PRODUCTION AND PURIFICATION OF TARTRATE DEHYDROGENASE - ROLE OF AQUEOUS 2-PHASE EXTRACTION

Tartrate dehydrogenase (TDH) is a stereospecific intracellular enzyme produced by Pseudomonas putida. Several methods for separation of nucl eic acids from the proteins in cell homogenate were compared in this s tudy. These methods included precipitation (using streptomycin sulfate , manganous sulfate, and protamine sulfate) and aqueous two-phase extr action. Under optimal conditions of separation, a single-step aqueous two-phase extraction followed by back-extraction of the enzyme from en zyme-rich PEG-phase resulted in 77% recovery of enzyme. This compared favorably with 50% enzyme recovery using protamine sulfate treatment. Furthermore, the remaining enzyme activity was accounted in the nuclei c acid-rich dextran phase and the spent-PEG phase, suggesting that a m ultistep extraction process would increase enzyme recovery even more. Under the conditions of aqueous two-phase extraction, the selectivity of proteins over nucleic acids was 30, indicating a high degree of sep aration of proteins and nucleic acids in this process. The experimenta l data and their implications are presented.