Immobilization of arabidopsis thaliana hydroxynitrile lyase (AtHNL) on EziG opal

José Coloma, Tim Lugtenburg, Muhammad Afendi, Mattia Lazzarotto, Paula Bracco, Peter Leon Hagedoorn, Lucia Gardossi, Ulf Hanefeld

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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.

Original languageEnglish
Article number899
Pages (from-to)1-14
JournalCatalysts
Volume10
Issue number8
DOIs
Publication statusPublished - 2020

Keywords

  • Arabidopsis thaliana
  • Batch
  • Continuous flow
  • His-tag
  • Hydroxynitrile lyase
  • Immobilization
  • Oxynitrilase

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