Search

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sir John Cornforth's work on the stereochemistry of enzyme reactions involved in the biosynthesis of squalene and cholesterol and in the formation and metabolism of a chiral methyl group in acetyl co-enzyme A, is reviewed.

The ongoing trends to process improvements, cost reductions and increasing quality, safety, health and environment requirements of industrial chemical transformations have strengthened the translation of global biocatalysis research work into industrial applications. One focus has been on biocatalytic single-step reactions with one or two substrates, the identification of bottlenecks and molecular as well as engineering approaches to overcome these bottlenecks. Robust industrial procedures have been established along classes of biocatalytic single-step reactions. Multi-step reactions and multi-component reactions (MCRs) enable a bottom-up approach with biocatalytic reactions working together in one compartment and recations hindering each other within different compartments or steps. The understanding of the catalytic functions of known and new enzymes is key for the development of new sustainable chemical transformations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A mixture of molecules can be regarded as a network if all the molecular components participate in some kind of interaction with other molecules--either physical or functional interactions. Template-assisted ligation reactions that direct replication processes can serve as the functional elements that connect two members of a chemical network. In such a process, the template does not necessarily catalyze its own formation, but rather the formation of another molecule, which in turn can operate as a template for reactions within the network medium. It was postulated that even networks made up of small numbers of molecules possess a wealth of molecular information sufficient to perform rather complex behavior. To probe this assumption, we have constructed virtual arrays consisting of three replicating molecules, in which dimer templates are capable of catalyzing reactants to form additional templates. By using realistic parameters from peptides or DNA replication experiments, we simulate the construction of various functional motifs within the networks. Specifically, we have designed and implemented each of the three-element Boolean logic gates, and show how these networks are assembled from four basic "building blocks". We also show how the catalytic pathways can be wired together to perform more complex arithmetic units and network motifs, such as the half adder and half subtractor computational modules, and the coherent feed-forward loop network motifs under different sets of parameters. As in previous studies of chemical networks, some of the systems described display behavior that would be difficult to predict without the numerical simulations. Furthermore, the simulations reveal trends and characteristics that should be useful as "recipes" for future design of experimental functional motifs and for potential integration into modular circuits and molecular computation devices.

IntroductionDeep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge.MethodsThis paper discusses the rational design of DES tailored to a given biocatalytic system through a combination of experimental screening and computational tools, guided by performance targets defined by solvent properties and process constraints. The efficacy of this approach is demonstrated by the reduction of CO2 to formate catalyzed by NADH-dependent formate dehydrogenase (FDH). By systematically analyzing FDH activity and stability, NADH stability (both long-term and short-term stability after solvent saturation with CO2), and CO2 solubility in initially selected glycerol-based DESs, we were able to skillfully guide the DES screening process.Results and discussionConsidering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly80%B) was identified as the most promising solvent system for a given reaction. Using ChCl:Gly as a co-solvent resulted in an almost 15-fold increase in FDH half-life compared to the reference buffer and stabilized the coenzyme after the addition of CO2. Moreover, the 20% addition of ChCl:Gly to the buffer improved the volumetric productivity of FDH-catalyzed CO2 reduction in a batch system compared to the reference buffer. The exceptional stability of the enzyme in this co-solvent system shows great potential for application in continuous operation, which can significantly improve process productivity. Additionally, based on easily measurable physicochemical solvent properties and molecular descriptors derived from COSMO-RS, QSAR models were developed, which successfully predicted enzyme activity and stability, as well as coenzyme stability in selected solvent systems with DESs.

In the past 20 years, unspecific peroxygenases (UPOs) have emerged as promising biocatalysts for various organic transformations. Particularly, we have witnessed great attention being paid to the screening of new enzymes and expansion of the substrates/products. However, challenges such as enzyme stability, low turnover numbers, and substrate specificity hinder their widespread utilization in practical organic synthesis. This review article provides a concrete and mini-overview of the challenges associated with using UPOs in organic synthesis and discusses strategies for enzyme engineering to overcome these limitations. The article highlights recent advancements in UPO research and presents potential solutions to enhance their catalytic efficiency, stability, substrate specificity, and regioselectivity. Additionally, the review outlines the current methodologies employed for directed evolution and protein engineering of UPOs, along with computational modeling approaches for rational enzyme design. By addressing the challenges and exploring avenues for enzyme engineering, this review aims to shed light on the prospects of UPOs in organic synthesis.

