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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.

2-Hydroxy-3-pentanone and 3-hydroxy-2-pentanone are flavor molecules present in various foods, such as cheese, wine, durian, and honey, where they impart buttery, hay-like, and caramel-sweet aromas. However, their utilization as flavoring agents is constrained by a lack of developed synthesis methods. In this study, we present their synthesis from simple starting compounds available in natural quality, catalyzed by previously characterized ThDP-dependent carboligases. Additionally, we demonstrate that newly discovered homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1), namely La PDH from Leclercia adecarboxylata , Cn PDH from Cupriavidus necator , and Tc PDH from Tanacetum cinerariifolium , exhibit promising potential for α-hydroxy pentanone synthesis in form of whole-cell biocatalysts. Enzyme stability at varying pH levels, kinetic parameters, and reaction intensification were investigated. Cn PDH, for example, exhibits superior stability across different pH levels compared to Ec PDH E1. Both α-hydroxy pentanones can be produced with Cn PDH in satisfactory yields (74% and 59%, respectively). • Synthesis of aroma constituents of wine, cheese and durian by ThDP-dependent carboligases from simple starting compounds. • Heterologous production and functional characterization of homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1). • Cupriavidus necator PDH is more stable across different pH levels compared to Ec PDH E1. • Product distribution of carboligations can be tuned by choice of enzyme and also by choice of donor and acceptor substrate. [ABSTRACT FROM AUTHOR]

The natural aroma compound (+)-nootkatone was obtained in selective conversions of up to 74 mol% from inexpensive (+)-valencene substrate by using a comparatively greener biocatalytic process developed based on modifications of the previously published Firmenich method. Buffer identity and concentration, pH, temperature and downstream work-up procedures were optimized to produce a crude product in which >90 % of (+)-valencene had been converted, with high chemoselectivity observed for (+)-nootkatone production. Interestingly, the biotransformation was carried out efficiently at temperatures as low as 21 ºC. Surprisingly, the best results were obtained when an acidic pH in the range of 3–6 was applied, as compared to the previously published procedure in which it appeared to be necessary to buffer the pH optimally and fixed throughout at 8.5. Furthermore, there was no need to maintain a pure oxygen atmosphere to achieve good (+)-nootkatone yields. Instead, air bubbled continuously at a low rate through the reaction mixture via a submerged glass capillary was sufficient to enable the desired lipoxygenase-catalyzed oxidation reactions to occur efficiently. No valencene epoxide side-products were detected in the organic product extract by a standard GCMS protocol. Only traces of the anticipated corresponding α- and β-nootkatol intermediates were routinely observed. [Display omitted] • A greener method was developed to produce (+)-nootkatone biocatalytically. • An unprecedentedly high conversion of 74 mol% (+)-nootkatone was obtained from inexpensive valencene. • Surprisingly, soy bean LOX isozymes were found to perform optimally in the pH range 4-6 and catalyzed the desired reaction at a pH as low as 2. [ABSTRACT FROM AUTHOR]

Standardization of the cultivation conditions is crucial for producing fungal biomass with effective whole-cell catalytic activity. Aspergillus flavus mutant was optimized for biomass transesterification and hydrolysis of waste frying oil (WFO) as a substrate. The glucose-polypeptone medium was selected among other culture media as the primary medium for the optimization of submerged cultivation. Biomass lipolytic activities and growth of A. flavus were higher under shaking conditions compared to static conditions, and 200 rpm was optimum. Olive oil was superior to soybean, rapeseed, and waste frying oils as biomass lipase and transesterification activities inducer. Replacing glucose with sugar cane molasses enhanced the growth but inhibited the biomass lipolytic capabilities. Urea improved the biomass lipolytic capabilities as an N-source feedstock compared to corn-steep liquor and whey. Plackett Burman screening of 9 factors showed that KH2PO4 (0.1–0.9 %, w/v), urea (0.1–1.3 %, w/v), and glucose (0.5–3.5 %, w/v) concentrations were significant factors with the highest main effect estimates 38.45, −19.04, and 11.82, respectively. These factors were selected for response surface methodology (RSM) optimization using central composite design (CCD). CCD models for growth, biomass lipase activity, and transesterification capability were significant, with R2 values of 0.93, 0.91, and 0.95, respectively. The optimum concentrations for growth and biomass transesterification capability were glucose (4.5 %, w/v), urea (0.7 %, w/v), and KH2PO4 (0.9 %, w/v), and those for lipase activity of the biomass were glucose (4.4 %, w/v), urea (0.7 %, w/v), and KH2PO4 (2.1 %, w/v). [ABSTRACT FROM AUTHOR]

