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Alternative TitleMicrobial fermentation production of shikimic acid using Bacillus subtilis
Thesis Advisor王云山 ; 苏志国
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword枯草芽孢杆菌   莽草酸   培养基优化
Abstract莽草酸是一种重要的化工原料和代谢中间体,可用于抗病毒药物磷酸奥司米韦(商品名达菲)及抗肿瘤药物二恶霉素、乙二醛酶抑制剂和生物体内芳香族氨基酸的合成。莽草酸生产方法有植物提取法、化学合成法和微生物发酵法三种,微生物发酵法与植物提取和化学合成法相比发展前景广阔,但目前生产工艺还不够成熟。本论文针对枯草芽孢杆菌莽草酸发酵工艺条件优化及发酵过程的实时监控开展了初步研究,主要研究结果如下:1. 建立了HPLC法快速测定发酵液中蔗糖、莽草酸和芳香族氨基酸含量的方法,为微生物发酵生产莽草酸的工艺控制提供了便捷。色谱条件为:Hypersil APS-2色谱柱 (5 μm,250 mm×4.6 mm),检测波长215 nm,示差检测器和紫外检测器联用。 流动相A和B分别为磷酸水溶液(pH 2.5)和乙腈,VA:VB=20:80,柱温30 ℃,流速1.0 mL/min,进样量10 μL。2. 利用单因素实验确定了发酵培养基中添加4 g/L的酵母粉可以满足枯草芽孢杆菌生长对芳香族氨基酸的需要;最佳碳源和氮源是蔗糖和尿素,其最适浓度分别是80 g/L和6 g/L,莽草酸产量可达3.0 g/L左右。 3. 通过三因素两水平正交实验考察了蔗糖、尿素和酵母粉浓度对发酵过程的影响,F检验表明,蔗糖浓度对发酵结果有显著效应,尿素含量、蔗糖和尿素的交互作用及酵母粉浓度的影响均不显著。4. 考察了发酵过程中莽草酸的反馈抑制作用,发现莽草酸浓度达到20 g/L时对整个发酵过程才会产生抑制作用。5. 利用5 L发酵罐进行了分批发酵和分批补料发酵实验,发酵过程中控制温度在37 ℃,氨水和磷酸调节pH 6.8~7.2之间,莽草酸产量为6.53 g/L和12.12 g/L,分别是摇瓶发酵的2.17倍和4.04倍。
Other AbstractShikimic acid was an important chemical material and metabolic intermediate which could be used for synthesis of antiviral drug oseltamivir phosphate (commodity called tamiflu), antitumor drug dioxolamycin and glyoxalase inhibitor and aromatic amino acids. Shikimic acid could be produced by extraction, chemical synthesis and microbial fermentation. Microbial fermentation had broad prospect in industrial production, but its productive technology was still immature. In this thesis, fermentation condition of shikimic acid was optimized and the real-time monitoring method of fermentation process was developed. The main results were as follows:1. To control the production process of shikimic Acid, the determination method of sucrose, shikimic acid and aromatic amino acids in fermentation liquor with HPLC was established. The analysis of above parameters was performed using a Hypersil APS-2 column (5 μm, 250 mm×4.6 mm), and an ultraviolet detector operated at 215 nm coupling a refractive index detector. The mobile phase consisted of phosphoric acid solution (pH 2.5) and acetonitrile, at a 20:80 ratio. The elution was carried out at 30 ℃ with a flow rate of 1.0 mL/min. The injection volume was 10 μL. 2. The single factor experiment showed that 4 g/L yeast extract powder added to fermentation liquor could meet the needs for aromatic amino acids of Bacillus subtilis, the best carbon source and nitrogen source were sucrose and urea, their optimum concentration respectively was 80 g/L and 6 g/L, Shikimic acid yield was up to about 3.0 g/L. 3. Through three factors and two levels orthogonal experiment, the effect on fermentation of sucrose, urea and yeast extract powder were investigated. F-test showed that sucrose concentration had significant effect on fermentation results, the influence of urea concentration, the interaction of sucrose and urea and yeast extract powder content were not significant.4. The feedback inhibition of shikimic acid in the process of fermentation was studied, it was found that shikimic acid did not have feedback inhibition on the whole fermentation process until its concentration reached 20 g/L. 5. The batch fermentation and fed-batch fermentation were carried out in a 5 L fermentation tank. Fermentation temperature was controled of 37 ℃, pH was adjusted from 6.8 to 7.2 used ammonia and phosphoric acid. The shikimic acid production were 6.53 g/L and 12.12 g/L, respectively was 2.17 times and 4.04 times than that of shake flask 
Document Type学位论文
Recommended Citation
GB/T 7714
徐晓娟. 枯草芽孢杆菌发酵生产莽草酸的研究[D]. 中国科学院研究生院,2013.
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