Abstract Unspecific peroxygenase from Agrocybe aegerite (AaeUPO) is a remarkable catalyst for the oxyfunctionalization of non‐activated C−H bonds under mild conditions. It exhibits comparable activity to P450 monooxygenase but offers the advantage of using H2O2 instead of a complex electron transport chain to reductively activate O2. Here, we demonstrate the successful oxidation of cyclohexane to cyclohexanol/cyclohexanone (KA‐oil) using sol‐gel encapsulated AaeUPO. Remarkably, cyclohexane serves both as a solvent and a substrate in this system, which simplifies product isolation. The ratio of cyclohexanone to cyclohexanol using this approach is remarkably higher compared to the oxidation using free AaeUPO in aqueous media using acetonitrile as a cosolvent. The utilization of sol‐gel encapsulated AaeUPO offers a promising approach for oxyfunctionalization reactions and improves the chances for this enzyme to be incorporated in the same pot with other chemical transformations.

Approximate models are a fast and most often precise tool for determining the effectiveness factor for heterogeneous catalysis processes that are realized in the real world. They are also frequently applied as robust transient models describing the work of a single catalyst pellet or as a part of a more complex model, for example, a reactor model, where mass balances for the gas phase and solid phase are necessary. So far, approximate models for diffusion and reaction processes have been presented for processes described by a single balance equation. In the present work, approximate models without the mentioned limitation are presented and discussed. In addition, simple rules are shown for the development of other complex approximate models without tedious derivation in the complex domain. The formulas considered in this work are typical long-time approximations of the transient process. The accuracy is good, especially in the range of small and intermediate Thiele modulus values.

Introduction: Biocatalysis, particularly through engineered enzymes, presents a cost-effective, efficient, and eco-friendly approach to compound synthesis. We sought to identify ketoreductases capable of synthesizing optically pure alcohols or ketones, essential chiral building blocks for active pharmaceutical ingredients.Methods: Using BioMatchMaker®, an in silico high-throughput platform that allows the identification of wild-type enzyme sequences for a desired chemical transformation, we identified a bacterial SDR ketoreductase from Thermus caliditerrae, Tcalid SDR, that demonstrates favorable reaction efficiency and desired enantiomeric excess.Results: Here we present two crystal structures of the Tcalid SDR in an apo-form at 1.9 Å and NADP-complexed form at 1.7 Å resolution (9FE6 and 9FEB, respectively). This enzyme forms a homotetramer with each subunit containing an N-terminal Rossmann-fold domain. We use computational analysis combined with site-directed mutagenesis and enzymatic characterization to define the substrate-binding pocket. Furthermore, the enzyme retained favorable reactivity and selectivity after incubation at elevated temperature.Conclusion: The enantioselectivity combined with the thermostability of Tcalid SDR makes this enzyme an attractive engineering starting point for biocatalysis applications.

Biohybrid materials and engineered living materials (ELMs) are a dynamic field of research at the interface of material science and synthetic biology. Recently, microorganisms have been described as both functional and structural building blocks of such materials. Dry materials with bacteria as structural building block have previously not been investigated for their ability to retain catalytic activity. Herein, it is shown that Saccharomyces cerevisiae biomass can act as a structural building block in dry, macroscopic materials while simultaneously providing functionality. A benign method for the preparation of both coatings and free‐standing, flexible films is presented. Notably, free‐standing films can be prepared solely from biomass and plasticizers without the need for other additives. During the process, the integrity of the cells and the catalytic activity within is preserved, which is demonstrated by biocatalytic H2O2 degradation. The film‐forming properties of S. cerevisiae biomass and the influence of plasticizing and polymeric additives on the mechanical properties are investigated in detail and compared to related materials. The work presented is a first step toward new, mechanically strong microorganism‐based functional coatings and films.

Abstract The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3‐hydroxyphenyl acetate to 2′,4′‐dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6−H of the substrate is rate‐limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds.

Abstract Chirality plays a significant role in the manufacture of pharmaceuticals and fine chemicals. The use of chemical catalysts to control stereoselectivity relies on the use of chiral catalysts with labor–intensive synthesis and purification. Natural enzymes offer inherent stereoselectivity, making them attractive catalysts for this purpose. We report here chiral biocatalytic oxidations in microemulsions driven by horseradish peroxidase coupled with a synthetic Cu2+‐polymer catalyst. This hybrid system features crosslinked layer–by–layer (LBL) films composed of polyions with Cu2+‐containing pyrene–labelled poly(2‐hydroxy‐3‐dipicolylamino) propyl methacrylate (Py−PGMADPA) to drive oxygen reduction to form hydrogen peroxide. Peroxide in turn activates horseradish peroxidase (HRP) crosslinked in LbL films on magnetic particle beads to biocatalytically oxidize styrene, ethylbenzene, and methyl phenylacetate to chiral products. R‐stereoisomers of these reactants were selectively formed with a high enantiomeric excess of ≥80 % at 90 °C. The enzyme films show high thermal stability at 90 °C in cetyltrimethylammonium bromide microemulsion. Reactions at 90 °C were essentially complete in 2 hr. This hybrid approach opens a door to new designs of biocatalytic syntheses using a separate electrocatalyst for enzyme activation.

