The purification of transketolase from Candida utilis

Abstract

Transketolase (EC 2.2.1.1) was purified 12-fold from an extract of dried Candida utilis by two fractionations with acetone followed by chromatography on DEAE-Sephadex. The fractions from the column were free from D-ribose 5-phosphate ketol isomerase and D-ribulose 5-phosphate 3-epimerase and could be used for the assay of D-xylulose 5-phosphate and of the epimerase. Three peaks of transketolase activity were eluted from the chromatography column; the material from each peak had a Michaelis constant for D-xylulose 5-phosphate close to the mean value for the three peaks of 6.8 × 10−5 M and gave the same two-banded pattern on cellulose acetate electrophoresis. When transaldolase was present in the material placed on the column, it appeared with transketolase in each of the three peaks. The enzyme could be stored for several months, in solution frozen at −20°. Dialysis against water caused a loss of activity which could be restored by the addition of thiamine pyrophosphate. The enzyme catalyzed the condensation of hydroxypyruvate with D-glyceraldehyde phosphate to form D-xylulose 5-phosphate.