One particular /3-galactosidase cleaves the compound lactose-lysine into ¡3-galactose and fructose-lysine. This activity is of interest because it has been suggested as a possible marker for the presence of bacteria, since it does not appear to be present in germ-free animals.
The reaction is followed by separation of the substrate, lactose-lysine, from the product, fructose-lysine, on a cation-exchange resin (Durrum DC6A) using an isocratic mobile phase of pyridine-acetic acid-water (6:60:176, v/v). o-Phthalaldehyde derivatives were formed and detected by fluorescence.
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Figure 9.64 Chromatogram obtained after injection of a /3-galactosidase incubation mixture onto a 45 mm X 3.6 mm cation-exchange column. Mobile phase: pyridine-acetic acid-water (6:60:176, v/v). Flow rate, 0.4 mL/min; temperature, 50°C. In front of lactose-lysine some free amino acids elute which are the result of proteolytic activity in the intestinal enzyme preparation. Peaks: 1, lactose-lysine; 2, j8-alanine (internal standard); 3, fructose-lysine. (From Schreuder and Welling, 1983.)
The reaction was carried out with e-lactose-lysine as the substrate using a sodium phosphate buffer (pH 7.5). The reaction was started by the addition of the enzyme; after incubation for 1 hour, it was terminated with methanol containing /3-alanine as an internal standard. Precipitated protein was removed by centrifugation, and samples of the supernatant solution were injected and analyzed by HPLC. A chromatogram showing the analysis of a reaction mixture is given in Figure 9.64.
The enzymes were obtained from mouse intestine. The intestinal segments were cut into pieces, homogenized with a glass rod, sonicated, and centrifuged. The supernatant solution was dialyzed and stored frozen for later use.
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