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Attached to many proteins are glycans\u2014complex strings of sugars\u2014that change how the whole unit functions. Removing them makes it easier to study both the proteins and glycans. But many glycans don\u2019t have a matching enzyme that can efficiently cleave them. This is especially true for O-glycans, which are attached to up to 83% of proteins secreted by cells.<\/p>\n
A new study led by Stephen Withers<\/a> from the University of British Columbia introduces a technique for engineering much more efficient enzymes. He and his team created a genetically\u00a0modified enzyme with an 840-fold improvement over the natural enzyme<\/a> in cleaving an O-glycan (ACS Cent. Sci. 2025, DOI: 10.1021\/acscentsci.5c01227).<\/p>\n To create an enzyme that cleaves the O-glycan sialyl T-antigen (STAg), Withers\u2019s team genetically modified an existing enzyme that naturally has a small amount of STAg-cleaving activity. The researchers modified it by introducing random mutations into the genes encoding that enzyme in Escherichia coli host cells and also by causing mutations in the DNA responsible for the enzyme\u2019s active binding site. Through this directed evolution process, they created tens of thousands of E. coli cells\u2014and hoped that one would contain an enzyme that would cleave the glycan efficiently.<\/p>\n To screen this library of enzymes, the team suspended tens of thousands of oil droplets in water, each of which contained one E. coli cell. The researchers mixed in a protein with both a STAg glycan attached to it and DNA that would cause blue fluorescence when the glycan was removed. After a few days, they measured enzyme activity by observing how much each droplet glowed.<\/p>\n Most of this is standard protocol. What Withers\u2019s team did differently was add DNA to the enzyme to make it fluoresce green. A brighter glow from the enzyme meant there was more of it. By comparing both colors of light, the researchers were able to compare the amount of each enzyme to the level of successful cleaving activity. This comparison allowed them to identify enzymes that are great at cleaving STAg but aren\u2019t expressed in high numbers by E. coli. Without this step, they might have overlooked potent enzymes. (Later, the team will be able to edit E. coli to better express the best enzymes.)<\/p>\n