Ohshiro T, Kuge Y, Igarashi A, Mochida K, Iwakura M, Uwajima T
Tokyo Research Laboratories, Kyowa Hakko Co., Ltd., Tokyo, Japan.
Biosci Biotechnol Biochem. 1992 Mar;56(3):437-40. doi: 10.1271/bbb.56.437.
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.
我们研究了携带高表达质粒pTP64-1的大肠杆菌生产二氢叶酸还原酶的培养条件。在罐式发酵罐中添加山梨醇和控制pH值对该酶的生产有效。通过DEAE-纤维素亲和柱色谱和Superose Prep12柱色谱从无细胞提取物中纯化该酶，其在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带。在厌氧条件下，使用1.5单位/毫升的纯化酶，200毫摩尔二氢叶酸还原为6(S)-四氢叶酸（l-亚叶酸合成的中间体）的反应在2小时内完成。
