Laminarinases of GH16 were only present in the GH16_3 subfamily based on phylogenetic analysis.

The hydrolysis mechanism of Lam092 was elucidated by the homology model and site-directed mutants.

The antioxidant activities of the laminarin hydrolysates from Lam1092 mutants were significantly improved.

Laminarinases from the glycoside hydrolase 16 (GH16) family are hydrolases that break β-1,3-glycosidic bonds in laminarin, which is the major storage polysaccharide present in brown algae or microalgae. We explored a laminarinase from the marine Flavobacteriaceae species Tamlana sp. PT2–4 at the structural and functional levels. Based on a homology model of Lam1092-substrate interactions, the large active groove crossing Lam1092 was deemed a reasonable pathway for the bent substrates for hydrolysis. Eight residues (Gly361, Asn364, Arg400, His466, Asp449, Glu452, Ser477 and Thr538) were selected for mutagenesis based on the interactions of Lam1092 in complex with Lam4/Lam6. Ultimately, we generated eight mutants of Lam1092, and the antioxidant activities of the hydrolysates of two mutants (G361A and H466A) showed significant improvement. These results show that the antioxidant activity of laminarin can be improved by laminarinase mutation, which will be beneficial for developing efficient approaches to engineer the substrate specificity of laminarinases and improve the application of bioactive laminarioligosaccharides.

Laminarinase

Active groove

Mutation

Antioxidant

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