Lc 2030c

The influent and effluent samples were collected daily and centrifuged at 10,000 rpm for 10 min, and the supernatant was taken to determine COD, ${\text{NH}}_4^{+}$ -N, and ${\text{PO}}_4^{3-}$ levels as previously described (Zhou et al., 2022 (link)). The DO and pH values were measured using a DO meter (YSI 550A, USA) and a pH meter (HQ30d, HACH, America). The supernatant samples were filtrated using a 0.22-μm filter membrane (PTFE) for SMZ quantification using an analytical liquid chromatograph (LC-2030C, Shimadzu, Japan), equipped with the SB-C18 chromatographic column (Agilent ZORBAX, 150 × 4.6 mm, 5 μm) at a detection wavelength of 270 nm, an injection volume of 20 μL, and a flow rate of 1 mL/min. A combination of two mobile stages was programmed with pure water (A) and acetonitrile (B), and the mobile stage elution gradient was 0–2 min 60% A, 2–4 min 70% A, and 4–6 min 75% A.

The content of ATP, ADP, and AMP, and the energy charge were determined with 5 g of pericarp tissue from 10 longan fruits based on a previous study (Chen et al., 2014 (link)), using a high-performance liquid chromatography (HPLC, LC-2030C, Shimadzu Corporation, Kyoto, Japan) equipped with an ultraviolet detector and a Megres^TM C18 column (4.6 × 250 mm). Energy charge was calculated by (ATP+1/2 ADP)/(ATP+ADP+AMP).

For the SL hydrolysis assays, 0.5 mL of PBS buffer (pH 7.3) containing 3.2 μM rac-GR24 (racemic mixture of a GR24^5DS and GR24^ent−5DS) or 5DS was mixed separately with 1.6 μM purified ShHTLs and ShD14 and incubated for 30 min at 30 °C. Next, 50 μL of 1 M HCl was added to each reaction solution, and the reaction solutions were extracted 3 times with 300 μL of ethyl acetate. The ethyl acetate layers were combined and dried in vacuo and were then dissolved in 50 μL of methanol. For each layer, 5 μL was applied to the reverse-phase HPLC (Shimadzu LC-2030C). The analytical column was a CAPCELL CORE C18 (Φ 2.1 × 100 mm, Shiseido). The analytes were eluted under gradient conditions at a flow rate of 0.20 mL min⁻¹ with a linear ramp of methanol to 90% methanol at 19 min, which was maintained for 6 min before resetting to the original conditions. The amount of rac-GR24 or 5DS was calculated by the peak area (detection wavelength: 254 nm) at retention times ranging from 12.2–12.4 min with the regression equation obtained from the calibration curve produced using a dilution series of rac-GR24 or 5DS solutions. The percentage of hydrolyzed SL was calculated and used for statistical analyses. One-tailed unpaired Student’s t- tests were used for comparison between buffer samples and enzyme samples.

Based on the protocols reported by Chen et al. (2018) and Zhang et al. (2019), 5 g of pericarp from 10 longan fruits were used to measure the contents of ADP, AMP, and ATP, and energy charge [30 (link),31 (link)], which was conducted by a high-performance liquid chromatography (LC-2030C, Shimadzu Corporation, Kyoto, Japan). The quantification of ADP, AMP, and ATP was expressed as mg/kg. Energy charge was computed using a formula: (ATP + 1/2 ADP)/(ATP + ADP + AMP).

The RP-HPLC system (LC 2030C, Shimadzu, Japan) was equipped with a reverse phase Symmetry C18 column (3.5 μm, 4.6 × 75 mm²) and an ultraviolet (UV) detector.