Specord 200 plus uv vis spectrophotometer

The total amount of phenolic compounds was determined by the Folin–Ciocalteu reagent [47 (link)]. A total of 200 μL of extract was dissolved in 10 mL of Folin–Ciocalteu solution and 8 mL of saturated sodium carbonate solution. After 90 min, the absorbance was recorded at 765 nm with a SPECORD-200 Plus UV–Vis spectrophotometer (Analytik Jena, Jena, Germany). Gallic acid was used as a standard. A calibration curve Y = 0.0131X + 0.0240 (R² = 0.9987) was obtained (see supporting information, Figure S3).
The total amount of flavonoids was determined using the aluminum trichloride (AlCl₃) reagent [47 (link)]. A volume of 1.5 mL (1 mg/mL) of extract was added to an equal volume of a 2% AlCl₃ solution. The mixture was vigorously shaken, and the absorbance was recorded at 367 nm after 10 min of incubation with a SPECORD-200 Plus UV–Vis spectrophotometer (Analytik Jena, Jena, Germany). Quercetin was used as a standard. A calibration curve Y = 0.0049X + 0.0167 (R² = 0.9954) was obtained (see supporting information, Figure S4).

Catalase activity (CAT) was determined in maize extracts according to the method described by Luck (1965) [29 ]. The extract was added to 3 mL of H₂O₂-phosphate buffer and the decrease of absorbance was measured at 240 nm. The Specord 200 Plus UV–VIS spectrophotometer (Analytik Jena, Jena, Germany) was used. The enzyme activity was expressed in terms of units/assay, one unit being defined as the amount of enzyme required to decrease the absorbance by 0.05.
Peroxidase activity (POD) was determined in maize extracts according to the method described by Reddy et al. (1995) [30 ]. The extract was added to 3 mL of 0.05 M pyrogallol and 0.5 mL H₂O₂. The change in absorbance was measured at 430 nm. The Specord 200 Plus UV–VIS spectrophotometer (Analytik Jena, Jena, Germany) was used. The enzyme activity was expressed in terms of units, one unit being defined as the change in absorbance/minute at 430 nm.

For expression of plant triterpene biosynthesis genes in Synechocystis, pVZ-based expression plasmids were transferred to strain Δshc by conjugation [55 ], whereupon exconjugants were selected and further cultivated on BG11 agar containing, 20 μg mL^-1 spectinomycin and 40 μg mL^-1 kanamycin_.For triterpene biosynthesis experiments, cells were grown under continuous illumination with white light (50 μmol photons m^–2 s^–1) at 30 °C and 150 rpm in a New Brunswick Innova42 incubator shaker (Eppendorf). The culture volume was 80 mL within 250 mL Erlenmeyer flasks. Precultures were inoculated in BG-11 medium containing 20 μg mL^-1 spectinomycin and 40 μg mL^-1 kanamycin, and 10 mM TES buffer (pH 8.0) without cobalt supply, in order to repress gene expression from promoter P_coaT. For induction, cultures were supplemented with 20 μM CoCl₂ at an optical density (OD_750nm) of ~0.4 (Specord 200 PLUS UV/Vis spectrophotometer, Analytik Jena). After cultivation for 4 days in cobalt-repleted medium, when cultures reached an OD_750nm of ~1.0, cells were harvested from 20 mL culture portions by centrifugation (10 min, 4 °C and 4800 g). Resulting pellets were frozen in liquid nitrogen and stored at -80 °C.