Ethyl hydrocinnamate is an ester with a sweet, fruity, honey-like scent commonly used as a flavor and fragrance agent. Due to its chemical structure, it can be easily obtained through enzymatic reactions without the need for harsh substances and processes. This study investigated the immobilization of the commercial lipase Sustine® 131 onto spent coffee grounds (SCG) as a low-cost support for the enzymatic synthesis of ethyl hydrocinnamate. Spent coffee grounds underwent pretreatment with water, hexane, and ethanol to serve as a lipase adsorption platform and extract valuable bioactive compounds such as polyphenols. The immobilized lipase displayed both hydrolytic and synthetic activities during 12 weeks of storage at room temperature. The optimal reaction conditions for the synthesis of ethyl hydrocinnamate were determined using a Box–Behnken plan. It was shown that the enzyme concentration and the temperature were crucial for achieving high yields of ethyl hydrocinnamate with a conversion rate above 92%. Specifically, at least 18% enzyme concentration and a temperature of 45 °C were necessary. This eco-friendly approach utilized abundant food waste residue as an inexpensive and renewable immobilization support, enabling efficient biocatalytic production of the high-value flavor ester ethyl hydrocinnamate.

The use of vegetable oils and their derivatives for polymer synthesis has been a major focus in recent years due to their universal availability, low production costs and biodegradability. In this study, the enzymatic synthesis of oligoesters of ricinoleic acid obtained from castor oil combined with three aromatic natural derivatives (cinnamyl alcohol, sinapic acid, and caffeic acid) was investigated. The formation of the reaction products was demonstrated by FT-IR, MALDI-TOF MS and NMR spectroscopy and for the oligo (ricinoleyl)-caffeate the thermal properties and biodegradability in sweet water were analyzed and a rheological characterization was performed. The successful enzymatic synthesis of oligoesters from ricinoleic acid and aromatic monomers using lipases not only highlights the potential of biocatalysis in green chemistry but also contributes to the development of sustainable and biodegradable methods for synthesizing products with potential applications as cosmetic ingredients.

Asymmetric catalysis stands at the forefront of modern chemistry, serving as a cornerstone for the efficient creation of enantiopure chiral molecules characterized by their high selectivity. In this review, we delve into the realm of asymmetric catalytic reactions, which spans various methodologies, each contributing to the broader landscape of the enantioselective synthesis of chiral molecules. Transition metals play a central role as catalysts for a wide range of transformations with chiral ligands such as phosphines, N-heterocyclic carbenes (NHCs), etc., facilitating the formation of chiral C-C and C-X bonds, enabling precise control over stereochemistry. Enantioselective photocatalytic reactions leverage the power of light as a driving force for the synthesis of chiral molecules. Asymmetric electrocatalysis has emerged as a sustainable approach, being both atom-efficient and environmentally friendly, while offering a versatile toolkit for enantioselective reductions and oxidations. Biocatalysis relies on nature’s most efficient catalysts, i.e., enzymes, to provide exquisite selectivity, as well as a high tolerance for diverse functional groups under mild conditions. Thus, enzymatic optical resolution, kinetic resolution and dynamic kinetic resolution have revolutionized the production of enantiopure compounds. Enantioselective organocatalysis uses metal-free organocatalysts, consisting of modular chiral phosphorus, sulfur and nitrogen components, facilitating remarkably efficient and diverse enantioselective transformations. Additionally, unlocking traditionally unreactive C-H bonds through selective functionalization has expanded the arsenal of catalytic asymmetric synthesis, enabling the efficient and atom-economical construction of enantiopure chiral molecules. Incorporating flow chemistry into asymmetric catalysis has been transformative, as continuous flow systems provide precise control over reaction conditions, enhancing the efficiency and facilitating optimization. Researchers are increasingly adopting hybrid approaches that combine multiple strategies synergistically to tackle complex synthetic challenges. This convergence holds great promise, propelling the field of asymmetric catalysis forward and facilitating the efficient construction of complex molecules in enantiopure form. As these methodologies evolve and complement one another, they push the boundaries of what can be accomplished in catalytic asymmetric synthesis, leading to the discovery of novel, highly selective transformations which may lead to groundbreaking applications across various industries.

