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The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzymes molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.

Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from Leuconostoc mesenteroides. These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 → 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with rule-of-three properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Repurposing the intrinsic properties of natural enzymes can offer a viable solution to current synthetic challenges through the development of novel biocatalytic processes. Although amino acid racemases are ubiquitous in living organisms, an amine racemase (AR) has not yet been discovered despite its synthetic potential for producing chiral amines. Here, we report the creation of an AR based on the serendipitous discovery that amine transaminases (ATAs) can perform stereoinversion of 2-aminobutane. Kinetic modeling revealed that the unexpected off-pathway activity results from stereochemically promiscuous futile cycles due to incomplete stereoselectivity for 2-aminobutane. This finding motivated us to engineer an S-selective ATA through in silico alanine scanning and empirical combinatorial mutations, creating an AR with broad substrate specificity. The resulting AR, carrying double point mutations, enables the racemization of both enantiomers of diverse chiral amines in the presence of a cognate ketone. This strategy may be generally applicable to a wide range of transaminases, paving the way for the development of new-to-nature racemases.

Enzymatic biocatalysis typically generates less waste, uses less water, and minimizes energy consumption compared to traditional chemical methods. Efficient, cell-free biosynthesis relies on the reuse of its valuable biocatalysts. Immobilization of enzymes on solid supports, such as enzyme carrier resins (ECRs), offers a reliable and widely deployed approach to maximize enzyme turnover in cell-free biosynthesis. We focus on two major bottlenecks associated with optimizing cell-free biocatalysis. First, we apply our lab's 3D-printed labware to screen ECRs in 96-well mini-reactors to optimize enzyme immobilization conditions. Second, we introduce inline infrared spectroscopy to monitor bioreactor output and maximize enzyme productivity. Urease provides a model system for examining immobilization conditions and continuous assessment of biocatalyst performance. As required for the high substrate concentrations to improve process efficiency and minimize waste, urease was studied in unusually high concentrations of its substrate – molar concentrations of urea. The optimized reactor processed 3.24 L of 4.00 M urea at an average volumetric productivity of 13 g ⋅ L−1 ⋅ h−1 over 18 h and achieved an estimated productivity number of >17.4 kg urea processed per g of immobilized urease Type-IX. This workflow can be generalized to most biocatalytic processes and could accelerate adoption of cell-free biosynthesis for greater chemical sustainability.

The effective design of combinatorial libraries to balance fitness and diversity facilitates the engineering of useful enzyme functions, particularly those that are poorly characterized or unknown in biology. We introduce MODIFY, a machine learning (ML) algorithm that learns from natural protein sequences to infer evolutionarily plausible mutations and predict enzyme fitness. MODIFY co-optimizes predicted fitness and sequence diversity of starting libraries, prioritizing high-fitness variants while ensuring broad sequence coverage. In silico evaluation shows that MODIFY outperforms state-of-the-art unsupervised methods in zero-shot fitness prediction and enables ML-guided directed evolution with enhanced efficiency. Using MODIFY, we engineer generalist biocatalysts derived from a thermostable cytochrome c to achieve enantioselective C-B and C-Si bond formation via a new-to-nature carbene transfer mechanism, leading to biocatalysts six mutations away from previously developed enzymes while exhibiting superior or comparable activities. These results demonstrate MODIFY's potential in solving challenging enzyme engineering problems beyond the reach of classic directed evolution.

Chemically inert hydrocarbons are the primary feedstocks used in the petrochemical industry and can be converted into more intricate and valuable chemicals. However, two major challenges impede this conversion process: selective activation of C−H bonds in hydrocarbons and systematic functionalization required to synthesize complex structures. To address these issues, we developed a multi‐enzyme cascade conversion system based on internal cofactor and H2O2 recycling to achieve the one‐pot deep conversion from heptane to chiral (S)‐2‐aminoheptanoic acid under mild conditions. First, a hydrogen‐borrowing‐cycle‐based NADH regeneration method and H2O2in situ generation and consumption strategy were applied to realize selective C−H bond oxyfunctionalization, converting heptane into 2‐hydroxyheptanoic acid. Integrating subsequent reductive amination driven by the second hydrogen‐borrowing cycle, (S)‐2‐aminoheptanoic acid was finally accumulated at 4.57 mM with eep>99 %. Hexane, octane, 2‐methylheptane, and butylbenzene were also successfully converted into the corresponding chiral amino acids with eep>99 %. Overall, the conversion system employed internal cofactor and H2O2 recycling, with O2 as the oxidant and ammonium as the amination reagent to fulfill the enzymatic conversion from chemically inert hydrocarbons into chiral amino acids under environmentally friendly conditions, which is a highly challenging transformation in traditional organic synthesis. [ABSTRACT FROM AUTHOR]

Triterpenoids have wide applications in the pharmaceutical and agricultural industries. The glycosylation of triterpenoids catalyzed by UDP‐glycosyltransferases (UGTs) is a crucial method for producing valuable derivatives with enhanced functions. However, only a few UDP‐glucosyltransferases have been reported to synthesize the rare triterpenoids with linear‐chain trisaccharide at C3−OH. This study revealed that the UGT91H subfamily primarily contributed to the 2"‐O‐glycosylation of triterpenoids with high regioselectivity, then the substrate scope was further expanded by ancestral sequence reconstruction (ASR). With ancestral enzyme UGT91H_A1 as a model, the sequence‐structure‐function relationship was explored. A RTAS loop (R212/T213/A214/S215) was identified to affect the substrate specificity of UGT91H_A1. Transferring this RTAS loop to the corresponding position of UGT91H enzymes successfully expanded their substrate spectra. The functional role of RTAS loop was further elucidated by molecular dynamics simulation and quantum mechanical computation. UGT91H_A1 was applied to the low‐cost synthesis of terpenoid rhamnosides with a linear trisaccharide in combining with a self‐sufficient UDP‐rhamnose regeneration system. Finally, we developed a phylogeny‐based platform to efficiently mining new UGT91Hs from plant genomic data. This study provided robust biocatalysts for synthesizing various triterpenoid glycosides with a linear trisaccharide and demonstrated ASR as an efficient tool in engineering the function of UDP‐glycosyltransferases. [ABSTRACT FROM AUTHOR]