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products. • Enzymatic production of hydroxy fatty acids for different application, such as flavors. • Screening of biocatalytic hydration of crude free fatty acids from waste cooking oils. • Design-of-experiment used to identify interactions between parameters. • Production of enzymatic cascade reactions. • Reproducibility of results on triglyceride-containing industrial feedstocks. [ABSTRACT FROM AUTHOR]

Inspired by the attractive structures and functions of natural matter (such as cells, organelles and enzymes), chemists are constantly exploring innovative material platforms to mimic natural catalytic systems, particularly liquid-phase hydrogenations, which are of great significance for chemical upgrading and synthesis. Hollow structured nanoreactors (HSNRs), featuring unique nanoarchitectures and advantageous properties, offer new opportunities for achieving excellent catalytic activity, selectivity, stability and sustainability. Notwithstanding the great progress made in HSNRs, there still remain the challenges of precise synthetic chemistry, and mesoscale catalytic kinetic investigation, and smart catalysis. To this extent, we provide an overview of recent developments in the synthetic chemistry of HSNRs, the unique characteristics of these materials and catalytic mechanisms in HSNRs. Finally, a brief outlook, challenges and further opportunities for their synthetic methodologies and catalytic application are discussed. This review might promote the creation of further HSNRs, realize the sustainable production of fine chemicals and pharmaceuticals, and contribute to the development of materials science. [ABSTRACT FROM AUTHOR]

Hydrophobic deep eutectic solvents (HDESs) are a new generation of water-immiscible solvents that have shown potential as green and sustainable reaction media for biocatalytic applications. In this study, esterification of tyrosol and hydroxytyrosol catalysed by Novozym 435 (Candida antarctica lipase B immobilised on acrylic resin) was taken as a model reaction to examine the feasibility of using HDESs as solvents for acylation reactions and the impacts of the components and physicochemical properties of the HDESs on the enzymatic performance. 21 HDESs were screened. Yields as high as 95.1% could be achieved within 2 h of reaction in an HDES, comparable to or even better than the results obtained in organic solvents. The enzymatic reactions were favoured in the HDES comprising a tertiary alcohol instead of a secondary/ primary one as the hydrogen bond acceptor (HBA). Inverse correlations were found between enzyme activity and the polarity and viscosity of the HDES. Carboxylic acid-based eutectic solvents can play a dual role as both a reaction medium and a substrate reservoir for the acylation reaction. These findings provide valuable guidelines for selecting HDESs for biocatalytic applications. [ABSTRACT FROM AUTHOR]

Catalytic esterification in water has been achieved with the aid of a commercially available polymer-supported lipase, Novozym-435. Remarkably, this well-known biocatalyst demonstrates significant activity towards esterification using just water as the reaction medium. This methodology features representative esterifications of complex acids and alcohols, in the presence of unprotected amines. The work disclosed leading to esters typically requires only two equivalents of alcohol, although conversion for water-soluble alcohols appears to require increased loadings. Recycling of both the aqueous medium and catalyst are documented, highlighting the potential of this new technology, especially using directed evolution on the enzyme involved. Pharmaceutically relevant compounds are efficiently esterified (e.g., Ibuprofen, Tolmetin, and Ticagrelor) and multi-step, one-pot chemoenzymatic sequences can be performed to demonstrate the robustness of this catalytic aqueous system. [ABSTRACT FROM AUTHOR]

The development of sustainable chemistry underlying the quest to minimize and/or valorize waste in the carbon‐neutral manufacture of chemicals is followed over the last four to five decades. Both chemo‐ and biocatalysis have played an indispensable role in this odyssey. in particular developments in protein engineering, metagenomics and bioinformatics over the preceding three decades have played a crucial supporting role in facilitating the widespread application of both whole cell and cell‐free biocatalysis. The pressing need, driven by climate change mitigation, for a drastic reduction in greenhouse gas (GHG) emissions, has precipitated an energy transition based on decarbonization of energy and defossilization of organic chemicals production. The latter involves waste biomass and/or waste CO2 as the feedstock and green electricity generated using solar, wind, hydroelectric or nuclear energy. The use of waste polysaccharides as feedstocks will underpin a renaissance in carbohydrate chemistry with pentoses and hexoses as base chemicals and bio‐based solvents and polymers as environmentally friendly downstream products. The widespread availability of inexpensive electricity and solar energy has led to increasing attention for electro(bio)catalysis and photo(bio)catalysis which in turn is leading to myriad innovations in these fields. [ABSTRACT FROM AUTHOR]

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology. [ABSTRACT FROM AUTHOR]