Enzymes are natural catalysts which are gaining momentum in chemical synthesis due to their exquisiteselectivity and their biodegradability. However, the cost-efficiency and the sustainability of the overall biocatalytic process must be enhanced to unlock completely the potential of enzymes for industrial applications. To reach this goal, enzyme immobilization and the integration into continuous flow reactors have been the cornerstone of our research. We showed key examples of the advantages of those tools for the biosynthesis of antivirals, anticancer drugs, and valuable fragrance molecules. By combining new strategies to immobilize biocatalysts, innovative bioengineering approaches, and process development, the performance of the reactions could be boosted up to 100-fold.

Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.

The CoA ligase from Metallosphaera sedula (MsACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of MsACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of MsACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction.

An efficient one-pot route leading to bis-indolizine symmetric compounds has been developed via a new approach from the dipyridinium heterocyclic compound, reactive halogenated derivative, and activated alkyne through biocatalysis. A set of local plants was evaluated for its catalytic potential in “one-pot” biocatalysis of these valuable fluorescent compound synthesis reactions. Most of these biocatalysts containing enzymes from the oxidoreductase class (peroxidase: 0.56–1.08 mmol purpurogallin‧g−1 fresh weight‧min−1, polyphenol oxidase (PPO) : 27.19–48.95 PPO units‧mg tissue−1, CAT: 3.27–21.71 µmol O2‧g−1 fresh weight‧min−1), were used as green catalysts in the multi-component cycloaddition reaction, in an aqueous buffer solution, for the production of bis-indolizine compounds in moderate to excellent yields (45–85%). The horseradish root (Armoracia rusticana) has been selected as the most promising biocatalyst source among the evaluated plants, and the obtained yields were greater than in the conventional synthesis method. The structures of indolizine derivatives were confirmed by nuclear magnetic resonance spectra, elemental analyses, as well as Fourier transform-infrared spectra. The cytotoxicity of the latter obtained indolizine compounds on the growth of the model microorganism, Saccharomyces cerevisiae MIUG 3.6 yeast strain, was also evaluated. Various parameters (number of generations, growth rate, generation time, dry matter yield, the degree of the budding yeast cells, and the degree of yeast autolysis, fermentation intensity), which describe the yeast growth, suggest that the nutrient broth supplemented with different concentrations of bis-indolizine compounds (10 and 1 µM) had no toxic effect on the yeast strain growth, under submerged cultivation conditions.

Surfactants are amphiphilic molecules with the ability to modify the surface tension between two surfaces. They can be obtained by various methods, the main one being synthetic, from petroleum-based substrates. Their universal use in a wide range of fields has created a global market and, consequently, ecological, and economic expectations for their production. Biocatalyzed processes, involving enzymes, can address this objective with processes complying with the principles of green chemistry: energy saving, product selectivity, monodispersity, and reduction in the use of solvents, with energy eco-efficiency. For example, fatty-acid carbohydrate esters are biobased surfactants that can be synthesized by lipases. In this work, we were interested in the synthesis of D-glucose lauric ester, which presents interesting properties described in the literature, with Aspergillus niger lipase, rarely described with sugar substrates. We optimized the synthesis for different parameters and reaction media. This lipase appeared to be highly selective for 6-O-lauroyl-D-glucopyranose. However, the addition of DMSO (dimethyl sulfoxide) as a co-solvent displays a duality, increasing yields but leading to a loss of selectivity. In addition, DMSO generates more complex and energy-intensive purification and processing steps. Consequently, a bio-sourced alternative as co-solvent with 2MeTHF3one (2-methyltetrahydrofuran-3-one) is proposed to replace DMSO widely described in the literature.

Modern societies rely heavily on centralized industrial processes to generate a multitude of products ranging from electrical energy to synthetic chemical building blocks to construction materials. To date, these processes have relied extensively on energy produced from fossil fuels, which has led to dramatically increased quantities of greenhouse gases (including carbon dioxide) being released into the atmosphere; the effects of the ensuing change to our climate are easily observed in day-to-day life. Some of the reactions catalyzed by these industrial processes can be catalyzed in nature by metal-containing enzymes (metalloenzymes) that have evolved over the course of up to 3.8 billion years to do so under mild physiological conditions using Earth-abundant metals. While such metalloenzymes could in principle facilitate the implementation of carbon-neutral processes around the globe, either in “bio-inspired” catalyst design or even by direct exploitation, many remaining questions surrounding their mechanisms often preclude both options. Here, our recent efforts in understanding and applying metalloenzymes that catalyze reactions such as dinitrogen reduction to ammonia or proton reduction to molecular hydrogen are discussed. In closing, an opinion on the question: “Can these types of enzymes really be used in new biotechnologies?” is offered.

