Optimization of the enzymatic one pot reaction for the synthesis of uridine 5'-diphosphogalactose

Bioprocess Biosyst Eng (2010) 33:7178 DOI 10.1007/s00449-009-0365-2

ORIGINAL PAPER

Optimization of the enzymatic one pot reaction for the synthesis of uridine 50-diphosphogalactose

Jae-Hun Lee Seung-Wook Chung Hwa-Jin Lee

Kyuong-Soon Jang Sun-Gu Lee Byung-Gee Kim

Received: 31 May 2009 / Accepted: 28 July 2009 / Published online: 28 August 2009 Springer-Verlag 2009

Abstract Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically xed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.

Keywords Uridine 5-diphosphogalactose

Optimization of biosynthetic process Recombinant E. coli

AbbreviationsAcetyl Pi Acetyl phosphateADP Adenosine 50-diphosphate

ATP Adenosine 50-triphosphate CMP-NeuAc Cytidine 50-monophosphate dNTP Deoxynucleoside-triphosphateE. coli Escherichia coliGal GalactoseGlc GlucosePi PhosphatePPi PyrophosphateUDP Uridine 50-diphosphate

UDP-Gal Uridine 50-diposphogalactose UDP-Glc Uridine 50-diphosphoglucose UMP Uridine 50-monophosphate UTP Uridine 50-triphosphate

Introduction

Oligosaccharides on the cell surface or secreted soluble oligosaccharides play important roles in various types of cell recognition or signal transduction processes. Such oligosaccharides can be used for investigation of biological functions of oligosaccharides as well as development of carbohydrate-based drugs or neutraceuticals [14]. The synthesis of oligosaccharides can be done using either chemical or biochemical methods. In general, biochemical methods using whole cell system is more advantageous for large-scale oligosaccharide synthesis, since high yields of the products can be achieved without chemical protections and deprotections of the substrate carbohydrates. One of the problems in the large-scale synthesis of oligosaccharides using biochemical methods, especially glycosyltransferase, is that it requires expensive substrates, sugar-nucleotides, usually NDP-sugars. A number of enzymatic processes have been developed to produce the sugar-nucleotides more

J.-H. Lee H.-J. Lee K.-S. Jang B.-G. Kim

Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University,Seoul 151-742, South Korea