Amine dehydrogenases (AmDHs) are NAD(P)H‐dependent oxidoreductases that catalyze the reductive amination between carbonyl compounds and ammonia as the amine donor yielding valuable amines, typically with excellent enantioselectivity. While nature has provided enzymes with inherent AmDH activities, protein engineering techniques allowed researchers to expand the toolbox of available AmDHs, extend their substrate scope, improve their catalytic activities and stability under synthetically relevant conditions and even enable new reactivity concepts. The biocatalytic synthesis of amines using AmDHs has matured to a point where hundreds of aldehydes or ketones, of varying steric demands and bearing diverse functional groups, can be efficiently transformed. This review offers an overview of the available AmDHs and their substrate spectrum, covering from structural and evolutionary analyses to diverse methods employing these enzymes. Depending on the catalytic activities of other enzymes as reaction partners, AmDHs were applied in kinetic resolution (KR) and deracemization processes, cascade reactions for the amination of alcohols and alkenes or for the synthesis of amines and amino alcohols featuring multiple stereogenic centers. Moreover, the synthetic potential of AmDHs in novel pathways, such as the synthesis of secondary amines or alcohols, presents exciting opportunities for expanding their catalytic repertoire. [ABSTRACT FROM AUTHOR]

With the industrialization and development of modern science, the application of enzymes as green and environmentally friendly biocatalysts in industry has been increased widely. Among them, lipase (EC. 3.1.1.3) is a very prominent biocatalyst, which has the ability to catalyze the hydrolysis and synthesis of ester compounds. Many lipases have been isolated from various sources, such as animals, plants and microorganisms, among which microbial lipase is the enzyme with the most diverse enzymatic properties and great industrial application potential. It therefore has promising applications in many industries, such as food and beverages, waste treatment, biofuels, leather, textiles, detergent formulations, ester synthesis, pharmaceuticals and medicine. Although many microbial lipases have been isolated and characterized, only some of them have been commercially exploited. In order to cope with the growing industrial demands and overcome these shortcomings to replace traditional chemical catalysts, the preparation of new lipases with thermal/acid-base stability, regioselectivity, organic solvent tolerance, high activity and yield, and reusability through excavation and modification has become a hot research topic. [ABSTRACT FROM AUTHOR]

The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept ̴ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined Km and Vmax were 0.1098 mg mL−1 and 273.7 U mL−1, respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs). [ABSTRACT FROM AUTHOR]

Nano-bienzyme conjugates (nano-BECs) for catalysing one-pot reactions can simplify the reaction process and improve the efficiency of biocatalytic reactions. In this work glucose dehydrogenase (GDH) and Candida parapsilosis secondary alcohol dehydrogenase (CpSADH) were immobilised with polyacrylic acid (PAA) to form two types of PAA-based nano-BECs (2-S, 2-P), which achieved in situ regeneration of NADH for the production of chiral alcohols in oil-water biphasic systems, such as the key chiral pharmaceutical intermediate of (S)-1-(3-trifluoromethylphenyl)ethanol ((S)-TMPE). At a substrate concentration of 100 mM 3-trifluoromethylacetophenone (TMAP), the conversion catalyzed by nano BEC was 98.2 % in dipentyl phthalate-phosphate buffer biphasic system. In contrast, the conversion was only 39.5 % and 38.9 % in the PB buffer and 8.4 % and 7.7 % in dipentyl phthalate system. The experiments on kinetic constants demonstrated that the catalytic efficiencies (K cat /K m) of nano-BECs (2-S, 2-P) were enhanced by 6.7-fold and 7-fold, respectively, compared to free CpSADH. Additionally, nano-BECs showed stronger coenzyme regeneration ability. Under optimal reaction conditions, nano-BECs achieved 96.2 % conversion of TMAP to (S)-TMPE in an organic-buffer system with e.e. > 99.6 % at 300 mM substrate concentration. This research expands the potential applications of polymers and proteins, and offers valuable insights into the synergistic effects of PAA on bienzyme catalytic systems in biocatalysis. [Display omitted] • Heterologous expression of Candida parapsilosis secondary alcohol dehydrogenase (CpSADH). • Co-immobilization of glucose dehydrogenase (GDH) and CpSADH to form nano-polymers. • One-pot efficient catalytic carbonyl asymmetric reduction by nano-polymers. • Preparation of (S)-1-(3-trifluoromethylphenyl)ethanol at high substrate concentration. [ABSTRACT FROM AUTHOR]

Chemical warfare agents represent a severe threat to mankind and their efficient decontamination is a global necessity. However, traditional disposal strategies have limitations, including high energy consumption, use of aggressive reagents and generation of toxic byproducts. Here, inspired by the compartmentalized architecture and detoxification mechanism of bacterial micro-compartments, we constructed oil-in-water Pickering emulsion droplets stabilized by hydrogen-bonded organic framework immobilized cascade enzymes for decontaminating mustard gas simulant (2-chloroethyl ethyl sulfide, CEES) under sweet conditions. Two exemplified droplet systems were developed with two-enzyme (glucose oxidase/chloroperoxidase) and three-enzyme (invertase/glucose oxidase/chloroperoxidase) cascades, both achieving over 6-fold enhancement in decontamination efficiency compared with free enzymes and >99% selectivity towards non-toxic sulfoxide. We found that the favored mass transfer of sugars and CEES from their respective phases to approach the cascade enzymes located at the droplet surface and the facilitated substrate channeling between proximally immobilized enzymes were key factors in augmenting the decontamination efficacy. More importantly, the robustness of immobilized enzymes enabled easy reproduction of both the droplet formation and detoxification performance over 10 cycles, following long-term storage and in far-field locations. [ABSTRACT FROM AUTHOR]

