Search

The production of active pharmaceutical ingredients (APIs) and fine chemicals is accelerating due to the advent of novel microreactors and new materials for immobilizing customized biocatalysts that permit long-term use in continuous-flow reactors. This work studied the scalability of a tunable U-shape magnetic nanoparticles (MNPs)-based microreactor. The reactor consisted of a polytetrafluoroethylene tube (PTFE) of various inner diameters (ID = 0.75 mm, 1.50 mm, or 2.15 mm) and six movable permanent magnets positioned under the tube to create reaction chambers allowing the fluid reaction mixture to flow through and above the enzyme-loaded MNPs anchored by permanent magnets. The microreactors with various tube sizes and MNP capacities were tested with the preparative scale kinetic resolution of the drug-like alcohols 4-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (±)-1a and 4-(3,4-dihydroquinolin-1(2H)-yl)butan-2-ol (±)-1b, utilizing Lipase B from Candida antarctica immobilized covalently onto MNPs, leading to highly enantioenriched products [(R)-2a,b and (S)-1a,b]. The results in the U-shape MNP flow reactor were compared with reactions in the batch mode with CaLB-MNPs using similar conditions. Of the three different systems, the one with ID = 1.50 mm showed the best balance between the maximum loading capacity of biocatalysts in the reactor and the most effective cross-section area. The results showed that this U-shaped tubular microreactor might be a simple and flexible instrument for many processes in biocatalysis, providing an easy-to-set-up alternative to existing techniques.

This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.

This study describes six yeast strains for stereoselective ketone reductions. The reaction conditions for the yeast strains (Pichia carsonii, Lodderomyces elongisporus, Candida norvegica, Candida guillermondi, Debaromyces fabryi and Candida parapsilosis) were optimized in a design of experiments for three ketones of different properties. The pH tolerance, temperature stability and productivity of the bioreductions with lyophilized cells of the yeast strains were characterized. In several cases, the optimized bioreductions resulted in enantiopure alcohols (ee > 99 %) with conversions ranging from moderate to excellent.

A novel phenylalanine ammonia-lyase of the psychrophilic yeast Pseudozyma antarctica (PzaPAL) was identified by screening microbial genomes against known PAL sequences. PzaPAL has a significantly different substrate binding pocket with an extended loop (26 aa long) connected to the aromatic ring binding region of the active site as compared to the known PALs from eukaryotes. The general properties of recombinant PzaPAL expressed in E. coli were characterized including kinetic features of this novel PAL with l -phenylalanine (S)-1a and further racemic substituted phenylalanines rac-1b-g,k. In most cases, PzaPAL revealed significantly higher turnover numbers than the PAL from Petroselinum crispum (PcPAL). Finally, the biocatalytic performance of PzaPAL and PcPAL was compared in the kinetic resolutions of racemic phenylalanine derivatives (rac-1a-s) by enzymatic ammonia elimination and also in the enantiotope selective ammonia addition reactions to cinnamic acid derivatives (2a-s). The enantiotope selectivity of PzaPAL with o-, m-, p-fluoro-, o-, p-chloro- and o-, m-bromo-substituted cinnamic acids proved to be higher than that of PcPAL.

The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomu...

In this work, we report a practical method for alkylation of saturated heterocycles with chloroacetone yielding prochiral heterocyclic ketones, including previously not described molecules. The desired building blocks were obtained with high yields in hydrochloric salt forms, without the need for chromatographic purification.

Transaminases (TAs) offer an environmentally and economically attractive method for the direct synthesis of pharmaceutically relevant disubstituted 1-phenylpropan-2-amine derivatives starting from prochiral ketones. In this work, we report the application of immobilised whole-cell biocatalysts with (R)-transaminase activity for the synthesis of novel disubstituted 1-phenylpropan-2-amines. After optimisation of the asymmetric synthesis, the (R)-enantiomers could be produced with 88–89% conversion and >99% ee, while the (S)-enantiomers could be selectively obtained as the unreacted fraction of the corresponding racemic amines in kinetic resolution with >48% conversion and >95% ee.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

In vitro mimicking of hepatic drug metabolism is a key issue in early-stage drug discovery. Synthetic metalloporphyrins show structural similarity with the heme type prosthetic group of cytochrome P450 as primary hepatic enzyme in oxidative drug biotransformation. Therefore, they can catalyze these oxidations. Concerning economical aspects and the poor stability of metalloporphyrin, their immobilization onto or into solid carriers can be promising solution. This study presents a novel immobilized metalloporphyrin nanocomposite system and its potential use as biomimetic catalysts. The developed two-step immobilization procedure consists of two main steps. First, the ionic binding of meso-tetra (parasulphonatophenyl) iron porphyrin onto functionalized magnetic nanoparticles is established, followed by embedding the nanoparticles into polylactic acid nanofibers by electrospinning technique. Due to the synergistic morphological and chemo-structural advantages of binding onto nanoparticles and embedding in polymeric matrices the biomimetic efficiency of metalloporphyrin can be remarkably enhanced, while substrate conversion value was tenfold larger than which could be achieved with classic human liver microsomal system.

As efforts in rational drug design are driving the pharmaceutical industry towards more complex molecules, the synthesis and production of these new drugs can benefit from new reaction routes. In addition to the introduction of new centers of asymmetry, complexity can be also increased by ring saturation, which also provides improved developability measures. Therefore, in this report, our aim was to develop transaminase (TA)-catalyzed asymmetric synthesis of a new group of potential chiral drug scaffolds comprising a saturated amine heterocycle backbone and an asymmetric primary amine sidechain (55a–g). We screened the Codex® Amine Transaminase Kit of 24 transaminases with the morpholine containing ketone 57a, resulting in one (R)-selective TA and three (S)-selective TAs operating at 100 mM substrate concentration and 25 v/v% isopropylamine (IPA) content. The optimized reaction conditions were than applied for asymmetric transamination of further six ketones (57b–g) containing various amine heterocycles, in which a strong effect of the substitution pattern of the γ-position relative to the substituted N-atom could be observed. Mediated by the most enantiotope selective (S)-TAs in scaled-up process, the (S)-amines [(S)-55a–g] were isolated with moderate-to-excellent yields (47–94%) in enantiopure form (>99% ee).

In this study, yeast strains were screened and immobilized in a form preserving the multifaceted biocatalytic activity. Immobilization of the silica-supported whole cells of various yeasts, such as...

A green and facile method has been developed for the preparation of in situ immobilized gold nanoparticles (AuNPs) using agarose as a reducing and stabilizing agent. The size of the synthesized AuNPs ranges between 10 and 100 nm, and their average size can be controlled by the concentrations of the agarose and gold salt. The agarose matrix as a mild and green reaction medium can provide a good dispersion environment for forming AuNPs, and the hydrogel can be well homogenized with polyacrylic macroporous microbeads as well, which can adsorb and stabilize the particles leading to the simultaneous synthesis and immobilization of AuNPs avoiding harmful inorganic compounds or organic solvents. The supported gold nanocatalyst was successfully applied as a catalyst in packed bed reactors for efficient NaBH4-mediated reduction of p-nitrophenol in continuous-flow mode.

Silica gel/alginate/poly(aspartic acid) composite beads were prepared for immobilization of lipase B enzyme from Candida antarctica (CaLB). CaLB was first adsorbed on functionalized mesoporous silica gel particles, which were then entrapped in the interpenetrating network of thiolated poly(aspartic acid) and alginate, cross-linked by zinc ions. Finally, the beads were chemically stabilized by poly(ethylene glycol) diglycidyl ether, a bisepoxide cross-linker. In this manner, spherical biocatalysts with a diameter of 3–4 mm were prepared and their biocatalytic performance was tested by kinetic resolution of racemic 1-phenylethanol. The activity of CaLB in the beads was comparable to that of CaLB physically adsorbed on silica gel particles. The composite beads were easy to recover after use and no loss of biocatalytic activity was observed even after five test reaction cycles. Furthermore, the CaLB in the composite beads showed sufficient thermal stability up to 90 °C, contrary to CaLB adsorbed only on silica gel particles.