Alpha‐ketoglutarate‐dependent dioxygenases (αKGDs) have recently emerged as useful biocatalysts for C−H oxidation and functionalization. In this work, we characterized a new αKGD from aculene biosynthesis, AneA, which displays broad promiscuity toward a number of substrates with different ring systems. Unexpectedly, AneA was found to be capable of both desaturation and hydroxylation and require an amino ester motif on its substrate for productive catalysis. Insights gathered from the functional characterization and substrate‐activity profiling of AneA enabled the development of a chemoenzymatic strategy toward several complex sesquiterpenoids. [ABSTRACT FROM AUTHOR]

Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His 6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His 6 -tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N -His 6 -ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL−1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His 6 -tagged ATA constructs. The highest turnover number within the indicated time was 7.23·106, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation. [Display omitted] • A microreactor with nonwoven nanofiber membrane was developed. • Nanofibers with various functional groups were tested for enzyme immobilization. • His6-tagged amine transaminase was immobilized on membranes with metal ions. • One-step purification and immobilization with very high enzyme loads was reached. • Long-term stability of microreactor system and high turnover numbers were obtained. [ABSTRACT FROM AUTHOR]

Autocatalysis, a self-sustained replication process where at least one of the products functions as a catalyst, plays a pivotal role in life's evolution, from genome duplication to the emergence of autocatalytic subnetworks in cell division and metabolism. Leveraging their programmability, controllability, and rich functionalities, DNA molecules have become a cornerstone for engineering autocatalytic circuits, driving diverse technological applications. In this tutorial review, we offer a comprehensive survey of recent advances in engineering autocatalytic DNA circuits and their practical implementations. We delve into the fundamental principles underlying the construction of these circuits, highlighting their reliance on DNAzyme biocatalysis, enzymatic catalysis, and dynamic hybridization assembly. The discussed autocatalytic DNA circuitry techniques have revolutionized ultrasensitive sensing of biologically significant molecules, encompassing genomic DNAs, RNAs, viruses, and proteins. Furthermore, the amplicons produced by these circuits serve as building blocks for higher-order DNA nanostructures, facilitating biomimetic behaviors such as high-performance intracellular bioimaging and precise algorithmic assembly. We summarize these applications and extensively address the current challenges, potential solutions, and future trajectories of autocatalytic DNA circuits. This review promises novel insights into the advancement and practical utilization of autocatalytic DNA circuits across bioanalysis, biomedicine, and biomimetics. [ABSTRACT FROM AUTHOR]

Levulinic acid, which can be obtained from biomass, has sparked great interest as a biologically-based chemical building block with wide versatility and potential. Its esterification with alcohols of different chain lengths is a promising valorization process for obtaining esters with various applications in the areas of biofuels/biolubricants, food and cosmetics, among others. In this work, the enzymatic esterification of levulinic acid and 1-octanol using a biocatalyst derived from Araujia sericifera latex was studied in systems with and without solvent. The influence of the molar ratio between alcohol and acid (ranging from 2:1–1:9), the biocatalyst loading (between 7.5 % and 17.5 % relative to the acid), the volume of n -heptane used as reaction solvent (from 0 to 4 ml), and the reaction time (6 hours) were investigated. The activity and stability of the biocatalyst in successive uses were also analyzed. A conversion of 49 % was achieved when the reaction was carried out in a solvent-free system, using an alcohol/acid molar ratio of 1:7 and after 5 h of reaction. On the other hand, the conversion was 65.1 % when the reaction was conducted in a system containing 1 ml of n -heptane as solvent, an alcohol/acid molar ratio of 1:8, and 5 h of reaction. In both cases, a temperature as low as 30 °C and an agitation speed of 300 RPM were used. • Enzymatic route for levulinic acid esterification using biocatalyst. • Araujia sericifera latex-derived biocatalyst demonstrates high activity. • Achieved 65.1 % conversion of levulinic acid in the synthesis of octyl levulinate. • Solvent-free and n-heptane solvent systems investigated. • Studied reaction parameters for optimal conversion. [ABSTRACT FROM AUTHOR]

β‐Branched chiral amines with contiguous stereocenters are valuable building blocks for preparing various biologically active molecules. However, their asymmetric synthesis remains challenging. Herein, we report a highly diastereo‐ and enantioselective biocatalytic approach for preparing a broad range of β‐branched chiral amines starting from their corresponding racemic ketones. This involves a dynamic kinetic resolution‐asymmetric reductive amination process catalyzed using only an imine reductase. Four rounds of protein engineering endowed wild‐type PocIRED with higher reactivity, better stereoselectivity, and a broader substrate scope. Using the engineered enzyme, various chiral amine products were synthesized with up to >99.9 % ee, >99 : 1 dr, and >99 % conversion. The practicability of the developed biocatalytic method was confirmed by producing a key intermediate of tofacitinib in 74 % yield, >99.9 % ee, and 98 : 2 dr at a challenging substrate loading of 110 g L−1. Our study provides a highly capable imine reductase and a protocol for developing an efficient biocatalytic dynamic kinetic resolution‐asymmetric reductive amination reaction system. [ABSTRACT FROM AUTHOR]

