polyester fabrics (∼2 mm thickness) were punched into discs of
30 mm in diameter and 25 g was tightly packed into a glass column of 240 mm
in length. To activate the fabrics in situ, the column was connected to a peristaltic
pump (set at 10 mL/min) and circulated with EDA (99%, 300 mL) for 2 h at room temperature,
washed with 2 L of water, then circulated with a glutaradehyde solution
(400 mL of 1% in phosphate buffer) for 2 h at room temperature and again flushed
with 2 L of water. The partially purified OpdA solution from above was then circulated
through the activated column overnight at 4 ◦C. The column was washed with
2 L of the phosphate buffer containing 0.1% Triton X-100 to remove any unbound
enzyme. The column was stored in the phosphate buffer containing 0.02% NaN3 at
4 ◦C when not in use. The void volume of the column was 170 mL.
Pesticide solutions were made in reverse osmosis water without the use of any
buffer, and all operations were conducted at room temperature (22 ◦C). Pesticide
solutions were fed to the column at pre-determined flow rates via a peristaltic pump.
The column flow though was collected using a fraction collector. Before and after
each run, the column was flushed with 1.5 L water. The degradation of MP was
quantified by measuring the absorbance of the degradation products, pNP, in the
fractions. The total MP concentrations (degraded and un-degraded) in the filtrate
was measured by treating the solutions with an excess of purified OpdA for several
hours, and then reading absorbance as above. Under the conditions used, the purified
OpdA was able to hydrolyze all the MP in the solutions tested. Degradation efficiency
was calculated as the percentage of pNP (already in the filtrate) vs. the total pNP (after
further treatment with purified OpdA) in the filtrate.