Biodiesel is synthesized by the transesterification of triglycerides of oils with short-chain alcohols, such as methanol and ethanol. According to the Renewable Energy Directive guidelines (RED II 2018/2001/EU) the contribution of advanced biofuels, which do not include edible oils, towards the overall EU target, is at 1% in 2025 and at least 3.5% in 2030. Bioprocesses that valorize non-edible oils for the production of second-generation biodiesel could play a critical role in achieving this goal. Immobilized lipases, as well as other enzyme classes, such as cutinases and acyltransferases, are utilized as biocatalysts for this process. For the sustainability of the process, renewable materials can be used as immobilization matrices, or even enzymes anchored on the cells as whole-cell biocatalysts. Membrane reactors can also be employed to facilitate the enzymatic transesterification by conducting a continuous enzymatic reaction and simultaneously separate the products in a single operation. The advances on the aforementioned fast-pacing fields are presented in this work.

Abstract The integration of enzymatic and electrochemical reactions offers a unique opportunity to optimize production processes. Recently, an increasing number of laboratory‐scale enzymatic electrosyntheses have shown impressive performance indicators, leading to scientific interest in technical implementation. However, important process parameters are missing in most of the relevant literature. On one hand, this is due to the large variety of relevant performance indicators. On the other hand, enzyme technologists and electrochemists use different parameters to describe a process. In this article, we review the most important performance indicators in electroenzymatic processes and suggest that in order to allow quantitative comparison, these indicators should be reported in all respective publications. In addition to quantitative parameters, non‐quantitative assessments often need to be included in a final evaluation. Examples of such parameters are sustainability, contribution to the UN Sustainable Development Goals or interactions with the overall process. We demonstrate the evaluation of processes using hydrogen peroxide‐dependent peroxygenases. The strength of the proposed evaluation system lies in its ability to identify weaknesses in a process at an early stage of development. Finally, it can be concluded that all evaluated enzymatic electrosynthesis do not yet meet typical industrial requirements for an enzyme‐based process.

Metal ions play a pivotal role in regulating and determining the functions of proteins and peptides in nature. This study aims to investigate the regulatory role of metal ions in peptide assembly and explore the influence of sequence variations and metal ions on the structure and function of resulting peptide nanoarchitectures. Dipeptide sequences with distinct charged properties (positive and negative) and functional groups (-COOH, -NH2, and phenolic hydroxyl) were meticulously selected and co-assembled with various metal ions (Au3+, Ag+, and Pt4+). The findings highlight the crucial functional role of the phenolic hydroxyl group of tyrosine in metal ion reduction, while positively charged groups promote metal ion accumulation through electrostatic forces, facilitating co-assembly. The formation of ordered structures in Au@Fmoc-YK and Au@Fmoc-YR nanoarchitectures further validates the significant interaction among metal ions, tyrosine-OH, and positively charged NH2. Notably, these nanoarchitectures possess the unique attribute of being prepared under physiological conditions, specifically at 37 °C, without the need for organic solvents or chemical modifications of peptides. This approach offers a straightforward means of constructing diverse functional nanoarchitectures based on peptides and metal ions. Moreover, Au@Fmoc-YR exhibits good performance as a nanoenzyme for detecting glucose in complex bodily fluids and plasma GSH in tumor patients, showcasing its promising potential for medical applications.

Abstract Adenosine‐5’‐triphosphate (ATP) plays a crucial role in many biocatalytic reactions and its regeneration can influence the performance of a related enzymatic reaction significantly. Here, we established electrochemically coupled ATP regeneration by pyruvate oxidase and acetate kinase (ACK) for the phosphorylation of mevalonate catalyzed by mevalonate kinase. A yield of 84 % for the product mevalonate phosphate was reached and a total turnover number for ADP of 68. These metrics are promising for the development of an economic feasible bioprocess and surpass many other enzymatic ATP regeneration systems. A comparison was made to polyphosphate kinases (PPKs), ACK, pyruvate kinase, and creatine kinase in terms of the phosphate donor properties and biocatalytic metrics of exemplary reactions. Furthermore, our system was expanded by a PPK that enables the phosphorylation of AMP, which can broaden the spectrum of applications even further.

Food safety has become a subject of major concern in controlling food contamination and supervision. Effective detection and analysis methods need to be urgently exploited to cope with this grand global challenge. Among conventional methods for food safety analysis, enzyme-based biosensing methods serve a critical role in the monitoring of food safety. However, some long-lasting challenges, like high cost and low stability, hinder enzyme-based biosensing systems. In this regard, nanozymes with superior enzyme-like activity and special physical and chemical characteristics gradually emerge as excellent tools for quality and safety analysis in agricultural fields. In this review, recent advances made in the food safety analysis based on nanozymes are summarized. Firstly, an introduction of nanozymes containing their definition, classification, and modulation is provided. Subsequently, four main sensing modes used in nanozyme-based biosensing are discussed in detail. Taking advantage of nanozyme-based biosensing platforms, great achievements made in food safety are highlighted, where biological hazards, heavy metal ions, antibiotics, pesticide residues, and additives are involved. At last, personal views of the progress and perspective in this evolving field are proposed. This review not only shows great reference significance for the construction and application of nanozyme-based analysis methods but also promotes solutions to some challenges existing in food safety.

The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis.

Introduction: The antimicrobial pyrrolnitrin from Pseudomonas strains is formed in four steps from tryptophan and comprises two flavin-dependent halogenases. Both PrnC and PrnA can carry out regioselective chlorination and bromination and are carrier protein-independent. Whilst the tryptophan halogenase PrnA has been studied in detail in the past, this study focuses on the pyrrole halogenating enzyme PrnC.Methods: The halogenating enzyme PrnC, as well as the essential electron suppliers, the flavin reductases, have been produced soluble in E. coli. Furthermore, a screening of a rational compound library revealed that the pyrrole is essential for substrate recognition; however, the substitution pattern of the benzene ring is not limiting the catalysis.Results and discussion: This renders PrnC to be a synthetically valuable enzyme for the synthesis of pyrrolnitrin congeners. For its natural substrate monodechloroaminopyrrolnitrin (MDA), the KM value was determined as 14.4 ± 1.2 µM and a kcat of 1.66 ± 0.02 min−1, which is comparable to other halogenases.

Enzymatic biotransformation of xenobiotics by the human microbiota mediates diet-drug-microbe-host interactions and affects human health. Most research on xenobiotics has focused on the gut microbiota while neglecting other body sites, yet over two-thirds of pharmaceuticals are primarily excreted in urine. As a result, the urinary microbiota is exposed to many xenobiotics in much higher concentrations than in the gut. Microbial xenobiotic biocatalysis in the bladder has implications for urinary tract infections and the emergence of antibiotic resistance. However, we have limited knowledge of biotransformations catalyzed by the urinary microbiota. In this perspective, we investigated differences in physicochemical conditions and microbial community composition between the gut and urinary tract. We used a comparative enzyme class mining approach to profile the distribution of xenobiotic-transforming enzyme homologs in genomes of urinary bacteria. Our analysis revealed a discontinuous distribution of enzyme classes even among closely related organisms. We detected diverse amidase homologs involved in pharmaceutical and dietary additive biotransformation pathways, pinpointing microbial candidates to validate for their involvement in xenobiotic transformations in urine. Overall, we highlight the biocatalytic potential of urinary tract bacteria as a lens to study how the human microbiota may respond and adapt to xenobiotic inputs.

Enantioselectivity has always been a key feature of enzymatic synthesis. In some cases, when enzymes are not strictly enantioselective, by tuning the reaction conditions it is possible to induce an enantioselective switch. A transaminase from Halomonas elongata (ω-HeWT), while generally S-selective, could be shifted towards generating the R-enantiomer at higher concentrations of amino acceptor or ionic strength, for example. Other enzymes are reported to have a similar behavior, and here we discuss some of them and their potential applications.

To support perfumers in their creation of olfactive signatures resulting in unique and instantly recognizable perfumes, there is a constant demand for the development of new odorant molecules and of novel processes for their production. Increasing the sustainability of both the molecules and the processes is a crucial activity at Givaudan. Biocatalysis has the potential to positively influence metrics applied at Givaudan that drive and measure our ambition to innovate responsibly, which is summarized in the FiveCarbon Path™. It targets an increased use of renewable carbon, carbon efficiency in synthesis, and the production of powerful and biodegradable odorant molecules while maximizing the use of upcycled carbon available from waste and side streams. This review illustrates with some examples how enzymes selected from the oxidoreductase and isomerase enzyme classes are applied at Givaudan for the preparation of odorant molecules both at laboratory and industrial scale.

