摘要
在E.coli BL21(DE3)中过量表达D-乳酸脱氢酶基因(D-ldh),并优化该重组菌全细胞转化苯丙酮酸钠合成苯基乳酸的条件。通过单因素实验和正交实验优化诱导表达条件,并在此基础上对全细胞转化苯丙酮酸钠合成苯基乳酸进行了优化。结果表明,菌体OD600为1.2时添加IPTG至终浓度0.2mmol/L,25℃诱导4h后收集菌体具有最佳转化活性;最优分批转化条件:p H7.0,8.0g/L苯丙酮酸钠,20g/L葡萄糖,1%(v/v)吐温-80,菌体浓度20g/L(干重),37℃,转速200r/min转化0.5h,苯基乳酸产量和转化率分别达到4.91g/L,56%。在上述优化条件上通过流加苯丙酮酸钠和葡萄糖,经6h转化,苯基乳酸最终产量达到17.23g/L,转化率为54%。研究结果表明该重组大肠杆菌成功转化苯丙酮酸合成苯基乳酸,具有较好的应用前景,为系统化代谢工程改造大肠杆菌生物合成苯基乳酸的进一步研究和应用提供了有用的技术参数。
D- lactate dehydrogenase was expressed in recombinant Escherichia coli BL21( DE3),the expression conditions and bioconversion medium were optimized for the production of PLA with whole- cell transformation.Single factor experiments and orthogonal experiments were used to optimize induction conditions and the transformation conditions with the engineered whole- cells transformation. The optimized induction condition for D- ldh expression was IPTG 0.2mmol / L and inducing for 4h at 25℃ and OD6001.2,the optimized batch reaction conditions in phosphate buffer( pH7.0) were: 8g / L PPA,20 g / L glucose,20 g / L( dry cell weight),1% Tween- 80,37℃,200 r / min for 0.5h with PLA production of 4.91 g / L and conversion ratio of 56%.Based on the above optimized conditions,fed- batch fermentation was conducted by intermittent feed PPA and glucose. After 6h transformation,the final PLA concentration reached 17.23 g / L with the conversion ratio of 54%. Results showed that PPA was efficiently transformed into PLA through engineered E.coli BL21( DE3) / p ET28a- D- ldh.This study not only showed a good industrial application prospect,but also laid a foundation for metabolic engineering of E. coli for the production of PLA.
出处
《食品工业科技》
CAS
CSCD
北大核心
2015年第9期147-151,157,共6页
Science and Technology of Food Industry
基金
科技部农业科技成果转化资金项目(2013GB2C100176)
江苏省自然科学青年基金项目(BK20130380)
江苏省高校自然科学研究项目(13KJB550002)
江苏省"六大高峰人才"资助计划项目(NY-021)
苏州市科技支撑计划(SNG201354)
常熟市科技发展计划(CN201220)
关键词
大肠杆菌
D-乳酸脱氢酶
苯基乳酸
全细胞生物转化
Escherichia coil
D-lactate dehydrogenase
phenyllactic acid
whole-cell bioconversion