1800 uv vis spectrophotometer

D-2-Hydroxyglutarate dehydrogenase (D2HGDH) activity was routinely measured by following the reduction of DCIP spectrophotometrically at 600 nm [14 (link)]. Reaction mixtures were incubated at 25 °C and contained 50 mM Tris-HCl buffer (pH 7.5), 200 μM phenazine ethosulfate (PES), 100 μM DCIP, 1 mM D-2-HGA (disodium salt), and cell extract (or pure enzyme) in a total volume of 1.0 mL. Phenazine ethosulfate (PES) was used instead of phenazine methosulfate (PMS) due to its higher stability, especially at increased pH and ionic strength [39 (link)]. After the determination of the pH optimum, D2HGDHPa was measured in 50 mM Tris-HCl buffer (pH 7.5), while D2HGDHEc was measured in 50 mM Tris-HCl buffer (pH 8.0). The reduction of DCIP was monitored at 600 nm with a thermostated Shimadzu-1800 UV-Vis spectrophotometer (Shimadzu Corp, Kyoto, Japan), converting the absorbance to concentration using a molar extinction coefficient of 22 mM⁻¹cm⁻¹. One unit (U) of activity was defined as 1 μmol of DCIP reduced per minute. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. All measured values indicate the means of at least three independent experiments.

The IDH activity was routinely measured by monitoring the reduction of NAD⁺ (or NADP⁺) at 340 nm. The reaction mixtures were incubated at 42°C and contained 100 mM Tris-HCl buffer (pH 7.5), 2 mM MnCl₂, 5.0 mM DL-isocitrate, 0.4 mM NAD⁺ or NADP⁺ and the enzyme, with a total volume of 1.0 mL. After determing their pH optima, NAD⁺-IDH activity was measured in 100 mM Tris-HCl buffer (pH 8.5), and NADP⁺-IDH activity was measured in 100 mM Bis-Tris-HCl buffer (pH 6.0). The increase in NAD(P)H concentration was determined by monitoring the absorbance at 340 nm with a thermostated Shimadzu-1800 UV-Vis spectrophotometer (Shimadzu Corp, Japan) and converting the absorbance to concentration using a molar extinction coefficient of 6.22 mM^-1 cm^-1. One unit (U) of activity was defined as 1 μmol of NAD(P)H formed per min. The protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, USA) with bovine serum albumin as the standard. All the reported values are the means of at least three independent experiments.

Folin–Ciocalteu’s phenol reagent, gallic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemicals (St. Louis, MO, USA). Mushroom β-glucan quantification kit was obtained from Megazyme Int. (Wicklow, Ireland). All of the chemicals and solvents were of analytical grade. L. casei (ATCC 334, KWIK-STICK) and E. coli (ATCC 25922, MECCOUNTI) were used in this study. The spectrophotometric measurements were performed using an 1800 UV–VIS spectrophotometer (SHIMADZU Inc., Kyoto, Japan) and a microplate reader (PowerWave XS2, BioTek, Winooski, VT, USA).