Search Results (12)

Ene reductases, belonging to the Old Yellow Enzyme (OYE) family, are widely used for biocatalysis. The OYE from Thermus scotoductus SA-01 (TsOYE) gained great attention due to its broad substrate scope, high stereoselectivity, thermostability, and catalytic versatility. Recently, the otherwise noncovalently bound flavin cofactor (FMN) was covalently anchored in several TsOYE mutants using the “flavin-fixing” method. However, the biochemical properties of these mutants remained unexplored. A detailed comparative study of wild-type (WT) TsOYE and the flavin-fixing variant F1 (F1 TsOYE) revealed that F1 TsOYE has a lower stability and poorer catalytic activity. Interestingly, both WT and F1 TsOYE have comparable redox potential values. These results suggest that the decrease in activity and stability is primarily caused by changes in structure and structural dynamics induced by the mutations and the covalent flavin-protein linkage. Replacing residues in the flavinylation recognition site did not result in significant repair of enzyme activity. Our findings highlight the sensitivity of TsOYE activity to covalent FMN incorporation and its associated mutations and underscore the necessity of structural insights for further rational design. This study also provides critical groundwork for optimizing the flavin-fixing strategy. Full article

Biocatalysis is fundamental to biological processes and sustainable chemical productions. Over time, the biocatalysis strategy has been widely researched. Initially, biomanufacturing and catalysis of high-value chemicals were carried out through direct immobilization and application of biocatalysts, including natural enzymes and living cells. With the evolution of green chemistry and environmental concern, hybrid photoelectro-biocatalysis (HPEB) platforms are seen as a new approach to enhance biocatalysis. This strategy greatly expands the domain of natural biocatalysis, especially for bio-based components. The selective valorization of renewable furan derivatives, such as 5-hydroxymethylfurfural (HMF) and furfural, is central to advancing biomass-based chemical production. Biocatalysis offers high chemo-, regio-, and stereo-selectivity under mild conditions compared with traditional chemical catalysis, yet it is often constrained by the costly and inefficient regeneration of redox cofactors like NAD(P)H. Photoelectrocatalysis provides a sustainable means to supply reducing equivalents using solar or electrical energy. In recent years, hybrid systems that integrate biocatalysis with photoelectrocatalysis have emerged as a promising strategy to overcome this limitation. This review focuses on recent advances in such systems, where photoelectrochemical platforms enable in situ cofactor regeneration to drive enzymatic transformations of furan-based substrates. We critically analyze representative coupling strategies, materials and device configurations, and reaction engineering approaches. Finally, we outline future directions for developing efficient, robust, and industrially viable hybrid catalytic platforms for green biomass valorization. Full article

Catalytic and bioelectrocatalytic properties of four white rot fungal laccases (Trametes hirsuta, ThL; Coriolopsis caperata, CcL; Steccherinum murashkinskyi, SmL; and Antrodiella faginea, AfL) from different orthologous groups were comparatively studied in homogeneous reactions of electron donor substrate oxidation and in a heterogeneous reaction of dioxygen electroreduction. The ThL and CcL laccases belong to high-redox-potential enzymes (E⁰_T1 = 780 mV), while the AfL and SmL laccases are medium-redox-potential enzymes (E⁰_T1 = 620 and 650 mV). We evaluated the efficiency of laccases in mediatorless bioelectrocatalytic dioxygen reduction by the steady-state potential (E_ss), onset potential (E_onset), half-wave potential (E_1/2), and the slope of the linear segment of the polarization curve. A good correlation was observed between the T1 center potential of the laccases and their electrocatalytic characteristics; however, no correlation with the homogeneous reactions of electron donor substrates’ oxidation was detected. The results obtained are discussed in the light of the known data on the three-dimensional structures of the laccases studied. Full article

Synthetic nicotinamide biomimetics (NCBs) have emerged as alternatives to the use of natural cofactors. The relatively low cost and ease of manufacture of NCBs may enable the scaling of biocatalytic reactions to produce bulk chemicals (e.g., biofuels and plastics). NCBs are also recognized by only a subset of NAD(P)/NAD(P)H-dependent enzymes, which potentially allows access to orthogonal redox cascades that can be run simultaneously within a single reactor. In the work presented here, a series of NCBs was prepared and tested for activity with alcohol dehydrogenases and ene-reductases. While the NCBs did not support enzymatic activity with the alcohol dehydrogenases, the observed rate of the ene-reductases with NCBs was greater than when incubated with the natural cofactor (consistent with previous observations). We obtained the structures of an ene-reductase and an alcohol dehydrogenase with an NCB bound in their active sites. While the NCB bound to the ene-reductases in a productive position and orientation for hydride transfer to the isoalloxazine ring of the flavin cofactor, the NCB failed to adopt a catalytically competent binding mode in the alcohol dehydrogenase. Full article

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c. Full article

Due to the complexity of heterogeneous reactions and heterogeneities of individual catalyst particles in size, morphology, and the surrounding medium, it is very important to characterize the structure of nanocatalysts and measure the reaction process of nanocatalysis at the single-particle level. Traditional ensemble measurements, however, only provide averaged results of billions of nanoparticles (NPs), which do not help reveal structure–activity relationships and may overlook a few NPs with high activity. The advent of dark-field microscopy (DFM) combined with plasmonic resonance Rayleigh scattering (PRRS) spectroscopy provides a powerful means for directly recording the localized surface plasmon resonance (LSPR) spectrum of single plasmonic nanoparticles (PNPs), which also enables quantitative measurements. In recent years, DFM has developed rapidly for a series of single-particle catalytic reactions such as redox reactions, electrocatalytic reactions, and DNAzyme catalysis, with the ability to monitor the catalytic reaction process in real time and reveal the catalytic mechanism. This review provides a comprehensive overview of the fundamental principles and practical applications of DFM in measuring various kinds of catalysis (including chemocatalysis, electrocatalysis, photocatalysis, and biocatalysis) at the single-particle level. Perspectives on the remaining challenges and future trends in this field are also proposed. Full article

