A Thermostable Multifunctional GH5 Endoglucanase From <i>Bacillus</i> sp. for Lignocellulosic Biomass Saccharification

The enzymatic conversion of lignocellulosic biomass to glucose remains a bottleneck for cost‑effective biofuel production, largely due to the complexity and expense of enzymes. Here we describe BsEG2, a multifunctional GH5‑like endoglucanase cloned from Bacillus sp. strain BS and heterologously expressed. BsEG2 displays optimal activity at pH 6.0 and 55°C and efficiently hydrolyzes both amorphous and crystalline cellulose. Kinetic analysis on carboxymethyl cellulose (CMC) indicates high substrate affinity and catalytic efficiency. Unlike typical endoglucanases, BsEG2 combines processive endoglucanase activity with broad substrate specificity. It degrades long cellulose chains and oligosaccharides and also shows β‑glucosidase‑like activity on cellobiose, yielding glucose across a wide range of chain lengths. BsEG2 is highly thermostable (a half-life of ~15 days at 55°C), tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM. It remains active in seawater, suggesting potential to reduce freshwater demand in biomass processing. These properties, namely, multifunctionality, stability, inhibitor tolerance, and halotolerance, make BsEG2 a strong candidate for simplified, cost‑effective enzyme formulations for glucose production from lignocellulosic feedstocks.