Transesterification reactions are fundamental transformations in organic chemistry, yet performing them in aqueous media is challenging because of the competing hydrolysis reaction. In this study, we describe a mutant of alcohol oxidase from Phanerochaete chrysosporium (PcAOx-VPN) that also exhibits transesterification activity. Moreover, PcAOx-VPN displays no detectable hydrolytic activity, owing to its hydrophobic active site, which effectively excludes water. These characteristics make PcAOx-VPN a promising catalyst for transesterification reactions in aqueous media, a context that is typically compromised by competing hydrolysis.

Enzymatic cascade systems enhance the efficiency of biocatalysis by mimicking natural metabolic pathways. However, conventional platforms face challenges such as ineffective enzyme distribution, uncontrolled stoichiometry, limited substrate accessibility, and a microenvironment that is suboptimal for enzyme performance. Inspired by cellular biomolecular condensates, we here present a design based on oppositely charged single enzyme nanogels (SENs) that phase separate into denser microdroplet coacervates, which thereby serve both as an immobilization scaffold and functional unit. These coacervates, the first fabricated exclusively from oppositely charged nanogels, form under broad environmental conditions and enable specific microenvironments by adjusting SEN ratios to match the requirements of coupled enzymes. Selective substrate enrichment and efficient diffusion of intermediates enhance cascade productivity. The applicability of the platform is demonstrated with pharmaceutically relevant enzymes, highlighting its potential for biosynthetic applications. This work establishes a new paradigm that integrates protein-polymer engineering with phase separation chemistry to overcome key limitations in cascade enzyme catalysis.

This study presents a three-step one pot enzymatic cascade for the synthesis of a δ-lactone. Utilising acetaldehyde, combining 2-deoxyribose-5-phosphate aldolase (DERA) with an alcohol dehydrogenase (ADH) and a cofactor regeneration system this δ-lactone is synthesised with the same stereochemistry as the statin side chain precursor. The initial stage in this cascade involves the double aldol reaction, catalysed by DERA to produce the chiral lactone precursor from the achiral substrate acetaldehyde. The main challenge at this stage is the instability of DERA in the presence of high acetaldehyde concentrations. Therefore, Lactobacillus brevis DERA with a high natural acetaldehyde tolerance was genetically engineered to further improve this property. LbDERA C42M E78K exhibited improved activity and stability (no activity loss over 2 h) compared to the wild type (20% activity loss). In the second stage of the cascade, the aldol product is selectively oxidised to the lactone. A commercially available ADH was identified to selectively catalyse this oxidation using NADP⁺ as electron acceptor. NADP⁺ regeneration was achieved using O₂ as substrate in two different ways: using either photo-activated flavin or NADPH oxidase (NOX). The lactone was successfully purified from the enzymatic cascades from a preparative scale reaction in 97% purity with an optical rotation [α]_D = +34.2° (c = 0.7), proving the feasibility in a multi-enzyme three-step one-pot cascade.

Contemporary Biocatalysis heavily relies on enzyme engineering as natural enzymes frequently lack the requisite attributes for effective organic synthesis. The inherent limitations in stability, catalytic activity, and selectivity of wild-type enzymes often hinder their suitability for chemical synthesis. Over the past 25 years, there has been an unprecedented advancement in protein engineering tools, empowering enzymologists to customise enzymes to precisely meet the demands of organic synthesis. In this discussion, we delineate some of the most crucial techniques in enzyme engineering and their significance in facilitating chemical synthesis.

Effectiveness of catalytic processes using heterogeneous biocatalysts depends not only on the activity of the enzyme, but also on the efficiency of the used reactor. In this paper, we present a novel design of a basket reactor with a stationary catalyst bed (StatBioChem). The developed design was compared to a commercially available rotating bed reactor (SpinChem®). The biocatalysts used were invertase and acyltransferase from Mycobacterium smegmatis (MsAcT) immobilised on macroporous silica supports. The obtained values of initial reaction rate, both in the reaction of saccharose hydrolysis and 2,2-dimethyl-1,3-propanediol (NPG) transesterification, were twice higher for the StatBioChem reactor. A similar relationship was also observed regarding the process efficiency expressed as STY.

S-Adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are highly chemoselective enzymes grouped in C-, N-, O-, S- and halide MTs, depending on the (hetero) atom that acts as the methyl group acceptor. So far, OMTs present the largest group, including many well investigated candidates. The catechol OMT from mammals such as from Rattus norvegicus (RnCOMT) is involved in the metabolism of neurotransmitters like dopamine. It is known to methylate the hydroxyl of the catechol ring in the 3 position. There are also reports showing that the regioselectivity of different COMTs can vary leading to different products with methyl groups in the 3 and or 4 positions. Nevertheless, there was only O-methylation reported for COMTs. Another related MT, the caffeate OMT involved in the lignin biosynthesis of plants has also been reported as a chemoselective enzyme. In nature, S-methylation is a rare phenomenon with different methyl donors being involved in the methyl transfer onto sulfur atoms. Several SAM-dependent MTs are identified as S-methyltransferases (SMTs), these are involved in salvaging pathways and xenobiotic metabolism of cells. Here, we report a new function of three OMTs; RnCOMT, a COMT from Myxococcus xanthus (MxSafC), and a CaOMT from Prunus persica (PpCaOMT) with acceptance towards different aromatic thiol substrates with up to full conversion.

