Uv 1202 spectrophotometer

The reduction of substrate was monitored at 440 nm on a Shimadzu UV 1202 spectrophotometer (Shimadzu, Germany). Total alcohol dehydrogenase activity was measured by the photometric assay with substrate (p-nitrosodimethylaniline)^{9 (link)}. The reaction mixture (2 ml) contained 1.8 ml of a 26 µM solution of substrate in 0.1 M of sodium phosphate buffer, (pH 8.5) and 0.1 ml of a mixture containing 5 mM NAD and 0.25 M n-butanol and 0.1 ml of serum.

Conjugates of a modified derivative of chloramphenicol, CAP-Su with BSA, were obtained through carbodiimide-succinimide method according to [25 (link)]. The molar ratio of the obtained CAP:BSA conjugate was 20:1. Concentration of the conjugate was determined by measuring its optical density at 280 nm and comparing with the initial BSA solution using the UV-1202 spectrophotometer (Shimadzu, Kyoto, Japan). The dialyzed conjugate was stored at −20 °C.

The strains and plasmids used in this work are listed in Table 1. C. glutamicum ATCC13032 was used as wild type (WT), for metabolic engineering the prophage-cured C. glutamicum MB001 (Baumgart et al., 2013 (link)) was used as platform strain. Precultivation of C. glutamicum strains was performed in LB medium or LB with glucose. For cultivation in CGXII medium (Eggeling and Reyes, 2005 ), precultivated cells were washed once with CGXII medium without carbon source and inoculated to an initial OD₆₀₀ of 1. Glucose was added as carbon and energy source to a concentration of 100 mM. Standard cultivations of C. glutamicum were performed at 30°C in a volume of 50 ml in 500 ml flasks with two baffles shaking at 120 rpm. The OD₆₀₀ was measured in dilutions using a Shimadzu UV-1202 spectrophotometer (Duisburg, Germany). Alternatively, cultivations were performed in 1 ml volume in microtiterplates at 1100 rpm at 30°C using Biolector^® micro fermentation system (m2p-labs GmbH, Baesweiler, Germany). For cloning, E. coli DH5α was used as host and cultivated in LB medium at 37°C. When appropriate, kanamycin or spectinomycin was added to concentrations of 25 and 100 μg ml⁻¹, respectively. Gene expression was induced by adding 50 μM and 1 mM IPTG, respectively, at inoculation of the main culture.

The method of determination relies on the production of a superoxide anion by the reaction of xanthine with xanthine oxidase (XOD), which, by reacting with 2-(4-iodophenyl)-3(4-nitrophenyl)-5-phenyltetrazole (INT) chloride, forms a red formazan dye. The measurement of the absorbance of the colored product determines the effectiveness of the above reaction. $$\text{Xantine}\stackrel{\text{XOD}}{⟶}\text{Uric}\text{acid}+{{\text{O}}_2}^{-}$$
$$\text{I}.\text{N}.\text{T}.\stackrel{{{\text{O}}_2}^{-}}{⟶}\text{Formazan}\text{dye}$$ CuZnSOD presented in the tested sample (biological material) inhibits this reaction by dismutating O₂⁻ to H₂O₂ and O₂. The degree of inhibition of this reaction is directly proportional to the activity of CuZnSOD. $${{\text{O}}_2}^{-}+{{\text{O}}_2}^{-}+2{\text{H}}^{+}\stackrel{\text{CuZnSOD}}{⟶}{\text{O}}_2+{\text{H}}_2{\text{O}}_2$$ CuZnSOD activity was determined spectrophotometrically using a standard RANSOD kit by Randox (United Kingdom), which includes the following reagents:

Substrate: Xanthine 0.05 mmol/L, INT 0.025 mmol/L;

Buffer: N-cyclohexyl-3-aminopropanesulfonic acid 40 mmol/L, pH 10.2, EDTA 0.94 mmol/L;

Xanthine oxidase 80 U/mL; and

Standard – CuZnSOD 5.5 U/mL.

