This is a review on the feasibility of monolithic porous supports in biocatalysis carried out in a continuous flow system. It discusses factors affecting the efficiency and stability of enzyme immobilisation, kinetic parameters of enzyme processes carried out inside a monolith, biocatalysis in single and two-phase systems, and cascade reactions including cofactor regeneration. It also covers materials engineering (monolith types) and issues related to the flow of reactants through the monolith (chemical engineering). Emphasis is placed on the fact that the application of (bio)catalysis improves selectivity and atom economy, thus lowering the E factor. However, biocatalysts need to be employed in a reactor, which can aid further improvement towards green chemistry goals. The application of enzymes in flow chemistry has been shown to lead to higher space time yields (STYs) compared to batch reactions. In particular, with monolithic reactors a drastic decrease in volume and thus solvent can be achieved. By immobilising very high densities of enzymes directly on the monolith, reaction times dwindle, improving STYs. The small reaction volumes enable excellent heat transfer, helping to save energy. The underlying principles of monolithic flow reactors and their application in mono- and bi-phasic biocatalytic systems will be examined.@en

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.

@en

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.

@en

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.

@en

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.

@en

Enzymes are supreme catalysts when it comes to high enantiopurities and their immobilization will pave the way for continuous operation. In this context, we show the covalent immobilization of hydroxynitrile lyases HbHNL (from Hevea brasiliensis) and MeHNL (from Manihot esculenta) in a siliceous monolithic microreactor for continuous operation. A thorough characterization of the immobilized HNLs on mesoporous silicates indicated the conditions essential for a successful immobilization. Their application in a continuous flow system enabled a remarkably fast (3.2 min) production of chiral cyanohydrins with high conversion (97%) and high ee (98%) using minimal enzyme loading (STY = 71 g L ^-1 h ^-1 mg _protein ^-1 ). MeHNL showed increased operational stability, possibly due to a structural difference. The continuous flow microreactor outperformed batch systems, demonstrating the advantage of the mesoporous/macroporous environment for the expression of enzyme activity and the favorable characteristics of the microreactor. Overall, the system shows great potential for future industrial application of biocatalytic asymmetric syntheses.

@en

The new application of acyltransferase, isolated from Mycobacterium smegmatis for the chemo-enzymatic Baeyer-Villiger oxidation of cyclic ketones to lactones was demonstrated. Acyltransferase exhibited high activity, and high stability under harsh reaction conditions, like oxidation with 60% aq. H₂O₂ at 45°C. This paves the way to a novel robust chemo-enzymatic method for lactone synthesis with high yields.

@en