Granulicella tundricola hydroxynitrile lyase (GtHNL) is a manganese dependent cupin that catalyzes the enantioselective synthesis of cyanohydrins. The analysis of its active site shows high similarity with the active site of cupin Tm1459 from Thermotoga maritima, an enzyme that catalyzes the oxidative cleavage of styrene derivatives. GtHNL (GtHNL-WT) was found to catalyze the oxidative cleavage of α-methyl styrene, too. The conversion of α-methyl styrene yielded 23.6 ± 0.8% of acetophenone after 20 h. On the other hand, Tm1459 was not able to catalyze the synthesis of cyanohydrins efficiently. A low yield of rac-mandelonitrile was obtained from benzaldehyde and HCN using either Tm1459-WT or Tm1459-C106L, a variant more active in oxidative catalysis. On the basis of the molecular analysis of GtHNL and Tm1459 active sites, the variants GtHNL-H96A, GtHNL-H96F, and GtHNL-A40H/V42T/H96A/Q110H were produced and evaluated for improved catalytic activity toward oxidative cleavage of styrenes. The amino acid substitution H96A liberates an additional manganese coordination position and enlarges the GtHNL-WT active site cavity. Similarly, the amino acid substitution H96F liberates a coordination site as described for the GtHNL-H96A variant but without enlarging the active site space. All variants were able to catalyze the oxidative cleavage of styrene derivatives. The best results were observed using GtHNL-H96A as catalyst. It displayed a higher yield of acetophenone (42%) as compared to GtHNL-A40H/V42T/H96A/Q110H (12%) and GtHNL-H96F (11%) after 20 h of reaction time. No oxidation of Mn(II) to Mn(III) could be detected by electron paramagnetic resonance (EPR), whereas evidence for a radical mechanism is presented. Control reactions using 0.1 and 0.5 mM of MnCl₂ in the absence of enzyme showed no significant oxidation reaction.

Enantiomerically pure cyanohydrins are of great importance in the chemical and pharmaceutical industries. Their synthesis is possible through the use of highly selective hydroxynitrile lyases. In this work, an R-selective hydroxynitrile lyase (AtHNL) from Arabidopsis thaliana was immobilized inside a silica microreactor with a tortuous and hierarchical pore structure. After immobilization, the enzyme activity was tested for benzaldehyde 1a, and its analogs 4-fluorobenzaldehyde 1b, 4-methoxybenzaldehyde 1c and 4-(trifluoromethyl)benzaldehyde 1d. With their different degrees of reactivity they also display a different susceptibility to the racemic chemical background reaction. It was shown that the use of a flow microreactor suppressed the background reaction even for the most susceptible substrate 1d. Furthermore, the use of a flow microreactor enabled high substrate conversion (90-95%) while maintaining a high enantiomeric excess (90-98%) with residence times of 3 to 30 min. The productivity, which depended on substrate reactivity and flow rate, was evaluated by space-time-yield (STY) and reached a value from 60 g L⁻¹ h⁻¹ to 1290 g L⁻¹ h⁻¹. Additionally it was demonstrated that the stability of the immobilized enzyme depends on the flow rates used and thus on the shear forces acting inside the microreactor and interfacial effects associated with them.

A sequential-type as well as a tandem-type chemoenzymatic flow cascade combining an organocatalytic aldol reaction and a biocatalytic reduction to form stereoselectively a 1,3-diol with two stereogenic centers were developed. Initially, a comprehensive screening of 24 alcohol dehydrogenases was carried out and the identified candidates were applied in different multi-step flow cascades. All four stereoisomers of the desired 1,3-diol product are accessible via a sequential flow approach with product formation-related conversions of up to 76 % over two steps, isolated yields of up to 64 % and enantiomeric excess of >99 % in all cases. In addition, a tandem-type flow process, performing both reaction steps simultaneously, was established leading to 51 % conversion with >99 % ee and 8 : 1 d.r. and representing a combination of the fields of asymmetric chemocatalysis, biocatalysis and flow chemistry.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M²⁺_R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M²⁺_A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M²⁺_A affords the overall lowest energy complex, which drives the transition from M²⁺_R to M²⁺_A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 g_product·L^-1·h⁻¹·mg_protein^-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN₃ solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed.