Biochemical characterization of the catalytic domain from a novel hyperthermophilic β-glucanase and its application for KOS production

Konjac oligosaccharide (KOS) exhibits various biological activities, and hyperthermophilic β-glucanases offer many advantages for KOS production from konjac glucanmannan (KGM). In this study, a novel β-glucanase, EG003, belonging to the glycosyl hydrolase (GH) subfamily 5_1, was predicted from the genome of the a Thermus strain. The recombinant EG003 and its catalytic domain, EG003A, were successfully expressed and characterized. EG003A displayed maximum activity at 95 °C and pH 8.0, with a specific activity of 1047.6 U/mg and retained approximately 50 % activity after 6 h at 90 °C. The enzyme exhibited both β-1,4-glucanase and β-1,3-1,4-glucanase activity with KGM and sodium carboxymethylcellulose (CMC), lichenan and oat β-glucan as substrates. Degree of polymerization (DP) 3 was the major oligosaccharide from the hydrolysis of KGM, while DP3 and DP4 were predominant products from the hydrolysis of oat β-glucan. Molecular docking analyses revealed that the catalytic mechanism of EG003A is consistent with those of other reported GH5_1 β-glucanases. Additionally, the viscosity of 500 mL solution of 1 % KGM decreased rapidly from 31,193 mPa.s to 4.50 mPa.s in 3 min with 30 U EG003A. This study provides an efficient hyperthermophilic β-glucanase with promising application for KOS production.