Lee D C, Lee S G, Hong S P, Sung M H, Kim H S
Department of Biotechnology, Korea Advanced Institute of Science and Technology, Yusung-Gu, Taejon, Korea.
Ann N Y Acad Sci. 1996 Oct 12;799:401-5. doi: 10.1111/j.1749-6632.1996.tb33232.x.
We cloned the thermostable D-hydantoinase gene from B. stearothermophilus SD-1 into E. coli. The cloned gene was constitutively expressed by its own promoter, and the enzyme was produced in its soluble form. The specific activity of the recombinant E. coli was 30 times higher than that of B. stearothermophilus SD-1. The cultivation conditions were investigated for the overproduction of the enzyme, and the temperature was found to affect the plasmid content and the expression level of the enzyme. Recombinant E. coli was cultivated in 30-L batch fermentation, the cell concentration reached 25 g-DCW/L, and the specific activity was about 20,000 units/g-DCW. D-Hydantoinase produced from the recombinant E. coli could be successfully applied to the synthesis of N-carbamoyl-D-amino acid from the 5-monosubstituted hydantoin derivative.
我们将嗜热脂肪芽孢杆菌SD-1的耐热D-海因酶基因克隆到大肠杆菌中。克隆的基因由其自身启动子组成型表达，并且该酶以可溶形式产生。重组大肠杆菌的比活性比嗜热脂肪芽孢杆菌SD-1高30倍。研究了用于过量生产该酶的培养条件，发现温度会影响质粒含量和酶的表达水平。重组大肠杆菌在30-L分批发酵中培养，细胞浓度达到25 g-DCW/L，比活性约为20,000单位/g-DCW。重组大肠杆菌产生的D-海因酶可成功应用于从5-单取代海因衍生物合成N-氨甲酰-D-氨基酸。
