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

Abstract

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 Bacteroides luhongzhouii to grow well in this medium. Several putative cellouronate lyases belonging to polysaccharide lyase family 38 from B. luhongzhouii were identified. Among these candidate enzymes, BlCUL1, which displayed the most similarity to authentic cellouronate lyases, was heterologously expressed and characterized. The recombinant BlCUL1 (rBlCUL1) 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 rBlCUL1 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 rBlCUL1 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.