Fig. 6: Characterization of enzyme activity immobilized on membrane.

a Percent of active enzymes immobilized (via batch method) on PMAA-PVDF (orange bar) and unfunctionalized PVDF400 (green striped bar) over time of dry storage with no hydration at 22–24 °C. Day 1 is the day enzyme immobilization occurred. The highest enzyme activity upon immobilization (Subtilisin-PMAA-PVDF) was considered 100% (0.19 mM product formed/min per mg of enzyme) and all subsequent values were normalized based on this activity. b Substrate interaction with Subtilisin-PMAA-PVDF (purple circle and line for Day 1, orange square and line for Day 3) and Subtilisin-PVDF membrane system (gray triangle and line for Day 1, yellow triangle for Day 3) 48 h after enzyme functionalization. Peptide concentration was 0.08–0.10 mM. c Minimal hydration (37 μL of solution/cm² of membrane = 0.02% water) test of Subtilisin-PMAA-PVDF and Subtilisin-PVDF membranes 120 h after enzyme functionalization. Reaction was allowed to proceed for 60 s. Activity measured using peptide (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) that, upon proteolysis, releases 4-nitroanaline, which absorbs light at wavelength of 410 nm. Initial concentration of peptide was 0.08–0.10 mM and reactions were conducted at 23 °C. Reactions occurred at pH ranging from 7–8. Error bars represent the standard deviation of 3 different measurements taken on the samples.