A unique highly efficient, thermostable and multi-substrate specific galactanase (AtGH53) from Acetivibrio...

The gene encoding endo-β-1,4-galactanase, AtGH53 from Acetivibrio thermocellus was cloned, expressed and the encoded soluble protein was biochemically characterized. Purified AtGH53 showed molecular mass of approximately, 36 kDa, an optimum temperature 70°C and half-life of 15 h at 70°C. AtGH53 displayed stability in acidic and alkaline pH ranges, with an optimum pH 7.5. AtGH53 activity increased over 30 % by Ni or Co²⁺ ions. AtGH53 exhibited broad substrate specificity, displaying the highest activity with potato pectic-galactan with V_max of 1432 U.mg⁻¹ and K_M of 1.2 mg.mL⁻¹ . TLC and HPLC analyses of potato galactan hydrolysis by AtGH53 showed initially the endo-lytic cleaving property and later shifting to exo-lytic mode. This was confirmed by the release of β-1,4-linked galacto-oligosaccharides of higher degrees of polymerization (DP>3) from potato galactan in first 2 h, followed by accumulation of galactobiose and galactose up to 24 h. In contrast, hydrolysis of larch-arabinogalactan by AtGH53 (specific activity, 148.6 U.mg^-1) resulted in the release of β-1,6-galactobiose from branches of the polymer. This indicated that AtGH53 also exhibits lower efficiency in hydrolyzing β-1,6-galactan of arabinogalactan via an exo-mode of action. The broad substrate specificity, pH stability and thermostability of AtGH53 make it a versatile enzyme for biotechnological applications.