Nowadays, there is a growing need for streamlined and sustainable strategies to access chiral nitrogen heterocycles, whose stereodefined frameworks underpin their broad functional and pharmaceutical relevance. Herein, we report a robust continuous flow enzymatic protocol for the stereoselective synthesis of the natural alkaloid (–)‐pinidinone and demonstrate its applicability to the preparation of a small family of structurally diverse chiral 2,6‐disubstituted piperidines. The strategy relies on the enantioselective transamination of tailor‐made α,β‐unsaturated ketones catalyzed by an immobilized form of ATA‐117, which triggers a spontaneous intramolecular aza‐Michael reaction, enabling rapid access to chiral cis‐(2R,6R)‐piperidine scaffolds under mild conditions. The enzyme, successfully immobilized on Eupergit C, showed good operational and storage stability while a dual‐flow feed configuration minimized the formation of imine and Michael‐type by‐products observed when a single stock solution of α,β‐unsaturated ketone acceptor and isopropylamine (IPA) amino donor was employed. The optimized conditions (20 mM substrate, 200 mM IPA, 60 min residence time, 30 °C) allowed for complete conversion and high stereoselectivity, while an integrated in‐line work‐up improved automation and reduced manual handling. This study establishes a robust biocatalytic flow platform for the asymmetric synthesis of valuable nitrogen‐containing heterocycles.

Cyrene (dihydrolevoglucosenone, (1R,5S)-7,8-Dioxabicyclo[3.2.1]octan-2-one, 1) and Levoglucosenone (2) represent renewable platform chemicals obtained from cellulose pyrolysis and have been attracting great interest as sustainable building blocks in organic synthesis. In the frame of an Italian project (acronym: SUST-CARB), the development of original methodologies for converting these two bio-based building blocks into complex high added-value fine chemicals have been investigated. Here we report on the catalytic behaviour of three imine reductases (IREDs) and of one reductive aminase (AspRedAm) when cyrene and levoglucosenone were used as substrates in the presence of allylamine or propargylamine. Compared to the chemical reductive amination, the three IREDs displayed the same selectivity in favour of the (S)-2-amino epimer. On the contrary, AspRedAm was not selective, thus allowing to isolate the unfavoured (R)-epimer. AspRedAm and the ancillary enzyme glucose dehydrogenase were also co-immobilized on activated agarose and the performances of this heterogeneous biocatalyst have been investigated.

2-Phosphoglycerate kinase (2PGK) and cyclic 2,3-diphosphoglycerate synthase (cDPGS) are key enzymes involved in the biosynthesis of cyclic 2,3-diphosphoglycerate (cDPG), an extremolyte known to stabilize proteins in hyperthermophilic Archaea. Using bioinformatics approaches, two candidate genes for each enzyme were identified from a range of thermophilic bacterial and archaeal genomes and metagenomes. Significantly, one gene pair derived from the Taman mud volcano metagenome represents the first indication of a bacterial cDPG biosynthesis pathway. The recombinant expression and purification of these enzymes paved the way to their biochemical and structural characterization. One 2PGK candidate displayed predominant ATPase activity, while the newly identified cDPGS variants demonstrated cDPG synthase activity. Moreover, one of the latter biocatalysts, Ts-cDPGS from the hyperthermophilic archaeon Thermococcus sibiricus, demonstrated a notable thermostability and its 3D structure was resolved at a resolution of 2.2 & Aring;. These findings broaden our understanding of extremophilic enzyme systems and lay the foundation for biotechnological applications involving extremolyte production.

Extremolytes – unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their considerable potential, only a limited number of extremolytes – most notably ectoine and hydroxyectoine – have achieved commercial relevance, primarily due to the absence of efficient production strategies for the majority of other extremolytes. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg−1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 min, using just 0.5 U of recombinant MfcDPGS at 55°C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

Capsaicin and simple capsaicinoids have been shown to possess multiple beneficial effects as antibacterials, anticancer agents, antioxidants or against obesity, just to name a few. Olvanil is one of the first synthetic capsaicinoid derivatives designed to activate the same receptor as natural capsaicin, without eliciting its powerful stinging or burning effects. The traditional synthetic approaches to olvanil involve the chemical conversion of vanillin into vanillylamine and a poorly atom-economic chemical amidation with an activated derivative of oleic acid. In this work, a simple biocatalytic two-step procedure has been developed and optimised to produce olvanil, starting from two biomass-derived synthons (oleic acid from vegetable oil soapstock waste and vanillin from lignin). The process employs only biobased reagents and catalysts, under mild conditions and without wasteful purifications, aligning well with the concepts of green chemistry and circular economy.

