Yeast strains
The killer yeast strains Torulaspora delbrueckii and Wickerhamomyces anomalus were isolated from soil—Basrah (Abu-Alkhaseeb and Al-Qurna)-Iraq.
Culture media
Yeast malt extract broth (YMB) with 1% SDS: yeast extract 3 g, malt extract 3 g, peptone 5 g, glucose 10 g, SDS 2 g, distilled water 1000 ml (Jorgensen et al. 2015); nutrient agar (Himedia, India) for antibacterial activity and activation of pathogenic bacteria, potato dextrose agar (Oxoid, UK) for the activation of yeast isolates.
Purification of killer toxins (TK, WK)
Fermentation medium
During this study, the best fermentation conditions were selected (medium of producing killer toxins), which is the medium of yeast malt extract broth (YMB) (Jorgensen et al. 2015) added to it is sodium dodecyl sulfate 0.2% (SDS) with the optimum conditions 30 C° for 3-5 days and rotated at 150 rpm.
Protein purification
Precipitation with ammonium sulfate salt
The precipitation total protein with ammonium sulfate salts at the concentration of 40% weight/volume (saturation ratio) of the crude protein extracted from the best liquid fermentation culture filtrate for two isolates Torulaspora delbrueckii and Wickerhamomyces anomalus after the elimination of the yeast cells by centrifugation at 6000 rpm/min for 20 min. Salt was added gradually with continuous stirring. After complete dissolving, the solution was left for an hour at 4 C°. For the purpose of obtaining the crude protein, the solution was centrifuged with the high-speed centrifuge (10,000 rpm/min for 10 min) modify in time and number of round per minute of centrifuge (Taguchi 1995).
Dialysis
The crude protein precipitate which was weighed 4.1 g for Torulaspora delbrueckii, obtained from the above stage was dissolved with 125 mL of the buffer solution (citrate buffer), (pH = 4.4) for the dialysis process using special dialysis bags to this purpose using (3,10) KDa dialysis bags. The process of dialysis was done at 4 °C for 48 h with dialysis solution (distilled water) changed almost every 12 h. The size of the protein extract was estimated with milliliter, and its protein concentration was estimated, then lyophilization is the purified protein, preserved it for biological activity.
Note: The purify of the protein was checked by using vertical electrophoresis SDS-PAGE. Purification of killer toxins from Wickerhamomyces anomalus by sephadex G-LH 20 (Villalba et al. 2016).
Fractionation of protein extract on G-20 sephadex
The gel chromatography was used for purification of protein extract into molecular size according to the method of Taguchi (1995) with modification as follows:
Sephadex G-20 2 g dry weight was activated in about 50 ml of ice methanol for 15 min to provide gel for a (1 × 15) cm column.
Sodium azide was added to column (NaN3) to prevent contamination; the gel was left after pouring in the column for 18 h to the purpose of stabilization and stacking, and then wash the gel by methanol.
Void volume (Vo) using the blue Dextran 2000 KDa dye at 0.01 g/ml concentration.
Measuring of protein concentration in purified killer toxin
The protein concentration of purified killer toxin was measured after pooling the tubes that form each peak together, the measurement was carried out using Lowry method. The Lowry protein assay is a biochemical test for defining the level of total protein in a solution. The concentration of total protein is displayed by the changes of sample solution color in ratio to the concentration of protein concentration that can be calculated using colorimetric techniques with using bovine serum albumin (BSA) as a standard protein with graduate concentrations (100 to 1000 μg/ml) (Lowery et al. 1951).
Polyacrylamide gel electrophoresis
The procedure according to the principle of (Laemmli 1970), SDS-PAGE depends on the separation of proteins and their molecular weight. In the present study, 12% resolving gel was made for analysis, (0.025 M Tris, 0.25 M glycine, 0.1% SDS) for 1× tank buffer for SDS-PAGE. The protein samples to be analyzed were loaded into the wells formed in the resolving gel with a fine tip. The samples were electrophoresed for 60 min at 80 volts then the voltage was increased to 100 volts for another 60 min. Coomassie blue was used for staining polyacrylamide gels after electrophoresis to visualize the resolved protein bands, destain solution is prepared as follows: methanol 10 ml, glacial acetic acid 10 ml, D.W. 80 ml.
Biological activity of purified killer toxins extracted as antifungal
The susceptibility test of the purified toxins was performed as described in (Al-Hilfy and Abu-Mejdad 2014), A 100 μl from the suspension of Candida albicans which obtained it from Al-Zubair Hospital, patient suffering from cutaneous candidiasis and identifying it by ITS1-5.8rRNA-ITS2 in mycological lab, then added suspension of yeast to the Sabourauds agar, spread it by L-shaped glass spreader. The plates were left to dry for 15 min at room temperature and then wells with 6 mm diameter were made using cork-borer. After that, a 100 μl of killer toxins (TK, WK) at concentration of 200 mg/ml, DW were added individually to each well and incubated at 37 C° for 24 h. The results were read by measuring the inhibition zone diameter in millimeter.
IR spectrum for killer toxins
These were measured using the FTIR spectra (KBr discs) that were recorded in the 4000-500 cm−1 range on a Shimadzu IRAffinity-1 spectrometer. This was processed at the Department of Chemistry\College of Science, University of Dhi Qar.
UV absorbance spectroscopy
UV absorption spectra for killer toxins were recorded in an aqueous solution (0.1 g/10 ml H2O) on a PG T90U UV-visible spectrophotometer using conventional quartz cell having an optical path length of 1 cm at 298 K. This was processed at the Department of Chemistry\College of Science, University of Dhi Qar (Silverstein et al. 1991).
