The preparation of transketolase free from d-ribulose-5-phosphate 3-epimerase

Add time:09/06/2019    Source:sciencedirect.com

A procedure for the purification from Candida utilis of transketolase (sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) free from d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was developed using acetone precipitation, elution from DEAE-cellulose, adsorption of epimerase by thiopropyl-Sepharose, and chromatography on d-ribose 5-phosphate-Sepharose and DEAE-Sephadex. The final product had a specific activity of 43 units/mg, a transketolase/epimerase activity ratio greater than 53 000 to 1, an apparent Km for d-xylulose 5-phosphate and d-ribose 5-phosphate of 77 and 430 μM, respectively, and ran as a single band using electrophoresis on polyacrylamide gel. It was inhibited by d-arabinose 5-phosphate and d-glucose 6-phosphate. During the purification by column chromatography, multiple forms of the enzyme were detected by gel electrophoresis but these gradually disappeared as the enzyme was further purified.

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