Novel, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase from Hymenobacter siberiensis PAMC 29290, a psychrotolerant bacterium from Arctic marine sediment

Endo-β-1,3-glucanase is a glycoside hydrolase (GH) that plays an essential role in the mineralization of β-glucan polysaccharides. In this study, the novel gene encoding an extracellular, non-modular GH16 endo-β-1,3-glucanase (GluH) from <i>Hymenobacter siberiensis</i> PAMC 29290 isolated from Arctic marine sediment was discovered through an <i>in silico</i> analysis of its whole genome sequence and subsequently overexpressed in <i>Escherichia coli</i> BL21. The 870-bp GluH gene encoded a protein featuring a single catalytic GH16 domain that shared over 61% sequence identity with uncharacterized endo-β-1,3-glucanases from diverse <i>Hymenobacter</i> species, as recorded in the National Center for Biotechnology Information database. The purified recombinant endo-β-1,3-glucanase (rGluH: 31.0 kDa) demonstrated peak activity on laminarin at pH 5.5 and 40°C, maintaining over 40% of its maximum endo-β-1,3-glucanase activity even at 25°C. rGluH preferentially hydrolyzed D-laminarioligosaccharides and β-1,3-linked polysaccharides, but did not degrade D-laminaribiose or structurally unrelated substrates, confirming its specificity as a true endo-β-1,3-glucanase without ancillary GH activities. The biodegradability of various substrate polymers by the enzyme was evaluated in the following sequence: laminarin > barley β-glucan > carboxymethyl-curdlan > curdlan > pachyman. Notably, the specific activity (253.1 U mg^-1) and catalytic efficiency (<i>k</i> _<i>cat</i> /<i>K</i> _<i>m</i> : 105.72 mg^-1 s^-1 mL) of rGluH for laminarin closely matched its specific activity (250.2 U mg^-1) and <i>k</i> _<i>cat</i> /<i>K</i> _<i>m</i> value (104.88 mg^-1 s^-1 mL) toward barley β-glucan. However, the <i>k</i> _<i>cat</i> /<i>K</i> _<i>m</i> value (9.86 mg^-1 s^-1 mL) of rGluH for insoluble curdlan was only about 9.3% of the value for laminarin, which correlates well with the observation that rGluH displayed weak binding affinity (< 40%) to the insoluble polymer. The biocatalytic hydrolysis of D-laminarioligosaccharides with a degree of polymerization between 3 and 6 and laminarin generally resulted in the formation of D-laminaribiose as the predominant product and D-glucose as the secondary product, with a ratio of approximately 4:1. These findings suggest that highly active rGluH is an acidic, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase, which can be exploited as a valuable biocatalyst for facilitating low temperature preservation of foods.