Heterologous Expression and Characterization of Cellouronate (β-1,4-Glucuronan) Lyase from a Human Intestinal Bacterium <i>Bacteroides luhongzhouii</i>

Cellouronate, β-1,4-glucuronan, is synthesized from regenerated cellulose via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical-mediated oxidation. Human intestinal bacteria were cultured in a medium containing cellouronate to evaluate its utilization. These experiments showed <i>Bacteroides luhongzhouii</i> to grow well in this medium. Several putative cellouronate lyases belonging to polysaccharide lyase family 38 from <i>B. luhongzhouii</i> were identified. Among these candidate enzymes, <i>Bl</i>CUL1, which displayed the most similarity to authentic cellouronate lyases, was heterologously expressed and characterized. The recombinant <i>Bl</i>CUL1 (r<i>Bl</i>CUL1) showed the highest activity at pH 8.0 and was deactivated by treatment at pH 3.0 for 24 h or heating above 50 °C for 10 min. Moreover, the activity of r<i>Bl</i>CUL1 was enhanced in the presence of Mg²⁺, Ca²⁺, or EDTA, but suppressed by Al³⁺ and completely inactivated by Fe³⁺. Analysis of the final reaction mixture generated from the r<i>Bl</i>CUL1 mediated degradation of cellouronate revealed an oligomer as the main product, but the monomer was barely detectable. This study is the first to report and characterize a cellouronate lyase from human intestinal bacteria.