High-cell-density fermentation of Komagataella phaffii enables the overproduction of a novel, broad-substrate endoglucanase, WcEG394 from Fuling (Wolfiporia cocos), for lignocellulose biorefining.
Previous transcriptomic analysis of W. cocos strain "Xiangjing 28" grown on filter paper as the sole carbon source identified a highly abundant endoglucanase, WcEG394, implicating its role in efficient cellulose degradation. To investigate the structural and functional basis of the enzyme and evaluate its potential for application, we employed an integrated approach involving bioinformatic analysis, high-cell-density fermentation, and enzymatic characterization. Sequence analysis and structural modeling classified WcEG394 into glycoside hydrolase family 5 (GH5), revealing its canonical (α/β)8 TIM barrel architecture. High-cell-density fed-batch fermentation in Komagataella phaffii enabled the secretory production of recombinant WcEG394 at a yield of 23 g/L. The crude enzyme exhibited carboxymethyl cellulase activity of 2217.6 U/mL and filter paper activity of 110.4 U/mL. The enzyme showed optimal activity at pH 4.0 and 50 °C, maintained stability under acidic and mesothermal conditions, and displayed notable tolerance to various metal ions. Using carboxymethyl cellulose as the substrate, the Km and specific activity were determined as 2.8 mg/mL and 113.7 U/mg, respectively. Recombinant WcEG394 efficiently hydrolyzed carboxymethyl cellulose, microcrystalline cellulose, and filter paper, and demonstrated broad substrate specificity toward xylans, pectin, chitin, agarose and agricultural residues such as corn cob and sugarcane bagasse. Scanning electron microscopy further confirmed that treatment with rWcEG394 resulted in smoother surface morphology of filter paper and cotton fibers while preserving their structural integrity. The combined properties of rWcEG394 demonstrate its potential as a viable biocatalyst for the scalable processing of cellulosic biomass.