AbstractBACKGROUNDAlginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance.RESULTSAn alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T, and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35–50 °C and pH 8.0–9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M‐specific enzyme (Aly17A). Ultra‐performance liquid chromatography–mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M‐blocks of oligosaccharide products, as well as the M‐blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides.CONCLUSIONA new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry.