This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: To produce pharmaceuticals, food ingredients, and flavors and fragrances, it is often necessary to oxidize alcohols. In conventional chemistry, this transformation typically relies on harsh oxidants and elevated temperatures and generates toxic waste.
But there is a more sustainable route: Living organisms, including fungi, produce galactose oxidases, proteins that can carry out the same reactions on alcohols and simple sugars under mild conditions, using oxygen. Researchers from Instituto de Tecnologia Química e Biológica António Xavier of NOVA University Lisbon (ITQB NOVA) in Portugal have expanded this toolbox by describing the first galactose oxidase in bacteria: PsGalOx. "For several years, the enzyme proved exceptionally difficult to produce in a soluble form and therefore to characterize," explains Lígia Martins, leader of the Microbial & Enzyme Technology laboratory at ITQB NOVA.
"A major turning point came with the emergence of AlphaFold, an AI system whose structural predictions helped us understand the enzyme's unusual architecture and identify changes that could improve its production," she adds. In the study, published in Chem & Bio Engineering, the research team shows that, unlike the best-characterized fungal galactose oxidases, their bacterial counterparts carry two nearly identical sugar-binding modules in addition to the catalytic module. This finding led the team to challenge the common simplification that sugar-binding modules are interchangeable.
"Although the duplicated modules share approximately 80% sequence identity, they have evolved distinct functions: one enhances binding to sugars, whereas the other primarily contributes to protein stability," explains Tomás Frazão, one of the paper's first authors and a doctoral student at ITQB NOVA. Building on this characterization, the research team recreated natural selection in the lab—a method known as directed evolution—introducing mutations and screening thousands of bacterial colonies to identify variants with improved performance. The evolution revealed something unexpected: The highly similar sugar-binding modules recombined into single hybrid modules, and the resulting variants produced more soluble protein.
The research team realized that increased activity during evolution did not necessarily mean that galactose oxidase had become better at speeding up reactions. Instead, much of the improvement resulted from cells producing more of it. This distinction was key to further improving the enzyme.
By the end, the team had developed a variant that was 40 to 1,800 times more efficient at processing a range of sugars and alcohols. As for the future, Martins explains that their plan is to "continue engineering PsGalOx to act on substrates of practical interest, creating a versatile intermediate for food, pharmaceutical and materials applications." Furthermore, the researchers want to explore the evolutionary diversity of these bacterial oxidases. "This will help us to understand how specificity evolved and to identify robust, versatile scaffolds for developing new biocatalysts," she concludes.
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