Development and Deployment of an Activity-Based N-Glycan Hydrolase Probe Suitable for Droplet-Based Screening

Area of Science:

• Glycomics
• Enzymology
• Biotechnology

Background:

• N-glycans are crucial for biological functions.
• Enzymes modifying N-glycans have therapeutic potential.
• Discovering these enzymes is challenging due to a lack of suitable substrates.

Purpose of the Study:

• To develop a sensitive, representative N-glycan substrate for enzyme discovery.
• To create a fluorescence-quenched (FQ) probe for monitoring enzymatic activity.
• To enable high-throughput screening of glycoside hydrolases (GHs).

Main Methods:

• Chemoenzymatic synthesis of an FQ N-glycan probe from egg-derived sialoglycopeptide (SGP).
• Utilizing the probe's 'OFF' to 'ON' fluorescence switch upon enzymatic cleavage.
• Employing fluorescence-activated single-droplet dispensing (FASD) for screening.

Main Results:

• The FQ N-glycan probe successfully detected activity from various GHs.
• The probe is compatible with single-emulsion droplet-based screening.
• A trial screen recovered N-glycan degrading clones from microbial populations.

Conclusions:

• The chemoenzymatic strategy provides a novel, fully representative glycan probe.
• This approach facilitates ultrahigh-throughput screening for enzyme discovery.
• The method advances glycoengineering and fundamental glycomics research.