Cloning and Characterization of the Novel Endoglucanase Identified in Deep Subsurface Thermal Well of Biragzang (North Ossetia) by Metagenomic Analysis

The phylum <i>Armatimonadota</i> represents one of the least characterized bacterial lineages, with only three formally described species despite its widespread distribution in various environments. Deep subsurface thermal environments harbor significant microbial diversity and represent promising sources for novel enzyme discovery through metagenomic approaches. This study reports the identification, cloning, and biochemical characterization of Cel7465, a novel endoglucanase from an uncultured GBS-DC family within the order <i>Fimbriimonadales</i>. The enzyme was identified through metagenomic analysis of microbial mats from the Biragzang deep thermal well (North Ossetia, Russia, 48 °C) and successfully expressed in cells of <i>Escherichia coli</i> strain ArcticExpress (DE3). Phylogenetic analysis assigned Cel7465 to glycoside hydrolase family 5 subfamily 36. The purified recombinant enzyme exhibited optimal activity at 55 °C and pH 8.0, with high specific activity of 4347 U/mg. The enzyme demonstrated broad pH tolerance (50% activity retained from pH 4.0 to 10.0) and notable thermal stability, retaining 60% activity after one hour at 80 °C and 20% after four hours. Remarkably, the presence of Mn²⁺ ions enhanced enzyme activity more than 7-fold, while Mg²⁺ and Ca²⁺ ions provided moderate activation. Cel7465 represents the first biochemically characterized glycoside hydrolase from the order <i>Fimbriimonadales</i>, expanding our understanding of enzymatic capabilities within the understudied phylum <i>Armatimonadota</i> and demonstrating the continued potential of extreme environments as sources of novel industrial biocatalysts.