Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency.

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2 s-1) compared to free cofactor (kdec=1.8 s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s-1. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.

Recyclable and Robust Optical Nanoprobes with Engineered Enzymes for Sustainable Serodiagnostics.

Recyclable fluorescence assays that can be stored at room temperature would greatly benefit biomedical diagnostics by bringing sustainability and cost-efficiency, especially for point-of-care serodiagnostics in developing regions. Here, a general strategy is proposed to generate recyclable fluorescent probes by using engineered enzymes with enhanced thermo-/chemo-stability, which maintains an outstanding serodiagnostic performance (accuracy >95%) after 10 times of recycling as well as after storage at elevated temperatures (37 °C for 10 days). With these three outstanding properties, recyclable fluorescent probes can be designed to detect various biomarkers of clinical importance by using different enzymes.

A high-performance electrochemical biosensor using an engineered urate oxidase.

We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. This biosensor showed a low limit-of-detection (9.16 nM), a high sensitivity (14 µA/µM), a wide range of linearity (50 nM-1 mM), and more than 28 days lifetime.

One-Pot Biocatalytic Synthesis of Primary, Secondary, and Tertiary Amines with Two Stereocenters from α,ß-Unsaturated Ketones Using Alkyl-Ammonium Formate.

The efficient asymmetric catalytic synthesis of amines containing more than one stereogenic center is a current challenge. Here, we present a biocatalytic cascade that combines ene-reductases (EReds) with imine reductases/reductive aminases (IReds/RedAms) to enable the conversion of α,ß-unsaturated ketones into primary, secondary, and tertiary amines containing two stereogenic centers in very high chemical purity (up to >99%), a diastereomeric ratio, and an enantiomeric ratio (up to >99.8:<0.2). Compared with previously reported strategies, our strategy could synthesize two, three, or even all four of the possible stereoisomers of the amine products while precluding the formation of side-products. Furthermore, ammonium or alkylammonium formate buffer could be used as the only additional reagent since it acted both as an amine donor and as a source of reducing equivalents. This was achieved through the implementation of an NADP-dependent formate dehydrogenase (FDH) for the in situ recycling of the NADPH coenzyme, thus leading to increased atom economy for this biocatalytic transformation. Finally, this dual-enzyme ERed/IRed cascade also exhibits a complementarity with the recently reported EneIRED enzymes for the synthesis of cyclic six-membered ring amines. The ERed/IRed method yielded trans-1,2 and cis-1,3 substituted cyclohexylamines in high optical purities, whereas the EneIRED method was reported to yield one cis-1,2 and one trans-1,3 enantiomer. As a proof of concept, when 3-methylcyclohex-2-en-1-one was converted into secondary and tertiary chiral amines with different amine donors, we could obtain all the four possible stereoisomer products. This result exemplifies the versatility of this method and its potential for future wider utilization in asymmetric synthesis by expanding the toolbox of currently available dehydrogenases via enzyme engineering and discovery.

Continuous Flow Biocatalytic Reductive Amination by Co-Entrapping Dehydrogenases with Agarose Gel in a 3D-Printed Mould Reactor.

Herein, we show how the merge of biocatalysis with flow chemistry aided by 3D-printing technologies can facilitate organic synthesis. This concept was exemplified for the reductive amination of benzaldehyde catalysed by co-immobilised amine dehydrogenase and formate dehydrogenase in a continuous flow micro-reactor. For this purpose, we investigated enzyme co-immobilisation by covalent binding, or ion-affinity binding, or entrapment. Entrapment in an agarose hydrogel turned out to be the most promising solution for this biocatalytic reaction. Therefore, we developed a scalable and customisable approach whereby an agarose hydrogel containing the co-entrapped dehydrogenases was cast in a 3D-printed mould. The reactor was applied to the reductive amination of benzaldehyde in continuous flow over 120 h and afforded 47 % analytical yield and a space-time yield of 7.4 g L day-1 using 0.03 mol% biocatalysts loading. This work also exemplifies how rapid prototyping of enzymatic reactions in flow can be achieved through 3D-printing technology.

High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from l-Phenylalanine via Linear and Divergent Enzymatic Cascades.

