Table 1. Systems and solutions utilized for the MLEE analyses of the S. aureus metabolic enzymes.
| Enzyme | Compound for staining | |||||||
|---|---|---|---|---|---|---|---|---|
| EC number | Name | Symbol | Substrate | Buffer | Salt | Coenzyme | Dye and Catalyser | |
| 1.1.1.1. | alcohol dehydrogenase | ADH | Ethanol (3mL) Isopropanol (2mL) | 200 mM Tris-HCl pH 8.0 (q.s.p. 50mL)a | NAD 1% (2 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | ||
| 1.1.1.17 | mannitol-1-phosphate dehydrogenase | M1P | Mannitol 1-phosphate (5mg) | 200 mM Tris-HCl pH 8.0 (q.s.p. 50 mL)a | NAD 1% (2 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | ||
| 1.1.1.37. | malate dehydrogenase | MDH | 2M Malic acid (6 mL) b | 200 mM Tris-HCl pH 8.0 (q.s.p. 50 mL)a | NAD 1% (2 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | ||
| 1.1.1.47 | glucose dehydrogenase | GDH | D-glucose (500 mg) | 200 mM Tris-HCl pH 8.0 (q.s.p. 50 mL)a | NAD 1% (2 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | ||
| 1.1.1.48 | D-galactose dehydrogenase | GLDH | Galactose (450mL) | Tris-HCl 100 mM pH 8.4 (q.s.p. 50 mL)c | NAD 1% (1 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | ||
| 1.1.1.49 | glucose-6-phosphate dehydrogenase | G6PDH | Glicose-6-phosphate disodium salt (100 mg) | 200 mM Tris-HCl pH 8.0 (q.s.p. 50 mL)a | 100 mM MgCl2 (1 mL)d | NADP 1% (1 mL) | PMS 1% (500 mL) MTT 1.25% (1 mL) | |
| 1.11.1.6 | catalase | CATe | ||||||
| 3.1.1.1. | α- and β- esterase | EST | α- and β- Naphthyl acetate (1% solution in acetone) (1.5ml) | 50mM Sodium phosphate pH 6.0 (q.s.p. 50mL)f | Fast Blue RR salt (25 mg) | |||
Electrode buffer: Tris–citrate pH 8.0 [83.2 g of C4H11NO3 (Tris), 33.09 g of C6H8O7.H2O (Citric acid), 1 L of H2O]; Gel buffer: Electrode buffer diluted 1:29. a 24.2 g of C4H11NO3 (Tris), 1 L of H2O (pH adjusted with HCl); b 26.8 g of C4H6O5 (DL-malic acid) and 16 g of NaOH in 0.1 L of H2O (caution: potentially explosive reaction); c 12.1 g of C4H11NO3 (Tris), 1 L of H2O (pH adjusted with HCl); d 2.03 g of MgCl2.6HCl (Magnesium chloride) in 0.1 L of H2O; e Incubate gel slice for 30 min at 0 oC in 50 mL of 0.1 M sodium phosphate pH 7.0 buffer, then pour off solution, and immerse it in 50 mL of 1.5% potassium iodide solution (KI) for 2 min. Therefore, rinse gel slice with water, and immerse it in 50 mL of 0.03% hydrogen peroxide (H2O2) solution. Mix gently and remove stain solution when white zones appear on dark-blue background; f Sodium phosphate buffer pH 7.0: mix equal parts of 27.6 g of NaH2PO4.H2O (monobasic) in 1 L of water and 53.6 g of Na2HPO4.7H2O in 1 L of water, then dilute the mixture 1:25 with water.