摘要
研究酶法合成叔亮氨酸的反应条件。利用产亮氨酸脱氢酶基因工程菌和产甲酸脱氢酶基因工程菌进行混合全细胞生物转化反应合成叔亮氨酸,考察了诱导剂IPTG浓度、诱导温度和诱导时间等不同诱导条件对产亮氨酸脱氢酶基因工程菌和产甲酸脱氢酶基因工程菌产酶比活力的影响,确定了两种基因工程菌优化的诱导表达条件;通过单因子试验(如转化pH值、转化温度、底物浓度和菌体浓度等)来研究不同反应条件对基因工程菌催化合成叔亮氨酸的影响。结果:工程菌催化合成叔亮氨酸的最适pH是9.5,适合的反应温度为30℃,且两种重组菌的反应湿重比为2∶1时,可达到最高的产量。较高的底物浓度会对反应产生抑制作用,适当提高菌体浓度可以降低底物的抑制作用。通过采用底物分批流加的添加方法,得到了更好的转化效果,最后可达到叔亮氨酸产量为42 mg/ml。
To study the transformation condition of L - tert- Leucine by E. coli. the genetic engineering Genetically engineered E. coli produced leucine dehydrogenase and fomate dehydrogenase which were used to biosynthesine L - tert - Leucine, and the substrate was trimethypyruvic acid. The biosynthesis conditions were investigated to obtain the highest product yield. The usual factors such as the optimum IPTG concentration, the inductive temperature and inductive time of the genetically engineered E. coli were determined. Factorial experiment design was employed to determine the effects of the reactive conditions （such as pH value, substrate concentration, and reactive temperature） on L-tert-Leucine biosynthesis. The results showed that pH9. 5 and 30℃ were the optimum conditions for L-tert-Leucine biosynthesis. L-tert-Leucine biosynthesis was inhibited by the high substrate concentration but it also was manifested that high density of recombinant cells can decrease this inhibitory effect.
出处
《药物生物技术》
CAS
CSCD
2009年第3期202-206,共5页
Pharmaceutical Biotechnology
关键词
叔亮氨酸
基因工程菌
诱导表达
生物合成
L-tert-Leucine, Genetically engineered bacteria, Expression, Biosynthesis
