Isolation, cloning, and characterization of a novel GH5 cellulase from yak rumen metagenome for enhanced lignocellulose hydrolysis in biofuel production and ruminant feed utilization.

Lignocellulosic biomass is a promising feedstock for biofuel production, but its complex structure, including cellulose and hemicellulose, challenges efficient enzymatic hydrolysis. CelyA, a novel cellulase from the yak rumen, has potential for improving biomass degradation and biofuel production. The CelyA gene was cloned, expressed, and purified. Biochemical characterization included assessments of pH, temperature, and salt tolerance. The enzyme's substrate specificity was tested on crystalline cellulose, CMC, and other polysaccharides. In vitro rumen fermentation was performed to evaluate its effect on fiber digestibility and microbial community composition. Biofuel production was tested by hydrolyzing maize, rice, and wheat straw. CelyA demonstrated optimal activity at pH 6.0 and 40.0 °C and maintained moderate stability across a wide pH range (3.0-12.0), retaining measurable activity even under strongly acidic and alkaline conditions. The enzyme demonstrated excellent salt tolerance, retaining 91.0 % activity in 1.0 M NaCl. CelyA efficiently degraded maize straw in hydrolysis assays, producing 7.2 µmol/L of reducing sugars. In vitro rumen fermentation with CelyA increased fiber digestibility by 8.3 % for maize straw, 14.5 % for rice straw, and 2.7 % for wheat straw. Gas production also increased significantly, with maize straw showing a 91.3 % increase. 16S rRNA sequencing revealed selective enrichment of Ruminococcus and Prevotella, key cellulolytic microbes. CelyA demonstrates strong potential for biofuel production, efficiently hydrolyzing lignocellulosic biomass and enhancing ruminal fiber digestibility. Its stability, salt tolerance, and substrate specificity make it a valuable enzyme for biofuel production and livestock feed optimization.