The dual functionalization of magnetic nanoparticles with inert (methyl) and reactive (aminopropyl) groups enables efficient immobilization of synthetic metalloporphyrins (such as 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin) via covalent or ionic interactions. The proportion of reactive function on the surface has significant effect on the biomimetic activity of metalloporphyrins. The optimized magnetic nanocatalyst containing porphyrin was successfully applied for biomimetic oxidation of antihypertensive drug Amlodipine in batch and continuous-flow reactors as well.

An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non-specific acid phosphatases bearing a His-tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni2+ as the most appropriate metal ion were applied as support. First, the His-tagged phosphatases were selectively bound to the metal-chelate functions of the support. Then, the enzyme-charged silica nanoparticles were further stabilized by forming a covalent linkage between nucleophilic moieties at the enzyme surface and free amino groups of the support using neopentylglycol diglycidylether as the most effective bifunctional linking agent. The phosphatase biocatalysts obtained by this method exhibited better phosphate transfer activity with a range of alcohols and PPi as phosphate donor in aqueous medium applying batch and continuous-flow modes than the ones immobilized on conventional supports. Furthermore, this novel strategy opens up novel possibility for efficient immobilization of other His-tagged recombinant enzymes.

Tailored mutants of phenylalanine ammonia‐lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non‐natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)‐arylacrylates. The wild‐type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well‐studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4‐methoxyphenyl)‐, (napthalen‐2‐yl)‐, ([1,1′‐biphenyl]‐4‐yl)‐, (4′‐fluoro‐[1,1′‐biphenyl]‐4‐yl)‐, and (5‐phenylthiophene‐2‐yl)alanines.

Ride the wave! Biocatalysis uses nature's catalysts, enzymes and whole cell systems, for synthetic purposes. In a biotransformation, the biocatalyst transforms a well-defined substrate to the desired product, in contrast to the fermentation process, which produces the desired product from a complex mixture of nutrients. Biocatalysis has reached an industrially established level through several waves of technological evolution; participants of the BioTrans 2017 conference in Budapest could witness the newest wave of this technology.

Immobilization of the recombinant, plant-derived Petroselinum crispum phenylalanine ammonia lyase (PcPAL) in electrospun matrices have the potential to create promising, easy-to-use biocatalysts. Polylactic acid (PLA) a biologically inert, commercial biopolymer, was chosen as the material of the carrier system. PLA could be electrospun properly only from water-immiscible organic solvents, which limits its application as a carrier of sensitive biological objects. The emulsion electrospinning is a proper solution to overcome this issue using non-ionic emulsifiers with different hydrophilic-lipophilic balance (HLB) values. The stabilized emulsion could protect the sensitive PcPAL dissolved in the aqueous buffer phase and improve fiber formation, plus help to keep the biocatalytic activity of enzymes. In this study, the first approach is described to produce PLA nanofibers containing PcPAL enzymes by emulsion electrospinning and to use the resulted biocatalyst in the ammonia elimination reaction from l-phenylalanine.

Aromatic amino acid ammonia‐lyases and aromatic amino acid 2,3‐aminomutases contain the post‐translationally formed prosthetic 3,5‐dihydro‐4‐methylidene‐5H‐imidazol‐5‐one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α‐ or β‐amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β‐position inhibited phenylalanine ammonia‐lyases (PAL), representing MIO enzymes. X‐ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene‐substituted substrate analogue as being a mirror image relation to the natural l‐phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)‐ and (S)‐enantiomers is possible by a mirror image packing.

An immobilization engineering approach using bioinformatics and experimental design tools was applied to improve the sol–gel enzyme entrapment methodology. This strategy was used for the immobilization of lipase B from Candida antarctica (CaLB), a versatile enzyme widely used even on the industrial scale. The optimized entrapment of CaLB in sol–gel matrices is reported by the response-surface methodology enabling efficient process development. The immobilized CaLBs characterized by functional efficiency and enhanced recovery provided economical and green options for flow chemistry. Various ternary mixtures of sol–gel precursors allowed the creation of tailored entrapment matrices best suited for the enzyme and its targeted substrate. The sol–gel-entrapped forms of CaLB were excellent biocatalysts in the kinetic resolutions of secondary alcohols and secondary amines with aromatic or aliphatic substituents both in batch and continuous-flow biotransformations.

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.

Biomimetic oxidation of drugs catalyzed by metalloporphyrins can be a novel and promising way for the effective and sustainable synthesis of drug metabolites. The immobilization of 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin (FeTPFP) and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin (FeTSPP) via stable covalent or rapid ionic binding on aminopropyl-functionalized magnetic nanoparticles (MNPs-NH2) were developed. These immobilized catalysts could be efficiently applied for the synthesis of new pharmaceutically active derivatives and liver related phase I oxidative major metabolite of an antiarrhythmic drug, amiodarone integrated in a continuous-flow magnetic chip reactor (Magnechip).

Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.

Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a&ndash, d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d.

A green and facile method has been developed for the preparation of

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial), the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

Immobilization of lipases from Pseudomonas fluorescens (Lipase AK), Burkholderia (Pseudomonas) cepacia (Lipase PS) and lipase B from Pseudozyma (Candida) antarctica (CaLB) was investigated by entrapment in electrospun poly(vinyl alcohol) (PVA) nanofibers. The activity and selectivity of the lipases entrapped in PVA nanofibers were characterized in kinetic resolution of racemic secondary alcohols using acylation in organic media. Potential bioimprinting effect of eight substrate-mimicking additives [polyethylene glycols (PEGs), non-ionic detergents (NIDs) and various organosilanes] was tested with the fiber-entrapped lipases. The nanofibrous lipase biocatalyst entrapped in the presence of the additives were also characterized by rheology, differential scanning calorimetry and scanning electron microscopy. In addition to the known lipase-bioimprinting agents (PEGs, NIDs), phenyl- and octyltriethoxysilane also enhanced substantially the biocatalytic properties of lipases in their electrospun PVA fiber-entrapped forms. The reasons of bioimprinting effect of several additives were rationalized by docking studies in the open and closed form of CaLB.

Alcalase® (Subtilisin A) was immobilized by simple hydrophobic adsorption onto various surface-grafted macroporous silica gels resulting in easy-to-prepare and stable biocatalysts enabling the efficient kinetic resolution (KR) and dynamic kinetic resolution (DKR) of racemic N-Boc-phenylalanine ethyl thioester with benzylamine. The immobilized Alcalase biocatalysts, which retained their activity and selectivity when stored at 4 °C for more than a year, were tested in enzymatic aminolysis in batch and continuous-flow KRs resulting in (S)-N-Boc-phenylalanine benzylamide in high enantiomeric purity. In KR of the racemic thioester by Alcalase-catalyzed aminolysis in a continuous-flow reactor, the productivity (specific reaction rate, rflow) and enantiomeric ratio (E) were studied in the 0–100 °C range. The effect of the temperature on base-catalyzed racemization of the non-transformed (R)-thioester in a continuous-flow reactor was also investigated in the 0–150 °C range. The continuous-mode DKR of the racemic thioester in a serial cascade system of six biocatalyst-filled columns at 50 °C for KR and five grafted silica gel-filled columns at 150 °C for racemization resulted in the formation of the (S)-benzylamide in 79% conversion, 8.17 g L−1 h−1 volumetric productivity and 98% ee. This is the first example of a dynamic kinetic resolution of an amino acid derivative in continuous-flow mode using an alternating cascade of packed-bed enzyme reactors and racemization reactors kept at different temperatures.

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).

In this study enantiomer selective isomerization of various racemic α- and β-arylalanines catalysed by phenylalanine 2,3-aminomutase from Pantoea agglomerans (PaPAM) was investigated. Both α- and β-arylalanines were accepted as substrates when the aryl moiety was relatively small, like phenyl, 2-, 3-, 4-fluorophenyl or thiophen-2-yl. While 2-substituted α-phenylalanines bearing bulky electron withdrawing substituents did not react, the corresponding substituted β-aryl analogues were converted rapidly. Conversion of 3- and 4-substituted α-arylalanines happened smoothly, while conversion of the corresponding β-arylalanines was poor or non-existent. In the range of pH 7–9 there was no significant influence on the conversion of racemic α- or β-(thiophen-2-yl)alanines, whereas increasing the concentration of ammonia (ammonium carbonate from 50 to 1000 mM) inhibited the isomerization progressively and decreased the amount of the by-product (i.e. (E)-3-(thiophen-2-yl)acrylic acid was detected). In all cases, the high ee values of the products indicated excellent enantiomer selectivity and stereospecificity of the isomerization except for (S)-2-nitro-α-phenylalanine (ee 92%) from the β-isomer. Substituent effects were rationalized by computational modelling revealing that one of the main factors controlling biocatalytic activity was the energy difference between the covalent regioisomeric enzyme–substrate complexes.

