Abstract
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−1 compared to 187 molproduct h−1 L−1 genzyme−1 achieved with the batch system.
Highlights
Hydroxynitrile lyases (HNLs) are enzymes that catalyze the synthesis of enantiopure cyanohydrins (Scheme 1), known building blocks for the production of fine chemicals, pharmaceuticals and cosmetics [1,2,3,4]
Arabidopsis thaliana hydroxynitrile lyase (AtHNL) was recombinantly produced with a His6 -tag to enable its purification and immobilization by metal-ion affinity
All batch reactions were performed with AtHNL-EziG Opal tightly packed into tea bags
Summary
Hydroxynitrile lyases (HNLs) are enzymes that catalyze the synthesis of enantiopure cyanohydrins (Scheme 1), known building blocks for the production of fine chemicals, pharmaceuticals and cosmetics [1,2,3,4]. Stability,activity activityand andselectivity selectivity immobilized enzymes have reported earlier. Improved productivity, and enhancedprocessing selectivity are reported benefits of performing reactions in flow [23,24,25,26].Reduced. His-tag/Fe and consumption they allow handling of toxic reactive such as cyanide compare its performance for the synthesis of (R)-mandelonitrile with the earlier reported successful [28]. EziG Opalparameters based on the and compare its performance for the in synthesis of flow (R)-mandelonitrile with the earlier reported rate and productivity were investigated batch and systems. Successful immobilization of AtHNL on Celite by adsorption [15] Important parameters such as stability, specific rate and productivity were investigated in batch and flow systems
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