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface‐exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high‐resolution structural data of flexible surface‐exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin‐based MIP that recognizes recombinant proteins via imprinting of the widely used Strep‐tag II affinity peptide (Strep‐MIP). Electrochemistry, surface‐sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep‐MIP electrosynthesis. The functionality of this novel platform was verified with two Strep‐tagged enzymes: an O2‐tolerant [NiFe]‐hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep‐MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology. [ABSTRACT FROM AUTHOR]

The biocatalytic asymmetric reduction of prochiral ketones offers a promising approach for producing optically active secondary alcohols. Alcohol dehydrogenases (ADHs) are enzymes that facilitate the reversible conversion of prochiral ketones into their corresponding optically active secondary alcohols, and thus are pivotal for this process. Most ADHs adhere to Prelog's rule in determining the stereopreference for the asymmetric reduction of prochiral ketones. This study focuses on the ΔP84/A85G mutation of TeSADH, demonstrating its capability to reduce aryl‐ring‐containing ketones to their corresponding alcohols in anti‐Prelog mode with high stereoselectivities. The study also highlights the crucial role of P84 in switching the stereopreference of TeSADH in the asymmetric reduction of aryl‐ring‐containing ketones. Furthermore, this mutant exhibits a broad substrate scope, including substrates accepted by the previously reported W110 mutants of TeSADH with opposite stereopreference. The ability to create mutants of the same enzyme with opposite stereopreferences presents intriguing opportunities for fascinating transformations, such as bienzymatic racemization, which can be utilized in dynamic kinetic resolution, and stereoinversion using two enantiocomplementary mutants of the same enzyme. [ABSTRACT FROM AUTHOR]

Formate dehydrogenases (FDHs) catalyze the oxidation of formate to CO2 while reducing NAD(P)+ to NAD(P)H and are classified into two main classes: metal‐dependent (Mo‐ or W‐containing) and metal‐independent FDHs. The latter are oxygen‐tolerant and relevant as a cofactor regeneration system for various bioprocesses and gained more and more attention due to their ability to catalyze the reverse CO2 reduction. This review gives an overview of metal‐independent FDHs, the recent advances made in this field, and their relevance for future applications in biocatalysis. This includes the exploitation of novel FDHs which have altered co‐substrate specificity as well as enzyme engineering approaches to improve process stability and general performance. [ABSTRACT FROM AUTHOR]

Structural diversity of diterpenes is mediated by the enigmatic family of diterpene synthases. The overall enzymatic contribution hereby lies in a carefully concerted chemistry of highly reactive carbocation intermediates mainly guided by aromatic and polar amino acid side chains and the pyrophosphate cofactor. To date several studies aimed to shed light on the mechanism underlining terpene synthases chemistry. Specifically, the diterpene synthase CotB2 serves as model enzyme for detailed mutagenesis studies. Here we investigate the catalytic mechanism of CotB2 variant V80L in a holistic, biochemical, structural, and computational biology approach. We were able to identify an altered product profile compared to CotB2WT for the substrates geranylgeranyl diphosphate and farnesyl diphosphate. Moreover, we solved the crystal structure, and shed further light on terpene synthase chemistry by modelling of the substrate and intermediate binding. [ABSTRACT FROM AUTHOR]

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselectivity and under milder conditions than traditional organochemical approaches. To expand the repertoire of well characterized ether synthases, we herein report on a promiscuous archaeal prenyltransferase from the scarcely researched family of geranylgeranylglyceryl phosphate synthases (GGGPSs or G3PSs). We show that the ultrastable Archaeoglobus fulgidus G3PS makes various (E)‐ and (Z)‐configured prenyl glycerol ethers from the corresponding pyrophosphates while exerting perfect control over the configuration at the glycerol unit. Based on experimental and computational data, we propose a mechanism for this enzyme which involves an intermediary prenyl carbocation equivalent. As such, this study provides the fundamental understanding and methods to introduce G3PSs into the biocatalytic alkylation toolbox. [ABSTRACT FROM AUTHOR]

Transitioning from both the direct and indirect use of fossil fuels to the renewable and sustainable resources of the near future demands a focal shift in catalysis research – from investigating catalytic reactions in isolation to developing coupled reactions for modern chemical value chains. In this Perspective, we discuss the status and emerging prospects of coupled catalytic reactions across various scales and provide key examples. Besides being a sustainable and essential alternative to current fossil‐based processes, the coupling of catalytic reactions offers novel and scalable pathways to value‐added chemicals. By emphasizing the specific requirements and challenges arising from coupled reactions, we aim to identify and underscore research needs that are critical to expedite their development and to fully unlock their potential for chemical and fuel production. [ABSTRACT FROM AUTHOR]

Introduction: Deep 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. Methods: This 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 discussion: Considering 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. [ABSTRACT FROM AUTHOR]

Light-responsive Pickering interfacial biocatalysis (LPIB) is desirable because of its convenient catalyst recovery, product separation and clean external stimuli trigger. However, the recyclability of LPIB is still limited due to its enzyme inactivation. Herein, lipase-entrapped colloidosomes as particulate emulsifiers and biocatalysts were fabricated using the one-step co-assembly of lipases and TiO2 nanoparticles (with light-responsive wettability). The obtained LPIB exhibited a 4.31-fold enhancement of enzyme activity compared with the traditional biphasic system. Impressively, the LPIB maintained nearly 90% of its initial enzyme activity even after 20 cycles, which is the highest among the currently reported LPIBs. It is attributed that the TiO2 nanoparticle layer on the colloidosome surface reflects UV light, thus protecting the enzyme from photodegradation. This green platform can be widely applied to construct recyclable and efficient biphasic biocatalytic systems. [ABSTRACT FROM AUTHOR]

