Discovery and characterization of a novel GH6 multifunctional enzyme from soil metagenomic library

Cellulases are crucial for converting biomass into renewable energy. Despite extensive research, there remains a significant industrial demand for novel cellulases, particularly those with multi-substrates catalytic activity. This study aimed to identify and characterize a novel cellulase from a high-altitude soil metagenome library using functional screening method. A novel 1218-bp GH6 family hydrolase gene, designated zfy1641, was identified from a Mount Everest soil library. Bioinformatics analysis indicated that it encoded a 405-amino-acid protein (43.7 kDa) and was classified into glycoside hydrolase family 6 (GH6). The target glycoside hydrolase gene was cloned and heterologously expressed, then the recombinant protein was purified, and its biochemical properties and kinetic parameters were characterized. The purified recombinant enzyme exhibited broad substrate specificity, demonstrating significant activity against carboxymethyl cellulose (CMC-Na; 69.87 ± 0.13 U/mg), locust bean gum (125.56 ± 0.18 U/mg) and chitin (77.06 ± 0.08 U/mg). ZFY1641 represented a novel member of the GH6 family, that exhibited detectable reducing sugar release from chitin-a function not previously documented for this family. Moreover, ZFY1641 demonstrated optimal activity at 50°C and pH 5.0, and exhibited moderate thermal stability, tolerance to selected metal ions, and halophilicity under the conditions tested. These characteristics suggest potential utility of ZFY1641 in industrial processes, though further validation is required. This work expanded the substrate diversity of GH6 family enzymes and provided a foundation for the development of new enzymatic preparations with a novel multi-functional GH6 family enzyme.