摘要
目的:对重组大肠杆菌E.coli BL21-pET28a(+)-ADH表达羰基还原酶的条件进行优化,以增加目的蛋白的表达量,提高生物合成(S)-3,5-双三氟甲基苯乙醇的转化率。方法:将重组质粒pET28a(+)-ADH转化E.coli BL21后,对诱导过程产生影响的4个因素,诱导时间、诱导温度、诱导剂浓度及摇菌转数,利用SPSS统计学软件进行正交实验设计,并对实验结果进行直观分析及方差分析,对实验得出的最佳搭配诱导条件进行验证。结果:直观分析中诱导温度的R值最大,其次为摇菌转数和诱导剂IPTG浓度,诱导时间的R值最小;方差分析表明诱导温度的P值小于0.05,而摇菌转数、IPTG浓度及时间因素的P值均大于0.05。利用优化后的菌种生物合成(S)-3,5-双三氟甲基苯乙醇,转化率达到95.16%。结论:本实验的最优诱导条件为,诱导温度25℃、诱导摇菌转数200r.min-1、诱导剂IPTG浓度0.5mmol.L-1、诱导时间6h。且重组菌对于(S)-3,5-双三氟甲基苯乙醇的转化率达到95.16%。
Objective： To optimize the recombinant expression of carbonyl reducase in E. coli BL21 induced by IPTG, and increase the conversion ratio of biosynthesis of （S）-3,5-bis （trifluoromethyl） phenethyl alcohol. Method： The plasmid pET28a （＋）-ADH converted E. coli BL21 was constructed by our laboratory. Four different factors, inducing time, temperature, agitation speed and concentration of IPTG, were studied by orthogonal experimental design. The data were analyzed by direct calculation and ANOVA method. Results： The R value of inducing temperature resulting from direct calculation was the highest among the four factors. The P value of inducing temperature was lower than 0.05. The P values of the other factors were higher than 0.05. The conversion ratio of biosynthesis of （S）-3,5-bis （trifluoromethyl） phenethyl alcohol with recombinant E. coli was 95.16%. Conclusion： The expression level is remarkablely affected by the inducing temperature. The expression level of target protein is the highest with inducing conditions at temperature 30℃, agitation rate 200 r-min^-1, IPTG 0.5mmol·L^-1 and inducing time 6 hours. And The conversion ratio of biosynthesis of （S）-3,5-bis（trifluoromethyl） phenethyl alcohol with recombinant E. coli is 95.16%.
出处
《药学与临床研究》
2010年第2期160-163,共4页
Pharmaceutical and Clinical Research
关键词
羰基还原酶
条件优化
正交试验
直观分析
方差分析
生物转化
Carbonyl Reducase
Optimization
variance
Biosynthesis Orthogonal experiment
Direct analysis
Analysis of