An enzymatic method for the selective hydroxylation of phenols using a peroxygenase from Aspergillus brasiliensis (AbrUPO) is reported. A broad range of phenolic starting materials can be selectively transformed into the corresponding hydroquinones. Semi‐preparative syntheses of several hydroquinones were realised without further optimization pointing out the applicability of this enzyme as biocatalyst. [ABSTRACT FROM AUTHOR]

Cells exhibit diverse structural formations such as biofilms and spores, enabling them to acquire novel functionalities. Many of these structures display biomacromolecules, including enzymes, tethered to cell walls to support various extracellular processes. Alternatively, encapsulating single cells with polymer coatings offers a strategy that circumvents the need for genetic engineering while imparting artificial functionalities to cells. Here, a universal method is presented for encapsulating single gram‐negative microbes with polymeric coatings based on the ancestral gall ink formed by tannic acid‐iron complexes. As a result, synthetic spores are achieved that selectively bind His‐tagged enzymes through the formation of unprecedented galloyl/imidazole‐Fe2+ complexes via ligand substitution demonstrated by density functional theory. These synthetic spores with a thickness of 41.5 ± 4.2 nm and a stiffness of 6.0 ± 3.5 GPa serve as biocatalytic materials for the one‐pot oxidative amination of diols into amino alcohols, facilitated by the cooperative catalysis between intracellular endogenous or recombinant oxidoreductases, and an extracellular transaminase from Pseudomonas fluorescens displayed at the spore surface. These spores maintain their performance in three consecutive batch cycles. Integrating isolated enzymes onto the surface of engineered microbes coated with polymers offers novel opportunities for synthetic biology, advancing the efficiency of biosynthetic cascades in solid‐state environments. [ABSTRACT FROM AUTHOR]

Nanoporous structured silica materials, namely f‐SiO2, SBA‐15, IITM‐41, and MCM‐41 were employed as the matrices for immobilizing endoxylanase by adsorption method. The equilibrium kinetics, activation energy, and thermodynamic parameters associated with endoxylanase adsorption were investigated. Our findings revealed a two‐phase adsorption mechanism: an initial phase featuring rapid adsorption rates, succeeded by a slower phase wherein adsorption gradually progressed until equilibrium was attained. Analysis of the adsorption kinetic data indicated a better fit with the pseudo‐second‐order model, suggesting chemisorption and highlighting its temperature dependency. The calculated activation energy (Ea) values fell within the range of physisorption, indicating the involvement of both types of adsorption processes. Thermodynamic assessments confirmed that the adsorption reactions were spontaneous, feasible, and endothermic. Notably, the immobilization process did not change the optimum pH of the enzyme, while the optimum temperature shifted slightly. Furthermore, immobilized enzymes show a higher reaction rate (Vmax) than the soluble XynC. All immobilized xylanases produced xylobiose (X2) to xylohexose (X6) by hydrolyzing the xylan substrate. Recycling studies showed that up to 80 % of the yield was retained after seven cycles of reuse. Our study demonstrates the potential of nanostructured silica as an effective immobilization matrix for enzymes of industrial significance. [ABSTRACT FROM AUTHOR]

One of the main goals of contemporary biotechnology has been the development of novel immobilized enzyme formulations. In the present study, the industrially important β‐galactosidase was trapped in a polyvinyl alcohol (PVA) gel to immobilize it. The optimization of immobilization method and characterization of the immobilized enzyme were studied. The results were compared with free enzymes. The results indicate that the optimal temperature range for the enzyme to be at following immobilization is between 40°C and 50°C. At pH 7, the optimal pH, the activity increased, the Vmax value increased from 1.936 to 2.495 U mg‒1, and the Km value decreased from 4.861 to 0.982 mM. Depending on how stable the immobilized enzyme when stored, β‐galactosidases immobilized on PVA gels showed 52.87% activity at the end of the seventh week and 58.86% activity at the end of the fifth week. Their initial activity subsided after three reuses. The final result was 66%. Therefore, one may argue that it increases the catalytic effect of the enzyme. As a result, it has been found that immobilized β‐galactosidase has more potent enzymatic properties than free β‐galactosidase, which may make it more advantageous for industrial processes. Further studies could delve deeper into the mechanistic aspects of the immobilization process in an effort to improve optimization and tailor the immobilized enzyme to specific industrial needs. [ABSTRACT FROM AUTHOR]

Despite considerable research efforts, lipase catalysis in a fluid milk system with aqueous multicomponent mixtures containing multiple microphases remains challenging. Pickering interfacial biocatalysis (PIB) platforms are typically fabricated with organic solvents or lipids and water. Whether a PIB with excellent catalytic performance can be constructed in complex milk mixtures remains unknown. Here, we challenged PIB with skim milk, a small amount of flaxseed oil, and phytosterols as a model system for transesterification and lipolysis to enhance quality and flavor. The amino-modified mesoporous silica spheres were employed as an emulsifier and carrier of lipase AYS. The conversion of phytosterol esters reached 75.5% at 1.5 h in prepared phytosterol ester-fortified milk with a content of 1.0 g/100 mL. The relative conversion rate remained above 70% after 6 cycles. In addition, the fortified milk showed an intensified and favorable effect on sensory traits through volatile flavor composition analysis. The findings provide a versatile alternative for PIB applications in complex environments, i.e., milk, which might inspire a new bioprocess strategy for dairy products. The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. [Display omitted] [ABSTRACT FROM AUTHOR]

