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

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.

Batch and Flow Nitroaldol Synthesis Catalysed by Granulicella tundricola Hydroxynitrile Lyase Immobilised on Celite R-633

“…This explains its higher productivity. This result is unexpected since flow systems commonly allowed process intensification [25,26,37,45,46]. However, the high concentration (1 M) and low polarity (log P = −0.27) of nitromethane might not maintain a constant a w on Celite-GtHNL-3V.…”

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry

“…This forms the basis of the EziG (EnginZyme AB Sweden) technology for immobilisation of a broad range of enzymes. 10,11,[35][36][37][38] It involves fusion of the His6-tag to target enzymes at the N or C terminus. Following the fermentation, the His-tagged recombinant enzyme is separated from cell lysate by affinity binding of the imidazole groups in His residues to surface Ni 2+ , Co 2+ or Fe 3+ ions coordinated to surface iminodiacetic acid functions on controlled porosity glass (CPG).…”

Immobilisation and flow chemistry: tools for implementing biocatalysis

“…The same AtHNL was also immobilised via the his-tag on the carrier EziG Opal. 52 This is a controlled porosity glass carrier bearing Fe 3+ on its surface. The availability of metal ions for the enzyme binding was guaranteed by using a molar ratio of monomeric AtHNL:Fe 3+ of 1:5.…”