A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Synthesis

Background:

• Hydroxynitrile lyases (Hnls) are crucial biocatalysts for producing optically pure cyanohydrins, key intermediates in fine chemical synthesis.
• Existing Hnl enzymes from Hevea brasiliensis and Prunus amygdalus require further optimization for industrial applications.
• Directed evolution is a primary strategy for enzyme improvement, with screening being a critical bottleneck.

Purpose of the Study:

• To develop a novel, high-throughput screening assay for hydroxynitrile lyases (Hnls).
• To apply this assay for identifying improved Hnl variants with enhanced industrial applicability.
• To establish a versatile screening method applicable to various Hnl substrates.

Main Methods:

• Development of a colorimetric assay based on the detection of hydrocyanic acid (HCN) produced by Hnls.
• Application of the assay for high-throughput screening of Escherichia coli mutant libraries.
• Validation of the screening method by identifying novel Hnl variants.

Main Results:

• A facile, sensitive, and reproducible screening assay for Hnls was successfully established.
• The assay enables rapid screening of large mutant libraries.
• New Hnl enzyme variants with novel substrate specificities were identified.

Conclusions:

• The developed screening assay is a powerful tool for directed evolution of Hnls.
• This method accelerates the discovery of improved biocatalysts for cyanohydrin synthesis.
• The assay's substrate-independent nature broadens its applicability in enzyme engineering.