Method scouting and optimisation for the separation and purification of alcohol dehydrogenase from Saccharomyces cerevisiae after alcoholic fermentation

RAYA-TONETTI, G; PEROTTI, N

LATIN AMERICAN APPLIED RESEARCH

PLAPIQUI(UNS-CONICET)

Lugar: Bahia Blanca; Año: 1999 vol. 29 p. 137 - 144

0327-0793

Abstract Alcohol dehydrogenate (ADH) has been partially purified from yeast cells after alcoholic fermentation by using different steps such as cell disruption by chemical treatment, protein precipitation wiíh ammonium sulfate, and affinity chromatography with reactive dyes immobilized on Sepharose CL4B in a packed bed. Two methods of cell disruption were tested to determine the optimal system. A chemical treatment with disodium phosphate produced 1286 units of ADH per gram of cells while a mechanical treatment yielded 211 units of ADH per gram of cells. ADH was precipitated at ammonium sulfate concentrations in the range of 30 to 60 % of the saturation limit. If the process had ío be interrupted, storage of the dialyzed homogenate at -20°C preserved the activity best. This homogenate was used in a series of pilot experiments at pre-determined binding conditions, to fmd the optimal conditions in packed beds by employing different dyes using non-specific elution. When non-specific elution was adopted, ADH could be recovered in an 84.7% yield with a purification factor of 15.7 when Procion Orange HER was used. ADH could be recovered in a 67.4% yíeld with a purification factor of 11.5 when Cibacron BÍue F3GA was used. Specific elution using NAD+ was successful in purifying ADH 68.1-fold in an 89.3% yield by Cibacron Blue F3GA. The ADH capacity of all the used affinity supports was estimated by frontal analysis to optimize the feed amount into the affinity chromatography columns. The overall recovery of the enzyme activity in affinity chromatography was higher than 85%, meaning that the packed bed approach to purification was non-denaturing.