CuZnSOD activity in the tested biological material was determined in a Shimadzu UV 1202 spectrophotometer (Kyoto, Japan), measuring the absorbance at 0 and 3 min at a wavelength of 505 nm.

Strains and plasmids used in this study are listed in Table S3. Chemicals were delivered by Carl Roth (Karlsruhe, Germany) if not stated differently. E. coli DH5α cells were used for cloning and were cultivated at 37 °C in LB medium^{66 (link)}. Precultures of C. glutamicum strains were grown in LB medium supplemented with 10 g L⁻¹ glucose overnight and addition of antibiotics. The main cultures of C. glutamicum for the screening of carotenoid production were grown in CGXII minimal medium⁶⁷ or in CGXII minimal media with optimized trace salts concentrations (25 g L⁻¹ FeSO₄ × 7H₂O, 2.5 g L⁻¹ MnSO₄ x H₂O, 1.18 g L⁻¹ ZnSO₄ × 7H₂O, 0.15 g L⁻¹ CuSO₄ × 5H₂O, 0.015 g L⁻¹ NiCl₂ × 6H₂O)⁵² , called CGXII_opt, both supplemented with 40 g L⁻¹ glucose and supplemented with 1 mM IPTG for induction if needed after washing in the minimal medium. Cultures were inoculated to an initial OD_600nm of 1 using a Shimadzu UV-1202 spectrophotometer (Duisburg, Germany). Cultivations of C. glutamicum were performed at 30 °C in a volume of 10 mL in 100 mL flasks with two baffles shaking at 120 rpm on a rotary shaker. Tetracycline, Kanamycin and Spectinomycin (VWR, Darmstadt, Germany) were added if appropriate to respective concentrations of 5 μg mL⁻¹, 25 μg mL⁻¹ and 100 μg mL⁻¹ in C. glutamicum cultures and in E. coli cultures.

As far as not mentioned specifically, C. glutamicum was precultured in LB medium (Sambrook, 2001 ) with 56 mM of glucose overnight, washed once with CGXII medium (Eggeling and Bott, 2005 ) without carbon source and inoculated in CGXII with 222 mM of glucose at initial optical density (OD) (λ=600 nm) of 1. The OD was measured with UV-1202 spectrophotometer (Shimadzu, Duisburg, Germany) with suitable dilutions. When appropriate, 25 μg/mL of kanamycin and IPTG were added as indicated in the text. Growth experiment with Biolector^® cultivation system (m2pLabs, Baesweiler, Germany) was performed in 1 mL of CGXII with 222 mM of glucose using FlowerPlate^® (m2pLabs, Baesweiler, Germany) at 30 °C, 1100 rpm. Growth experiment with flask was performed in 50 mL of CGXII 222 mM of glucose using 500 mL of baffled flask at 30 °C, 120 rpm. For growth rate calculation, cell growth was monitored online every 10 min for 48 h with Biolector^®. Maximum growth rate μ (h⁻¹) was calculated from 20 measuring points of arbitrary unit of the backscattering light (620 nm). Plate image was scanned with Perfection V750-M Pro scanner (Epson, Ludwigshafen am Rhein, Germany). Consumption of glucose was tested with Diabur 5000 glucose test stripes (Roche Diagnostics, Mannheim, Germany) with a detection limit of 0.005% glucose.

The antioxidant activity was determined using a method involving DPPH free radicals [57 (link)]. One milliliter of the DPPH methanol solution (300 μM) was mixed with 4 mL of a methanol solution of α-T or CT (25 μM). The mixture was then incubated for a 30 min at room temperature in the dark. During incubation, a reduction of part of the DPPH free radical occurred, and the absorbance at 517 nm just after mixing, after 15 min of incubation, and after 30 min of incubation was measured. The absorbance was measured against methanol as a blank using a Shimadzu UV 1202 spectrophotometer (Shimadzu, Kyoto, Japan).