The use of enzymes or microorganisms for the synthesis of aroma compounds is a valuable strategy to meet the growing consumers' demand for natural products. Short-to-medium chain esters are widely used in the industry to provide fruity and floral flavors and fragrances to foods, cosmetics and pharmaceutical products. We present an extensive bibliometric analysis of the literature between 2000 and 2023 dealing with the biotechnological synthesis of aroma esters based on the review of 644 scientific documents retrieved from the Scopus database. The VOSviewer software was applied to analyze the bibliometric indicators of quantity and quality. The results showed the strategic distribution map of the different journals and countries where research groups developed this topic. It became evident that the research focused mainly on isoamyl acetate, 2-phenylethyl acetate and other acetate esters. We review recent progress regarding native microorganisms and recombinant enzymes that produce esters by esterification, Baeyer-Villiger oxidation, alcohol acylation and hemiacetal dehydrogenation. As a concluding remark, it is worth mentioning that lipases are the most frequently reported biocatalysts for the synthesis of aroma esters but the use of other enzymes has grown significantly in the last years, especially as part of cascades or modified metabolic pathways. • Short-to-medium chain esters are molecules with fruity and floral fragrances. • Aroma esters are used in foods, cosmetics, cleaning and pharmaceutical products. • Lipases, acyltransferases and Baeyer-Villiger monooxygenases can produce esters. • Biotechnological synthesis of aroma esters has the potential to become competitive. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of polyethylene terephthalate (PET) for surface modification of polyester fibers has attracted considerable research attention in recent years. However, the high crystallinity of polyester fibers, combined with limited enzyme activity and stability, challenges the surface modification study of enzymes. Deep eutectic solvents (DES) can create a favorable environment for proteins and are a new generation of biodegradable solvents. Few studies have been conducted on the use of DES to enhance enzymatic degradation. Therefore, we attempted to hydrolyze PET with DES-activated enzymes to increase the hydrolysis yield and thus improve PET modification. Using betaine and choline chloride as hydrogen bond acceptors and polyols as hydrogen bond donors, we investigated the effects of DES type, molar ratio, and concentration on enzymatic hydrolysis. Humicola insolens cutinase (HiC) was used as the biocatalyst for PET fabric hydrolysis, the role of DES as an additive in improving the yield of enzymatically hydrolyzed PET fabric was investigated. The results showed that under the conditions of an enzyme concentration of 6.5% v/v (volume of enzyme on the total volume), a temperature of 60 °C, and a reaction time of 72 h, the low concentration (20% v/v) of DES (betaine: sorbitol; 1:2 molar ratio) increased the hydrolysis yield by more than 1.5 times. Enzymatic hydrolysis in DES aqueous solution (betaine: sorbitol; 1:2 molar ratio) and DES single-component aqueous solution (betaine, sorbitol without synthesis of DES) indicated that the increase in hydrolysis yield was mainly due to the formation of hydrogen bonds between betaine and sorbitol, rather than the superposition of individual components. Further analysis revealed that HiC exhibited high relative enzyme activity and stability at low DES concentrations. Additionally, CD spectroscopy and fluorescence spectroscopy analyses demonstrated the effective preservation of HiC structure by DES. Our work provides insights into the development of efficient and sustainable methods to enhance HiC hydrolysis of PET fabric, unlocking new opportunities and potential for the comprehensive utilization of DES in the bio-modification of PET fabric. [ABSTRACT FROM AUTHOR]

Encapsulating natural enzymes in metal—organic frameworks (MOFs) can maintain the original biological functions of enzymes in harsh environments. However, the nature of interfacial interactions between a MOF and enzyme is currently unclear, rendering effective regulation of the biocatalytic activity of the enzyme@MOF composite difficult. Differences in the hydrophilicity of MOF carriers are closely related to the conformational changes and catalytic properties of the enzyme. In this study, the catalytic activity, stability, and conformational changes of alkaline phosphatase (ALP) encapsulated in hydrophilic zeolite imidazolate framework-90 (ZIF-90) and hydrophobic ZIF-8 were systematically investigated using experimental methods and molecular dynamics simulations. The results demonstrated that hydrophilic ZIF-90-encapsulated ALP exhibited superior stability and was 2.22-fold more retained catalytically active than hydrophobic ALP@ZIF-8 after 20 cycles of utilization. Moreover, the hydrophilic interface provided by ZIF-90 effectively regulated the structure of ALP to maintain the optimal catalytic conformation of its active center. The practical application of highly bioactive ALP@ZIF-90 was demonstrated by employing it in a self-calibrated colorimetric/fluorescence dual-mode sensing method for the efficient, reliable, and accurate detection of methyl paraoxon. This study provides new insights for improving enzyme immobilization strategies and promoting the rapid development of enzyme@MOF composites for catalytic and sensing applications. [ABSTRACT FROM AUTHOR]

The field of biocatalysis is envisioned as an important contributor to the development of bioprocesses producing molecules that can replace those derived from oil, while maintaining the durability and resistance characteristics offered by petroleum-based materials. In the search for substitutes for petroleum derivatives, the compounds belonging to the furan platform appear among the best known due to their reactivity, as they present a mono- (Furfural) or disubstituted (5-HMF) furan ring in their structure, identifying them among the molecules with the greatest applications in the synthesis of new fuels and polymer precursors. In this context, taking advantage of the metabolic diversity of microorganisms, biocatalytic and fermentative methods for the bioconversion of furans have been explored, proposing processes of lower cost and low environmental impact. This review presents the oxidation and reduction products of furfural and 5-HMF obtained by biological processes, using cells or enzymes. [ABSTRACT FROM AUTHOR]

A growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97 % in 6 h for formation of N‐benzylfuranamide as the model reaction. We demonstrate PestE's potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L F289A and F33L. F33L F289A increased conversion of N‐benzylfuranamide by 1.2‐fold, and F33L gave a 4‐fold increase in conversion to tamibarotene. [ABSTRACT FROM AUTHOR]

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min−1 mM−1. Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds. Key points: • Identification and characterization of two novel bacterial aryl-alcohol oxidases • Crystal structures reveal wide-open active-site pockets, impacting substrate scope • Total turnover numbers and cell-free extracts demonstrate the synthetic potential [ABSTRACT FROM AUTHOR]

AbstractIn this study, biocatalytic transesterification reaction using Novozyme 435® (N435) lipase was employed to enhance the hydrophobicity of esculin, aiming to improve its solubility for commercial applications and enhance its bioactivity and oral viability. The acylation reaction of esculin with vinyl acetate was conducted at 60 °C and 200 rpm for 24 h. After chromatographic and spectroscopic analysis, two products were identified: the first one was monoacylated at the 6′-OH position of the glucosyl moiety of esculin (TR: 10.3 min and m/z 382.93 [M + H]+), and the second one was diacylated at the 6′-OH and 3′-OH positions (TR: 13.0 min and m/z 424.93 [M + H]+). The latter was the major product, with a conversion rate of 53.550 ± 0.368%, while the monoacetylated one showed 8.715 ± 0.064%. Both products were isolated by high-speed counter-current chromatography (HSCCC) using a two-phase system HEMWat 1:9:1:9 and characterized by NMR. In this way, these results improve the practical application of esculin, through the obtention of esculin mono and diacetates by fast and efficient biocatalysis reaction. [ABSTRACT FROM AUTHOR]

Nanostructured materials represent promising substrates for biocatalyst immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme‐based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on enzymatic CNCs and on their TEMPO oxidized derivatives (TO‐CNCs), comparing immobilization yields, material properties, and enzymatic activities. The materials were characterized by X‐ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR‐FTIR), bicinchoninic acid (BCA) assay, field‐emission scanning electron microscopy (FE‐SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO‐CNCs, based on the success of covalent bonding and on the stability of the isolated bioconjugates. [ABSTRACT FROM AUTHOR]

Protein photocatalysts provide a modular platform to access new reaction pathways and affect product outcomes, but their use in polymer synthesis is limited because co‐catalysts and/or co‐reductants are required to complete catalytic cycles. Herein, we report using zinc myoglobin (ZnMb), an inherently photoactive protein, to mediate photoinduced electron/energy transfer (PET) reversible addition‐fragmentation chain transfer (RAFT) polymerizations. Using ZnMb as the sole reagent for catalysis, photomediated polymerizations of N,N‐dimethylacrylamide in PBS were achieved with predictable molecular weights, dispersity values approaching 1.1, and high chain‐end fidelity. We found that initial apparent rate constants of polymerization increased from 4.6×10–5 s−1 for zinc mesoporpyhrin IX (ZnMIX) to 6.5×10–5 s−1 when ZnMIX was incorporated into myoglobin to yield ZnMb, indicating that the protein binding site enhanced catalytic activity. Chain extension reactions comparing ZnMb‐mediated RAFT polymerizations to thermally‐initiated RAFT polymerizations showed minimal differences in block copolymer molecular weights and dispersities. This work enables studies to elucidate how protein modifications (e.g., secondary structure folding, site‐directed mutagenesis, directed evolution) can be used to modulate polymerization outcomes (e.g., selective monomer additions towards sequence control, tacticity control, molar mass distributions). [ABSTRACT FROM AUTHOR]

Many industrial processes are performed using harmful chemicals. The current technical synthesis of N-acyl-amino acids relies on acyl chlorides, which are typically obtained from phosgene chemistry. A greener alternative is the application of whole cells or enzymes to carry out synthesis in an environmentally friendly manner. Aminoacylases belong to the hydrolase family and the resolution of racemic mixtures of N-acetyl-amino acids is a well-known industrial process. Several new enzymes accepting long-chain fatty acids as substrates were discovered in recent years. This article reviews the synthetic potential of aminoacylases to produce biobased N-acyl-amino acid surfactants. The focus lays on a survey of the different types of aminoacylases available for synthesis and their reaction products. The enzymes are categorized according to their protein family classification and their biochemical characteristics including substrate spectra, reaction optima and process stability, both in hydrolysis and under process conditions suitable for synthesis. Finally, the benefits and future challenges of enzymatic N-acyl-amino acid synthesis with aminoacylases will be discussed. Key points: • Enzymatic synthesis of N-acyl-amino acids, biobased surfactants by aminoacylases. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Plastics are ubiquitous in our ecosystems, and microplastic accumulation in the environment is an emerging global health concern. Since available recycling technologies are not economically competitive with primary plastic production, global use is expected to reach 1231 megatons by 2060, with 493 megatons leeching into the environment each year. To identify new nylon‐recycling biotechnologies, targeted genome mining was used to identify thermostable enzymes capable of degrading polyamides. Here, we describe the characterization of a novel protein sourced from Thermovenabulum gondwanense: TvgC. TvgC is extremely stable, exhibiting a melting temperature of 93 °C and no detectable losses in hydrolytic activity after one week at 60 °C. While nylonases primarily process nylon‐6, TvgC catalysed the degradation of both nylon‐6 and nylon‐6,6 films, which are considerably more difficult to degrade. Finally, conversion experiments demonstrate that TvgC achieves a 1.2 wt % conversion of nylon‐6 film, comparable to that of the most highly engineered nylonases. This novel hyperthermostable protein represents an excellent starting point for future engineering of increasingly efficient nylonases. [ABSTRACT FROM AUTHOR]

In this study, we report an environment‐friendly protocol by integrating the nonnatural catalytic activity of lipase with electrocatalysis for synthesizing C‐3 alkylated oxindoles, which are part of many natural and pharmaceuticals products. Gratifyingly, Candida antarctica lipase B (CALB) is found to be highly active and regioselective for catalyzing the nonnatural C‐3 alkylation reaction at indole when combined with an electrochemical C‐2 oxidation process in the same vessels. Further, the generality and feasibility of the developed protocol are shown by employing several functional groups on the indole moiety and obtaining the desired products in moderate to good yield. Besides, the control experiments are set up along with the molecular docking studies to substantiate the role of the active site of Candida antarctica lipase B (CALB) in carrying out the regioselective C‐3 alkylation reaction. In addition, control experiments and cyclic voltammetry are performed to get insight into the electrochemical C‐2 oxidation process and as a result, a plausible mechanism for the integrated process is presented. [ABSTRACT FROM AUTHOR]

Biocatalysis has emerged in recent decades toward a widely applied catalysis technology in the chemical industry. In particular the fine chemicals and pharmaceuticals industries benefit from the advantages of biocatalysis, which made its way to a dominating industrial core technology for manufacturing chiral molecules. However, often biocatalysis is still to a certain extent away from having solved all challenges being needed for a "perfect industrial process technology". Among existing challenges are, e.g., stability under process conditions and easy separation of the catalyst from the reaction mixture with the additional option of recyclability. Here metal‐organic framework (MOF) structures offer unique advantages, which can be beneficial for biocatalysis. A particular valuable option is integration of enzymes into MOF‐subunits, thus having a potential positive impact on stability (by reducing the tendency of unfolding) and enabling compartmentalization as a beneficial strategy in, e.g., chemoenzymatic synthesis. In this "Perspective"‐article, first the state of the art in biocatalysis is briefly summarized together with current challenges. Then, a short overview about current research achievements in "merging" MOF chemistry and biocatalysis is given as well as an outlook written from a biocatalysis practitioner's view, how MOF‐enzyme hybrid systems can play a major role in future process development to overcome existing hurdles of enzymatic catalysis. [ABSTRACT FROM AUTHOR]

Macrolactonization is a challenging process where high dilution and temperatures can extend reaction times and promote reagent degradation. Biocatalysis is a versatile strategy for synthesis but not traditionally associated within the toolbox of organic chemists for macrocyclization. Macrolactonization has been investigated using modern methods employing both catalysis and stoichiometric activation strategies on 20 different substrates with differing ring sizes and types (cyclophanes, macrolactones, macrodiolides) and structural features at the reaction site (central vs. planar chirality; primary vs. secondary alcohols). The data demonstrates that of all protocols examined, the biocatalytic route was superior, providing the highest average yields across all classes of macrocycles studied. From the stoichiometric activation strategies investigated, the Yamaguchi macrolactonization was the most versatile in terms of ring size and nature. Despite the advantages of biocatalytic macrolactonization, advances in developing supported, versatile non-enantioselective lipases would actually represent a useful tool in molecular synthesis. [ABSTRACT FROM AUTHOR]

Xylanase is a natural and high-efficiency catalyst for the degradation of xylan to reducing sugar. However, the catalytic performance of xylanase is greatly limited by its poor activity and stability under harsh conditions. To overcome this issue, covalent immobilization of xylanase on a magnetic porous MOF material with a core–shell structure, UIO-66-NH2 carrier, was conducted to improve the catalytic activity and stability of xylanase. Several tools were employed to monitor the preparation and immobilization processes, magnetic properties, and structural/chemical characteristics of the biocatalyst. It was found that 77.9% of the activity of xylanase was retained after immobilization. Moreover, the thermal stability in terms of half-life of the immobilized enzyme was three times higher than that of the free form; the immobilized enzyme also showed higher catalytic efficiency for hydrolysis of xylan. Furthermore, stable activity at higher temperatures and a wider pH range was achieved for the immobilized xylanase compared with the free catalyst. Notably, the biocatalyst exhibited both excellent storage stability and could be easily separated using a magnet, which is conducive to its reusability. Moreover, nearly 70% of its initial activity was retained after seven recycles for the hydrolysis of corn straw. These features indicate that the present immobilization method is promising for the industrial application of enzymes. [ABSTRACT FROM AUTHOR]

Aiming at sustainable solutions suitable for industrial‐scale catalysis catalytic phase containing lipase and deep eutectic solvent (DES) was designed. To achieve this, both physical and chemical immobilizations of lipases were performed on the surface of silica and carbon materials. The catalytic activity of developed biosystem was tested in biotransformation of α‐angelica lactone into butyl levulinate at 60 °C. The best results were achieved for Candida antarctica lipase B adsorbed on fumed silica suspended in choline chloride: glycerol (1:2) with the addition of 20 wt% of water. Under these conditions 99.9% conversion of α‐angelica lactone and 100% selectivity to butyl levulinate were obtained after 45 min. The biocatalytic system maintained its activity for up to five consecutive reaction cycles with full conversion of lactone to ester. The heterogenization of the enzyme allowed the biocatalyst to be integrated into the bulk of the DES, which includes essential water. This combination resulted in the formation of a stable and easy‐to‐operate catalytic phase where reagents formed a second organic phase. The integration of cost‐effective biocatalyst with process efficiency provides a greener alternative with significant potential for industrial use. [ABSTRACT FROM AUTHOR]

