prior to mixing, and for an additional 30 min at the same
temperature after mixing (Liu and others 2010b). The reaction
was terminated by adding 60 μL 1 M HCL before injecting the
sample into the HPLC sample loop to quantify the hippuric acid
produced by the enzymatic hydrolysis of the substrate hippuryll-
histidyl-l-leucine. The isocratic mobile phase consisted of
25% acetonitrile in deionized water (v:v) with 0.5% TFA. It was
filtered through 0.45 μm cellulose filters and degassed by ultrasound
for 30 min. An aliquot of 10 μL of the reaction mixture
was analyzed on the reverse phase HPLC. Hippuryl-l-histidyll-
leucine and hippuric acid were detected at 228 nm when the
mobile phase was controlled at 0.5 mL/min.