Document Type

Article

Publication Date

12-12-2005

Publication Title

Biotechnology and Bioengineering

Abstract

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy- D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4- keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6- dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1- phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identifiedbyHPLC, ESI-MS,andNMR,showingabout95%purity.

Keywords

dTDP-L-rhamnose; dTDP-4-keto-6-deoxy-Dglucose; dTDP-4-keto-6-deoxy-D-glucose 3, 5 epimerase; dTDP-4-keto-rhamnose reductase

Volume

93

Issue

1

DOI

https://doi.org/10.1002/bit.20648

ISSN

1097-0290

Comments

Originally published in Biotechnology and Bioengineering, Volume 93, Issue 1, 12 December 2005, Wiley.

https://doi.org/10.1002/bit.20648

Download

Share

COinS