Hydroxynitrile lyases covalently immobilized in continuous flow microreactors

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) mgprotein(-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.