Activity assessment of Pn3Pase. (A) Time course assay of recombinant Pn3Pase depolymerized tritium radioisotope labeled Pn3P separated by size exclusion chromatography, measured by counts per min in each 1 ml fraction. (B) Optimization of recombinant Pn3Pase in three different buffer conditions, measured by concentration of glucuronic acid reducing end generated in μg/ml. (C) Metal dependence determination with Mg2+ and Ca2+ reaction supplements, measured by concentration of glucuronic acid reducing end generated in ug/ml. (D) Temperature dependence of recombinant Pn3Pase, measured by concentration of glucuronic acid reducing end generated in μg/ml. Statistical significance was determined with the two-tailed Student t-test, ***P < 0.001, **P < 0.01; ns, not significant.