Characterization of Cellulase from the Rumen Metagenome and Evaluation of its Hydrolytic Potential for Agricultural Wastes

Abstract

The cellulase RuCel224, derived from the rumen metagenome, was successfully expressed in Escherichia coli BL21(DE3), with a molecular weight of ~43 kDa, as confirmed by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Substrate specificity assays revealed the highest activity against sodium carboxymethyl cellulose (CMC-Na) (0.861 ± 0.011 U/mg), followed by wheat straw xylan (0.150 ± 0.050 U/mg). RuCel224 exhibited optimal activity at pH 5.0 and 35 °C, retaining over 70% activity between pH 5.0 and 7.0 and 80% activity within the temperature range of 20 to 40 °C. Thermal stability tests showed that RuCel224 retained 80% activity after 30 min at 50 °C. Metal ion analysis demonstrated that Mn²⁺ significantly enhanced RuCel224 activity by 68.5% and 83.1% at 1 mM and 5 mM concentrations, respectively. Hydrolysis efficiency on lignocellulosic substrates revealed the highest reducing sugar release from corn stalk (206.21 ± 19.11 μg/mL), followed by wheat bran (106.63 ± 20.08 μg/mL) and rapeseed straw (93.83 ± 3.57 μg/mL). Overall, RuCel224 is a highly versatile cellulase under mild conditions, demonstrating strong adaptability to agricultural residues. Its superior performance on corn stalk highlights its potential for bioethanol production and other biomass valorization processes.