The increasing atmospheric carbon dioxide (CO2) concentration has exposed a series of crises in the earth's ecological environment. How to effectively fix and convert carbon dioxide into products with added value has attracted the attention of many researchers. Cell-free enzyme catalytic system coupled with electrical and light have been a promising attempt in the field of biological carbon fixation in recent years. In this review, the research progresses of photoenzyme catalysis, electroenzyme catalysis and photo-electroenzyme catalysis for converting carbon dioxide into chemical products in cell-free systems are systematically summarized. We focus on reviewing and comparing various coupling methods and principles of photoenzyme catalysis and electroenzyme catalysis in cell-free systems, especially the materials used in the construction of the coupling system, and analyze and point out the characteristics and possible problems of different coupling methods. Finally, we discuss the major challenges and prospects of coupling physical signals and cell-free enzymatic catalytic systems in the field of CO2 fixation, suggesting possible strategies to improve the carbon sequestration capacity of such systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi‐enzyme system is extremely difficult and relies on a combinatorial trial‐and‐error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96‐well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python‐based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four‐enzyme system to transform vinyl acetate into (S)‐3‐hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi‐enzyme pathways for chemical manufacturing. [ABSTRACT FROM AUTHOR]

Aldolases are powerful C−C bond‐forming enzymes with high stereoselectivity and broad substrate scope in biocatalysis, but their ability to stereoselectively construct tertiary alcohols has not been fully explored. Herein, we demonstrate that vicinal diones and α‐keto esters are electrophiles that can be accepted by both natural and computationally designed aldolases via various catalytic mechanisms. This method allows for the efficient asymmetric synthesis of small molecules with tertiary alcohols, including noncanonical amino acids. This study presents the first types of generic nonnatural substrates of aldolases and reveals new opportunities for the use of aldolases in the synthesis of versatile chiral synthons. [ABSTRACT FROM AUTHOR]

The enzyme CbA5H, a [FeFe]‐hydrogenase from Clostridium beijerinckii, has previously been shown to survive exposure to oxygen, making it a promising candidate for biotechnological applications. Thus far [NiFe]‐hydrogenases are typically considered for such applications, due to the superior O2‐tolerance and therefore simplified enzyme handling. However, methods for production of [FeFe]‐hydrogenases are generally more successful than for other classes of hydrogenases, therefore in this work we focus on demonstrating scalable CbA5H production, and report results with active enzyme prepared in bioreactors (up to 10 L) with >20‐fold improvement in purified enzyme yield. We then go on to confirm excellent H2/H+‐cycling activity of the air‐purified protein, highlighting that CbA5H can be prepared and isolated without the need for complex and expensive infrastructure. Next, we demonstrate good stability of the air‐purified CbA5H both in solution assays, and as a heterogenous catalyst system when immobilized on a carbon support. Finally, we successfully implement this enzyme within previously demonstrated biotechnologies for flavin and NADH recycling, highlighting its relevance in chemical synthesis, and we demonstrate production of an important API precursor, 3‐quinuclidinol at >0.4 g scale in standard benchtop hydrogenation infrastructure, with >100,000 CbA5H turnovers over 18 operational hours. [ABSTRACT FROM AUTHOR]

Single-atom nanozymes (SAzymes) represent a cutting-edge advancement in nanomaterials, merging the high catalytic efficiency of natural enzymes with the benefits of atomic economy. Traditionally, natural enzymes exhibit high specificity and efficiency, but their stability are limited by environmental conditions and production costs. Here we show that SAzymes, with their large specific surface area and high atomic utilization, achieve superior catalytic activity. However, their high dispersibility poses stability challenges. Our review focuses on recent structural and preparative advancements aimed at enhancing the catalytic specificity and stability of SAzymes. Compared to previous nanozymes, SAzymes demonstrate significantly improved performance in biomedical applications, particularly in tumor medicine. This progress positions SAzymes as a promising tool for future cancer treatment strategies, integrating the robustness of inorganic materials with the specificity of biological systems. The development and application of SAzymes could revolutionize the field of biocatalysis, offering a stable, cost-effective alternative to natural enzymes. [ABSTRACT FROM AUTHOR]