Enantiomerically pure 1,2-amino alcohols are important compounds due to their biological activities and wide applications in chemical synthesis. In this work, we present two multienzyme pathways for the conversion of l-phenylalanine into either 2-phenylglycinol or phenylethanolamine in the enantiomerically pure form. Both pathways start with the two-pot sequential four-step conversion of l-phenylalanine into styrene via subsequent deamination, decarboxylation, enantioselective epoxidation, and enantioselective hydrolysis. For instance, after optimization, the multienzyme process could convert 507 mg of l-phenylalanine into (R)-1-phenyl-1,2-diol in an overall isolated yield of 75% and >99% ee. The opposite enantiomer, (S)-1-phenyl-1,2-diol, was also obtained in a 70% yield and 98-99% ee following the same approach. At this stage, two divergent routes were developed to convert the chiral diols into either 2-phenylglycinol or phenylethanolamine. The former route consisted of a one-pot concurrent interconnected two-step cascade in which the diol intermediate was oxidized to 2-hydroxy-acetophenone by an alcohol dehydrogenase and then aminated by a transaminase to give enantiomerically pure 2-phenylglycinol. Notably, the addition of an alanine dehydrogenase enabled the connection of the two steps and made the overall process redox-self-sufficient. Thus, (S)-phenylglycinol was isolated in an 81% yield and >99.4% ee starting from ca. 100 mg of the diol intermediate. The second route consisted of a one-pot concurrent two-step cascade in which the oxidative and reductive steps were not interconnected. In this case, the diol intermediate was oxidized to either (S)- or (R)-2-hydroxy-2-phenylacetaldehyde by an alcohol oxidase and then aminated by an amine dehydrogenase to give the enantiomerically pure phenylethanolamine. The addition of a formate dehydrogenase and sodium formate was required to provide the reducing equivalents for the reductive amination step. Thus, (R)-phenylethanolamine was isolated in a 92% yield and >99.9% ee starting from ca. 100 mg of the diol intermediate. In summary, l-phenylalanine was converted into enantiomerically pure 2-phenylglycinol and phenylethanolamine in overall yields of 61% and 69%, respectively. This work exemplifies how linear and divergent enzyme cascades can enable the synthesis of high-value chiral molecules such as amino alcohols from a renewable material such as l-phenylalanine with high atom economy and improved sustainability.

Bacterial Peroxidase on Electrochemically Reduced Graphene Oxide for Highly Sensitive H2 O2 Detection.

Peroxidase enzymes enable the construction of electrochemical sensors for highly sensitive and selective quantitative detection of various molecules, pathogens and diseases. Herein, we describe the immobilization of a peroxidase from Bacillus s. (BsDyP) on electrochemically reduced graphene oxide (ERGO) deposited on indium tin oxide (ITO) and polyethylene terephthalate (PET) layers. XRD, SEM, AFM, FT-IR and Raman characterization of the sensor confirmed its structural integrity and a higher enzyme surface occupancy. The BsDyP-ERGO/ITO/PET electrode performed better than other horseradish peroxidase-based electrodes, as evinced by an improved electrochemical response in the nanomolar range (linearity 0.05-280 µM of H2 O2 , LOD 32 nM). The bioelectrode was mechanically robust, active in the 3.5-6 pH range and exhibited no loss of activity upon storage for 8 weeks at 4 °C.

High Regio- and Stereoselective Multi-enzymatic Synthesis of All Phenylpropanolamine Stereoisomers from ß-Methylstyrene.

We present a one-pot cascade for the synthesis of phenylpropanolamines (PPAs) in high optical purities (er and dr up to >99.5 %) and analytical yields (up to 95 %) by using 1-phenylpropane-1,2-diols as key intermediates. This bioamination entails the combination of an alcohol dehydrogenase (ADH), an ω-transaminase (ωTA) and an alanine dehydrogenase to create a redox-neutral network, which harnesses the exquisite and complementary regio- and stereo-selectivities of the selected ADHs and ωTAs. The requisite 1-phenylpropane-1,2-diol intermediates were obtained from trans- or cis-ß-methylstyrene by combining a styrene monooxygenase with epoxide hydrolases. Furthermore, in selected cases, the envisioned cascade enabled to obtain the structural isomer (1S,2R)-1-amino-1-phenylpropan-2-ol in high optical purity (er and dr >99.5 %). This is the first report on an enzymatic method that enables to obtain all of the four possible PPA stereoisomers in great enantio- and diastereo-selectivity.

Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity.

The l-lysine-ϵ-dehydrogenase (LysEDH) from Geobacillus stearothermophilus naturally catalyzes the oxidative deamination of the ϵ-amino group of l-lysine. We previously engineered this enzyme to create amine dehydrogenase (AmDH) variants that possess a new hydrophobic cavity in their active site such that aromatic ketones can bind and be converted into α-chiral amines with excellent enantioselectivity. We also recently observed that LysEDH was capable of reducing aromatic aldehydes into primary alcohols. Herein, we harnessed the promiscuous alcohol dehydrogenase (ADH) activity of LysEDH to create new variants that exhibited enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes to primary alcohols. Notably, these novel engineered dehydrogenases also catalyzed the reductive amination of a variety of aldehydes and ketones with excellent enantioselectivity, thus exhibiting a dual AmDH/ADH activity. We envisioned that the catalytic bi-functionality of these enzymes could be applied for the direct conversion of alcohols into amines. As a proof-of-principle, we performed an unprecedented one-pot "hydrogen-borrowing" cascade to convert benzyl alcohol to benzylamine using a single enzyme. Conducting the same biocatalytic cascade in the presence of cofactor recycling enzymes (i.e., NADH-oxidase and formate dehydrogenase) increased the reaction yields. In summary, this work provides the first examples of enzymes showing "alcohol aminase" activity.

Kinetic Resolution of Racemic Primary Amines Using Geobacillus stearothermophilus Amine Dehydrogenase Variant.

A NADH-dependent engineered amine dehydrogenase from Geobacillus stearothermophilus (LE-AmDH-v1) was applied together with a NADH-oxidase from Streptococcus mutans (NOx) for the kinetic resolution of pharmaceutically relevant racemic α-chiral primary amines. The reaction conditions (e. g., pH, temperature, type of buffer) were optimised to yield S-configured amines with up to >99 % ee.

Generation of amine dehydrogenases with increased catalytic performance and substrate scope from ε-deaminating L-Lysine dehydrogenase.

Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.

Efficient synthesis of enantiopure amines from alcohols using resting E. coli cells and ammonia.

α-Chiral amines are pivotal building blocks for chemical manufacturing. Stereoselective amination of alcohols is receiving increased interest due to its higher atom-efficiency and overall improved environmental footprint compared with other chemocatalytic and biocatalytic methods. We previously developed a hydrogen-borrowing amination by combining an alcohol dehydrogenase (ADH) with an amine dehydrogenase (AmDH) in vitro. Herein, we implemented the ADH-AmDH bioamination in resting Escherichia coli cells for the first time. Different genetic constructs were created and tested in order to obtain balanced expression levels of the dehydrogenase enzymes in E. coli. Using the optimized constructs, the influence of several parameters towards the productivity of the system were investigated such as the intracellular NAD+/NADH redox balance, the cell loading, the survival rate of recombinant E. coli cells, the possible toxicity of the components of the reaction at different concentrations and the influence of different substrates and cosolvents. In particular, the cofactor redox-balance for the bioamination was maintained by the addition of moderate and precise amounts of glucose. Higher concentrations of certain amine products resulted in toxicity and cell death, which could be alleviated by the addition of a co-solvent. Notably, amine formation was consistent using several independently grown E. coli batches. The optimized E. coli/ADH-AmDH strains produced enantiopure amines from the alcohols with up to 80% conversion and a molar productivity up to 15 mM. Practical applicability was demonstrated in a gram-scale biotransformation. In summary, the present E. coli-ADH-AmDH system represents an important advancement towards the development of 'green', efficient and selective biocatalytic processes for the amination of alcohols.

Regio- and stereoselective multi-enzymatic aminohydroxylation of ß-methylstyrene using dioxygen, ammonia and formate.

