Immobilization of Arabidopsis thaliana Hydroxynitrile Lyase (AtHNL) on EziG Opal

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 genzyme-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 genzyme -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 molproduct h-1 L-1 genzyme - compared to 187 mol product h-1 L-1 genzyme -1 achieved with the batch system.