Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

Area of Science:

• Biocatalysis
• Enzyme Engineering

Background:

• Enzymatic cyanohydrin synthesis typically requires low pH to minimize side reactions.
• High pH conditions pose challenges for enzyme stability and substrate conversion.

Purpose of the Study:

• Investigate the application of unusual high pH values in enzymatic cyanohydrin synthesis.
• Evaluate the performance of wild-type and variant hydroxynitrile lyase under these conditions.
• Develop a continuous spectroscopic assay for product monitoring.

Main Methods:

• Utilized (S)-selective hydroxynitrile lyase from Manihot esculenta.
• Performed enzymatic cyanohydrin synthesis at pH values above 6.
• Employed a new continuous spectroscopic assay to monitor 3-phenoxy-benzaldehyde cyanohydrin formation.

Main Results:

• Achieved excellent conversion and high enantiomeric excess (97%) for (S)-3-phenoxy-benzaldehyde cyanohydrin using the wild-type enzyme at high pH.
• The MeHNL-W128A variant showed higher activity but reduced enantioselectivity (85% e.e.(S)).
• Successfully developed and implemented a novel continuous spectroscopic assay.

Conclusions:

• High pH conditions can be effectively employed in enzymatic cyanohydrin synthesis with appropriate enzymes.
• The wild-type enzyme demonstrates superior enantioselectivity under high pH compared to the tested variant.
• The developed assay provides a valuable tool for real-time monitoring of cyanohydrin synthesis.