The fortieth anniversary of biocatalysis started at Ciba-Geigy and later at Novartis is a great time to pause and reflect on development of science and technology in this field. Enzyme-based synthesis became a highly valued enabling tool for pharmaceutical research and development over the last decades. In this perspective we aim to discuss how the scientific approaches and trends evolved over the time and present future challenges and opportunities.

Detailed preclinical characterization of metabolites formed in vivo from candidate drug substances is mandatory prior to the initiation of clinical trials. Therefore, inexpensive and efficient methods for drug metabolite synthesis are of high importance for rapid advancement of the drug development process. A large fraction of small molecule drugs is modified by monooxygenase cytochrome P450 3A4 produced in the human liver and intestine. Therefore, this enzyme is frequently employed to catalyze metabolite synthesis in vitro, making 3A4 availability a critical requirement in early drug development. Unfortunately, the recombinant production of this enzyme in microbial hosts is notoriously difficult. Maintaining low oxygen transfer rates and the use of rich media for host cultivation are required for P450 3A4 production. However, detailed studies on the relationship between oxygen supply and P450 3A4 space-time yields are missing. We describe an improved biotechnological process for the heterologous expression of P450 3A4 together with its redox partner, cytochrome P450 reductase, in Escherichia coli. Enzyme production was most efficient under so-called “late microaerobic” growth conditions, in which the cells have just not yet made the switch to anaerobic metabolism, characterized by a limited oxygen supply leading to oxygen concentrations in the liquid phase that are far below the detection limit of standard oxygen electrodes. Furthermore, feeding the carbon source glycerol as well as controlling cellular acetate formation improved process productivity. The presented protocol resulted in the formation of functional recombinant 3A4 at concentrations up to 680 nmol L-1.

In nonribosomal peptide synthesis, condensation (C) domains are key catalytic domains that most commonly link carrier protein bound substrates to form peptides or depsipeptides. While adenylation domains have been well characterized due to their role in the selection of monomers and hence as gate keepers in nonribosomal peptide biosynthesis, C-domains have been the subject of debate as they do not have apparent “A-domain like” side chain selectivity for their acceptor substrates. To probe the selectivity and specificity of C-domains, here we report our biochemical and structural characterization of the C3-domain from the biosynthesis of the siderophore fusachelin. Our results show that this C-domain is not broadly flexible for monomers bearing significantly alternated side chains or backbones, which suggests there can be a need to consider C-domain specificity for acceptor substrates when undertaking NRPS engineering.

To develop a biocatalyst with high efficiency, stability and reusability for the degradation of organic pollutant acridine in wastewater, a novel biocatalyst named bienzyme-inorganic hybrid nanoflowers beads were successfully prepared in the present work, designated as biE-HNFs-beads. This bienzyme biocatalyst composed of bienzyme-inorganic hybrid nanoflowers and traditional beads carriers were characterized by SEM, TEM, FT-IR, XRD, and EPR. The biE-HNFs-beads showed better stability, recyclability and improved catalytic activity for acridine degradation than that of free bienzyme. Furthermore, biE-HNFs-beads could reduce the diffusion and decomposition of the intermediate H2O2 through the bienzyme cascade reactions, which not only improved the stability of the bienzyme, but also accelerated the degradation of acridine. Under optimized conditions, the degradation rate of acridine by biE-HNFs-beads could reach 99.9 %. The stability study indicated that the biE-HNFs-beads exhibited enhanced stability, reusability and storage stability. Besides, the bioconversion products analysis of acridine degraded by biE-HNFs-beads biocatalyst were identified by LC-MS, and the enzymatic degradation pathway of acridine was inferred for the first time.

Naturally occurring sinapic acid is a valuable synthon for many high-value applications (e.g. cosmetic, pharmaceutic) that can be found in numerous plants, especially Brasiccacea species (e.g. rapeseed, mustard). It has been demonstrated that alkaline extraction of matter rich in sinapic acid resulted in the conversion of sinapic acid into 6-hydroxy-5,7-dimethoxy-2-naphtanoic acid (DMNA). Although DMNA could be a potential alternative to petro-based naphtanoic acid used in polymers, untill now, no reliable (bio)synthetic procedure existed to synthesize it at the preparative-scale. Herein, starting from sinapic acid, we describe a very simple one-pot two-step chemo-enzymatic pathway to DMNA – and reaction intermediaries bislactone (BL) and thomasidioic acid (TA) – at gram scale using a simple filtration as purification step. Green metrics – Process Mass Index and EcoScale – have been determined to assess the sustainability of this new process. Finally, the antiradical activities of DMNA, BL and TA have been evaluated.

