Enzyme-Modified Microelectrode for Simultaneous Local Measurements of O and pH.

The use of miniaturized probes opens a new dimension in the analysis of (bio)chemical processes, enabling the possibility to perform measurements with local resolution. In addition, multiparametric measurements are highly valuable for a holistic understanding of the investigated process. Therefore, different strategies have been suggested for simultaneous local measurements of various parameters.

Unlocking Lignin's Potential: Engineered Bacterial Laccases to Produce Biologically Active Molecules.

Laccases are biocatalysts with immense potential in lignocellulose biorefineries to valorize emerging lignin monomers for sustainable chemicals. Despite reduced costs over the past two decades, enzymes remain a major expense in biorefining. Protein engineering can enhance enzyme properties and lower costs further.

Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis.

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile .

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.

Spectroelectrochemistry for determination of the redox potential in heme enzymes: Dye-decolorizing peroxidases.

Dye-decolorizing peroxidases (DyPs) are heme-containing enzymes that are structurally unrelated to other peroxidases. Some DyPs show high potential for applications in biotechnology, which critically depends on the stability and redox potential (E°') of the enzyme. Here we provide a comparative analysis of UV-Vis- and surface-enhanced resonance Raman-based spectroelectrochemical methods for determination of the E°' of DyPs from two different organisms, and their variants generated targeting E°' upshift.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates.

Evolution and separation of actinobacterial pyranose and -glycoside-3-oxidases.

FAD-dependent pyranose oxidase (POx) and -glycoside-3-oxidase (CGOx) are both members of the glucose-methanol-choline superfamily of oxidoreductases and belong to the same sequence space. Pyranose oxidases had been studied for their oxidation of monosaccharides such as D-glucose, but recently, a bacterial -glycoside-3-oxidase that is phylogenetically related to POx and that reacts with -glycosides such as carminic acid, mangiferin or puerarin has been described. Since these actinobacterial CGOx enzymes belong to the same sequence space as bacterial POx, they must have evolved from the same ancestor.

The quest for new robust bacterial monoamine oxidases.

Microbial enzymes are versatile, cost-effective, and sustainable tools, making them a preferred choice for enzymatic processes. Santema et al. harnessed AlphaFold, a cutting-edge structure prediction tool, to discover new thermophilic monoamine oxidases (MAO) that could be relevant for drug development and use in biotechnology fields.

Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O to HO. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step.

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance.

Dye decolorizing peroxidases (DyP) have attracted interest for applications such as dye-containing wastewater remediation and biomass processing. So far, efforts to improve operational pH ranges, activities, and stabilities have focused on site-directed mutagenesis and directed evolution strategies. Here, we show that the performance of the DyP from can be drastically boosted without the need for complex molecular biology procedures by simply activating the enzyme electrochemically in the absence of externally added HO.

Biocatalysis for biorefineries: The case of dye-decolorizing peroxidases.

Dye-decolorizing Peroxidases (DyPs) are heme-containing enzymes in fungi and bacteria that catalyze the reduction of hydrogen peroxide to water with concomitant oxidation of various substrates, including anthraquinone dyes, lignin-related phenolic and non-phenolic compounds, and metal ions. Investigation of DyPs has shed new light on peroxidases, one of the most extensively studied families of oxidoreductases; still, details of their microbial physiological role and catalytic mechanisms remain to be fully disclosed. They display a distinctive ferredoxin-like fold encompassing anti-parallel β-sheets and α-helices, and long conserved loops surround the heme pocket with a role in catalysis and stability.

Corrigendum to "Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase" Comput Struct Biotechnol J, 20 (2022), 3899-3910.

[This corrects the article DOI: 10.1016/j.csbj.

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase ( 273 s for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate.

A wide array of lignin-related phenolics are oxidized by an evolved bacterial dye-decolourising peroxidase.

Lignin is the second most abundant natural polymer next to cellulose and by far the largest renewable source of aromatic compounds on the planet. Dye-decolourising peroxidases (DyPs) are biocatalysts with immense potential in lignocellulose biorefineries to valorize emerging lignin building blocks for environmentally friendly chemicals and materials. This work investigates the catalytic potential of the engineered PpDyP variant 6E10 for the oxidation of 24 syringyl, guaiacyl and hydroxybenzene lignin-phenolic derivatives.

The gastrointestinal microbiome of browsing goats (Capra hircus).

Despite the growing interest in the ruminants' gastrointestinal tract (GIT) microbiomes' ability to degrade plant materials by animal husbandry and industrial sectors, only a few studies addressed browsing ruminants. The present work describes the taxonomic and functional profile of the bacterial and archaeal communities from five different gastrointestinal sections (rumen, omasum-abomasum, jejunum, cecum and colon) of browsing Capra hircus, by metabarcoding using 16S rRNA genes hypervariable regions. The bacterial communities across the GITs are mainly composed of Bacillota and Bacteroidota.

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase.

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn and Fe, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of DyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained.

Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin.

Biocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type.

Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases.

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of HO or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from (BsDyP; Class I) and (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of DyP from .

 DyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of DyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme.

Wasteful Azo Dyes as a Source of Biologically Active Building Blocks.

In this work, an environment-friendly enzymatic strategy was developed for the valorisation of dye-containing wastewaters. We set up biocatalytic processes for the conversion of azo dyes representative of the main classes used in the textile industry into valuable aromatic compounds: aromatic amines, phenoxazinones, phenazines, and naphthoquinones. First, purified preparations of PpAzoR azoreductase efficiently reduced mordant, acid, reactive, and direct azo dyes into aromatic amines, and CotA-laccase oxidised these compounds into phenazines, phenoxazinones, and naphthoquinones.

Laccases: Versatile Biocatalysts for the Synthesis of Heterocyclic Cores.

Laccases are multicopper oxidases that have shown a great potential in various biotechnological and green chemistry processes mainly due to their high relative non-specific oxidation of phenols, arylamines and some inorganic metals, and their high redox potentials that can span from 500 to 800 mV vs. SHE. Other advantages of laccases include the use of readily available oxygen as a second substrate, the formation of water as a side-product and no requirement for cofactors.

Developing a Sustainable and Circular Bio-Based Economy in EU: By Partnering Across Sectors, Upscaling and Using New Knowledge Faster, and For the Benefit of Climate, Environment & Biodiversity, and People & Business.

This paper gives an overview of development of the EU-bioeconomy, 2014-2020. The Vision of the new Circular Bio-based Economy, CBE is presented: Unlocking the full potential of all types of sustainably sourced biomass, crop residues, industrial side-streams, and wastes by transforming it into value-added products. The resulting product portfolio consists of a wide spectrum of value-added products, addressing societal and consumer needs.

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases.

Dye decolorizing peroxidases (DyPs) are novel haem-containing peroxidases, which are structurally unrelated to classical peroxidases. They lack the highly conserved distal histidine that acts as an acid-base catalyst in the catalytic reaction of classical peroxidases, which implies distinct mechanistic properties. Despite the remarkable catalytic properties and recognized potential for biotechnology applications, the knowledge of DyP's structural features in solution, which govern the reactivity and catalysis, is lagging behind.