The Optimization for the Isolation and Purification of Alcohol Dehydrogenase Utilizing Aqueous Polyphase Solutions

RM 216

Aqueous two-phase polymer systems have been utilized as a common research method for the separation of proteins, viruses, and DNA (Albertsson, 1971). Albertsson has also described the methods for generating poly-phase systems where three or more aqueous polymer solutions were utilized. Eleven different polymers were tested for aqueous twophase compatibility at maximum solubility in water. The best polymers, poly(ethylene glycol) (Mw=4600), polyvinylpyrrolidone, hydroxypropyl cellulose and poly(acrylic acid sodium salt), were placed in a polyphase column, made in-house, and allowed to form the poly-phase system. Alcohol dehydrogenase (ADH) is a common enzyme in many different organisms, especially S. cerevisiae or baker’s yeast, and it is readily available lyophilized from multiple scientific companies. A method for salting-out ADH from Baker’s yeast was optimized. The salted-out ADH was then placed in the chosen polymers separately for activity, according to Racker’s method (1949). The ADH was active in all polymers chosen separately, and the poly-phase system was created and optimized. The purification of the ADH using the polymers was evaluated using SDS PAGE and native electrophoresis, which was optimized for strongest bands. The main issue of separating the polymers from the proteins was necessary due to streaking of the electrophoresis bands. Common methods were tried to separate the polymer from the protein with little success except for an electrophoresis method described by Albertsson using a multiple U-shaped glass apparatus. Computational visualization of the ADH was also performed using YASARA.