Acetyl esterases play an important role in the deacetylation of hemicellulose. This study aimed to identify and characterize a novel carbohydrate esterase family 16 (CE16) acetyl esterase from the saprophytic fungus Stropharia rugosoannulata and evaluate its potential for enhancing the enzymatic depolymerization of natural lignocellulosic substrates under mild conditions. A putative CE16 family gene was identified through genomic mining and heterologously expressed in Escherichia coli. The recombinant enzyme, SrAEA, was purified and biochemically characterized. The synergistic effect of SrAEA with xylanase was evaluated on beechwood powder, corncob powder, and rice bran. SrAEA exhibited optimal catalytic activity at 20 °C and pH 7.0, defining it as a cold-adapted enzyme. Structural analysis showed that it possesses a non-canonical Ser-His-Asn catalytic triad and a conserved SGNH-hydrolase fold. The enzyme exhibited the highest catalytic efficiency toward the short-chain substrate 4-methylumbelliferyl acetate, with no detectable activity on longer-chain substrates. Sequential treatment with SrAEA and xylanase increased reducing-sugar release in a substrate-dependent manner. These findings identify SrAEA as a fungal CE16 acetyl esterase with activity at 20 °C and show that its sequential use with xylanase can increase reducing-sugar release under the tested conditions. Its process utility and energy requirements remain to be evaluated.
