Biochemical characterization and catalytic features of a GH5 endo-β-1,4-mannanase from Aspergillus niger code 1234.

Abstract

Endo-β-1,4-mannanases (EC 3.2.1.78) hydrolyze β-1,4-mannan backbones, enabling the conversion of mannan-rich biomass into value-added products such as mannose and mannan-oligosaccharides (MOS). In this study, an endo-β-1,4-mannanase produced by Aspergillus niger code 1234 under submerged cultivation was purified and biochemically characterized. The purification strategy included ultrafiltration (10 kDa), followed by size-exclusion and ion-exchange chromatography using Sephadex® G-75 and DEAE-Sephacel™, respectively. The purified enzyme (AnMan5A-like) was confirmed by SDS-PAGE and mass spectrometry. AnMan5A-like has an estimated molecular mass of 41.2 kDa, an optimum activity at 62 ºC and pH 3.9, and a specific endo-β-1,4-mannanase activity of 79.30 IU/g of protein. The enzyme exhibited remarkable thermostability, retaining more than 95% of its residual activity after 72 h at 50 °C and 60 °C, respectively. Kinetic analysis revealed a Km of 2.52 mg/mL and a Vmax of 2.20 µmol/min/mL using Locust Bean Gum as the substrate. Enzymatic hydrolysis assays demonstrated the production of MOS with degrees of polymerization up to six. Structural analysis suggested a catalytic cleft capable of accommodating five subsites and favoring mannopentaose as a transient substrate that is rapidly cleaved into mannobiose and mannotriose. These results highlight the potential of functional oligosaccharides production via conversion of mannan-rich biomass using a stable endo-β-1,4-mannanases from A. niger code 1234. Its high catalytic performance, combined with thermostability under acidic conditions, makes AnMan5A-likea promising biocatalyst for industrial applications in the food, animal feed, and biorefinery sectors.