Figure 5.

Activity measurement of Hyal-1 displayed on E. coli F470 at different pH values. E. coli F470 displaying Hyal-1 was suspended in formate buffer, pH ranges 3–5 and mixed with the substrate solution resulting in a final OD₅₇₈ of 10 and HA final concentration of 0.11 mg/mL. After an incubation time of 5 min the cells were removed by a centrifugation step at 4 °C. 25 µL of the supernatant was applied to the wells of a 96-microplate and mixed with “stains-all” solution following deionized water. The absorbance was immediately measured at 650 nm. The highest decrease in absorbance due to the cleavage of HA was set 100% activity. Mean values are given. There were no significant differences in activity at pH 3 and 3.5 as well as in activity at pH 4.5 and 5 (n = 3, error bars ± SD, *** p < 0.05, unpaired t test).