Isolation, expression, and characterization of potato (Solanum tuberosum) GH family 17 β-1,3-glucanase (Stglu) for exploring its potential as an antifungal agent.

Plant glucanases, including potato glucanase, are pivotal in biological processes such as cell growth, development, and defense against pathogens. These enzymes hold substantial promises in biotechnological applications, especially genetic engineering for enhancing crop disease resistance and stress tolerance. In this study, from Solanum tuberosum, glycosyl hydrolases family 17 (GH-17) β-1,3-glucanase (Stglu) was cloned, expressed, characterized and its antifungal activity was evaluated. The gene was isolated from infected potato plants and cloned into the pDrive and subsequently into the pET32a (+) protein expression vector. Sequence analysis revealed a 1044 bp open reading frame encoding a 347 amino acid protein with an anticipated molecular weight of 38 kDa and a signature motif (-IEIIVSESGWPSEG-) of the GH-17 family. The recombinant β-1,3-glucanase (Stglu) protein was expressed in E. coli Rosetta-gami 2 (DE3) cells. After recovery from inclusion bodies using urea buffer solubilization and refolding by dialysis, expression of Stglu protein was confirmed by Western blot analysis using an anti-His antibody. Enzymatic assays were performed to characterize β-1,3-glucanase activity which showed its maximum activity at pH 7.0 and 37 °C. Plate assays for substrate specificity showed that the enzyme hydrolyzed azo-barley β-glucan and laminarin. The metal ions strongly affected the enzyme's activity; Ca2+ acted as a weak activator. Plate assays further indicated the antifungal activity of Stglu against the plant pathogen Fusarium solani, showing a biotechnological potential tool in controlling fungal pathogenicity in crop plants.