We report here a mild process to access diversely functionalized enantioenriched β‐gem‐difluorinated (S)‐alcohols by using biocatalytic reduction in >99% yield and enantiomeric excesses ranging from 96% to >99%. This novel approach involves enzymatic reduction of α‐gem‐difluorinated heterocyclic and carbocyclic ketone derivatives as well as an acyclic gem‐difluorinated β‐ketosulfone and proceeds under mild conditions at 30 °C under aqueous conditions to produce a range of high added value enantioenriched difluorinated alcohols including 3,3‐difluorochromanol, 3,3‐difluorothiochromanol, 3,3‐difluoro‐dihydroquinolinol, and α‐difluorinated β‐hydroxysulfone derivatives. A comparative study between enzymatic reduction and metal‐catalyzed asymmetric transfer hydrogenation was carried out as well. [ABSTRACT FROM AUTHOR]

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. Key points: • A novel neuraminic acid synthase was characterized. • The effect of cofactors on NeuS activity was elucidated. • Kinetic and stability characterization of two neuraminic acid synthases was performed. [ABSTRACT FROM AUTHOR]

Deep eutectic solvents (DESs) are gaining rapid prominence in numerous biocatalysis processes due to their green characteristics, biodegradability, low cost, and simple preparation compared to harsh organic solvents and toxic ionic liquids. However, natural enzymes tend to show activity reduction, even inactivation in the presence of many DESs. Here, we present the first rational design approach to achieve enzymes resistant to both DESs and high temperatures. Using the interaction pattern between DESs and BSLA (Bacillus subtilis lipase A, our model enzyme) derived from all-atom molecular dynamics (MD) simulations, we formulated a solvation-guided engineering strategy. This was established after assessing 33 structural, solvation, and energy observables. We rationally designed and experimentally tested 36 single substitutions, of which 28 (a 77.78% success rate) exhibited improvements in at least two DES cosolvents: choline chloride (ChCl) : acetamide, tetrabutylphosphonium bromide (TBPB) : ethylene glycol (EG), and ChCl : EG. Additionally, through stepwise recombination, we identified two robust BSLA variants, D64H/R107L/E171Y and D64H/R142L, showing stability improvements of up to 4.4-fold and 3.2-fold in three DESs and at 50 °C, respectively. Further MD studies demonstrated that (i) the restricted overall structure but fine-tuned local flexibility and (ii) increased water but decreased DES molecules at substituted sites are two main factors contributing to the enhanced multiple DES resistance. Overall, solvation-guided engineering offers an efficient and rational approach for designing lipases tolerant to DES cosolvents and elevated temperatures, with a considerable potential for adaptation to other enzymes. [ABSTRACT FROM AUTHOR]

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK. [ABSTRACT FROM AUTHOR]

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R² > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes. [ABSTRACT FROM AUTHOR]

Cellulose nanocrystals are an important class of bio‐based crystalline nanostructures, finding application in several technological fields, including paper and textile coating, biocomposite engineering, biocatalysts immobilization, etc. This study explores enzymatic hydrolysis of Avicel, using endoglucanase from Aspergillus niger, to find an environmentally friendly method to extract cellulose nanocrystals from cellulose sources. Enzymatic hydrolysis has the advantage of reduced energy consumption and higher environmental friendliness compared to acid hydrolysis. In this work, we report for the first time very high nanocrystals yield by combining mechanical pretreatment of the cellulose starting material with a ball miller and endoglucanase hydrolysis, as a result of an extensive optimization of reaction conditions. In particular, a ball milling pretreatment carried out for 50 minutes at 3 Hz, allowed to isolate enzymatic CNCs with 76 % yield and with crystallinity as high as 75 %. The materials were characterized by X‐Ray diffractometry, attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy, dynamic light scattering, zeta potential and field emission scanning electron microscopy (FE‐SEM). Their characteristics were compared with the properties of sulfated CNCs, prepared from Avicel by sulfuric acid hydrolysis. Our results are technologically relevant, as they contribute to the accessibility and sustainability of CNCs for a wide range of applications in various industries. [ABSTRACT FROM AUTHOR]

Fluorinated cyclopropanes are highly desired pharmacophores in drug discovery owing to the rigid nature of the cyclopropane ring and the beneficial effects of C−F bonds on the pharmacokinetic properties, cell permeability, and metabolic stability of drug molecules. Herein a biocatalytic strategy for the stereoselective synthesis of mono‐fluorinated and gem‐difluoro cyclopropanes is reported though the use of engineered myoglobin‐based catalysts. In particular, this system allows for a broad range of gem‐difluoro alkenes to be cyclopropanated in the presence of diazoacetonitrile with excellent diastereo and enantiocontrol (up to 99 : 1 d.r. and 99 % e.e.), thereby enabling a transformation not currently accessible with chemocatalytic methods. The synthetic utility of the present approach is further exemplified through the gram‐scale synthesis of a key gem‐difluorinated cyclopropane intermediate useful for the preparation of fluorinated bioactive molecules. [ABSTRACT FROM AUTHOR]

Consistent introduction of novel enzymes is required for developing efficient biocatalysts for challenging biotransformations. Absorbing catalytic modes from organocatalysis may be fruitful for designing new‐to‐nature enzymes with novel functions. Herein we report a newly designed artificial enzyme harboring a catalytic pyrrolidine residue that catalyzes the asymmetric Michael addition of cyclic ketones to nitroolefins through enamine activation with high efficiency. Diverse chiral γ‐nitro cyclic ketones with two stereocenters were efficiently prepared with excellent stereoselectivity (up to 97 % e.e. >20 : 1 d.r.) and good yield (up to 86 %). This work provides an efficient biocatalytic strategy for cyclic ketone functionalization, and highlights the usefulness of artificial enzymes for extending biocatalysis to further non‐natural reactions. [ABSTRACT FROM AUTHOR]

Polyphosphate kinases (PPKs) are valuable biocatalysts for ATP cofactor regeneration as well as for the phosphorylation of non‐canonical nucleotides. The versatility of PPKs in the latter application is defined by their substrate scope. In this study, we investigate the substrate spectrum of the PPK2‐III enzyme from Meiothermus ruber (MrPPK) for the conversion of canonical and non‐canonical (deoxy)‐ribonucleotides. The enzyme shows high substrate promiscuity for purine and to minor degree pyrimidine substrates, with an overall preference for the native substrate AMP. With structure guided rational amino acid exchanges in the active site, we produced an MrPPK variant with improved activity for a broad variety of purine nucleotides. While the preference for AMP is lost, conversion of nucleotides without a 6‐amino function at the purine moiety is increased. This MrPPK variant is a versatile biocatalyst for the synthesis of non‐canonical nucleotides and could also be useful as a GTP cofactor regeneration system. [ABSTRACT FROM AUTHOR]

The synergistic combination of biocatalysis and photocatalysis is emerging as powerful tool for the development of sustainable and atom‐efficient synthetic concepts facilitating an enormous portfolio of possible reactions which even goes beyond the capabilities found in nature. Here, a cyclic deracemization process is presented tailored for the synthesis of optically pure sulfoxides which are versatile structural motifs in asymmetric synthesis as well as in bioactive compounds. Enantioselective enzyme‐catalyzed reduction of rac‐sulfoxides was combined with a stereo‐unselective photocatalytic oxidation of the corresponding sulfide intermediate. The utilization of the readily accessible and rather inexpensive photocatalyst Eosin Y increases the usability of this synthetic method. To overcome the incompatibility between the photocatalyst Eosin Y and the biocatalytic step, the cyclic deracemization process was performed in a step‐wise fashion via alternated reduction in the darkness and oxidation under illumination. This modular system allowed precise adjustments of reaction parameters yielding the desired sulfoxide targets with up to >99 % ee. Evaluation of the substrate scope including a range of structurally diverse molecules demonstrated its broad applicability. [ABSTRACT FROM AUTHOR]

Thiamine diphosphate (ThDP) dependent carboligases catalyze the formation of carbon‐carbon bonds. These enzymes are prominent biocatalysts for the production of valuable α‐hydroxy carbonyl compounds, which serve as key building blocks in the synthesis of various pharmaceuticals and fine chemicals. Carboligases act in selective manner to afford regio‐ and stereochemically defined products from simple starting materials. This review explores the catalytic prowess of carboligases for synthetic purposes and is aimed at providing a selection tool of enzymes for particular sets of substrates or desired products. The comprehensive overview encompasses structural insights, relationships of the currently known sequence space and practical applications with a focus on recent literature, showcasing carboligases as potent tools for sustainable and efficient synthesis. [ABSTRACT FROM AUTHOR]

Known for its tunable conductivity and stability, Polyaniline (PANI) is a valuable polymer for electronics and sensing devices. Challenges in solubility have been addressed by creating sulfonated PANI (SPANI), enhancing its practical use. Synthesizing SPANI from sulfonated aniline is intricate, but laccase biocatalysis offers an eco-conscious solution, effective even against high redox potential obstacles. This research monitored the Trametes versicolor laccase-induced oxidation of 3-ABSa via UV-vis spectroscopy, with a notable peak at 565 nm signifying SPANI synthesis, effective even at suboptimal pH. Mediators further boost this process. Moreover, NMR and spectroelectrochemistry confirm the green synthesis of SPANI by laccase, hinting that pH fine-tuning could improve yields, alongside the concurrent creation of azobenzene derivatives. [ABSTRACT FROM AUTHOR]

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Å from the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90–100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications. [Display omitted] • NOV1 catalyzes the one-step coenzyme-free formation of vanillin from isoeugenol. • Computational tools predicted beneficial mutations distal from active site. • Distal mutations significantly enhance NOV1 activity and stability properties. • Distal mutations improved immobilization efficiency in metal affinity resins. • Our study emphasizes distal interactions' role in enhancing enzymatic properties. [ABSTRACT FROM AUTHOR]

The combination of chemo- and photocatalyses with biocatalysis, which couples the flexible reactivity of the photo- and chemocatalysts with the highly selective and environmentally friendly nature of enzymes in one-pot linear cascades, represents a powerful tool in organic synthesis. However, the combination of photo-, chemo- and biocatalysts in one-pot is challenging because the optimal operating conditions of the involved catalyst types may be rather different, and the different stabilities of catalysts and their mutual deactivation are additional problems often encountered in one-pot cascade processes. This review explores a large number of transformations and approaches adopted for combining enzymes and chemo- and photocatalytic processes in a successful way to achieve valuable chemicals and valorisation of biomass. Moreover, the strategies for solving incompatibility issues in chemo-enzymatic reactions are analysed, introducing recent examples of the application of non-conventional solvents, enzyme–metal hybrid catalysts, and spatial compartmentalization strategies to implement chemo-enzymatic cascade processes. [ABSTRACT FROM AUTHOR]

Protein engineering often targets binding pockets or active sites which are enriched in epistasis--nonadditive interactions between amino acid substitutions--and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable ß-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences--a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling. [ABSTRACT FROM AUTHOR]

Enantioselective reduction of cyclobutenones to optically active cyclobutanones has been achieved by using whole‐cell overexpressing Seqenzym ene‐reductases (EREDs). By using these enzymes, trans‐cyclobutanones were isolated in good yields and with enantiomeric excesses up to 99%. [ABSTRACT FROM AUTHOR]

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity. [ABSTRACT FROM AUTHOR]

Metagenomic fosmid libraries were prepared from soil and water samples collected from diverse places. Clones in the range of (1.1–2.8) × 103 were isolated from these samples and used for screening and isolation of D-amidase producer using dyes bromthymol green and phenol red. Maximum number of metagenomic libraries were obtained from soil rich in fishery waste while minimum number of libraries were obtained from tap water genome. Five positive clones after two-stage functional screening for D-alanine amidase were isolated on the basis of change in colour of growth medium due to enzyme production. Selected isolate on the basis of maximum enzyme production (2 U/mL) was cloned and overexpressed. Selected isolate from fishery waste metagenomes was found to be constitutive producer of D-amidase enzyme which after optimization led to 32% increase in enzyme activity. Isolated novel enzyme showed catalytic promiscuity with amides of D-Leu, D-Pro, D-Met and D-Val. Optimization of reaction conditions leading to production of 72% D-Ala and 65% D-Leu from their D-amide salts, respectively, in 8 h were confirmed by HPLC. Isolated homologous and putative genes after genomic screening were confirmed to be matching with Rhodococcus and Pseudomonas aeruginosa amidases. Enzyme also had acyl transferase activity with various amide substrates. [ABSTRACT FROM AUTHOR]

Enzymes are specialized proteins that act as catalysts, facilitating chemical reactions by either building up or breaking down molecules in all living organisms. They are distinguished by their specificity, a unique characteristic which has been recognized as a valuable tool by various industries. This specificity allows industries to target very particular processes. For instance, in the food industry, enzymes are used in processes like cheese making, brewing, and baking, where their specific actions are required to produce desired flavours, textures, or shelf-life improvements. While industries highly appreciate the specific functionalities of enzymes, their primary focus lies on the commercial form of the enzyme, which demands to function in rigorous industrial settings, not interfere the processes, and be compatible within the chemical environment. Stability is the deciding factor that greatly influences the enzymatic activity, shelf-life, and consequently, the overall viability and commercial success of enzymes. Thus, incorporating enzymes into industrial processes necessitates their preparation or formulation in a form that meets the specific needs of the intended use. The present work focuses on reviewing the fundamental aspects, requirements, and specifications involved in the preparation of enzymes for selected industrial sectors. [ABSTRACT FROM AUTHOR]

Conventional biomineralization strategies are constructed using microparticles, suffering the inhibition of protease bio-availability and required rigorous bio-evaluation. The use of nanomineralization technology could apply control over the size, shape, and composition of the materials and provide a steadier maintenance environment for the bio-enzyme. In this study, stable and nanoscale copper phosphate (Cu3(PO4)2) bio-nanomineralization materials were constructed to encapsulate free bio-enzyme. By optimizing the biomineralization process, Cu3(PO4)2 was demonstrated to enable the facile coordination of various bio-enzymes (e.g., catalase or β-glucosidase) for efficient bio-enzyme transportation and maintaining the bio-enzyme activity in harsh condition. The current nanomineralization strategy could provide new ideas and methods for the development of biocatalysis, biosensing, biomedicine, and other related fields. [ABSTRACT FROM AUTHOR]

