Production of dihydrofolate reductase by cloned Escherichia coli and its application to asymmetric synthesis of l-leucovorin.
We have investigated culture conditions for production of dihydrofolate reductase by Escherichia coli harboring a high expression plasmid, pTP64-1. Sorbitol addition and pH control were effective for the production of the enzyme in a jar fermentor. The enzyme was purified from a cell-free extract by column chromatographies on DEAE-Cellulofine and Superose Prep12 and showed a single band on SDS-polyacrylamide gel electrophoresis. The reduction of 200 mM dihydrofolate to 6(S)-tetrahydrofolate, an intermediate for l-leucovorin synthesis, was complete in 2 hr under anaerobic conditions, using 1.5 units/ml of the purified enzyme.
Tokyo Research Laboratories, Kyowa Hakko Co., Ltd., Tokyo, Japan.
T
T
Ohshiro
Y
Y
Kuge
A
A
Igarashi
K
K
Mochida
M
M
Iwakura
T
T
Uwajima
D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002955:Leucovorin / Q000096:biosynthesis*; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis*
C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin