Hydroxynitrile lyase from cassava, Manihot esculenta, (MeHNL) catalyzes the formation of (S)‐cyanohydrins from HCN and aldehydes or ketones. Four differently immobilized MeHNLs were prepared: by noncovalent immobilization (celite R‐633), covalent immobilization (cross‐linked enzyme aggregates, CLEA), encapsulation [in a poly(vinyl alcohol) hydrogel Lentikats] and a combination of the above, an unusual immobilization, CLEA encapsulated in a poly(vinyl alcohol) hydrogel. A comparative study of the recyclability of each immobilized MeHNL was performed. Particular attention was paid to the stability and activity of the new immobilized MeHNL–CLEA–Lentikats and to the minimum enzyme loading required to achieve high yields and enantiomeric excesses. MeHNL–CLEA stability was slightly improved by encapsulation into Lentikats and good recyclability rates at low enzyme loading were obtained. However, MeHNL immobilized on celite R‐633 exhibited the best recyclability, giving >95 % conversion and an enantiomeric excess of 99 % during 12 cycles.

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.Catalysts 2020, 10, 308 2 of 17 lipase A (BSLA) [9]. BSLA is a small (181 amino acids, 19 kDa) serine hydrolase ( Figure 1). It is neither interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not water-miscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low a w [1,3].Catalysts 2019, 9, x FOR PEER REVIEW 2 of 17 interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not watermiscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low aw [1,3].