Optimization of the enzymatic one pot reaction for the synthesis of uridine 5'-diphosphogalactose.
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 fixed, 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 mMUMP.
Lee
Jae-Hun
JH
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, South Korea.
Chung
Seung-Wook
SW
Lee
Hwa-Jin
HJ
Jang
Kyuong-Soon
KS
Lee
Sun-Gu
SG
Kim
Byung-Gee
BG
D002474:Cell-Free System / Q000201:enzymology; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D014539:Uridine Diphosphate Sugars / Q000096:biosynthesis* / Q000737:chemistry
D004798:Enzymes; D029968:Escherichia coli Proteins; D014539:Uridine Diphosphate Sugars