Ultrospec 2100 pro

Growth of isolates in medium containing 6.5% (w/v) NaCl solid powder was determined, as previously described [14 (link)]. Briefly, isolates were inoculated (1.0%, v/v) into MRS broth containing 6.5% NaCl solid powder and then anaerobically incubated (HB-103S, Hanbaek, Bucheon, Korea) for 21 days at 25 °C. Growth was measured at 600 nm (A₆₀₀; Ultrospec 2100 Pro, Biochrom, Cambridge, UK).

The free iron, as a measure of fructation-induced iron release in the reaction mixture after 10, 20 and 30 days of incubation, was estimated according to the method of Panter [47 (link)]. Briefly, to 250 μL of the reaction mixture, 250 μL of ice cold TCA (20%) was added and centrifuged in a refrigerated centrifuge at 15,000 rpm for 4 min. To 250 μL of the supernatant, 2.5 mL of iron buffer (1.5% hydroxylamine hydrochloride in acetate buffer, pH 4.5) and 50 μL iron colour reagent (0.85% ferrozine in iron buffer) were added. The resultant mixture was incubated at 37 °C for 30 min and the absorbance was measured at 560 nm using a UV-visible spectrophotometer (Ultrospec 2100 Pro, Biochrom). The concentration of liberated free iron was calculated as follows: $$\begin{array}{l}\text{Concentration of free iron}\left(\mathrm{\mu g}/\mathrm{dL}\right)=\\ \left(\text{Absorbance of test}/\text{Absorbance of standard}\right)\mathrm{X}\text{Concentration of standard}\left(\mathrm{\mu g}/\mathrm{dL}\right)\end{array}$$

Standard UV-Vis spectra detection was performed on a UV-Vis and Vis instrument (Ultrospec 2100 pro, Biochrom, UK) at room temperature. MtMbnBC and RrMbnBC proteins were adjusted to 5 mg/mL (nearly 100 μM), and VcMbnBC was adjusted to a concentration of 1 mg/mL (nearly 20 μM). The corresponding substrates, VcMbnA, RrMbnA, MtMbnA, and their variants (Supplementary information, Table S5), were dissolved in distilled water and diluted to a working concentration of 5 mM with buffer B. The reaction was initiated when 100 μL MbnBC proteins and 5 μL MbnA peptides were applied to the reaction system, and the reaction was diluted up to 1 mL with buffer B. The mixture was then added into a quartz cuvette and recorded at 20-s intervals for a total of 50 reads at room temperature. For assays using anaerobically prepared MbnBC proteins, the measurements were immediately recorded by adding the anaerobically prepared MbnBC into the reaction system, with peptides pre-mixed in the quartz cuvette. The buffer B used in the assay was O₂-saturated at room temperature.
For the stopped-flow UV-Vis spectroscopic analysis, 10 μM MtMbnBC or RrMbnBC or 2 μM VcMbnBC was incubated with 250 μM MbnA peptides at room temperature for 1 h, followed by detection on a UV-Vis and Vis instrument (Nanodrop One, Thermo Scientific, MA). Data were processed by Graphpad Prism version 8.3.

The parameter of main interest was the fraction of CO-Hb in percent. It was measured using a blood gas analyser (ABL800 Flex, Radiometer Medical ApS, Brønshøj, Denmark), which was also used for measuring the pH-value.
Hemolysis was determined by photometric measurement of the plasma free hemoglobin (Ultrospec 2100 Pro, Biochrom, Berlin, Germany). The analysis of hemolysis was performed according to DIN 58,931 [27 ] by means of the cyanmethemoglobin method (Hemoglobin FS, DiaSys, Germany) according to manufacturers’ instructions. For this, the plasma of each blood sample was separated from the cells by double centrifugation at 1500× g for 15 min.

All strains and mutants in this study are based on the S. cerevisiae strain BY4741 (MATa; his3Δ-1; leu2Δ-0; met15Δ-0; and ura3Δ-0). The collection analyzed here, called the SGA-V2 collection, contains about 4,200 mutants (with mutations in non-essential genes), and was a gift provided by Prof. Charles Boone (University of Toronto, Canada; Tong et al., 2001 (link), 2004 (link)). Mutant his3Δ::KanR in this collection was designed as the control strain and added as a border around four edges of each plate (Tong and Boone, 2007 ). The strains were grown at 30°C, in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) with 200 mg·L⁻¹ G418 (YPD + G418). NaHCO₃-containing plates were made with YPD agar medium that was supplemented with 40 mM NaHCO₃ and 200 mg·L⁻¹ G418. For the yeast growth curve, the optical density at 600 nm (OD₆₀₀) absorbance value of yeast BY4741 under different NaHCO₃ concentration treatments was measured using Ultrospec 2100 pro (Biochrom, United Kingdom) spectrophotometer. The measurement was performed every 2 h and the growth curve was drawn.

Overnight-cultured LAB isolates were used to inoculate (1.0%, v/v) MRS broth and then incubated at 5, 10, 15, 25, or 30 °C for 24–288 h. Growth was measured every 4 or 8 h by measuring absorbance at 600 nm (A₆₀₀; Ultrospec 2100 Pro, Biochrom, Cambridge, UK). In addition, viable counts at maximum A₆₀₀ were determined using the plate method [26 (link)].

Cell concentration was determined by turbidimetry using a spectrophotometer (Ultrospec 2100 pro (Biochrom, Holliston, MA, USA)), correlating the measured optical density (OD) at 600 nm with cell dry weight (g/L) through a previously determined calibration curve. Cell viability was quantified using methylene blue staining technique [33 ], followed by counting viable cells in a Neubauer’s chamber. To release the immobilized cells for viability assays, the biocatalyst beads were suspended in 8% (w/v) sodium citrate buffer (100 mg_beads/mL) under magnetic stirring to solubilize the alginate gel [15 (link)]. Cell viability was defined as the ratio between viable cells and total cells counted in a defined space of the chamber.