Uncovering the β-1,6-galactan-degrading machinery: Biochemical and functional insights into a processive exo-β-1,6-galactanase, RfGH30 from Ruminococcusflavefaciens

Abstract

The glycoside hydrolase family 30 (GH30) enzyme RfGH30, originating from the ruminal bacterium Ruminococcus flavefaciens, was biochemically characterized to elucidate its catalytic properties and substrate specificity. Although initially classified as endoxylanase, experimental analysis confirmed RfGH30 as processive exo-β-1,6-galactanase targeting galactan-rich polysaccharides. Zymogram analysis revealed a clear hydrolytic zone at ∼54 kDa on larchwood arabinogalactan (LWAG), confirming its specificity for β-1,6-glycosidic linkage containing galactose units. The enzyme exhibited optimum pH 5.5 and temperature 40 °C, retaining stability across pH 3.8-7.0 and temperature range, 4-70 °C. Among natural substrates, RfGH30 displayed highest specific activity, 8.1 U mg^-1 against LWAG followed by arabinan 4.6 U mg^-1 against sugarbeet arabinan. RfGH30 also hydrolysed synthetic substrates, pNPG and oNPG consistent with an exo-mode of catalysis. Kinetic analysis showed a V_max of 9.8 U mg^-1 and K_M of 1.45 mg mL^-1 against LWAG. Enzyme activity was enhanced by Ca²⁺ and Mg²⁺, but inhibited by Fe²⁺ and the chelating agent EGTA. RfGH30 hydrolysed LWAG product profile by TLC, HPLC and HRMS revealed the predominant release of β-1,6-galactobiose along with minor galactose, demonstrating strict cleavage of β-1,6-galactosyl linkages. RfGH30 showed strong processivity with LWAG, with the high processivity index of 21 and a low, 4.7 with sugarbeet arabinan. This study reports the first biochemical evidence of a GH30 processive exo-β-1,6-galactanase from R. flavefaciens, providing insights into its role in ruminal degradation of plant arabinogalactans and its potential applications in biomass valorization.

Keywords: Arabinan; Arabinogalactan; Exo-galactanase; Zymogram; β-1,6-galactobiose.