20a series instrument

HPLC analysis was carried out on a Shimadzu-20A series instrument (Shimadzu, Japan) connected to a fluorescence detector (RF-10A xl, Shimadzu, Japan). The signals from the fluorescence system were transmitted to LC Solution software for the purposes of analyzing and calculating the peak areas. Chromatographic separation was carried out on an RPAQUEOUS-AR-5 (4.6 × 250 mm) column. The HPLC was operated with a gradient mobile phase system consisting of water containing 0.1% formic acid (phase A) and methanol (phase B) at a flow rate of 1 mL/min. The pump was programmed as follows: phase B was maintained at 40% for the first 5 min, increased from 40% to 80% within the next 25 min, maintained at 80% for the next 10 min, increased to 100% for 10 min, and then decreased back to 40% for 10 min (total gradient time: 60 min). A 50 μL sample was injected into the system with the auto-sampler and conditioned at 4 °C, and the column temperature was maintained at 30 °C.
The standard curve was constructed in the concentration range of 20–640 ng/mL of zonarol, which was dissolved with methanol by plotting the peak-area (y) of the zonarol against the concentration of zonarol (x). Zonarol samples were analyzed in triplicate. The regression parameters of slope and correlation coefficient were also calculated.

The ZEN degradation ability of StDyP was first evaluated in the presence of Mn²⁺ or 1-HBT. The degradation of ZEN was performed in 50 mM malonate buffer (pH 4.0 or 5.0) containing 0.003 mM ZEN, 1 mM MnSO₄ or 1-HBT, 0.1 mM H₂O₂, and 1 U/mL StDyP for 48 h at 30 °C.
Secondly, the time course of ZEN degradation by StDyP was determined in 50 mM malonate buffer (pH 5.0) containing 0.003 mM ZEN, 1 mM MnSO₄ or 1-HBT, 0.1 mM H₂O₂, and 1 U/mL StDyP for 6, 12, 24, 36, and 48 h at 30 °C. In addition, the effect of substrate concentration on the degradation of ZEN was assessed in 50 mM malonate buffer (pH 5.0) containing 0.003, 0.015, 0.03, 0.06 or 0.15 mM ZEN, 1 mM MnSO₄ or 1-HBT, 0.1 mM H₂O₂, and 1 U/mL StDyP for 48 h at 30 °C.
HPLC analysis of ZEN degradation was performed by using a SHIMADZU 20A series instrument (Kyoto, Japan) equipped with a UV/Vis detector and RF-20A fluorescence detector with a Waters XBridge C18 column (5 µm, 4.6 mm × 150 mm). The elution condition for ZEN was set as follows: 45% acetonitrile (ACN) at a flow rate of 0.75 mL/min; ZEN was monitored at 274 nm excitation wavelength and 440 nm emission wavelength.

Enzyme activity was determined by measuring GABA production rate using HPLC analysis, with some modifications [46 (link), 47 (link)]. The reaction mixture comprised 400 µL of Na₂HPO₄-citric acid buffer (80 mM, pH 6.0), 500 µL of l-Glu (50 mM), 50 µL of PLP (0.02 mM), and 50 µL of purified enzyme. Ice-chilled 80% ethanol was added in an equal volume (1 mL) to stop the reaction after 30 min at 40 °C. The reaction supernatant (500 µL) was mixed with 100 µL of NaHCO₃ (2.5 g/L) and 200 µL of 4-N, N-dimethylaminoazobenzene-4′-sulfonyl chloride (DABS-Cl) (0.25 g/L, dissolved in acetonitrile), and incubated at 70 °C for 20 min. This was followed by analysis using a SHIMADZU 20 A series instrument (Shimadzu, Kyoto, Japan) and an Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 150 mm) (Agilent, Santa Clara, CA, USA). The mobile phase was a solution of 35% (v/v) acetonitrile solution and 65% 50 mM sodium acetate. The flow rate and column temperature were 1 mL/min and 30 °C, respectively; the injection volume was 10 µL; and the detection wavelength was 436 nm. The GABA content in the test solutions was calculated by taking into consideration the peak areas observed with the standard. One enzyme activity unit was defined as the amount of enzyme required for the release of 1 mM free GABA in 1 min. Three parallel wells were set up per group.