ABSTRACT
Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).
Subject(s)
Aspergillus/enzymology , Chemical Fractionation/methods , Chromatography, Affinity/methods , Chromatography, Agarose/methods , Glycoside Hydrolases/biosynthesis , Glycoside Hydrolases/isolation & purification , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/isolation & purification , Triazines , beta-Glucosidase/biosynthesis , beta-Glucosidase/isolation & purification , Adsorption , Coloring Agents , Enzyme Activation , Glycoside Hydrolases/chemistry , Hydrogen-Ion Concentration , Multienzyme Complexes/chemistry , Temperature , beta-Glucosidase/chemistryABSTRACT
Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A4R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.
