Search Results (1,182)

The application of immobilized enzymes often plays a key role in successfully implementing an economically feasible biocatalytic process at an industrial scale. Designing an immobilized biocatalyst involves solving several tasks, from the selection of the carrier and immobilization method to the characterization of the kinetic properties of the immobilized enzyme. In this study, we focused on the kinetic properties of free and immobilized ß-N-acetylhexosaminidase (Hex), a promising enzyme for application in the field of biotechnology, especially for the synthesis of bioactive carbohydrates. Hex was immobilized via covalent binding in methacrylate particles. The effect of immobilizing Hex from Penicillium oxalicum into porous particles on kinetic properties was investigated, and mathematical and experimental modeling showed that the kinetic behavior of the enzyme was significantly influenced by diffusion in the particles. Along with the study on kinetics, a simple method was developed to investigate the reversible inhibition of the immobilized enzyme in a continuous-flow system. The method is suitable for application in cases where a chromogenic substrate is used, and here it was applied to demonstrate the inhibitory effects of N-acetyl-glucosaminyl thiazoline (NAG-thiazoline) and O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate ((Z)-PugNAc) on Hex. Full article

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts. Full article

Using free microorganisms for industrial processes has some limitations, such as the extensive consumption of substrates for growth, significant sensitivity to the microenvironment, and the necessity of separation from the product and, therefore, the cyclic process. It is widely acknowledged that confining or immobilizing cells in a matrix or support structure enhances enzyme stability, facilitates recycling, enhances rheological resilience, lowers bioprocess costs, and serves as a fundamental prerequisite for large-scale applications. This report summarizes the various cell immobilization methods, including several synthetic (polyvinylalcohol, polyethylenimine, polyacrylates, and Eudragit) and natural (gelatin, chitosan, alginate, cellulose, agar–agar, carboxymethylcellulose, and other polysaccharides) polymeric materials in the form of thin films, hydrogels, and cryogels. Advancements in the production of well-known antibiotics like penicillin and cephalosporin by various strains were discussed. Additionally, we highlighted cutting-edge research related to strain producers of peptide-based antibiotics (polymyxin B, Subtilin, Tyrothricin, varigomycin, gramicidin S, friulimicin, and bacteriocin), glusoseamines, and polyene derivatives. Crosslinking agents, especially covalent linkers, significantly affect the activity and stability of biocatalysts (penicillin G acylase, penicillinase, deacetoxycephalosporinase, L-asparaginase, β-glucosidase, Xylanase, and urease). The molecular weight of polymers is an important parameter influencing oxygen and nutrient diffusion, the kinetics of hydrogel formation, rigidity, rheology, elastic moduli, and other mechanical properties crucial for long-term utilization. A comparison of stability and enzymatic activity between immobilized enzymes and their free native counterparts was explored. The discussion was not limited to recent advancements in the biopharmaceutical field, such as microorganism or enzyme immobilization, but also extended to methods used in sensor and biosensor applications. In this study, we present data on the advantages of cell and enzyme immobilization over microorganism (bacteria and fungi) suspension states to produce various bioproducts and metabolites—such as antibiotics, enzymes, and precursors—and determine the efficiency of immobilization processes and the optimal conditions and process parameters to maximize the yield of the target products. Full article

This research emphasizes the effect of using Eisenia foetida in vermicompost for power generation in microbial fuel cells (MFCs). By accelerating the organic decomposition, the bioenergy generation is improved. A vermicompost-microbial fuel cell employing electrogenic microorganisms was used to convert chemical energy into electrical energy. In this work, substrates of black soil, tree bark, leaves, eggshells, and ground tomatoes were used. The vermicompost MFC has a copper cathode and a stainless steel anode. In this study, the performance of MFCs was evaluated using different numbers of Eisenia foetida specimens, with three specimens (MFC_W3), five specimens (MFC_W5), and seven specimens (MFC_W7). Our key findings show that by increasing the number of Eisenia foetida specimens does not bring higher power densities; as a result, the best power density was observed in MFC_W3 and MFC_W5 at the end of the fourth week, both presenting a total of five Eisenia foetida specimens with a power density of 192 mW m⁻². Therefore, optimal results were found when 330 g of substrate and five Eisenia foetida specimens were used to achieve a maximum current density of 900 mW m⁻² and a maximum power density of 192 mW m⁻². This type of microbial fuel cell can be considered as an alternative for power generation with a significantly reduced environmental impact, considering the use of organic waste. It can be considered a game-changer in waste management and bioenergy projects. Full article

Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution. Full article

This study describes the production of solketal esters from used soybean cooking oil (USCO) via enzymatic hydroesterification. This process consists of the complete hydrolysis of USCO into free fatty acids (FFAs) catalyzed by crude lipase extract from Candida rugosa (CRL). The resulting FFAs were recovered and utilized as the raw material for an esterification reaction with solketal, which was achieved via an open reaction. For this purpose, lipase Eversa^® Transform 2.0 (ET2.0) was immobilized via physical adsorption on treated epicarp particles from Acrocomia aculeata (macauba), a lignocellulosic residue. A protein loading of 25.2 ± 1.3 mg g⁻¹ with a support and immobilization yield of 64.8 ± 2.5% was achieved using an initial protein loading of 40 mg g⁻¹ of support. The influence of certain parameters on the esterification reaction was evaluated using a central composite rotatable design (CCRD). Under optimal conditions, a FFAs conversion of 72.5 ± 0.8% was obtained after 150 min of reaction at 46 °C using a biocatalyst concentration of 20% wt. and a FFAs–solketal molar ratio of 1:1.6. The biocatalyst retained 70% of its original activity after ten esterification batches. This paper shows the conversion of two agro-industrial waste into valuable materials (enzyme immobilization support and solketal esters). Full article

This study aimed to optimize the kinetic resolution of building blocks for the synthesis of β-blockers using Candida rugosa lipases, which could be potentially used to synthesize enantiomerically pure β-blockers further. Reaction mixtures were incubated in a thermostated shaker. Qualitative and quantitative analyses of the reaction mixtures were performed using chiral stationary phases and the UPLC-IT-TOF system. Of the 24 catalytic systems prepared, a system containing lipase from Candida rugosa MY, [EMIM][BF₄] and toluene as a two-phase reaction medium and isopropenyl acetate as an acetylating agent was optimal. This resulted in a product with high enantiomeric purity produced via biotransformation, whose enantioselectivity was E = 67.5. Using lipases from Candida rugosa enables the enantioselective biotransformation of the β-blockers building block. The biocatalyst used, the reaction environment, and the acetylating agent significantly influence the efficiency of performer kinetic resolutions. The studies made it possible to select an optimum system, a prerequisite for obtaining a product of high enantiomeric purity. As a result of the performed biotransformation, the (S)-enantiomer of the β-blocker derivative was obtained, which can be used to further synthesize enantiomerically pure β-blockers. Full article

Heterocyclic scaffolds are often present in many natural and non-natural products with important biological activity, such as synthetic intermediates used to synthesise many drugs. Among others, heterocycles based on a pyrimidine ring may have antioxidant, antibacterial, antiviral, antifungal, antituberculosis, and anti-inflammatory properties. The present study investigated commercially available microbial biocatalysts in the enzymatic desymmetrization reaction of bulky–bulky ketones derived from pyrimidine bases. The influence of some parameters on the efficiency of biocatalysis, i.e., the substrate concentration and pH of the reaction medium, was evaluated. In the one-step bioreduction catalysed by Saccharomyces cerevisiae, secondary alcohols with a defined absolute configuration were obtained with high enantiomeric excess up to 99% ee and moderate conversion. Biocatalysis offers economic and environmental benefits as an alternative to conventional methods, becoming a powerful tool in the synthesis of crowded alcohols. Full article

Extracellular electron transfer (EET) is key to the success of microbial fuel cells (MFCs). Clostridium sp. often occurs in MFC anode communities, but its ability to perform EET remains controversial. We have employed Clostridium pasteurianum DSM 525 as a biocatalyst in a glycerol-fed MFC, designated MFC_DSM. We have also followed the EET of this biocatalyst in the presence of a mediator, namely soluble neutral red (NR), soluble methyl viologen (MV), neutral red film (FNR), or methyl viologen film (FMV). MFC_DSM provided power and current densities (j) of 0.39 μW·cm⁻² and 2.47 μA·cm⁻², respectively, which evidenced that the biocatalyst performs direct electron transfer (DET). Introducing 150.0 µM NR or MV into the MFC_DSM improved the current density by 7.0- and 3.7-fold (17.05 and 8.45 μA·cm⁻²), respectively. After 20 cyclic voltammetry (CV) cycles, the presence of FNR in the MFC_DSM anodic chamber provided an almost twofold higher current density (30.76 µA·cm⁻²) compared to the presence of NR in the MFC_DSM. Introducing MV or FMV into the MFC_DSM anodic chamber gave practically the same current density after 10 CV cycles. The MFC_DSM anodic electrode might interact with FMV weakly than with FNR, so FNR is more promising to enhance C. pasteurianum DSM 525 EET within MFC_DSM. Full article

Since the 2005 discovery of the first enzyme capable of depolymerizing polyethylene terephthalate (PET), an aromatic polyester once thought to be enzymatically inert, extensive research has been undertaken to identify and engineer new biocatalysts for plastic degradation. This effort was directed toward developing efficient enzymatic recycling technologies that could overcome the limitations of mechanical and chemical methods. These enzymes are versatile molecules obtained from microorganisms living in various environments, including soil, compost, surface seawater, and extreme habitats such as hot springs, hydrothermal vents, deep-sea regions, and Antarctic seawater. Among various plastics, PET and polylactic acid (PLA) have been the primary focus of enzymatic depolymerization research, greatly enhancing our knowledge of enzymes that degrade these specific polymers. They often display unique catalytic properties that reflect their particular ecological niches. This review explores recent advancements in marine-derived enzymes that can depolymerize synthetic plastic polymers, emphasizing their structural and functional features that influence the efficiency of these catalysts in biorecycling processes. Current status and future perspectives of enzymatic plastic depolymerization are also discussed, with a focus on the underexplored marine enzymatic resources. Full article

The increasing disposal of emerging contaminants in the environment is a worldwide concern due to environmental impacts, such as toxicity, hormonal disorders, and bioaccumulation. The persistence of these pollutants in water bodies makes conventional pollutant removal techniques inefficient or partial, thus requiring the development of new, more effective, sustainable remediation technologies. Therefore, chitosan-based materials have emerged as a promising alternative for application in catalysis and contaminant removal. The biopolymer has functional properties that make it an excellent adsorbent capable of removing more specific pollutants, such as pharmaceuticals, microplastics, agricultural pesticides, and perfluoroalkyl and poly-fluoroalkyl substances, which are increasingly in evidence today. Therefore, this review of recent and advanced research into using chitosan to manufacture catalytic and adsorption materials offers an innovative approach to treating contaminants in aqueous environments, significantly reducing their presence and impact. It discusses the advantages of using chitosan as an adsorbent and catalyst and its role as a support for catalysts and biocatalysts. In addition, the review highlights the diversity of the physical forms of chitosan, such as particles, membranes, and hydrogels, and its possible chemical modifications, highlighting its effectiveness in catalytic applications and the removal of a wide range of emerging contaminants. Full article

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 °C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. Full article

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni⁺² agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations. Full article

Methylotrophic yeast Ogataea polymorpha BKM Y-2559 was immobilized in organosilicon sol–gel matrices using precursors isobutyltriethoxysilane (iBTES) and tetraethoxysilane (TEOS) to create an effective biocatalyst. The analytical and metrological performance of the biosensor permitted the determination of the optimum ratio of iBTES and TEOS, which was found to be 20/80 vol.%. The results of the scanning electron microscopy method demonstrated the formation of organosilicon material around microorganisms, as well as the ease with which metabolic products of yeast cells and substrates could diffuse through the obtained pores. A laboratory model of the biofilter was developed, exhibiting an oxidative capacity that varied from 0.14 to 1.25 gO₂/(m³ × cycle) in accordance with the initial level of water pollution and the degree of purification of moderately polluted water. The latter was found to be 20%, which aligns with the norm for drip biofilters operating in cyclic mode. Full article

This study explores the reasons behind the variations in the enantioselectivity of the sulfoxidation of methyl phenyl sulfide by marine-derived vanadium-dependent haloperoxidases (VHPOs). Twelve new VHPOs of marine organisms were overexpressed, purified, and tested for their ability to oxidize sulfide. Most of these marine enzymes exhibited nonenantioselective behavior, underscoring the uniqueness of AnVBPO from the brown seaweed Ascophyllum nodosum and CpVBPO from the red seaweed Corallina pilulifera, which produce (R)- and (S)-sulfoxides, respectively. The enantioselective sulfoxidation pathway is likely due to direct oxygen transfer within the VHPO active site. This was demonstrated through molecular docking and molecular dynamics simulations, which revealed differences in the positioning of sulfide within AnVBPO and CpVBPO, thus explaining their distinct enantioselectivities. Nonenantioselective VHPOs probably follow a different oxidation pathway, initiating with sulfide oxidation to form a positively charged radical. Further insights were gained from studying the catalytic effect of VO₄³⁻ on H₂O₂-driven sulfoxidation. This research improves the understanding of VHPO-mediated sulfoxidation and aids in developing biocatalysts for sulfoxide synthesis. Full article