生物转化2-苯乙醇辅因子NADH/NAD+的检测研究

摘要：探究酵母摇瓶发酵过程中辅因子NADH/NAD+的主要影响因子。通过单因素实验探讨各因素对辅酶提取得率的影响。在此基础上，采Plackett-Burman 设计对影响酵母细胞内辅因子NADH/NAD+提取得率的因素进行筛选，PB 试验设计与统计学分析表明,超声时间、提取液的加入量和震荡次数是影响酵母细胞内辅因子NADH/NAD+提取得率的4个关键因素。通过拟合得到响应曲面函数，获得了最佳的提取条件为：超声功率：300瓦、超声时间：20min、提取液的加入量：3ml、震荡次数：10次，在此条件下酵母细胞内辅因子NADH/NAD+的提取得率最高，这表明从实验结果可以看出单因素法、Plackett-Burman试验设计结合响应面分析法，可以很好地优化酵母细胞内辅因子NADH/NAD+的提取工艺。4924
关键词：PB设计；响应面优化；NADH/NAD+；超声波；高效液相色谱
Detection of cofactor NADH / NAD + during the process of biotransformation of producing 2 – Phenylethanol
   Abstract ：The research aimed to explore main influencing factors of extraction NADH / NAD + by yeast in shaking flask fermentation. Single factor test of all kinds of factors for the extraction rate of NADH / NAD + produced by yeast. On this basis, Plackett- Burman (PB) design were applied to screen and optimize influential factors for extraction process of total flavonoids . The ultrasonic time, the amount of added extracts, number of shocks, as three key factors, were found to significantly influence the yields of extraction NADH / NAD + by yeast via PB design and the following statistic analysis, With the hybrid design, response surfaces were optimized and response surfaces function was obtained. Three statistically significant parameters were ultrasonic power：300W、ultrasonic time ：20 min、the amount of added extracts：3mL、numbers of shocks：10 times. Under the optimal levels of the extraction conditions the content of NADH / NAD + was the highest. It can be seen from the experimental results show that Single factor test、Plackett-Burman (PB) design with the response surface methodology can optimize the extraction process of cofactor NADH / NAD + in yeast cells .

key words：Plackett–Burman design; response surface methodology; Ultrasonic NADH / NAD + ; HPLC

目录

1前言   1
1.1国内外研究进展   1
1.2酵母细胞内辅因子NAD+、NADH代谢机制   2
1.3辅因子对发酵过程的影响   3
2 材料与方法   4
2.1材料与仪器设备   4
2.1.1仪器设备   4
2.1.2培养基及实验试剂   4
2.2方法   5
2.2.1实验方法   5
2.2.2分析方法   6
3实验条件优化   6
3.1单因素实验   6
3.1.1.细胞破碎法   6
3.1.2提取液配比   8
3.1.3 超声功率   8
3.2 Plackett-Burman实验设计筛选关键因素   9
3.3相应面实验设计   9
3.4 NAD+/NADH检测条件优化   10
3.4.1 高效液相色谱法   10
3.4.2 酶循环法   12
3.4.3 试剂盒法   13
4结果与分析   14
4.1单因素结果分析   14
4.1.1.细胞破碎法   14
4.1.2 提取液配比   15
4.2 PB设计结果分析   17
4.3响应面结果分析   19
５.实验结果分析   22
文献引用   23 生物转化2-苯乙醇辅因子NADH/NAD+的检测研究:http://www.751com.cn/shengwu/lunwen_1743.html