Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis

CALLONI, RODRIGO D.; MUCHUT, ROBERTINO J.; GARAY, SERGIO A.; ARIAS, DIEGO G.; IGLESIAS, ALBERTO A.; GUERRERO, SERGIO A.

Rosario

Workshop; V Encuentro y II Workshop de la Red Argentina de Tecnología Enzimática; 2023

Red Argentina de Tecnología Enzimática

Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg.ml-1,kcat 1.20 s-1) and, but with less enzymatic efficiency, paramylon (KM 20.2 mg.ml-1,kcat 0.23 ml.mg-1.s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg.ml-1, kcat 0.88 ml.mg-1.s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein’s greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Using western blot, we evidenced the presence of this protein in extracts of E. gracilis cells grown under heterotrophic conditions and anaerobic fermentation. We did not observe signal in cells grown in autotrophy. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.