Expression and characterization of acetylxylan esterase 2 from Fusarium graminearum

Xylan, one of the sugar polymer components of biomass, is complex in the number and types of glycosidic bonds that comprise its structure. Due to this complexity, xylan hydrolysis requires many different enzymatic activities to fully release available sugars for subsequent fermentation to fuels and chemicals. As pretreatment severities are decreased to prevent degradation of biomass carbohydrates, the extent of hydrolysis of xylan bonds is also decreased. Due to the limited hydrolysis associated with lower severity treatments, esterases will be needed to hydrolyze acetyl-, feruloyl-, or coumaryl- groups that can block access to the sugar polymers. Acetylxylan esterases represent a class of enzymes that have the capability to hydrolyze acetyl- groups from xylan. A candidate fungal gene from Fusarium graminearum, XP_011319679.1, was expressed heterologously in Komagataella phaffii. The secreted protein was purified in a multi-step procedure consisting of ammonium sulfate precipitation, anion-exchange chromatography, acetone precipitation, and size exclusion chromatography. The purified protein was 25 kDa as determined by SDS-PAGE. The expressed protein liberated acetate from partially acetylated birchwood xylan but did not hydrolyze the artificial substrate pNP-acetate. The enzyme, herein designated FgAxe2, had kinetic values of: K_m 6.4 mM ± 0.7; k_cat 12.9 ± 0.6 s^-1; V_max 24.8 ± 1.1 µmol/min/ml acting on partially acetylated birch xylan. The enzyme had activity between pH 3.0 and 7.0 and temperatures 10°C-90°C, with optima at pH 5.0 and 37 °C. Additional studies evaluating the specificity of the enzyme using ¹H NMR and LC-MS were also performed.