We report an enzymatic route for the formal regio- and stereoselective aminohydroxylation of ß-methylstyrene that consumes only dioxygen, ammonia and formate; carbonate is the by-product. The biocascade entails highly selective epoxidation, hydrolysis and hydrogen-borrowing alcohol amination. Thus, ß-methylstyrene was converted into 1R,2R and 1S,2R-phenylpropanolamine in 59-63% isolated yields, and up to >99.5: <0.5 dr and er.

Mechanistic Insight into the Catalytic Promiscuity of Amine Dehydrogenases: Asymmetric Synthesis of Secondary and Primary Amines.

Biocatalytic asymmetric amination of ketones, by using amine dehydrogenases (AmDHs) or transaminases, is an efficient method for the synthesis of α-chiral primary amines. A major challenge is to extend amination to the synthesis of secondary and tertiary amines. Herein, for the first time, it is shown that AmDHs are capable of accepting other amine donors, thus giving access to enantioenriched secondary amines with conversions up to 43 %. Surprisingly, in several cases, the promiscuous formation of enantiopure primary amines, along with the expected secondary amines, was observed. By conducting practical laboratory experiments and computational experiments, it is proposed that the promiscuous formation of primary amines along with secondary amines is due to an unprecedented nicotinamide (NAD)-dependent formal transamination catalysed by AmDHs. In nature, this type of mechanism is commonly performed by pyridoxal 5'-phosphate aminotransferase and not by dehydrogenases. Finally, a catalytic pathway that rationalises the promiscuous NAD-dependent formal transamination activity and explains the formation of the observed mixture of products is proposed. This work increases the understanding of the catalytic mechanism of NAD-dependent aminating enzymes, such as AmDHs, and will aid further research into the rational engineering of oxidoreductases for the synthesis of α-chiral secondary and tertiary amines.

Hydrolases-mediated transformation of oleuropein into demethyloleuropein.

Phenolic compounds present in extra virgin olive oil have recently attracted considerable attention due to their pharmacological activities. Among them oleacein (3,4-DHPEA-EDA), structurally related to oleochantal (4-HPEA-EDA), is one of the most studied. 3,4-DHPEA-EDA has been synthesized through decarboxylation of demethyloleuropein catalyzed by Er(OTf)3. Demethyloleuropein is extracted from black olives drupes in very limited amounts and only in particular periods of the year. The availability of demethyloleuropein could be increased by a selective hydrolysis of the methyl ester moiety of oleuropein, a secoiridoid present in large amount in olive leaves. In this work we describe a new enzymatic method for carrying out a selective hydrolysis of oleuropein via the screening of a panel of hydrolases (lipases, esterases and proteases). Among all the enzymes tested the best results was obtained using α-chymotrypsyn from bovine pancreas as biocatalyst, thus revealing a classic example of catalytic enzyme promiscuity.

Highly efficient production of chiral amines in batch and continuous flow by immobilized ω-transaminases on controlled porosity glass metal-ion affinity carrier.

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100 mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG3-AsR-ωTA could be recycled for at least 16 consecutive cycles (15 min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG3-AsR-ωTA was tested in a continuous flow packed-bed reactor (157 µL reactor volume), which produced more than 5 g of (S)-α-MBA (>49% conversion, >99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L-1 h-1.

Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of Nitriles from Alcohols, Air, and Ammonia.

We report an unprecedented catalytically promiscuous activity of the copper-dependent enzyme galactose oxidase. The enzyme catalyses the one-pot conversion of alcohols into the related nitriles under mild reaction conditions in ammonium buffer, consuming ammonia as the source of nitrogen and dioxygen (from air at atmospheric pressure) as the only oxidant. Thus, this green method does not require either cyanide salts, toxic metals, or undesired oxidants in stoichiometric amounts. The substrate scope of the reaction includes benzyl and cinnamyl alcohols as well as 4- and 3-pyridylmethanol, giving access to valuable chemical compounds. The oxidation proceeds through oxidation from alcohol to aldehyde, in situ imine formation, and final direct oxidation to nitrile.

Hydrogen-Borrowing Alcohol Bioamination with Coimmobilized Dehydrogenases.

