Uv vis recording spectrophotometer

After the cell density had been adjusted to approximately 1.0 × 10⁸ CFU ml^–1 (Inorganic salt medium), the aliquots (5%, v/v) were inoculated into 500-ml conical flasks containing 100 ml of MSM supplemented with 300 mg L^–1 diazinon. Samples of MSM supplemented with diazinon but free of bacterial inoculation were retained as controls. Conical flasks were incubated at 30°C on a rotary shaker at 160 rpm, in the dark to avoid photodegradation of diazinon. Samples of liquid medium were aseptically withdrawn at regular intervals to assess both bacterial growth (OD_{600 nm}) and diazinon degradation; diazinon degradation efficiency was estimated by the loss of diazinon from the culture. DI-6 cell growth was also investigated to determine whether the strain could utilize diazinon as the sole carbon source. Cell density was monitored by measuring the absorbance at 600 nm using a SHIMADZU UV–vis recording spectrophotometer.

Serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), immunoglobulin (Ig) A, IgG, and IgM were analyzed by enzyme-linked immunosorbent assay (ELISA) according to the instructions of manufacturers (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the T-AOC was detected using a spectrophotometer (LengGuang SFZ1606017568, Shanghai, China) at 520 nm measuring the development of stable and colored chelates. The SOD activity was quantified with the xanthine superoxide anion radical product and oxidase reaction system, and the MDA concentration was quantified with the thiobarbituric acid reactive substances method. The activity of GSH-Px was quantified by measuring the consumption rate of reduced glutathione in the enzymatic reaction. The CAT activity was detected at 240 nm based on the consumption of H₂O₂. According to the optical density values of standards, IgA, IgG, and IgM were measured with UV-2401PC at 700 nm, 340 nm, and 340 nm, respectively (UV–vis recording spectrophotometer; SHIMADZU Corporation, Kyoto, Japan). The immunoglobulin concentration of the samples (g/L) was calculated with the standard curve.

Assays were carried out in a total volume of 200 µL with 20 mM MOPS, pH 7.0, 150 µM NADPH, 6 µL DMSO and 10–3000 µM substrate (8-oxogeranial, citral or progesterone). 8-oxogeranial was synthesized according to the published protocols^{4 (link)}, citral and progesterone were purchased from Sigma. Substrate concentration was varied between 10 µM and 3000 µM for citral and 8-oxogeranial and 10 µM and 200 µM for progesterone. The progesterone concentration was not increased above 200 µM due to its limited solubility in aqueous buffer. For determination of kinetic constants for the wild type enzymes (PmMOR and 5ß–POR) substrate concentrations were varied. Specific activity assays were performed using a substrate concentration of 1 μM and 0.1 μg of protein, well above the K_m as determined for the wild type proteins. The reaction was monitored by the reduction in absorbance of NADPH at 340 nm and measured using a Shimadzu UV-VIS recording spectrophotometer according to Geu-Flores^{4 (link)}. All assays were performed in quadruplicate. Results were analysed using Kaleidagraph software.