A novel assay system for the measurement of transketolase activity using xylulokinase from Saccharomyces cerevisiae.

Abstract:

:The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 x 10(-4)unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.

journal_name

Biotechnol Lett

journal_title

Biotechnology letters

authors

Lee JY,Cheong DE,Kim GJ

doi

10.1007/s10529-007-9616-y

subject

Has Abstract

pub_date

2008-05-01 00:00:00

pages

899-904

issue

5

eissn

0141-5492

issn

1573-6776

journal_volume

30

pub_type

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