重组大肠杆菌产NAD激酶发酵条件的优化研究  

Optimization of fermentation conditions for recombinant E. coli producing NAD Kinase

作　　者：侯堃[1] 李红梅[1] 

机构地区：[1]上海理工大学医疗器械与食品学院,上海杨浦200093

出　　处：《工业微生物》2015年第3期41-47,共7页Industrial Microbiology

基　　金：上海市研究生创新基金(编号:54-13-115-103)

摘　　要：本研究将重组大肠杆菌E.coli BL21(DE3)/p ET30α(+)-NADK作为NAD激酶生产菌种,对其产酶发酵培养基及发酵条件进行优化。采用Placket-Burman(PB)设计先筛选出影响重组菌产NAD激酶的三个主要因素:葡萄糖浓度、Mg SO4浓度和诱导表达时间,试验结果表明,增加葡萄糖和Mg SO4的浓度及缩短诱导表达时间对产酶有利。根据中心组合实验设计(Central Composite Design,CCD)原理,利用PB设计确定的这三个显著影响因素,通过最陡爬坡实验逼近最大响应区域,挑选出实验范围内的最优点,以此作为响应面中心组合设计的中心点,用NAD激酶酶活作为响应值,使用Design Expert 8.0软件设计中心组合实验,通过对实验数据进行分析,得出最佳发酵培养基成分及发酵条件为:葡萄糖14.24 g/L、酵母粉8 g/L、胰蛋白胨8 g/L、Mg SO40.94 g/L、Na Cl 5g/L、NH4Cl 2 g/L、KH2PO42 g/L、K2HPO49 g/L,诱导表达时间8.34 h,接种量2%。在此最佳条件下,NAD激酶酶活实验验证值可达10.17 U/mg,与优化前相比提高了2.77倍。对诱导表达结束后的细胞上清液进行SDS-PAGE分析也证明优化取得了显著的效果。In this study,E. coli BL21（ DE3） / p ET30α（ ＋）-NADK was used for NAD kinase production. Fermentation medium and culture conditions for enzyme production were optimized. Three factors（ induction time,the concentration of glucose and MgSO4） significantly affecting the enzyme production were firstly sifted out by using Placket-Burman experiment. Based on the three identified factors,the most advantage value within the test scope was then selected through the steepest uphill experiment which approached the maximum response region. Finally,optimization experiment was designed through Design Expert 8. 0 software by using this selected value as center of Central Composite Design（ CCD） and using the activity of NAD kinase as response value. The optimal fermentation medium and culture conditions obtained from experiment data analysis were as follows： glucose 14. 24 g / L,yeast powder 8 g / L,tryptone 8 g / L,MgSO40. 94 g / L,NaCl5 g / L,NH4 Cl 2 g / L,KH2PO42 g / L,K2HPO49 g / L,induction time 8. 34 h and inoculum size 2%. Under the optimum conditions,the capacity of recombinant E. coli cells producing NAD kinase reached 10. 17 U / mg,which was 2. 77 times compared with that without optimization. SDS-PAGE analysis of cell supernatant after expression also proved the significant effects of optimization.

关 键 词：重组大肠杆菌 NAD激酶 PB设计 响应面设计 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]