Excessive signaling by various GPCRs underlies a variety of human disorders. Suppression of GPCRs by "enhanced" arrestin mutants was proposed as therapy. We hypothesized that GPCR binding of endogenous arrestins can be increased by small molecules stabilizing pre-activated conformation. Using molecular dynamics, we identified potentially druggable pockets in pre-activated conformation of arrestin-3 and discovered a compound targeting one of these pockets. Saturation-transfer difference NMR data showed that the compound binds at the back loop of arrestin-3. FRET- and NanoBiT-based assays in living cells showed that the compound increased in-cell arrestin-3, but not arrestin-2, binding to basal β2-adrenergic receptor and its phosphorylation-deficient mutant, but not to muscarinic M2 receptor. These experiments demonstrated the feasibility of enhancing the binding of endogenous wild type arrestin-3 to GPCRs in a receptor-specific and arrestin-subtype selective manner.

A (S)-selective amine transaminase from a Streptomyces strain, Sbv333-ATA, is a biocatalyst showing both high thermostability with a melting temperature of 85 °C and broad substrate specificity for the amino acceptor. This enzyme was further characterized both biochemically and structurally. The Sbv333-ATA is stable in the presence of up to 20

Lactonases, a class of metalloenzymes that exhibit catalytic promiscuity, have been extensively studied from a biological perspective, yet their application as biocatalysts remains underexplored. In this study, we disclose the biocatalytic activity of lactonase enzymes in the hydrolysis and deracemisation of chiral C3-substituted-γ-thiolactones and the asymmetric synthesis of γ-thio-α-substituted-carboxylic acids. The thiolactonase activity of lactonases from different protein superfamilies was investigated. The biocatalyst GcL, from the metallo-β-lactamase-like lactonase family, catalysed the enzymatic kinetic resolution (EKR) of homocysteine (Hcy) thiolactones with excellent enantioselectivity (E-value up to 136), yielding enantioenriched Hcy thiolactones and γ-thio-α-amino-carboxylic acids with high ees. Additionally, the biocatalyst N9 Y71G, a rationally engineered variant of the reconstructed ancestral paraoxonase enzyme N9, catalysed the dynamic kinetic resolution (DKR) of C3-thio-γ-thiolactones, yielding γ-thio-α-thio-carboxylic acids in enantioselective manner with high ees (up to >99%) and yields (up to >99%). Insights on the mechanism and the stereoselectivity of the lactonase biocatalysts were gained through computational and site-directed mutagenesis studies.

The development of immobilized enzymes both for batch and continuous flow biocatalytic processes has gained significant traction in recent years, driven by the need for cost-effective and sustainable production methods in the fine chemicals and pharmaceutical industries. Enzyme immobilization not only enables the recycling of biocatalysts but also streamlines downstream processing, significantly reducing the cost and environmental impact of biotransformations. This review explores recent advancements in enzyme immobilization techniques, covering both carrier-free methods, entrapment strategies and support-based approaches. At this regard, the selection of suitable materials for enzyme immobilization is examined, highlighting the advantages and challenges associated with inorganic, natural, and synthetic organic carriers. Novel opportunities coming from innovative binding strategies, such as genetic fusion technologies, for the preparation of heterogeneous biocatalysts with enhanced activity and stability will be discussed as well. This review underscores the need for ongoing research to address current limitations and optimize immobilization strategies for industrial applications.