Baeyer–Villiger monooxygenases (BVMOs) are flavin-dependent oxidative enzymes capable of catalyzing the insertion of an oxygen atom between a carbonylic Csp² and the Csp³ at the alpha position, therefore transforming linear and cyclic ketones into esters and lactones. These enzymes are dependent on nicotinamides (NAD(P)H) for the flavin reduction and subsequent reaction with molecular oxygen. BVMOs can be included in cascade reactions, coupled to other redox enzymes, such as alcohol dehydrogenases (ADHs) or ene-reductases (EREDs), so that the direct conversion of alcohols or α,β-unsaturated carbonylic compounds to the corresponding esters can be achieved. In the present review, the different synthetic methodologies that have been performed by employing multienzymatic strategies with BVMOs combining whole cells or isolated enzymes, through sequential or parallel methods, are described, with the aim of highlighting the advantages of performing multienzymatic systems, and show the recent advances for overcoming the drawbacks of using BVMOs in these techniques. Full article

A surface-engineered nano-support for enzyme laccase-immobilization was designed by grafting the surface of halloysite nanotubes (HNTs) with Fe₃O₄ nanoparticles and chitosan. Herein, HNTs were magnetized (HNTs-M) by a cost-effective reduction-precipitation method. The synthesized HNTs-M were grafted with 0.25%, 0.5%, 1%, and 2% chitosan (HNTs-M-chitosan), respectively. Synthesized HNTs-M-chitosan (0.25%), HNTs-M-chitosan (0.5%), HNTs-M-chitosan (1%) and HNTs-M-chitosan (2%) were linked with glutaraldehyde (GTA) for laccase immobilization. Among these formulations, HNTs-M-chitosan (1%) exhibited the highest laccase immobilization with 95.13% activity recovery and 100.12 mg/g of laccase loading. The optimized material was characterized thoroughly by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), X-ray powder diffraction (XRD), thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM) analysis. The immobilized laccase (HNTs-M-chitosan (1%)-GTA-Laccase) exhibited higher pH, temperature, and storage stabilities. The HNTs-M-chitosan (1%)-GTA-Laccase possesses excellent reusability capabilities. At the end of 10 cycles of the reusability experiment, HNTs-M-chitosan (1%)-GTA-Laccase retained 59.88% of its initial activity. The immobilized laccase was utilized for redox-mediated degradation of sulfamethoxazole (SMX), resulting in 41%, 59%, and 62% degradation of SMX in the presence of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), guaiacol (GUA), and syringaldehyde (SA), respectively. Repeated SMX degradation (57.10% after the sixth cycle) confirmed the potential of HNTs-M-chitosan (1%)-GTA-Laccase for environmental pollutant degradation. Thus, we successfully designed chitosan-based, rapidly separable super-magnetic nanotubes for efficacious enhancement of laccase biocatalysis, which can be applied as nano-supports for other enzymes. Full article

Deep eutectic solvents (DESs) have been applied as cosolvents in various biocatalytic processes during recent years. However, their use in combination with redox enzymes has been limited. In this study, we have explored the beneficial effects of several DES as cosolvents on the performance of 5-hydroxymethylfurfural oxidase (HMFO), a valuable oxidative enzyme for the preparation of furan-2,5-dicarboxylic acid (FDCA), and other compounds, such as carbonyl compounds and carboxylic acids. The use of natural DESs, based on glucose and fructose, was found to have a positive effect. Higher conversions are obtained for the synthesis of several oxidized compounds, including FDCA. Depending on the type of DES, the stability of HMFO could be significantly improved. As the use of DES increases the solubility of many substrates while they only mildly affect dioxygen solubility, this study demonstrates that biocatalysis based on HMFO and other redox biocatalysts can benefit from a carefully selected DES. Full article

Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein–material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD⁺(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis–Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)⁺/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions. Full article

The asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F₄₂₀ also has electrochemical properties that make it suitable as an alternative to nicotinamide cofactors for use in asymmetric reduction reactions. However, cofactor F₄₂₀-dependent enzymes remain under-explored as a resource for biocatalysis. This review considers the cofactor F₄₂₀-dependent enzyme families with the greatest potential for the discovery of new biocatalysts: the flavin/deazaflavin-dependent oxidoreductases (FDORs) and the luciferase-like hydride transferases (LLHTs). The characterized F₄₂₀-dependent reductions that have the potential for adaptation for biocatalysis are discussed, and the enzymes best suited for use in the reduction of oxidized cofactor F₄₂₀ to allow cofactor recycling in situ are considered. Further discussed are the recent advances in the production of cofactor F₄₂₀ and its functional analog F_O-5′-phosphate, which remains an impediment to the adoption of this family of enzymes for industrial biocatalytic processes. Finally, the prospects for the use of this cofactor and dependent enzymes as a resource for industrial biocatalysis are discussed. Full article

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer–Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications. Full article