The move towards sustainable syntheses is a widespread effort which sees academia and industry developing new strategies and solutions. Flow chemistry, and in general the flow set up, with the compartmentalization of different steps in dedicated reactors, offers new possibility to integrate biocatalytic steps within a chemical cascade, often without the need to redesign the whole pathway. Here we report key examples in the field over the past few years.

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron (II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (−)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst.

The application of phenylalanine ammonia lyases (PALs) for the amination of a variety of cinnamic acids has been shown to be a cost-efficient method to produce a variety of phenylalanine analogues. Nonetheless, as many other biocatalytic tools, the process intensification, especially due to the high equivalents of ammonia needed, and the cost-efficiency of the catalyst production and use have been key points to further prove their usefulness. Here, we investigated the use of previously characterized PALs (AvPAL and PbPAL) for the amination of a series of substituted cinnamic acids. To enhance the process scalability and the reusability of the catalyst, we investigated the use of covalent immobilization onto commercially available supports, creating a heterogeneous catalyst with good recovered activity (50%) and excellent stability. The immobilized enzyme was also incorporated in continuous flow for the synthesis of 3-methoxy-phenyl alanine and 4-nitro-phenylalanine, which allowed for shorter reaction times (20 min of contact time) and excellent conversions (88% ± 4% and 89% ± 5%) respectively, which could be maintained over extended period of time, up to 24 h. This work exemplifies the advantages that the combination of enzyme catalysis with flow technologies can have not only in the reaction kinetics, but also in the productivity, catalyst reusability and downstream processing.

A visible-light-driven photocatalytic decarboxylation of palmitic acid and related fatty acids is described in this study. Remarkable decarboxylation rates have been observed with full conversion in less than 20 min. In this study, we have demonstrated that sunlight irradiation, even on cloudy days, can deliver similar results to traditional LED lamps while using much less energy and minimizing environmental impact. The findings indicate that the process of enzymatic decarboxylation could be useful for the production of different biofuels in the future.

Transglucosylation of resveratrol by the Q345F variant of sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) was extensively studied during the last decade. Indeed, Q345F is able to catalyze the synthesis of resveratrol-3-O-⍺-D-glucoside (RES-3) with yield up to 97% using a cost-effective glucosyl donor, sucrose (Kraus et al., Chemical Communications, 53(90), 12182–12184 (2017)). Despite the fact that two further products were detectable in low amounts after glucoside synthesis, they were never identified. Here, we isolated and fully characterized one of those two minor products: resveratrol-3,4′-O-⍺-D-diglucoside (RES-3,4′). This original compound had never been described before. Using bioinformatics models, we successfully explained the formation of this diglucosylated product. Indeed, with RES-3 as acceptor substrate, Q345F is able to transfer a glucosyl moiety in position 4′-OH, what had been reported as impossible in the literature. The low yield observed is due to the steric hindrance into the catalytic site between RES-3 and residues Tyr132 and Tyr344. Nevertheless, the substrate orientation in the active site is favored by stabilizing interactions. Ring A of RES-3 bearing the diol moiety is stabilized by hydrogen bonds with residues Asp50, Arg135, Asn347 and Arg399. Hydroxyl group OH-4′ shares hydrogen bonds with the catalytic residues Asp192 and Glu232. Multiple hydrophobic contacts complete the stabilization of the substrate to favor the glucosylation at position 4′. Understanding of the mechanisms allowing the glucosylation at position 4′ of resveratrol will help the development of enzymatic tools to target and control the enzymatic synthesis of original ⍺-glucosylated polyphenols with high added value and better biodisponibility.

Nitrile hydratase activity has been reported as an exciting alternative for the industrial production of a variety of compounds overwhelming its chemical counterpart; despite this, until now, a few enzymes have been thoroughly studied. Efficient expression of nitrile hydratase enzymes has been the bottleneck to explore this activity. Here, we report the cloning and expression of Rhodococcus erythropolis ATCC 4277 nitrile hydratase (α- and β-subunits) and the correspondent activator gene. Furthermore, substrate scope with whole cells of recombinant E. coli demonstrates that this Fe-type NHase could hydrate a wide range of aliphatic and aromatic nitrile with high conversion rates and moderate enantiomeric excess.