GH53 endo-beta-1,4 galactanase of Bacillus licheniformis: biochemical characterization applied to biomass degradation

Endo-beta-1,4-galactanases hydrolyze β-1,4 galactan in the hairy region of pectin. The aim of this work was to understand the mode of action of the GH53 endo-beta-1,4 galactanase (BLicGal) for supplementation of commercial cocktails. The gene encoding the BLicGal was cloned and expressed in E. coli, and after purification the catalyitic properties of the enzyme were characterized. The results indicate that the optimum pH and temperature range were 6.5 to 8.0 and 40 to 45˚ C, respectively. The catalytic activity of the enzyme remains stable over two hours at temperatures  between 25-45°C over a wide pH range, which is favorable for biotechnological applications. Increased enzyme activity was observed in the presence of KCl, CoCl₂, NiCl₂, MnCl₂, LiCl, NaCl, CaCl2 and strongly inhibition was observed with Cu²⁺, Zn²⁺ e Fe³⁺. The catalytic efficiency was evaluated and the value of kcat/Km against potato galactan at the optimum pH and temperature conditions was 241,27 s^-1mg^-1mL. The pattern of potato galactan degradation by BLicGal innitialy produces galactotetraose as the main product and after 16 hours galactotriose, galactobiose and galactose are present. Secondary structure characterization using circular dichroism (CD) revealed that the BLicGal had a predominantly α-helix profile, which is in agreement with the crystallographic structure. Thermal unfolding data as monitored by CD reveal a melting temperature  of 52.9˚C. Based ion these results, we suggest the use of BLicGal to supplemet commercial cocktails in lignocelullosic biomass saccarification to produce biofuels and biobased products.