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.

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.

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.

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.

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.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing.

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for HO detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of HO biosensors, since they exhibit a good dynamic response range (1-200 μM HO), short response times (2 s) and a superior sensitivity (1.

Decolorization and detoxification of textile dyes using a versatile laccase-natural mediator system.

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.

An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants.

PpDyP from Pseudomonas putida MET94 is an extremely versatile B-type dye-decolourising peroxidase (DyP) capable of efficient oxidation of a wide range of anthraquinonic and azo dyes, phenolic substrates, the non-phenolic veratryl alcohol and even manganese and ferrous ions. In reaction with H2O2 it forms a stable Compound I at a rate of (1.4±0.

Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology.

The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms.

Improving kinetic or thermodynamic stability of an azoreductase by directed evolution.

Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (≈10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50°C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94.

New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

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 kinetic role of carboxylate residues in the proximity of the trinuclear centre in the O2 reactivity of CotA-laccase.

Multicopper oxidases catalyze the four-electron reduction of dioxygen to water without the release of any reactive oxygen intermediate species. The role of carboxylate residue Asp116 located at the exit channel for water molecules of CotA-laccase has been investigated by site-saturation mutagenesis. A total of 300 clones was picked and screened for activity.

The role of Asp116 in the reductive cleavage of dioxygen to water in CotA laccase: assistance during the proton-transfer mechanism.

Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole.

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes.

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions.