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The biocatalytic oxidative deamination‐ reductive amination process for the production of L‐phosphinothricin (L‐PPT) from D,L‐phosphinothricin (D,L‐PPT) is a green and environmentally friendly approach with significant development potential. In this study, the adopted technological route involves the oxidative deamination of the D‐PPT component in D,L‐PPT to 2‐oxo‐4‐(hydroxymethylphosphinyl)butanoic acid (PPO) by utilizing D‐amino acid oxidase (DAAO), followed by the catalytic reductive amination of PPO to L‐PPT using phosphinothricin dehydrogenase (PpDH). In order to enhance the catalytic rate and reduce the inhibitory effect of the intermediate product PPO on the catalytic process, optimization of both oxidative deamination and reductive amination phases was conducted through separate strategies of constant‐pressure oxygen supply and substrate feeding, respectively. A "two‐step one‐pot" method was employed to link the optimized reactions, significantly shortening the production time. Furthermore, the "two‐step one‐pot" catalytic strategy was optimized, ultimately achieving a high 97.7% yield of L‐PPT with ee of L‐PPT>99.9% after 5.5 h of reaction under 800 mM D,L‐PPT catalytic conditions, D‐PPT and PPO could not bedetected in the reaction system, further enchanced the catalytic efficiency, production efficiency and product quality. The aforementioned results demonstrate that this proposed approach has great potential for industrial‐scale production of L‐PPT as well as significant competitive advantages in terms of economic feasibility. [ABSTRACT FROM AUTHOR]

The synthetic pathway for Etiracetam depends on alpha‐ethyl‐2‐oxo‐1‐pyrrolidineacetic acid (AEOPA) as a crucial intermediate. This paper presents an enzymatic synthesis approach for producing (R)‐AEOPA. Through database mining, we discovered a tauriopine dehydrogenase capable of catalyzing the reaction between γ‐aminobutyric acid and 2‐oxobutyric acid, resulting in the synthesis of (R)‐4‐(carboxypropylamino) butyric acid with a specific activity of 6.74 U/mg. By enhancing substrate affinity and catalytic efficiency of CgTaDH through protein engineering, we achieved a 5.9‐fold increase in enzyme activity compared to the wild type. Further optimization led to a space‐time yield (STY) of 3.95 g/L/h for (R)‐4‐(carboxypropyl amino) butyric acid and a high yield of 73.0 % for the final product, (R)‐AEOPA. This study demonstrates a novel synthesis method for (R)‐AEOPA and highlights the potential of biocatalysis in improving the production of Etiracetam through successful enzymatic processes. [ABSTRACT FROM AUTHOR]

N-Acetylaspartylglutamate (NAAG) is the most common dipeptide in brain cells, which is synthesized using the enzyme N-acetylaspartylglutamate synthase. In this study, we utilize bioinformatics methods to predict the protein structure based on the primary sequence of the coding gene, classical molecular dynamics to obtain a stable protein complex with N-acetylaspartate and glutamate ligands within the trajectory, and machine learning methods to analyze, describe, and select potential reactive and nonreactive conformations of the model system describing the enzyme–substrate complex. Molecular dynamics trajectories are obtained for a set of selected conformations within the framework of the method of combined quantum and classical molecular mechanics, and the active site of the protein–ligand complex and potential reaction mechanism are characterized. [ABSTRACT FROM AUTHOR]

Chronic diabetic wounds are the most common complication for diabetic patients. Due to high oxidative stress levels affecting the entire healing process, treating diabetic wounds remains a challenge. Here, we present a strategy for continuously regulating oxidative stress microenvironment by the catalyst-like magnesium-gallate metal-organic framework (Mg-GA MOF) and developing sprayable hydrogel dressing with sodium alginate/chitosan quaternary ammonium salts to treat diabetic wounds. Chitosan quaternary ammonium salts with antibacterial properties can prevent bacterial infection. The continuous release of gallic acid (GA) effectively eliminates reactive oxygen species (ROS), reduces oxidative stress, and accelerates the polarization of M1-type macrophages to M2-type, shortening the transition between inflammation and proliferative phase and maintaining redox balance. Besides, magnesium ions adjuvant therapy promotes vascular regeneration and neuronal formation by activating the expression of vascular-associated genes. Sprayable hydrogel dressings with antibacterial, antioxidant, and inflammatory regulation rapidly repair diabetic wounds by promoting neurovascular network reconstruction and accelerating re-epithelialization and collagen deposition. This study confirms the feasibility of catalyst-like MOF-contained sprayable hydrogel to regulate the microenvironment continuously and provides guidance for developing the next generation of non-drug diabetes dressings.

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A “one-pot” system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

This study presents a novel approach to producing activated carbon from agro-industrial residues, specifically cocoa fruit peel, using solid-state fermentation (SSF) with Aspergillus niger. The process effectively degrades lignin, a major impediment in traditional activated carbon production, resulting in a high-quality carbon material. This carbon was successfully utilized for enzyme immobilization and aroma synthesis, showcasing its potential as a versatile biocatalyst. The study meticulously evaluated the physical and chemical attributes of activated carbon derived from fermented cocoa peel, alongside the immobilized enzymes. Employing a suite of analytical techniques—electrophoresis, FTIR, XRD, and TG/DTG the research revealed that fermentation yields a porous material with an expansive surface area of 1107.87 m2/g. This material proves to be an excellent medium for lipase immobilization. The biocatalyst fashioned from the fermented biomass exhibited a notable increase in protein content (13% w/w), hydrolytic activity (15% w/w), and specific activity (29% w/w), underscoring the efficacy of the fermentation process. The significant outcome of this research is the development of a sustainable method for activated carbon production that not only overcomes the limitations posed by lignin but also enhances enzyme immobilization for industrial applications. The study's findings have important implications for the agro-industrial sector, promoting a circular economy and advancing sustainable biotechnological processes. [ABSTRACT FROM AUTHOR]

Allergies towards gluten and legumes (such as, soybean, peanut, and faba bean) are a global issue and, occasionally, can be fatal. At the same time, an increasing number of households are shifting to plant protein ingredients from these sources, which application and consumption are limited by said food allergies. Children, the elderly, and people with immune diseases are particularly at risk when consuming these plant proteins. Finding ways to reduce or eliminate the allergenicity of gluten, soybean, peanut, and faba bean is becoming crucial. While thermal and pH treatments are often not sufficient, chemical processes such as glycation, polyphenol conjugation, and polysaccharide complexation, as well as controlled biochemical approaches, such as fermentation and enzyme catalysis, are more successful. Non-thermal treatments such as microwave, high pressure, and ultrasonication can be used prior to further chemical and/or biochemical processing. This paper presents an up-to-date review of promising chemical, biochemical, and non-thermal physical treatments that can be used in the food industry to reduce or eliminate food allergenicity. [ABSTRACT FROM AUTHOR]

[Display omitted] The key feature of living cells is multicompartmentalization for enzymatic reactions. Artificial cell-like multicompartments with micro domains are appealing to mimic the biological counterparts. In addition, establishing a sustainable, efficient, and controllable reaction system for enzymatic hydrolysis is imperative for the production of natural fatty acids from animal and plant-based fats. Reverse Janus emulsion microreactors, i.e. (W 1 + W 2)/O, is constructed through directly using natural fats as continuous phase and aqueous two-phase solutions (ATPS) as inner phases. Enzyme is confined in the compartmented aqueous droplets dominated by the salt of Na 2 SO 4 and polyethylene glycol (PEG). Enzyme catalyzed ester hydrolysis employed as a model reaction is performed under the conditions of agitation-free and mild temperature. Regulation of reaction kinetics is investigated by diverse droplet topology, composition of inner ATPS, and on-demand emulsification. Excellent enzymatic activity toward hydrolysis of plant and animal oils achieves 88.5 % conversion after 3 h. Compartmented micro domains contribute to condense and organize the enzymes spatially. Timely removal of the products away from reaction sites of oil/water interface "pushed" the reaction forward. Distribution and transfer of enzyme in two aqueous lobes provide extra freedom in the regulation of hydrolysis kinetics, with equilibrium conversion controlled freely from 14.5 % to 88.5 %. Reversible "open" and "shut" of hydrolysis is acheived by on-demand emulsification and spontaneous demulsification. This paper paves the way to advancing progress in compartmentalized emulsion as a sustainable and high-efficiency platform for biocatalytic applications. [ABSTRACT FROM AUTHOR]

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A "one-pot" system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated S N 2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1 H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology. CtMaT1, a plant malonyltransferase with both unprecedented substrate promiscuity and malonylation regiospecificity, was functionally and structurally identified, and employed in universal enzymatic synthesis of diverse malonylated glycosides with potential druggabilities. [Display omitted] [ABSTRACT FROM AUTHOR]

The combined quantum mechanics/molecular mechanics method is most often used to describe the molecular mechanisms of enzymatic reactions. The review discusses the main methodological issues, gives practical recommendations, and also illustrates the progress of the method over the past 20 years using an important example of the reaction of guanosine triphosphate hydrolysis by a protein complex. [ABSTRACT FROM AUTHOR]

We report an enzyme‐catalyzed dynamic kinetic resolution protocol for the one‐pot asymmetric synthesis of substituted purine derivatives. The controllable N‐9/N‐7 substitution, which posed formidable challenges in organic synthesis, could be accomplished by choosing different types of enzymes as catalysts. This method was proved scalable and was used to prepare a series of prodrugs of medicinally active acids. In addition, the recyclability test of the biocatalysts indicated the potential of this strategy for applications in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Galactomannans (GM) are hemicellulosic polysaccharides composed of D-mannopyranose chains linked by β (1 → 4) glycosidic linkages with branches of D-galactopyranose linked by α (1 → 6) linkages. This polysaccharide is recognized for its hydrophilic character, as it is rich in hydroxyl groups (-OH). This chemical characteristic, combined with the absence of ionic charges, enables structural modifications such as transesterification of the fatty acid chains (FA), which provides a strategy for obtaining amphiphilic structures. The enzyme-catalyzed syntheses were carried out in DMSO with GM decanoate (GMD) and GM palmitate (GMP) at different molar ratios (0.5 and 1.0) and the resulting structures were evaluated with infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (CP/MAS 13C NMR) and differential scanning calorimetry (DSC). The FTIR spectrum confirmed the transesterification of GM with the appearance of a CO band (1730-1750 cm−1). These results were confirmed by the signals observed at 177 and 30 ppm in the CP/MAS 13C NMR spectrum, which corresponded to the CO groups of the esters and the terminal –CH3 groups of the FA chains, respectively. Finally, DSC showed glass transition temperatures (Tg) in the range 43–51 °C, while the melting temperatures (Tm) of the GM esters (59 °C) were not affected by different degrees of esterification (DE) for GMD (0.37 and 0.71) and GMP (0.47 and 0.57).

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. Key points: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. [ABSTRACT FROM AUTHOR]

In this study, lactic acid bacteria (LAB) fermentation and β-galactosidase catalysis methods were combined to increase the lactulose concentration and reduce the galactose and lactose content in a hot-alkaline-based system. The optimal conditions for chemical isomerization were 70 °C for 50 min for lactulose production, in which the concentration of lactulose was 31.3 ± 1.2%. Then, the selection and identification of LAB, which can utilize lactose and cannot affect lactulose content, were determined from 451 strains in the laboratory. It was found that Lactobacillus salivarius TM-2–8 had weak lactulose utilization and more robust lactose utilization. Lactobacillus rhamnosus grx.21 was weak in terms of lactulose utilization and strong in terms of galactose utilization. These two strains fermented the chemical isomerization system of lactulose to reduce the content of lactose and galactose. The results showed that the lactose concentration was 48.96 ± 2.92 g/L and the lactulose concentration was 59.73 ± 1. 8 g/L for fermentation lasting 18 h. The β-galactosidase was used to increase the content of lactulose in the fermented system at this time. The highest concentration of 74.89 ± 1.68 g/L lactulose was obtained at an enzymatic concentration of 3 U/mL and catalyzed at 50 °C for 3 h by β-galactosidase. [ABSTRACT FROM AUTHOR]

Dynamic conformational fluctuations of single catalase molecules on the sub-millisecond timescale is probed by label-free single-entity electrochemical measurements. This work introduces a new complementary approach to existing single-molecule enzymology, giving further insight into the enzymatic reaction mechanism. [Display omitted] The conformational motions of enzymes are crucial for their catalytic activities, but these fluctuations are usually spontaneous and unsynchronized and thus difficult to obtain from ensemble-averaged measurements. Here, we employ label-free single-entity electrochemical measurements to monitor in real time the fluctuating enzymatic behavior of single catalase molecules toward the degradation of hydrogen peroxide. By probing the electrochemical signals of single catalase molecules at a carbon nanoelectrode, we were able to observe three distinct current traces that could be attributed to conformational changes on the sub-millisecond timescale. Whereas, nearly uniform single long peaks were observed for single catalase molecules under a moderate magnetic field due to the restricted conformational changes of catalase. By combining high-resolution current signals with a multiphysics simulation model, we studied the catalytic kinetics of catalase with and without a magnetic field, and further estimated the maximum catalytic rate and conformational transition rate. This work introduces a new complementary approach to existing single-molecule enzymology, giving further insight into the enzymatic reaction mechanism. [ABSTRACT FROM AUTHOR]

Utilizing electric fields to catalyze chemical reactions is not a new idea, but in enzymology it undergoes a renaissance, inspired by Warhsel's concept of electrostatic preorganization. According to this concept, the source of the immense catalytic efficiency of enzymes is the intramolecular electric field that permanently favors the reaction transition state over the reactants. Within enzyme design, computational efforts have fallen short in designing enzymes with natural-like efficacy. The outcome could improve if long-range electrostatics (often omitted in current protocols) would be optimized. Here, we highlight the major developments in methods for analyzing and designing electric fields generated by the protein scaffolds, in order to both better understand how natural enzymes function, and aid artificial enzyme design.

Bisphenol A (BPA) is one of the most widely used chemical products, which is discharged into rivers and oceans, posing great hazards to organisms such as reproductive toxicity, hormone imbalance and cardiopathy induction. With the expansion harm of BPA, people have paid more attention to the environmental effects. In this paper, the degradation of BPA from the synthetic wastewater using the immobilization of horseradish peroxidase membrane reactor (HPR) was investigated. The immobilized HRP microporous membrane was prepared by the porous calcium alginate method. In addition, the reuse of the immobilized HPR membrane and the measurement of membrane flux showed that the membrane has good activity and stability. Finally, the experimental parameters including reaction time, pH, the concentration of BPA and the dosage of H2O2 were optimized to remove the BPA, and about 78% degradation efficiency of BPA was achieved at the optimal condition as follows: H2O2 to BPA molar ratio of 1.50 with an initial BPA concentration of 0.1 mol/L, the HPR dosage of 3.84 u/mL, the initial solution pH of 7.0, a temperature of 20 °C and a contact time of 10 min. HIGHLIGHTS Horseradish peroxidase (HRP) can be effectively immobilized on the membrane.; The immobilized HRP can maintain high activity and be circulation utilization.; The optimum conditions for the mole rate of H2O2/BPA dosage and the pH value are 1.5 and 7, respectively.;

Allergies towards gluten and legumes (such as, soybean, peanut, and faba bean) are a global issue and, occasionally, can be fatal. At the same time, an increasing number of households are shifting to plant protein ingredients from these sources, which application and consumption are limited by said food allergies. Children, the elderly, and people with immune diseases are particularly at risk when consuming these plant proteins. Finding ways to reduce or eliminate the allergenicity of gluten, soybean, peanut, and faba bean is becoming crucial. While thermal and pH treatments are often not sufficient, chemical processes such as glycation, polyphenol conjugation, and polysaccharide complexation, as well as controlled biochemical approaches, such as fermentation and enzyme catalysis, are more successful. Non-thermal treatments such as microwave, high pressure, and ultrasonication can be used prior to further chemical and/or biochemical processing. This paper presents an up-to-date review of promising chemical, biochemical, and non-thermal physical treatments that can be used in the food industry to reduce or eliminate food allergenicity.

Sugar esters are non-ionic biosurfactants. In comparison to traditional surfactants, sugar ester biosurfactants have superior emulsifying, antibacterial, and biodegradable properties, and are widely utilized in foods, cosmetics, medicine, and other sectors. Compared to sucrose esters, the most investigated sugar esters, lactose esters (LEs) exhibit superior physicochemical and biological properties and have more promising applications. This article focuses on recent advances in the manufacture of LEs using chemical and biological methods, as well as their application as emulsifiers and antibacterial materials. The application potential of LEs in the fields of bacterial photodynamic inactivation and photodynamic therapy is summarized. In addition, some problems with the preparation and application of LEs as well as their future prospects are discussed in order to provide a reference for intensive research and development of LEs.

Tetrahydroprotoberberines (THPBs) are plant-specific alkaloids with significant medicinal value. They are present in trace amounts in plants and are difficult to chemically synthesize due to stereoselectivity and an unfavorable environment. In this study, a selective methylation strategy was developed for the biocatalysis of seven high-value-added THPB compounds using 4'-O-methyltransferase (Cj4'OMT), norcoclaurine 6-O-methyltransferase (Cj6OMT), and (S)-scoulerine 9-O-methyltransferase (SiSOMT and PsSOMT) in engineered E. coli. The methyltransferases Cj4'OMT, Cj6OMT, PsSOMT, and SiSOMT were expressed heterologously in E. coli. Compound 1 (10-methoxy-2,3,9-tetrahydroxyberbine) was synthesized using the recombinant E. coli strain Cj4'OMT and the substrate 2,3,9,10-tetrahydroxyberbine. Compound 2 (9-methoxy-2,3,10-tetrahydroxyberbine) was produced in the recombinant Escherichia coli (E. coli) strain PsSOMT, and compounds 2 and 3 (discretamine) were produced in the recombinant E. coli strain SiSOMT. Compounds 4 (9,10-methoxy-2,3-tetrahydroxyberbine) and 5 (corypalmine) were obtained by co-culturing the recombinant strains Cj4'OMT and SiSOMT with substrate. Compounds 6 (scoulerine) and 7 (isoscoulerine) were produced by co-culturing the substrate with the recombinant strains Cj4'OMT and Cj6OMT. To increase the yield of novel compound 2, the flask culture conditions of the engineered SiSOMT strain were optimized, resulting in the production of 165.74 mg/L of this compound. This study thus presents an enzymatic approach to the synthesis of high-value-added THPBs with minimum environmental wastage. [ABSTRACT FROM AUTHOR]

Biocatalysis has emerged as a transformative approach in sustainable drug synthesis. This article provides an extensive overview of key enzymes harnessed in biocatalysis and their diverse applications in eco-friendly drug development. Cytochrome P450 enzymes enable the hydroxylation of drug precursors, metabolite generation for toxicity assessment and chiral drug intermediate synthesis. Lipases contribute to asymmetric ester prodrug synthesis and racemic mixture resolution, while proteases are instrumental in peptide bond formation and peptide-based drug synthesis. Transaminases facilitate chiral amine synthesis and amino acid derivative production for drugs. Dehydrogenases allow stereoselective ketone reduction and chiral alcohol production. Aldolases enable enantioselective aldol reactions and key drug intermediate formation. DNA ligases aid in DNA-based drug development and targeted drug delivery. Nitrilases promote nitrile hydrolysis and green amide synthesis. Kinases play roles in prodrug activation and drug target validation. Methyltransferases are vital for small molecule methylation and epigenetic drug discovery. Oxidoreductases enable redox reactions and aldehyde-ketone interconversion. Glycosyltransferases contribute to drug candidate glycosylation and bioconjugation. Biocatalysis offers a sustainable alternative, aligning with pharmaceutical industries’ commitment to environmentally responsible drug discovery and production, as supported by the referenced literature. [ABSTRACT FROM AUTHOR]

β‐Lactam antibiotics are one of the most commonly prescribed drugs to treat bacterial infections. However, their use has been somehow limited given the emergence of bacteria with resistance mechanisms, such as β‐lactamases, which inactivate them by degrading their four‐membered β‐lactam rings. So, a total knowledge of the mechanisms governing the catalytic activity of β‐lactamases is required. Here, we report a novel Zn‐based metal–organic framework (MOF, 1), possessing functional channels capable to accommodate and interact with antibiotics, which catalyze the selective hydrolysis of the penicillinic antibiotics amoxicillin and ceftriaxone. In particular, MOF 1 degrades, very efficiently, the four‐membered β‐lactam ring of amoxicillin, acting as a β‐lactamase mimic, and expands the very limited number of MOFs capable to mimic catalytic enzymatic processes. Combined single‐crystal X‐ray diffraction (SCXRD) studies and density functional (DFT) calculations offer unique snapshots on the host‐guest interactions established between amoxicillin and the functional channels of 1. This allows to propose a degradation mechanism based on the activation of a water molecule, promoted by a Zn‐bridging hydroxyl group, concertedly to the nucleophilic attack to the carbonyl moiety and the cleaving of C−N bond of the lactam ring. [ABSTRACT FROM AUTHOR]

Enzymatic catalysis is characterized by multistage chemical reactions from enzyme-substrate complexes to products. In a number of cases, in the course of experimental studies, it is possible to characterize the structures and properties of intermediates of complex chemical reactions in proteins. The use of modern computer simulation methods makes it possible to significantly supplement the knowledge of the mechanisms of enzymatic catalysis reactions and provide detailed data on reaction intermediates, including structures with atomic resolution. The materials accumulated to date make it possible to create a unique dat-abase called ENIAD (ENzyme-In-Action-Databank). The article describes the principles of building the ENIAD database, as well as a multiplatform web interface for accessing data (https://lcc.chem.msu.ru/eniad/). [ABSTRACT FROM AUTHOR]

In this work, the catalytic mechanism of loganic acid methyltransferase was characterized at the molecular level. This enzyme is responsible for the biosynthesis of loganin, which is a precursor for a wide range of biologically active compounds. Due to the lack of detailed knowledge about this process, the aim of this study was the analysis of the structure and activity of loganic acid methyltransferase. Using molecular dynamics (MD) simulations, the native structure of the complex was reconstructed, and the key interactions between the substrate and loganic acid methyltransferase were investigated. Subsequently, the structures obtained from the simulations were used for quantum chemical (QM) calculations. The QM calculations allowed for the exploration of the energetic aspects of the reaction and the characterization of its mechanism. The results obtained in this study suggest the existence of two patterns of interactions between loganic acid methyltransferase and the substrate. The role of residue Q38 in the binding and orientation of the substrate's carboxyl group was also demonstrated. By employing a combined MD and QM approach, the experimental reaction barrier was reproduced, and detailed insights into the enzymatic activity mechanism of loganic acid methyltransferase were revealed. [ABSTRACT FROM AUTHOR]

Utilizing electric fields to catalyze chemical reactions is not a new idea, but in enzymology it undergoes a renaissance, inspired by Warhsel's concept of electrostatic preorganization. According to this concept, the source of the immense catalytic efficiency of enzymes is the intramolecular electric field that permanently favors the reaction transition state over the reactants. Within enzyme design, computational efforts have fallen short in designing enzymes with natural-like efficacy. The outcome could improve if long-range electrostatics (often omitted in current protocols) would be optimized. Here, we highlight the major developments in methods for analyzing and designing electric fields generated by the protein scaffolds, in order to both better understand how natural enzymes function, and aid artificial enzyme design.

Aiming to reduce the toxicity and operational costs often associated to chemical processes, the enzymatic synthesis is applied herein as a sustainable route for producing polyesters. The use of NADES' (Natural Deep Eutectic Solvents) components as a source of monomers for the synthesis of polymers through lipase‐catalyzed esterification in an anhydrous medium is detailed for the first time. Three NADES composed by glycerol and an organic base, or acid, were used to produce polyesters, through polymerization reactions catalyzed by Aspergillus oryzae lipase. High polyester conversion rates (above 70 %), containing at least 20 monomeric units (glycerol:organic acid/base (1 : 1)), were observed by matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) analysis. The NADES monomers′ capacity for polymerization, along with their non‐toxicity, cheap cost, and simplicity of production, sets up these solvents as a greener and cleaner approach for the synthesis of high value‐added products. [ABSTRACT FROM AUTHOR]

Simple Summary: Traditionally, enzymes have been studied in standardized buffers, but in real life, inside cells, they have to function under conditions far from "ideal". The intracellular milieu is crowded by numerous molecules with various physical and chemical properties. What consequences does it have for enzymatic reactions? How do enzymes deal with disturbing neighbors? To examine this issue, we studied the mechanisms of influence of seven model substances with different molecular sizes on the reaction catalyzed by bacterial luciferase. This reaction passes through several stages and ends with light emission. We analyzed the responses rate in different stages to the addition of alcohols, saccharides, and biopolymers and found the counteraction of two effects. On the one hand, increased viscosity slows down many of the stages, both associative and dissociative. On the other hand, it is accompanied by an increase in the catalytic constant of luciferase, which compensates for kinetic disadvantages. We assumed that both effects could be caused by the same property of the added substances—their ability to interact with water. Our findings serve not only to shed light on the organization of the cellular metabolism, but also to improve enzyme-based biotechnologies using the recipes of nature. A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water–cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site. [ABSTRACT FROM AUTHOR]

Worldwide pollution of waters and soils is a rising health issue calling for advanced decontamination methods such as adsorption of contaminants on biochar because biochar is carbon-negative material displaying many adsorptive properties. Biochar has been improved by chemical, physical and magnetic modifications, and by surface decoration, yet the recent development of biochar-supported composites appears more promising. Here we review biochar-supported composites with focus on preparation, mechanisms, biochar-clays, biochar-microorganisms, biochar-enzymes, and applications to adsorb metals, nutrients and organic contaminants. [ABSTRACT FROM AUTHOR]

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Enantioselective reduction reactions are privileged transformations for the construction of trisubstituted stereogenic centers. While these include established synthetic strategies, such as asymmetric hydrogenation, methods based on the enantioselective addition of hydridic reagents to electrophilic prochiral substrates have also gained importance. In this context, the asymmetric conjugate reduction (ACR) of α,β‐unsaturated compounds has become a convenient approach for the synthesis of chiral compounds with trisubstituted stereocenters in α‐, β‐, or γ‐position to electron‐withdrawing functional groups. Because such activating groups are diverse and amenable of further derivatizations, ACRs provide a general and powerful synthetic entry towards a variety of valuable chiral building blocks. This Review provides a comprehensive collection of catalytic ACR methods involving transition‐metal, organic, and enzymatic catalysis since its first versions dating back to the late 1970s. [ABSTRACT FROM AUTHOR]

The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non–trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1.

All catalysts have unique abilities. This is especially true for microbial, enzymatic, and organometallic catalysis, which are often seen as competitive approaches preventing the exploitation of their complementarity. An increasing number of examples show, how using the complete catalytic spectrum can open roads from new substrates to new products. C1‐compounds such as formate, formaldehyde, methanol, or methane from CO2 in combination with green H2 are likely to be future sources of carbon feedstock. This short review highlights how combinations of different catalyst types can facilitate integrated reaction sequences with biogenic substrates to form "bio‐hybrid" fuels and products. [ABSTRACT FROM AUTHOR]

Approximately 400 billion PET bottles are produced annually in the world, of which from 8 to 9 million tons are discarded in oceans. This requires developing strategies to urgently recycle them. PET recycling can be carried out using the microbial hydrolysis of polymers when monomers and oligomers are released. Exploring the metabolic activity of fungi is an environmentally friendly way to treat harmful polymeric waste and obtain the production of monomers. The present study addressed: (i) the investigation of potential of strains with the potential for the depolymerization of PET bottles from different manufacturers (crystallinity of 35.5 and 10.4%); (ii) the search for a culture medium that favors the depolymerization process; and (iii) gaining more knowledge on fungal enzymes that can be applied to PET recycling. Four strains (from 100 fungal strains) were found as promising for conversion into terephthalic acid from PET nanoparticles (npPET): Curvularia trifolii CBMAI 2111, Trichoderma sp. CBMAI 2071, Trichoderma atroviride CBMAI 2073, and Cladosporium cladosporioides CBMAI 2075. The fermentation assays in the presence of PET led to the release of terephthalic acid in concentrations above 12 ppm. Biodegradation was also confirmed using mass variation analyses (reducing mass), scanning electron microscopy (SEM) that showed evidence of material roughness, FTIR analysis that showed band modification, enzymatic activities detected for lipase, and esterase and cutinase, confirmed by monomers/oligomers quantification using high performance liquid chromatography (HPLC-UV). Based on the microbial strains PET depolymerization, the results are promising for the exploration of the selected microbial strain. [ABSTRACT FROM AUTHOR]

A fast and highly efficient method for the synthesis of functionalized quinazolinones by combining enzymatic catalysis and photocatalysis is reported. The α-Chymotrypsin catalyzed the cyclization of aldehyde and 2-aminobenzamide, which was subsequently followed by White LED-induced oxidation of 2-phenyl-2, 3-dihydroquinazolin-4(1H)-one to obtain quinazolinone. The reaction process was highly efficient with a reaction yield of 99% in just 2 h, and a wide range of quinazolinones could be synthesized. Furthermore, the plausible mechanism was investigated by control experiments and DFT calculations. This protocol provides an alternative synthetic route for the preparation of quinazolinone derivatives. [ABSTRACT FROM AUTHOR]

Plants have been used by humans since ancient times due their antimicrobial and medicinal properties. Essential oils (EOs) are complex mixtures of secondary plant metabolites, including terpenoids, phenylpropanoids, and other aromatic compounds. Cinnamomun verum and Thyme vulgaris EOs and their organic extracts exert numerous biological activities because of their major compounds, particularly thymol, carvacrol, eugenol, and benzoic and cinnamic acid. The structural motifs presented by these phytochemicals are responsible for their biological activities. Modification or hybridization of these structures could lead to new bio-based compounds with improved efficacy or multiple modes of action. In this work, we aimed to develop reliable methods of obtaining six hybrid molecules from the major constituents of C. verum and T. vulgaris EOs. For the first time, we tested their efficacy in the inhibition of the mycelium growth and spore germination of two of the most important phytopathogenic fungi, Fusarium oxysporum and Colletotrichum gloeosporioides, and one opportunistic human pathogen, Aspergillus niger. The cytotoxic activity of the obtained hybrids was assessed using the brine shrimp lethality assay. In addition, we report for the first time a biocatalytic process for the obtention of these bioactive hybrid molecules. The results of this work enable the possibility of using hybrid molecules based on the major constituents of EOs as active ingredients in strategic industries such as agriculture, aquaculture, and pharmaceuticals. [ABSTRACT FROM AUTHOR]

Amide bond is often seen in value-added nitrogen-containing heterocyclic compounds, which can present promising chemical, biological, and pharmaceutical significance. However, current synthesis methods in the preparation of amide-containing N-heterocyclic compounds have low specificity (large amount of by-products) and efficiency. In this study, we focused on reviewing the feasible enzymes (nitrogen acetyltransferase, carboxylic acid reductase, lipase, and cutinase) for the amidation of N-heterocyclic compounds; summarizing their advantages and weakness in the specific applications; and further predicting candidate enzymes through in silico structure-functional analysis. For future prospects, current enzymes demand further engineering and improving for practical industrial applications and more enzymatic tools need to be explored and developed for a broader range of N-heterocyclic substrates. [ABSTRACT FROM AUTHOR]

A biocatalyst is an enzyme that speeds up or slows down the rate at which a chemical reaction occurs and speeds up certain processes by 108 times. It is used as an anticancer agent because it targets drug activation inside the tumor microenvironment while limiting damage to healthy cells. Biocatalysts have been used for the synthesis of different heterocyclic compounds and is also used in the nano drug delivery systems. The use of nano-biocatalysts for tumor-targeted delivery not only aids in tumor invasion, angiogenesis, and mutagenesis, but also provides information on the expression and activity of many markers related to the microenvironment. Iosmapinol, moclobemide, cinepazide, lysine dioxygenase, epothilone, 1-homophenylalanine, and many more are only some of the anticancer medicines that have been synthesised using biocatalysts. In this review, we have highlighted the application of biocatalysts in cancer therapies as well as the use of biocatalysts in the synthesis of drugs and drug-delivery systems in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Driven by the massive demand in recent years, the production of β-alanine has significantly progressed in chemical and biological ways. Although the chemical method is relatively mature compared to biological synthesis, its high cost of waste disposal and environmental pollution does not meet the environmental protection standard. Hence, the biological method has become more prevalent as a potential alternative to the chemical synthesis of β-alanine in recent years. As a result, the aspartate pathway from l-aspartate to β-alanine (the most significant rate-limiting step in the β-alanine synthesis) catalyzed by l-aspartate-α-decarboxylase (ADC) has become a research hotspot in recent years. Therefore, it is vital to comprehensively understand the different enzymes that possess a similar catalytic ability to ADC. This review will investigate the exploratory process of unique synthesis features and catalytic properties of ADC/ADC-like enzymes in particular creatures with similar catalytic capacity or high sequence homology. At the same time, we will discuss the different β-alanine production methods which can apply to future industrialization. [ABSTRACT FROM AUTHOR]

Introduction: β-Alanine is the only β-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of β-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method.Methods: In this study, we constructed an Escherichia coli recombinant strain for efficient β-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome.Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of β-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the β-alanine content. The key cellulosome elements, dockerin (docA) and cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. β-alanine production reached 7.439 mg/L and 25.87 mg/L in the two engineered strains. The β-alanine content reached 755.465 mg/L in a 5 L fermenter.Discussion: The content of β-alanine synthesized by constructed β-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of β-alanine using a cellulosome multi-enzyme self-assembly system.

In this study, lactic acid bacteria (LAB) fermentation and β-galactosidase catalysis methods were combined to increase the lactulose concentration and reduce the galactose and lactose content in a hot-alkaline-based system. The optimal conditions for chemical isomerization were 70 °C for 50 min for lactulose production, in which the concentration of lactulose was 31.3 ± 1.2%. Then, the selection and identification of LAB, which can utilize lactose and cannot affect lactulose content, were determined from 451 strains in the laboratory. It was found that Lactobacillus salivarius TM-2–8 had weak lactulose utilization and more robust lactose utilization. Lactobacillus rhamnosus grx.21 was weak in terms of lactulose utilization and strong in terms of galactose utilization. These two strains fermented the chemical isomerization system of lactulose to reduce the content of lactose and galactose. The results showed that the lactose concentration was 48.96 ± 2.92 g/L and the lactulose concentration was 59.73 ± 1. 8 g/L for fermentation lasting 18 h. The β-galactosidase was used to increase the content of lactulose in the fermented system at this time. The highest concentration of 74.89 ± 1.68 g/L lactulose was obtained at an enzymatic concentration of 3 U/mL and catalyzed at 50 °C for 3 h by β-galactosidase.

The phosphorolysis of cello-oligosaccharides is a critical process played in the rumen by Ruminococcus albus to degrade cellulose. Cellodextrins, made up of a few glucosyl units, have gained lots of interest by their potential applications. Here, we characterized a cellobiose phosphorylase (RalCBP) and a cellodextrin phosphorylase (RalCDP) from R. albus 8. This latter was further analyzed in detail by constructing a truncated mutant (Ral∆N63CDP) lacking the N-terminal domain and a chimeric protein by fusing a CBM (RalCDP-CBM37). RalCBP showed a typical behavior with high activity on cellobiose. Instead, RalCDP extended its activity to longer soluble or insoluble cello-oligosaccharides. The catalytic efficiency of RalCDP was higher with cellotetraose and cellopentaose as substrates for both reaction directions. Concerning properties of Ral∆N63CDP, results support roles for the N-terminal domain in the conformation of the homo-dimer and conferring the enzyme the capacity to catalyze the phosphorolytic reaction. This mutant exhibited reduced affinity toward phosphate and increased to glucose-1-phosphate. Further, the CBM37 module showed functionality when fused to RalCDP, as RalCDP-CBM37 exhibited an enhanced ability to use insoluble cellulosic substrates. Data obtained from this enzyme’s binding parameters to cellulosic polysaccharides agree with the kinetic results. Besides, studies of synthesis and phosphorolysis of cello-saccharides at long-time reactions served to identify the utility of these enzymes. While RalCDP produces a mixture of cello-oligosaccharides (from cellotriose to longer oligosaccharides), the impaired phosphorolytic activity makes Ral∆N63CDP lead mainly toward the synthesis of cellotetraose. On the other hand, RalCDP-CBM37 remarks on the utility of obtaining glucose-1-phosphate from cellulosic compounds.

Polyesters are important materials with a wide range of applications, but there has been increasing concern over their sustainability. One example is the need for safer, bio-derived solvents to replace those currently in use for the polymer’s synthesis and processing. In this work, several variants of the bio-based cellulose/levoglucosenone derived solvent Cyrene, namely the ketal derivatives dioxolane Cygnet, dioxane Cygnet and dioxepane Cygnet were synthesized and tested as media for enzymatic polycondensation reactions using bio-based building blocks. Dioxolane Cygnet and dioxepane Cygnet were found to be suitable solvents for enzymatic polycondensation reactions, with dioxolane Cygnet being the preferred solvent, yielding polymers with a Mn >22 kDa. In addition, these solvents were tested in the biocatalyzed synthesis of levoglucosenone-based polyesters. The alternative solvents gave superior yields to those previously observed, demonstrating the versatility of these solvents in enzymatic polycondensation reactions, representing the first synthetic polymer-solvent system fully derived from cellulose. [ABSTRACT FROM AUTHOR]

Glycerol is a common by‐product of industrial biodiesel syntheses. Due to its properties, availability, and versatility, residual glycerol can be used as a raw material in the production of high value‐added industrial inputs and outputs. In particular, products like hydrogen, propylene glycol, acrolein, epichlorohydrin, dioxalane and dioxane, glycerol carbonate, n‐butanol, citric acid, ethanol, butanol, propionic acid, (mono‐, di‐, and triacylglycerols), cynamoil esters, glycerol acetate, benzoic acid, and other applications. In this context, the present study presents a critical evaluation of the innovative technologies based on the use of residual glycerol in different industries, including the pharmaceutical, textile, food, cosmetic, and energy sectors. Chemical and biochemical catalysts in the transformation of residual glycerol are explored, along with the factors to be considered regarding the choice of catalyst route used in the conversion process, aiming at improving the production of these industrial products. [ABSTRACT FROM AUTHOR]

2-Mercaptobenzothiazole (MBT) poses an unignorable risk to the aquatic environment and human health. Traditional analysis and prevention methods are unsuitable for broader applications due to their complexity and high-cost instruments. To address these challenges, a novel CuO@MnO 2 (CM) nanozyme has been designed to establish absorbance-colorimetric dual-mode sensing and modulate the content of MBT through reactive oxygen species (ROS) mediated oxidative degradation. CM nanozyme exhibits notable catalytic ability with K m 13.6 μM and V max 0.763 μM/s. Furthermore, by integrating the generated 1O 2 , O 2 •- and •OH in the presence of O 2 , the absorbance and colorimetric method for MBT sensing is developed, offering a linear detection range from 0.1 to 15 μM through the color change of 3,3′,5,5′-tetramethylbenzidine (TMB) and TMBox. Besides, the CM nanozyme can oxidatively decompose MBT, significantly reducing its biotoxicity without needing photocatalytic auxiliary equipment. This work presents a promising MBT detection and degradation approach in future applications. [Display omitted] • CM nanozyme exhibits superior catalytic properties with higher substrate affinity and faster reaction rate. • The absorbance and colorimetric dual-channel mode are established for 2-mercaptobenzothiazole determination. • The photocatalytic equipment-free 2-mercaptobenzothiazole degradation is realized. • CM nanozyme could mitigate 2-mercaptobenzothiazole's biotoxicity. [ABSTRACT FROM AUTHOR]

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9–285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e− oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H + /2e − oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO 2 to regulate the pH in the acidic environment. We identified the catalytic intermediates of Helicobacter pylori catalase. The [Fe(IV) = O Por•+] intermediate, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285 GHz/10T-EPR spectrum. We demonstrated that moonlighting by the H. pylori catalase includes formate oxidation to CO 2. [Display omitted] • High-valent intermediates in H. pylori heme catalase were identified by 9-285 GHz EPR spectroscopy • The [Fe(IV) = O Por•+] species showed pH dependence on the exchange-coupling of the porphyrin radical • Trp• (pH ≤ 6) and Tyr• intermediates were discerned by their g-values in the 285-GHz EPR spectrum. • The stabilization of an [Fe(IV) = O Trp•] species in HPC is unprecedented in monofunctional catalases • 1H+/2e− oxidation of formate to CO 2 by the [Fe(IV) = O Por•+] in a peroxygenase reaction is proposed. [ABSTRACT FROM AUTHOR]

Tetrahydroprotoberberines (THPBs) are plant-specific alkaloids with significant medicinal value. They are present in trace amounts in plants and are difficult to chemically synthesize due to stereoselectivity and an unfavorable environment. In this study, a selective methylation strategy was developed for the biocatalysis of seven high-value-added THPB compounds using 4’-O-methyltransferase (Cj4’OMT), norcoclaurine 6-O-methyltransferase (Cj6OMT), and (S)-scoulerine 9-O-methyltransferase (SiSOMT and PsSOMT) in engineered E. coli. The methyltransferases Cj4’OMT, Cj6OMT, PsSOMT, and SiSOMT were expressed heterologously in E. coli. Compound 1 (10-methoxy-2,3,9-tetrahydroxyberbine) was synthesized using the recombinant E. coli strain Cj4’OMT and the substrate 2,3,9,10-tetrahydroxyberbine. Compound 2 (9-methoxy-2,3,10-tetrahydroxyberbine) was produced in the recombinant Escherichia coli (E. coli) strain PsSOMT, and compounds 2 and 3 (discretamine) were produced in the recombinant E. coli strain SiSOMT. Compounds 4 (9,10-methoxy-2,3-tetrahydroxyberbine) and 5 (corypalmine) were obtained by co-culturing the recombinant strains Cj4’OMT and SiSOMT with substrate. Compounds 6 (scoulerine) and 7 (isoscoulerine) were produced by co-culturing the substrate with the recombinant strains Cj4’OMT and Cj6OMT. To increase the yield of novel compound 2, the flask culture conditions of the engineered SiSOMT strain were optimized, resulting in the production of 165.74 mg/L of this compound. This study thus presents an enzymatic approach to the synthesis of high-value-added THPBs with minimum environmental wastage.

C-glycosides are an important class of bioactive natural products, and the C-glycosidic bonds are usually catalyzed by C-glycosyltransferases. In this work, an efficient and rare CGT, LaCGT1, was discovered from the aquatic plant Lemna aequinoctialis. LaCGT1 could accept five sugar donors (UDP-Glc/-Xyl/-Gal/-GlcNAc/-Ara) to catalyze C-glycosylation, and showed strong preference to uridine 5′-diphosphate xylose (UDP-Xyl). LaCGT1 catalyzed at least six substrates using UDP-Xyl as sugar donor, with conversion rates of > 95%. Three xylosides were obtained by scaled-up enzymatic catalysis, and their structures were identified by 1D NMR, 2D NMR, and HR-ESIMS data analyses. Molecular modeling and site-directed mutagenesis indicated that R271, W357, D378, and Q379 residues were key amino acids contributing to sugar donor recognition of UDP-Xyl. LaCGT1 could be a promising catalyst to prepare bioactive flavonoid C-xylosides.