The amination of alcohols is an important transformation in chemistry. The redox-neutral (i.e., hydrogen-borrowing) asymmetric amination of alcohols is enabled by the combination of an alcohol dehydrogenase (ADH) with an amine dehydrogenase (AmDH). In this work, we enhanced the efficiency of hydrogen-borrowing biocatalytic amination by co-immobilizing both dehydrogenases on controlled porosity glass FeIII ion-affinity beads. The recyclability of the dual-enzyme system was demonstrated (5 cycles) with total turnover numbers of >4000 and >1000 for ADH and AmDH, respectively. A set of (S)-configured alcohol substrates was aminated with up to 95% conversion and >99%ee (R). Preparative-scale amination of (S)-phenylpropan-2-ol resulted in 90% conversion and 80% yield of the product in 24 h.

A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation.

The styrene monooxygenase (SMO) system from Pseudomonas sp. consists of two enzymes (StyA and StyB). StyB catalyses the reduction of FAD at the expense of NADH. After the transfer of FADH2 from StyB to StyA, reaction with O2 generates FAD-OOH, which is the epoxidising agent. The wastage of redox equivalents due to partial diffusive transfer of FADH2 , the insolubility of recombinant StyB and the impossibility of expressing StyA and StyB in a 1:1 molar ratio reduce the catalytic efficiency of the natural system. Herein we present a chimeric SMO (Fus-SMO) that was obtained by genetic fusion of StyA and StyB through a flexible linker. Thanks to a combination of: 1) balanced and improved expression levels of reductase and epoxidase units, and 2) intrinsically higher specific epoxidation activity of Fus-SMO in some cases, Escherichia coli cells expressing Fus-SMO possess about 50 % higher activity for the epoxidation of styrene derivatives than E. coli cells coexpressing StyA and StyB as discrete enzymes. The epoxidation activity of purified Fus-SMO was up to three times higher than that of the two-component StyA/StyB (1:1, molar ratio) system and up to 110 times higher than that of the natural fused SMO. Determination of coupling efficiency and study of the influence of O2 pressure were also performed. Finally, Fus-SMO and formate dehydrogenase were coexpressed in E. coli and applied as a self-sufficient biocatalytic system for epoxidation on greater than 500 mg scale.

A biocatalytic method for the chemoselective aerobic oxidation of aldehydes to carboxylic acids.

Herein, we present a study on the oxidation of aldehydes to carboxylic acids using three recombinant aldehyde dehydrogenases (ALDHs). The ALDHs were used in purified form with a nicotinamide oxidase (NOx), which recycles the catalytic NAD+ at the expense of dioxygen (air at atmospheric pressure). The reaction was studied also with lyophilised whole cell as well as resting cell biocatalysts for more convenient practical application. The optimised biocatalytic oxidation runs in phosphate buffer at pH 8.5 and at 40 °C. From a set of sixty-one aliphatic, aryl-aliphatic, benzylic, hetero-aromatic and bicyclic aldehydes, fifty were converted with elevated yield (up to >99%). The exceptions were a few ortho-substituted benzaldehydes, bicyclic heteroaromatic aldehydes and 2-phenylpropanal. In all cases, the expected carboxylic acid was shown to be the only product (>99% chemoselectivity). Other oxidisable functionalities within the same molecule (e.g. hydroxyl, alkene, and heteroaromatic nitrogen or sulphur atoms) remained untouched. The reaction was scaled for the oxidation of 5-(hydroxymethyl)furfural (2 g), a bio-based starting material, to afford 5-(hydroxymethyl)furoic acid in 61% isolated yield. The new biocatalytic method avoids the use of toxic or unsafe oxidants, strong acids or bases, or undesired solvents. It shows applicability across a wide range of substrates, and retains perfect chemoselectivity. Alternative oxidisable groups were not converted, and other classical side-reactions (e.g. halogenation of unsaturated functionalities, Dakin-type oxidation) did not occur. In comparison to other established enzymatic methods such as the use of oxidases (where the concomitant oxidation of alcohols and aldehydes is common), ALDHs offer greatly improved selectivity.