Expression and characterization of acetylxylan esterase 2 from Fusarium graminearum

Authors: Bowman, Michael; WALLS, KAITLYN - Former ARS Employee; Naumann, Todd; Vermillion, Karl

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/6/2026
Publication Date: 7/7/2026
Citation: Bowman, M.J., Walls, K.N., Naumann, T.A., Vermillion, K.E. 2026. Expression and characterization of acetylxylan esterase 2 from Fusarium graminearum. Enzyme and Microbial Technology. https://doi.org/10.1016/j.enzmictec.2026.110933.
DOI: https://doi.org/10.1016/j.enzmictec.2026.110933

Interpretive Summary: The United States generates about 1.5 billion tons of plant material each year from crops, crop residues, and processing byproducts that includes materials such as corn stalks, corn fiber, and soybean hulls. Much of this plant material is currently underused even though it contains valuable natural sugars, in the form of polysaccharides, which can be converted into renewable fuels and other biobased chemicals. However, these sugars must first be released by using enzymes that break down the polysaccharides into their individual sugars. This process can be challenging because certain chemical groups bound to these polysaccharides often interfere with the desired enzyme degradation, resulting in low sugar recovery. In this study, USDA researchers in Peoria, Illinois, identified a new type of enzyme that efficiently removes these interfering compounds, allowing more sugar to be released through enzymatic degradation of materials like soybean hulls, sorghum, and corn fiber. By improving sugar release, this enzyme can lower manufacturing costs and increase the value of agricultural residues. The technology also supports domestic production of renewable fuels and chemicals and is needed to develop new markets for unused crop residues and increase revenue streams for U.S. farmers.

Technical Abstract: 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: Km 6.4 mM; kcat 12.9 s-1; Vmax 24.8 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.