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.

The question of how to distinguish between lipases and esterases is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic solvents as a criterion is probed on a minimal α/β hydrolase fold enzyme, the Bacillus subtilis lipase A (BSLA), and compared to Candida antarctica lipase B (CALB), a proven lipase. Both hydrolases show activity in dry solvents and this proves BSLA to be a lipase. Overall, this demonstrates the value of this additional parameter to distinguish between lipases and esterases. Lipases tend to be active in dry organic solvents, while esterases are not active under these circumstances.

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.

The enzymatic oxidation of amino alcohols was studied to address the long-standing problem of product stability. Amino aldehydes, highly sought and unstable compounds, can be generated under mild conditions if they are immediately protected. Utilizing a range of alcohol dehydrogenases (ADHs) and semicarbazide as a scavenger, the enantioselective synthesis of protected amino aldehydes is possible. Glycerol dehydrogenase from Gluconobacter oxydans (GoGDH) displayed excellent enantioselectivity but limited substrate scope, whereas horse liver ADH catalyzed a broad range of conversions with low enantioselectivities.