Systematic Exploration of GH94 Sequence Space Reveals a New Chain Length Boundary in Glycoside Phosphorylase Specificities

Since the turn of the century, the number of available sequences within glycoside hydrolase (GH) families has rapidly expanded, presenting ever-increasing opportunities for the discovery of novel enzyme activities. Here, we expanded our previous strategy to explore the sequence–function relationship of the GH94 family by sequence similarity network analysis. Experimental screening of putative enzymes from 21 unknown clusters led to the discovery of an enzyme from the marine bacterium Verrucomicrobia bacterium S94 with a novel 4-O-β-d-glucosyl-laminaribiose phosphorylase specificity (VbGLamP). Biochemical characterization unveiled unique selectivity toward its trisaccharide substrate in phosphorolysis, representing a new chain-length boundary within β-glycoside phosphorylases (β-GPs). Its high chain-length selectivity during synthesis was exploited in a multienzyme cascade enabling selective, cost-effective synthesis of β-glucotriose from sucrose and glucose. Our current work highlights the power of rational sequence space exploration for novel enzyme discovery and demonstrates the potential of GPs as sustainable biocatalysts for the synthesis of specialty carbohydrates.