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.

Granulicella tundricola hydroxynitrile lyase (GtHNL) catalyses the synthesis of chiral (R)‐ cyanohydrins and (R)‐β‐nitro alcohols. The triple variant GtHNL‐A40H/V42T/Q110H (GtHNL‐3V) was immobilised on Celite R‐633 and used in monophasic MTBE saturated with 100 mM KPi buffer pH 7 for the synthesis of (R)‐2‐nitro‐1‐phenylethanol (NPE) in batch and continuous flow systems. Nitromethane was used as a nucleophile. A total of 82% of (R)‐NPE and excellent enantioselectivity (>99%) were achieved in the batch system after 24 hours of reaction time. GtHNL‐3V on Celite R‐ 633 was successfully recycled five times. During more recycling steps a significant decrease in yield was observed while the enantioselectivity remained excellent over eight cycles. The use of a flow system enabled the continuous synthesis of (R)‐NPE. A total of 15% formation of (R)‐NPE was reached using a flow rate of 0.1 mL min⁻¹; unfortunately, the enzyme was not stable, and the yield decreased to 4% after 4 hours on stream. A similar yield was observed during 15 hours at a rate of 0.01 mL min⁻¹. Surprisingly the use of a continuous flow system did not facilitate the process intensification. In fact, the batch system displayed a space‐time‐yield (STY/mgenzyme) of 0.10 g L⁻¹ h⁻¹ mgenzyme⁻¹ whereas the flow system displayed 0.02 and 0.003 g L⁻¹ h⁻¹ mgenzyme⁻¹ at 0.1 and 0.01 mL min⁻¹, respectively. In general, the addition of 1 M nitromethane potentially changed the polarity of the reaction mixture affecting the stability of Celite‐GtHNL‐3V. The nature of the batch system maintained the reaction conditions better than the flow system. The higher yield and productivity observed for the batch system show that it is a superior system for the synthesis of (R)‐NPE compared with the flow approach.

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.

The merger of enzyme immobilisation and flow chemistry has attracted the attention of the scientific community during recent years. Immobilisation enhances enzyme stability and enables recycling, flow chemistry allows process intensification. Their combination is desirable for the development of more efficient and environmentally friendly biocatalytic processes. In this feature article, we aim to point out important metrics for successful enzyme immobilisation and for reporting flow biocatalytic processes. Relevant examples of immobilised enzymes used in flow systems in organic, biphasic and aqueous systems are discussed. Finally, we describe recent developments to address the cofactor recycling hurdle.

Arabidopsis thaliana hydroxynitrile lyase (AtHNL) catalyzes the selective synthesis of (R)-cyanohydrins. This enzyme is unstable under acidic conditions, therefore its immobilization is necessary for the synthesis of enantiopure cyanohydrins. EziG Opal is a controlled porosity glass material for the immobilization of His-tagged enzymes. The immobilization of His₆-tagged AtHNL on EziG Opal was optimized for higher enzyme stability and tested for the synthesis of (R)-mandelonitrile in batch and continuous flow systems. AtHNL-EziG Opal achieved 95% of conversion after 30 min of reaction time in batch and it was recycled up to eight times with a final conversion of 80% and excellent enantioselectivity. The EziG Opal carrier catalyzed the racemic background reaction; however, the high enantioselectivity observed in the recycling study demonstrated that this was efficiently suppressed by using citrate/phosphate buffer saturated methyl-tert-butylether (MTBE) pH 5 as reaction medium. The continuous flow system achieved 96% of conversion and excellent enantioselectivity at 0.1 mL min⁻¹ . Lower conversion and enantioselectivity were observed at higher flow rates. The specific rate of AtHNL-EziG Opal in flow was 0.26 mol h⁻¹ g_enzyme⁻¹ at 0.1 mL min⁻¹ and 96% of conversion whereas in batch, the immobilized enzyme displayed a specific rate of 0.51 mol h⁻¹ g_enzyme⁻¹ after 30 min of reaction time at a similar level of conversion. However, in terms of productivity the continuous flow system proved to be almost four times more productive than the batch approach, displaying a space-time-yield (STY) of 690 mol_product h⁻¹ L⁻¹ g_enzyme⁻¹ compared to 187 mol_product h⁻¹ L⁻¹ g_enzyme⁻¹ achieved with the batch system.

Granulicella tundricolahydroxynitrile lyase (GtHNL) is a manganese dependent cupin which catalyses the enantioselective synthesis of (R)-cyanohydrins. TheGtHNL triple variant A40H/V42T/Q110H, previously reported to exhibit a high activity and stability, was immobilised on Celite R-633 by adsorption. The synthesis of (R)-mandelonitrile catalysed by immobilised enzyme in a rotating bed reactor was compared to a continuous flow reactor. A batch reaction was used as reference system and organic solvent (MTBE) was used as reaction medium to suppress the chemical background reaction, ensuring the synthesis of enantiopure cyanohydrin. The rotating bed reactor, designed to boost conversion rates due to enhanced mass transfer, did not greatly enhance the reaction displaying a rate 1.7 times higher than the reference batch model. Moreover, similar conversion (96% after 4 hours) and recyclability were observed as compared to the reference system. The continuous flow reactor displayed rates 2 and 3 times higher than the rotating bed and the reference batch systems, respectively. Good conversions were achieved within minutes (97% conversion in 4 minutes at 0.1 mL min⁻¹). The immobilised enzyme displayed excellent enantioselectivity and high operational stability under all evaluated conditions. Overall,GtHNL triple variant A40H/V42T/Q110H immobilised on Celite R-633 is an excellent catalyst for the synthesis of (R)-mandelonitrile with a great potential for continuous flow production of cyanohydrins.