Nor(pseudo)ephedrines (N(P)Es), vicinal amino alcohols possessing sympathomimetic biological activity, constitute valuable intermediates and chiral building blocks for the organic synthesis of several active pharmaceutical ingredients (APIs). Due to the presence of two chiral centers, their conventional chemical asymmetric syntheses often involve long, multi‐step procedures, frequently with the aid of expensive and harmful metal catalysts, making it challenging to achieve high yields and optical purities overall. A two‐steps biocatalytic synthetic sequence for the preparation of (1 S )‐N(P)E analogues was therefore designed and carried out, consisting of a benzoin‐type condensation catalysed by the ( S )‐selective acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and a transamination mediated by either an ( S )‐ or ( R )‐selective amine transaminase (ATA). A multistep chemical synthesis of racemic N(P)Es was also optimised in order to obtain reference material for evaluating the performance of the biocatalysed reactions. The novel bi‐enzymatic synthesis provided the desired products with acceptable yields and good diastereo‐ and enantiomeric excesses, thereby paving the way for greener production of these important building blocks.

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 (R) 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 %).

Hydroxysteroid dehydrogenases (HSDHs) are NAD(P)H-dependent alcohol dehydrogenases (ADHs) known for their exceptional stereo- and regioselectivity when acting on natural substrates, including neutral steroids, bile acids, and various steroid derivatives. Notably, in recent studies this specific subfamily of oxidoreductases has displayed intriguing substrate promiscuity, exhibiting the capacity to accommodate a diverse array of substrates, such as sterically hindered ketones and even alpha-keto esters. Herein, the promiscuous nature of HSDHs was further explored by investigating their catalytic activity with representative 1,2-diketones. This set encompasses symmetric aliphatic/aromatic diketones - namely, 3,4-hexandione and benzil - as well as the asymmetric synthon 1-phenyl-1,2-propanedione. In the case of 3,4-hexandione, substrate conversion and selectivity closely resembled that previously observed with aliphatic alpha-keto esters. On the contrary, a more heterogeneous behavior was observed in the case of aromatic substrates, with diverse performances in terms of conversions and stereo- or regioselectivity. Additionally, docking studies were carried out to get a deeper insight in the stereochemistry of 1,2-diketones reduction catalyzed by the broad substrate scope and steroid-active ketoreductase Is2-SDR. Selected hydroxysteroid dehydrogenases (HSDHs) were screened against 1,2-diketones to investigate their promiscuous character. This study focused on the asymmetric synthon 1-phenyl-1,2-propanedione as well as the symmetric diketones 3,4-hexandione and benzil. The substrate conversion and selectivity for 3,4-hexandione nearly matched those previously noted for aliphatic alpha-keto esters, while HSDHs showed a more heterogeneous response with aromatic substrates, exhibiting different conversion rates and stereo- or regioselectivity.image

As a rich source of biological active compounds, marine natural products have been increasingly screened as candidates for developing new drugs. Among the several marine products and metabolites, (+)-Harzialactone A has drawn considerable attention for its antitumor and antileishmanial activity. In this work a chemoenzymatic approach has been implemented for the preparation of the marine metabolite (+)-Harzialactone A. The synthesis involved a stereoselective, biocatalyzed reduction of the prochiral ketone 4-oxo-5-phenylpentanoic acid or the corresponding esters, all generated by chemical reactions. A collection of different promiscuous oxidoreductases (both wild-type and engineered) and diverse microorganism strains were investigated to mediate the bioconversions. After co-solvent and co-substrate investigation in order to enhance the bioreduction performance, T. molischiana in presence of NADES (choline hydrochloride-glucose) and ADH442 were identified as the most promising biocatalysts, allowing the obtainment of the (S)-enantiomer with excellent ee (97% to >99% respectively) and good to excellent conversion (88% to 80% respectively). The successful attempt in this study provides a new chemoenzymatic approach for the synthesis of (+)-Harzialactone A.

In a metagenome mining-based search of novel thermostable hydroxysteroid dehydrogenases (HSDHs), enzymes that are able to selectively oxidize/reduce steroidal compounds, a novel short-chain dehydrogenase/reductase (SDR), named Is2-SDR, was recently discovered. This enzyme, found in an Icelandic hot spring metagenome, shared a high sequence similarity with HSDHs, but, unexpectedly, showed no activity in the oxidation of the tested steroid substrates, e.g., cholic acid. Despite that, Is2-SDR proved to be a very active and versatile ketoreductase, being able to regio- and stereoselectively reduce a diversified panel of carbonylic substrates, including bulky ketones, α- and β-ketoesters, and α-diketones of pharmaceutical relevance. Further investigations showed that Is2-SDR was indeed active in the regio- and stereoselective reduction of oxidized steroid derivatives, and this outcome was rationalized by docking analysis in the active site model. Moreover, Is2-SDR showed remarkable thermostability, with an apparent melting temperature (TM) around 75 °C, as determined by circular dichroism analysis, and no significant decrease in catalytic activity, even after 5 h at 80 °C. A broad tolerance to both water-miscible and water-immiscible organic solvents was demonstrated as well, thus, confirming the potential of this new biocatalyst for its synthetic application.