This chapter focuses on nanoiber fabrication by electrospinning techniques for the efective immobilization of biomolecules (such as enzymes or active pharmaceutical ingredients—APIs). In this chapter, the development of precursor materials (from commercial polymer systems to systematically designed biopolymers), entrapment protocols, and precursor-nanoiber characterization methods are represented. The entrapment ability of poly(vinyl alcohol) and systematically modiied polyaspartamide nanoibers was investigated for immobilization of two diferent lipases (from Candida antarctica and Pseudomonas luorescens) and for formulation of the antibacterial and antiviral agent, rifampicin. The encapsulated biomolecules in electrospun polymer ibers could be promising nanomaterials for industrial biocatalysis to produce chiral compound or in the development of smart drug delivery systems.

Carboxylated single‐walled carbon nanotubes (SwCNTCOOH) were used as a support for the covalent immobilization of phenylalanine ammonia‐lyase (PAL) from parsley by two different methods. The nanostructured biocatalysts (SwCNTCOOH‐PALI and SwCNTCOOH‐PALII) with low diffusional limitation were tested in the batch‐mode kinetic resolution of racemic 2‐amino‐3‐(thiophen‐2‐yl)propanoic acid (1) to yield a mixture of (R)‐1 and (E)‐3‐(thiophen‐2‐yl)acrylic acid (2) and in ammonia addition to 2 to yield enantiopure (S)‐1. SwCNTCOOH‐PALII was a stable biocatalyst (>90 % of the original activity remained after six cycles with 1 and after three cycles in 6 m NH3 with 2). The study of ammonia addition to 2 in a continuous‐flow microreactor filled with SwCNTCOOH‐PALII (2 m NH3, pH 10.0, 15 bar) between 30–80 °C indicated no significant loss of activity over 72 h up to 60 °C. SwCNTCOOH‐PALII in the continuous‐flow system at 30 °C was more productive (specific reaction rate, r flow=2.39 μmol min−1 g−1) than in the batch reaction (r batch=1.34 μmol min−1 g−1).

The Chan-Lam-Evans coupling is the copper(II) acetate promoted reaction of phenols and boronic acids producing diaryl ethers. In this paper a modification of the original reaction is reported using copper(II) fluoride reagent which gives higher yields than copper(II) acetate. The most significant increase in yield was observed in formation of sterically hindered phenols. We hypothesize that fluoride ions help to cleave the boron-carbon bond. The novel method is suitable for selective, efficient and economical synthesis of diaryl ether compounds under mild conditions.

The activity of Bioorganic Chemistry Group (BCG) within Department of Organic Chemistry and Technology at Budapest University of Technology and Economics is related to various areas of synthetic chemistry, biotechnology and enzymology. This review gives an overview on the research activity of the group covering development of synthetic organic chemistry methods; stereoselective biotransformations with lipases, ammonia-lyases and further biocatalysts in batch and continuous-flow reactions; novel enzyme immobilization methods; and enzyme structural and mechanistic studies by experimental and computational techniques.

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the L-enantiomers of racemic styrylalanines rac-1a–d – which are less studied and synthetically challenging unnatural amino acids – by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from L-styrylalanine (L-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, L-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of L-styrylalanine, L-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform L-styrylalanine with comparable activity to that of the wt-PcPAL with L-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a–d) providing access to D-1a–d by kinetic resolution, even though the D-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a–d could be rationalized by molecular modeling, indicating the more relaxed enzyme–substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a–d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a–d, and the non-productive binding states of the unsaturated ligands 2a–d within the active sites of either wt-PcPAL or F137V-PcPAL – as indicated by molecular modeling – might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.