Bioactive dimeric (pre‐)anthraquinones are ubiquitous in nature and are found in bacteria, fungi, insects, and plants. Their biosynthesis via oxidative phenol coupling (OPC) is catalyzed by cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well‐known, the respective enzymes in mushroom‐forming fungi (Basidiomycota) are unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A1 and B1 of the mushroom Cortinarius odorifer. The biosynthesis of these unsymmetrically 7,10'‐homo‐coupled dihydroanthracenones was heterologously reconstituted in the mold Aspergillus niger. Methylation of the parental monomer atrochrysone to its 6‐O‐methyl ether torosachrysone by the O‐methyltransferase CoOMT1 precedes the regioselective homocoupling to phlegmacin, catalyzed by the enzyme CoUPO1 annotated as an "unspecific peroxygenase" (UPO). Our results reveal an unprecedented UPO reaction, thereby expanding the biocatalytic portfolio of oxidative phenol coupling beyond the commonly reported enzymes. The results show that Basidiomycota use peroxygenases to selectively couple aryls independently of and convergently to any other group of organisms, emphasizing the central role of OPC in natural processes. [ABSTRACT FROM AUTHOR]

Many bacterial natural products contain C‐branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C‐branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)‐dependent enzymes. Chiral tertiary α‐hydroxy ketones are formed in this process. The ThDP‐dependent enzymes that catalyze C‐branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α‐ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α‐ketoacid dehydrogenase is responsible for C‐branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α‐hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP‐dependent enzymes catalyzing the C‐branching of keto sugars and subsequent modifications. [ABSTRACT FROM AUTHOR]

Deep learning tools for enzyme design are rapidly emerging, and there is a critical need to evaluate their effectiveness in engineering workflows. Here we show that the deep learning‐based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially relevant non‐native functions. This superfamily has diverse catalytic functions and could provide a rich new source of biocatalysts for synthesis and industrial processes. Through systematic comparisons of directed evolution trajectories for a non‐native, remote C(sp3)−H hydroxylation reaction, we demonstrate that the stabilized redesign can be evolved more efficiently than the wild‐type enzyme. After three rounds of directed evolution, we obtained a 6‐fold activity increase from the wild‐type parent and an 80‐fold increase from the stabilized variant. To generate the initial stabilized variant, we identified multiple structural and sequence constraints to preserve catalytic function. We applied these criteria to produce stabilized, catalytically active variants of a second Fe(II)/αKG enzyme, suggesting that the approach is generalizable to additional members of the Fe(II)/αKG superfamily. ProteinMPNN is user‐friendly and widely accessible, and our results provide a framework for the routine implementation of deep learning‐based protein stabilization tools in directed evolution workflows for novel biocatalysts. [ABSTRACT FROM AUTHOR]

N‐heterocyclic compounds have a broad range of applications and their selective synthesis is very appealing for the pharmaceutical and agrochemical industries. Herein we report the usage of the flavin‐dependent nitroreductase BaNTR1 for the photoenzymatic synthesis of various anthranils and quinolines from retro‐synthetically designed o‐nitrophenyl‐substituted carbonyl substrates, achieving high conversions (up to >99 %) and good product yields (up to 96 %). Whereas the effective production of anthranils required the inclusion of H2O2 in the reaction mixtures to accumulate the needed hydroxylamine intermediates, the formation of quinolines required the use of anaerobic or reducing conditions to efficiently generate the essential amine intermediates. Critical to our success was the high chemoselectivity of BaNTR1, performing selective reduction of the nitro group without reduction of the carbonyl moiety or the activated carbon‐carbon double bond. The results highlight the usefulness of an innocuous chlorophyll‐ and nitroreductase‐based photoenzymatic system for the tailored synthesis of diverse N‐heterocycles from simple nitro compounds. [ABSTRACT FROM AUTHOR]

Nor(pseudo)ephedrines [N(P)Es] are naturally occurring compounds showing sympathomimetic activity that have many applications especially in the chemical industry and in the pharmaceutical field. Recently a two‐step biocatalytic cascade for the preparation of (1S)‐N(P)E was designed, consisting of a benzoin‐type condensation catalyzed by the (S)‐selective acetoin:dichlorophenolindophenol oxidoreductase (Ao : DCPIP OR) followed by a transamination mediated by either a (S)‐ or (R)‐selective amine transaminase (ATA). In this study, we successfully immobilized both Ao : DCPIP OR and two ATAs of opposite enantioselectivity and used them in the biosynthetic cascade to N(P)Es. Immobilization yield, activity recovery, and stability of the immobilized enzymes, both after use (enzyme recycling) and storage (shelf‐life), were assessed. Ao : DCPIP OR immobilized on glyoxyl‐agarose exhibited remarkable stability under storage conditions throughout the 30‐days monitoring period. Moreover, it was successfully reused in the synthesis of (S)‐phenylacetylcarbinol [(S)‐PAC] yielding high conversion (>94 %) and enantiomeric excess (>99 %). The (R)‐selective At‐ATA from Aspergillus terreus, immobilized on Eupergit® C, demonstrated a slightly higher storage stability when compared to the (S)‐selective Sbv333‐ATA from Streptomyces sp. Bv333 immobilized on the same carrier. Remarkably, both enzymes were effectively reused for ten reaction cycles in the synthesis of N(P)Es, starting from the enzymatically synthesized (S)‐PAC, achieving complete conversions and excellent diastereoselectivity (>99 %). [ABSTRACT FROM AUTHOR]