The combination of a biocatalytic asymmetric C-C reduction with a simple sequence of chemical transformations was implemented in a new chemoenzymatic synthesis of various substituted aryloxyalkanoic acids, used as weed-killing agrochemicals or chiral precursors. By careful selection of the biocatalyst, either enantiomer of the product could be obtained in good yield and moderate to good ee. The method relies on the use of simple and commercially available starting materials, and requires neither purified enzymes nor chromatographic separations.

The valorization of biomass residuals constitutes a key aspect of circular economy and thus a major challenge for the scientific community. Among industrial wastes, plant residuals could represent an attractive source of bioactive compounds. In this context, a residue from the industrial extraction of Cucurbita pepo L. seeds, whose oil is commercialized for the treatment of genito-urinary tract pathologies, has been selected. Supercritical CO2 technology has been employed as a highly selective "green" methodology allowing the recovery of compounds without chemical degradation and limited operational costs. Free fatty acids have been collected in mild conditions while an enrichment in sterols has been selectively obtained from sc-CO2 extracts by appropriate modulation of process parameters (supercritical fluid pressure and temperature), hence demonstrating the feasibility of the technique to target added-value compounds in a selective way. Obtained fatty acids were thus converted into the corresponding ethanol carboxamide derivatives by lipase-mediated biocatalyzed reactions, while the hydroxylated derivatives of unsaturated fatty acids were obtained by stereoselective hydration reaction under reductive conditions in the presence of a selected FADH2-dependent oleate hydratase.

Ginger is among the most widespread and widely consumed traditional medicinal plants around the world. Its beneficial effects, which comprise e. g. anticancer and anti-inflammatory activities as well as gastrointestinal regulatory effects, are generally attributed to a family of non-volatile compounds characterized by an arylalkyl long-chained alcohol, diol, or ketone moiety. In this work, ginger active components have been successfully recovered from industrial waste biomass of fermented ginger. Moreover, their recovery has been combined with the first systematic study of the stereoselective reduction of gingerol-like compounds by isolated alcohol dehydrogenases (ADHs), obtaining the enantioenriched sec-alcohol derivatives via a sustainable biocatalytic path in up to >99 % conversions and >99 % enantiomeric/diastereomeric excesses.

Three Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (TM = 85 °C) only slightly lower compared to the TM of the most thermostable transaminases described to date (87–88 °C) and Sbv286-LAC being even thermoactivated at temperature >60 °C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of β-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts.

Laccases are multicopper oxidases, which have been widely investigated in recent decades thanks to their ability to oxidize organic substrates to the corresponding radicals while producing water at the expense of molecular oxygen. Besides their successful (bio)technological applications, for example, in textile, petrochemical, and detoxifications/bioremediations industrial processes, their synthetic potentialities for the mild and green preparation or selective modification of fine chemicals are of outstanding value in biocatalyzed organic synthesis. Accordingly, this review is focused on reporting and rationalizing some of the most recent and interesting synthetic exploitations of laccases. Applications of the so-called laccase-mediator system (LMS) for alcohol oxidation are discussed with a focus on carbohydrate chemistry and natural products modification as well as on bio- and chemo-integrated processes. The laccase-catalyzed Csp(2)-H bonds activation via monoelectronic oxidation is also discussed by reporting examples of enzymatic C-C and C-O radical homo- and hetero-couplings, as well as of aromatic nucleophilic substitutions of hydroquinones or quinoids. Finally, the laccase-initiated domino/cascade synthesis of valuable aromatic (hetero)cycles, elegant strategies widely documented in the literature across more than three decades, is also presented.