Biocatalytic performance of immobilized ficin onto silica-coated iron oxide magnetic nanoparticles (MNPs) was examined. FTIR study indicated the successful immobilization of ficin onto silica-coated MNPs. The MNPs were synthesized by an ultrasonic-assisted sol-gel method, resulting in coarse silica-coated MNPs with an amorphous or nanocrystalline phase. Results showed that the affinity of ficin to the substrate decreased due to the immobilization. However, the immobilized ficin exhibited enhanced stability and catalytic activity, with a wider pH range and higher thermal stability compared to free ficin. The optimum pH for immobilized ficin activity was found to be shifted from 7.0 to 6.0, making it a suitable candidate for slight acidic conditions. After five days of storage, immobilized ficin retained its initial activity, while free ficin showed a 30% decrease in activity. The immobilized ficin also exhibited a substantially higher activity than its free form after multiple cycles of usage. These findings suggest that immobilized ficin onto silica-coated iron oxide MNPs is a promising candidate for biocatalysis and industrial processes in a wide range of temperature and solutions with high reusability and stability. Highlights: Iron oxide magnetic nanoparticles were successfully coated with silica. The immobilized ficin exhibited enhanced stability and catalytic activity. Immobilized ficin was found to be a suitable candidate for slight acidic conditions. The immobilized ficin exhibited higher activity than its free form after multiple cycles of usage. Immobilized ficin onto silica-coated iron oxide MNPs is a promising candidate for biocatalysis and industrial processes. [ABSTRACT FROM AUTHOR]

Rhodococcus rhodochrous strain М8 is a platform for development of the biotechnologies for biocatalytic production of acrylic monomers, the raw material for synthesis of acrylic heteropolymers. A genetic system for investigation of the cobalt-dependent transcription of nitrile hydratase genes in this strain was constructed, based on the reporter gene of the metal-independent acylamidase from Rhodococcus qingshengii TA37. The cobalt-regulated promoter was shown to be located at a significant distance (~0.5 kb) from nitrile hydratase genes. Excision of the region between the promoter and the nitrile hydratase genes decreased significantly both the promoter activity and the degree of regulation by cobalt. Our results improve the possibilities for rational design of regulated expression cassettes using the promoter of nitrile hydratase genes in Rhodococcus, and for further development of biocatalysts based on these bacteria. [ABSTRACT FROM AUTHOR]

Amines, crucial components in various industries, play a pivotal role in the synthesis of pharmaceuticals, agrochemicals, and specialty chemicals. Recognizing the environmental impact of conventional methods for their preparation, our study centers on the utilization of abundantly available natural molecules, specifically amino acids, as precursors for short chain amine synthesis. This paper focuses on the biocatalyst, L‐valine decarboxylase from Streptomyces viridifaciens (VlmD), delving into the substrate scope, catalytic activity, and cost‐effective scalability of an enzymatic process for amine synthesis. Additionally, we investigate the feasibility of immobilizing VlmD, aiming to pave the way for its effective use in industrial applications. Our study exploits the SpinChem system and provides a comprehensive understanding of the potential and limitations of this biocatalyst. Notably, our yields for key amines (8.42 g ⋅ d−1 for isobutylamine, 5.23 g ⋅ d−1 for isoamylamine, 5.16 g ⋅ d−1 for (S)‐2‐methylbutylamine, 3.78 g ⋅ d−1 for 3‐(methylthio)propylamine, and 10.52 g ⋅ d−1 for (R)‐1‐amino‐2‐propanol) demonstrate the process efficiency and potential for industrial scalability. [ABSTRACT FROM AUTHOR]

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations. [ABSTRACT FROM AUTHOR]

Inspired by the layered structure, light absorption, and charge carrier pathway of chloroplast thylakoids in natural photosynthesis, we propose a novel artificial photosynthesis platform, which is composed of layered structured vaterite as the scaffold with gold nanoparticles (AuNPs), photosensitizer eosin Y (EY), and redox enzyme L-glutamate dehydrogenase (GDH) as the functional components. The EY exhibited significantly enhanced light absorption and charge carrier generation due to the localized surface plasmon resonance (LSPR) around the AuNPs and light refraction within the layers. This artificial photosynthesis platform can regenerate reduced nicotinamide adenine dinucleotide (NADH) under visible light and promote the rapid conversion of α-ketoglutarate to L-glutamate (0.453 Mm/h). The excellent biocompatibility of layered vaterite significantly enhances the resistance of GDH to harsh conditions, including high pH (pH = 10) and elevated temperatures (37–57 °C). [ABSTRACT FROM AUTHOR]