preserving their functional properties. A combined system using the recombinant CotA laccase from Bacillus subtilis mediated by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) followed by a H 2 O 2 oxidation step was settled. ABTS boosted the laccase performance resulting in light brown chitosan-genipin films that were further decolorized upon immersion in a 5% H 2 O 2 solution at pH 11.0 and 40 °C for 30 min. The applied methodology leads to films that sustain both acidic stability and antioxidant capacity of the pristine films. Overall, the combined CotA laccase-ABTS/hydrogen peroxide developed methodology efficiently produce active and decolorized chitosan-genipin films with potential for application as eco-friendly transparent material which may have application as food packaging.

BsDyP 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 H2O2 exhibits saturation behaviour consistent with a two-step mechanism involving the formation of an E–H2O2 intermediate (K1 = (12 ± 1) × 10−6 M) followed by the formation of Compound I (k1 = 22 ± 1 s−1). We demonstrate that k1obs is pH-dependent and controlled by an ionisable group with a pKa of 4.3, suggesting the involvement of distal Asp. The reaction of Compound I with guaiacol obeys second-order kinetics (k3′ = (0.21 ± 0.01) × 106 M−1 s−1) while the reaction of Compound II with guaiacol shows saturation kinetics (K4 = (22 ± 5) × 10−6 M and k4 = 0.13 ± 0.01 s−1) and is the rate-limiting step in the BsDyP catalytic cycle. Furthermore, we use transient and steady-state kinetics as well as 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 K1 (and Km) for H2O2 leading to catalytic efficiencies (kcat/Km) of only one to two orders of magnitude lower than those in the wild-type. Notably, a significant improvement in the catalytic efficiency for reducing substrates is observed in the variants. We conclude that the Asp and Arg residues are important for the proper binding of H2O2 to the haem but none is individually indispensable for promoting H2O2 (de)protonation and O–O bond cleavage. The obtained kinetic data suggest an important role of distal Asn in modulating the acid–base 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 the subfamily-dependent features of these enzymes.

The increasing interest in clinical bacterial photodynamic inactivation has led to the search for photosensitizers with higher bactericidal efficiency and less side effects on the surrounding tissues. We present a novel nonionic porphyrin, the 5,10,15-tris(2,6-dichlorophenyl)-20-[4-N-(6-amino-hexyl) sulfonamido)phenyl]-porphyrin (ACS769F4) with substantial improvements in the efficiency of nonionic sensitizers. This porphyrin causes eradication of both Escherichia coli and Staphylococcus aureus by the photodynamic effect but in higher concentrations compared with 5,10,15,20-tetrakis (4-N,N,N- trimethylammoniumphenyl)-porphyrin p-tosylate (TTAP 4+), a known bactericidal tetracationic porphyrin. More important, under such conditions, ACS769F4 proved to be harmless to two mammalian cells lines (human embryonic and baby hamster kidney), causing no reduction in their viability or negative impact on their cytoskeleton, despite its accumulation in cellular structures. On the contrary, TTAP 4+ is shown to accumulate in the nucleus of mammalian cells, in association to DNA, causing chromatin condensation after exposure to light. Furthermore, dark incubation with TTAP 4+ was shown to have a deleterious effect on the microtubule network. Based on its bactericidal efficiency, also observed without exposure to light, and on the low tendency to be harmful or genotoxic to mammalian cells, ACS769F4 should be looked at as an interesting photosensitizer to be evaluated for clinical purposes. A new nonionic porphyrin was synthesized and its properties as antibacterial agent by photodynamic effect were tested against Escherichia coli and Staphylococcus aureus. More relevant, this porphyrin proved to be harmless to two lines of mammalian cells (HEK and BHK cells), causing no reduction in their viability or negative impact on their cytoskeleton, despite its accumulation in cellular structures. On the contrary, a cationic porphyrin with a high efficiency as an antibacterial agent, under the same conditions, was shown to accumulate in the nucleus of mammalian cells, in association to DNA, causing chromatin condensation after exposure to light.

In this study we provide the first biochemical characterization of a bacterial pyranose 2-oxidase (AsP2Ox) from Arthrobacter siccitolerans. The enzyme catalyzes the oxidation of several aldopyranoses at the C-2 position, coupling it to the reduction of dioxygen to hydrogen peroxide. Pyranose 2-oxidases belong to the glucose-methanol-choline oxidoreductase family. A structural model based on the known X-ray structure of P2Ox from Phanerochaete chrysosporium supports that AsP2Ox shares structural features with well-characterized fungal P2Oxs. The gene coding for AsP2Ox was cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme is a 64-kDa monomer containing a non-covalently bound flavin adenine dinucleotide (FAD) cofactor, distinct features as compared with fungal counterparts that are ∼ 270 kDa homotetramers with covalent-linked FAD. AsP2Ox exhibits a redox potential of −50 mV, an optimum temperature of 37 °C and an optimum pH at 6.5. AsP2Ox oxidizes d -glucose at the highest efficiency, using additionally d -galactose, d -xylose, l -arabinose and d -ribose as electron donors, coupling their oxidation to the reduction of both dioxygen and 1,4-benzoquinone. AsP2Ox shows a relatively low thermal stability with a melting temperature (Tm) of 43 °C and a half-life (t1/2) at 40 °C of 25 min. This work expands the repertoire of bacterial oxidoreductases with importance in biotechnological and diagnostic applications.

This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 μM at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles.

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 (Em) of the immobilised protein (Em −300 mV) is observed with respect to that in solution (Em,sol −260 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.