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

CALLONI RD; MUCHUT RJ; GARAY AS; ARIAS DG; IGLESIAS AA; GUERRERO SA

BIOCHIMIE

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Lugar: Paris; Año: 2023 vol. 208 p. 117 - 128

0300-9084

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 also, 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. 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.