Uv 260 spectrophotometer

The ACC deaminase activity was measured spectrophotometrically following a protocol based on the accumulation of α-ketobutyrate [53 (link)], with slight modifications. The bacterial culture was grown until the mid-exponential phase, the cells were gathered by centrifugation and the pellet was resuspended in DF medium supplemented with 3 mM 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole nitrogen source. After 24 h incubation at 30 °C, the cells were pelleted and resuspended in 1.5 mL Tris-HCl pH 7.0 buffer. After one wash of the cells with the same buffer, they were lysed by adding 30 µL of toluene. For the enzymatic assay, 200 µL of cell lysate was incubated 15 min at 30 °C with 20 µL ACC 0.5 M. The reaction was stopped by the addition of 1 mL HCl 0.56 M. The mix was centrifuged for 5 min at 20,000× g, and 1 mL supernatants were incubated at 30 °C with 800 µL HCl 0.56 M and 300 µL 2,4-dinitrophenylhydrazine. After 30 min of incubation, 2 mL NaOH 2M were added, and the A₅₄₀ was measured in a Shimadzu UV-260 spectrophotometer, Kioto, Japan. The α-ketobutyrate produced was determined using a calibration curve built with known concentrations of α-ketobutyrate. The protein concentration of the cell extracts was determined using the Bradford method [54 (link)].

Column chromatography was conducted using silica gel (SiO₂, 200–300 µm mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (20–100 µm; Pharmacia, Uppsala, Sweden) as packing materials. Silica GF₂₅₄ (10–40 mm) for TLC was supplied by the Qingdao Marine Chemical Factory, Qingdao, China. All TLC spots were visualized under UV light (254 nm) and stained with a 10% H₂SO₄ solution in ethanol, followed by heating. Optical rotations were recorded with a 341 polarimeter (Perkin-Elmer, Waltham, MA, USA) in a 1 dm cell. The UV spectra were measured on a UV-260 spectrophotometer (Shimadzu, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto, Japan) and the IR spectra were obtained on a NEXUS 670 FT-IR spectrometer (Nicolet, Madison, WI, USA). Nuclear magnetic resonance spectra were recorded on INOVA-400 (Varian, Palo Alto, CA, USA) and AVANCE III-400 (Bruker, Billerica, MA, USA) spectrometers. Chemical shifts were given on a δ (ppm) scale using tetramethylsilane as the internal standard. High-resolution electrospray ionization (ESI) mass spectrometry (MS) was carried out on a APEX II mass spectrometer (Bruker Daltonics, Billerica, MA, USA) and ESI-MS spectra were determined on a Bruker Daltonics Esquire 6000 spectrometer. The ABTS and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) used were obtained from Aladdin Industrial Co. (Shanghai, China).

The optical rotations were measured using a PerkinElmer model 341 polarimeter. A Bruker Tensor spectrometer was used for the measurement of the IR spectra. A Shimadzu UV-260 spectrophotometer was used to measure the UV spectra. ¹H, ¹³C, and 2D NMR spectra were recorded on a Bruker AVANCE III-400, a Bruker AVANCE NEO 600, or a Varian Mercury-600BB spectrometer. The HRESIMS data were acquired utilizing a Bruker Daltonics APEX II spectrometer. The ECD curves were obtained on an Olis DSM-1000 spectrometer. The compounds were purified by a macroporous resin HP-20 and a semipreparative HPLC with a reversed-phase C₁₈ (150 × 10 mm, 10 μm) column. Silica gel (200–300 mesh) for column chromatography and Silica gel GF254 (10–40 mm) for TLC were purchased from the Qingdao Marine Chemical Factory, Qingdao, China.

The E. coli and Azoarcus strains, as well as the plasmids used in this study, are detailed in Table 1. E. coli strains were grown at 37°C in Lysogeny Broth (LB) mediun (Bertani, 1951 (link)). Azoarcus sp. strain CIB and its derivatives were grown anaerobically under nitrate reducing conditions (10 mM nitrate) at 30°C in MC medium as previously described (López-Barragán et al., 2004 (link)). Aromatic hydrocarbons such as toluene, xylenes, styrene were supplied at 250 mM in an inert carrier phase of 2,2,4,4,6,8,8-heptamethylnonan (HMN). Benzylsuccinate was added at 4 mM to the culture medium as inducer. Organic acids such as succinate or pyruvate were added at 0.2% (w/v). Azoarcus sp. CIB cells were also cultivated aerobically in MC medium in the absence of nitrate. When using toluene aerobically as sole carbon source, it was added directly to the culture medium at 1 mM. Where appropriate, antibiotics were added to the culture medium at the following concentrations: ampicillin, 100 μg/ml; gentamicin, 10 μg/ml; kanamycin, 50 μg/ml. Growth was determined by measuring the optical density at 600 nm (OD₆₀₀) in a Shimadzu UV-260 spectrophotometer.

Lyophilized extracts were dissolved in K₂HPO₄/KH₂PO₄ buffer (0.1 M, pH 7.4), and stock solutions with concentrations in the reaction volume of 0.19 (Samples 1 and 3), 0.16 (Sample 2), 0.23 (Sample 4), and 0.21 (Sample 5) mg/mL were prepared. From these stock solutions, serial dilutions were made to test 6 dilutions of each sample. H₂O₂ scavenging activity was evaluated according to a previously described methodology [19 (link)]. The absorbance at 230 nm was read with a spectrophotometer (Shimadzu UV-260 spectrophotometer, Kyoto, Japan) after 10 min incubation. Three assays were performed, each assay in triplicate (n = 3), and the results obtained were compared with Ascorbic acid (AA) (IC50 = 51.2 ± 4.6 µg/mL).