The biochemical and structural characteristics of Pt Lam, a laminarinase from deep-sea Planctomycetota , have been extensively elucidated, unveiling the fundamental molecular mechanisms governing substrate recognition and enzymatic catalysis. Pt Lam functions as an exo-laminarinase with the ability to sequentially hydrolyze laminarin, cleaving glucose units individually. Notably, Pt Lam exhibits proficient transglycosylation capabilities, utilizing various sugar alcohols as acceptors, with lyxose, in particular, yielding exclusively transglycosylated products. Structural analysis of both apo- Pt Lam and its laminarin oligosaccharide-bound complex revealed significant conformational alterations in active residues upon substrate binding. Moreover, pivotal residues involved in substrate recognition were identified, with subsequent mutation assays indicating the contribution of positive subsites in modulating exo-hydrolysis and transglycosidic activities. These results enhance our comprehension of laminarin cycling mechanisms by marine Planctomycetota , while also providing essential enzyme components for laminarin hetero-oligosaccharide synthesis.IMPORTANCEThe ubiquitous Planctomycetota , with distinctive physiological traits, exert a significant influence on global carbon and nitrogen fluxes. Their intimate association with algae suggests a propensity for efficient polysaccharide degradation; however, research on glycoside hydrolases derived from Planctomycetota remains scarce. Herein, we unveil the GH16 family laminarinase Pt Lam from deep-sea Planctomycetota , shedding light on its catalytic mechanisms underlying hydrolysis and transglycosylation. Our findings elucidate the enzymatic pathways governing the marine laminarin cycle orchestrated by Planctomycetota, thereby fostering the exploration of novel polysaccharide hydrolases with promising practical implications.