Identification and Biochemical Characterization of a Novel β-1, 3-glucanase for Efficient Preparation of Laminarin Oligosaccharides

Abstract

Endo-β-1, 3-glucanase, also called laminarinase, is a key enzyme tool for laminarin biomass biorefinery and pretreatment in industry by hydrolyzing the laminarin polysaccharide into oligosaccharides. Cel2C, a novel kind of endo-β-1, 3-glucanase, was identified and characterized in this study. The name of Cel2C originates from the genus abbreviation of its source strain, Cellulophaga lytica (Cel), combined with an internal sequence identifier (2 C). It consisted of three domains formed by 507 amino acids, namely the N-terminal helix domain, the uncharacterized region and the TIM-like catalytic domain. Cel2C exhibited degrading activities to laminarin, β-glucan and sodium carboxymethyl cellulose (CMC-Na). Notably, it exhibited maximal activity (1.062 × 10⁴ nkat/mg, equivalent to ~ 636.203 U/mg) toward laminarin at pH 6.0 and 50 °C, and retained approximately 80% of its maximal activity below 40 °C within a pH range of 6.0–8.0. The Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the recombinant Cel2C for laminarin were determined as 7.433 mg/mL and 575.820 nkat/mg (equivalent to ~ 34.54 U/mg), respectively. HPLC and ESI-MS analysis indicated that Cel2C can degrade laminarin and β-glucan into oligosaccharides with degrees of polymerization (DPs) of 1–7. According to structural characteristics and molecular docking analysis, the catalytic mechanism of Cel2C is similar to Glycoside Hydrolase Family 5, with the highly conserved residues Glu336 and Glu420 acting as the proton donor and nucleophile, which is essential for the degradation of laminarin and β-glucan. These properties highlight the potential application of Cel2C as an endo-β-1, 3-glucanase in the biorefinery of laminarin biomass in related industries.