# Reshaping of a Glycoside Hydrolase Active Site through Expression-Compensated Droplet-Based Microfluidic Screening Provides Useful Tools for Glycomics

## TL;DR

Researchers used microfluidics to evolve an enzyme that can better cleave a specific type of sugar chain, improving tools for studying glycoproteins.

## Contribution

A new microfluidic screening strategy was developed to rapidly evolve glycoside hydrolases with enhanced activity and specificity for mucin-type O-glycans.

## Key findings

- Variants with 840-fold higher activity than the wild-type enzyme were identified in two rounds of screening.
- New enzyme specificities were discovered, expanding the range of enzymatic tools available for glycomics.
- Fluorescent fusion and ratiometric gating improved the identification of active enzymes despite expression variability.

## Abstract

The glycosylation of proteins endows them with distinct
biophysical
properties and allows them to play fundamental roles in cellular communication.
Much of our understanding of glycoproteins has derived from the ability
to enzymatically manipulate glycan structures. In particular, selective
cleavage of glycans from proteins simplifies the analysis of glycoproteins
and the determination of structure–activity relationships.
However, limited enzymatic tools are available for the study of mucin-type
O-glycans. To address this, we carried out the directed evolution
of a glycoside hydrolase to increase its ability to cleave the sialyl
T-antigen, a ubiquitous O-glycan structure in humans. We employed
ultrahigh-throughput droplet-based microfluidics to rapidly screen
vast libraries of variants in pL-sized droplets, thus minimizing the
quantities of complex substrate required. Furthermore, by use of fluorescent
protein-fusion and ratiometric gating during droplet sorting we could
account for varying expression levels and identify highly active hits
that could have been overlooked due to lower expression levels. Within
just two rounds of screening, we uncovered variants with 840-fold
enhancements in activity and new specificities compared to those of
the WT enzyme. This campaign highlights the versatility of glycoside
hydrolases and provides a broadly applicable strategy to engineer
enzymatic tools for glycomics through microfluidic screening.