Endoglucanase plays a vital role in lignocellulose degradation, yet the functional diversity of glycosyl hydrolase 12 (GH12) endoglucanases remains underexplored. In this study, TpCel12a, a novel GH12 endoglucanase from Talaromyces pinophilus Y117 was identified and characterized. It exhibited optimal activity at pH 4.0 and 40 °C with a preference for xyloglucan. Size exclusion chromatography revealed a monomeric state of TpCel12a. Despite classification into the promiscuous subfamily I, TpCel12a showed strict specificity for xyloglucan and carboxymethyl cellulose, yielding XXXG-type oligosaccharides and glucose/cellobiose, respectively. A loop 3 insertion mutant, designated as TpCel12a(+TQA), enhanced substrate affinity (5-fold lower Km) but reduced activity, suggesting a trade-off between binding and catalysis. Surprisingly, TpCel12a inhibited the hydrolytic efficiency of native T. pinophilus crude cellulase on corn cob powder, coinciding with its undetectable secretion under microcrystalline cellulose induction. This study highlights TpCel12a’s unique biochemical properties and its unexpected antagonistic role in native cellulase systems, offering insights for engineering fungal
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