【作者】 徐小玲；

【导师】 汤亚杰；

【作者基本信息】 湖北工业大学 ， 生物化工， 2008， 硕士

【摘要】 本文首次建立了4’-去甲基表鬼臼毒素转化为4’-去甲基表鬼臼酸的生物转化过程,并对该生物转化过程进行了动力学研究和过程优化,提高了生物转化过程的效率。首先,从8株菌株中筛选得到对4’-去甲基表鬼臼毒素具有转化能力的4株菌株,即Bacillus fusiformis CICC 20463、Bacillus subtilis CCTCC AB93174、Pseudomonas aeruginosa CCTCC AB 93 066和Pseudomonas oleovorans CGMCC1.1641,HPLC图谱说明在生物转化过程中有转化产物的产生。其中B.fusiformis对4’-去甲基表鬼臼毒素的转化能力较强,转化率达到63.3%,因此选用B.fusiformis进行后续研究。采用大孔吸附树脂D312从生物转化基质中分离得到产物晶体,将生物转化产物与4’-去甲基表鬼臼毒素的核磁共振和质谱图对比分析,确定转化产物为4’-去甲基表鬼臼酸,从而建立了4’-去甲基表鬼臼毒素转化为4’-去甲基表鬼臼酸的生物转化过程。然后,对上述建立的生物转化过程进行了动力学研究及过程优化。氮源全因子实验结果表明,在酵母膏、蛋白胨分别为5.0和10.0 g/L时,4’-去甲基表鬼臼酸浓度达到最高值(2.81±0.21 mg/L)。在蔗糖浓度考察范围(如0、10、20、40 g/L)内,4’-去甲基表鬼臼酸的浓度随着蔗糖浓度的上升而增加,在40 g/L时达到最高值(2.94±0.17 mg/L)。3 g/L的氯化钠不利于细胞的生长,但有利于4’-去甲基表鬼臼酸的产生(4.10±0.18 mg/L)。100 mg/L的4’-去甲基表鬼臼毒素起始浓度有利于4’-去甲基表鬼臼酸的产生(6.47+0.35 mg/L)。在pH值考察范围(8.0、8.5、9.0)内,pH值的上升促进4’-去甲基表鬼臼毒素的转化和4’-去甲基表鬼臼酸的形成,在pH值为9.0时,4’-去甲基表鬼臼酸浓度达到最高值(38.78 mg/L),较过程优化初始值2.01mg/L提高了18.3倍。本文首次采用生物转化技术对4’-去甲基表鬼臼毒素进行分子结构修饰研究,得到水溶性提高的生物转化产物4’-去甲基表鬼臼酸,建立了4’-去甲基表鬼臼毒素生物转化过程。通过过程优化,显著提高了4’-去甲基表鬼臼酸浓度。该研究为4’-去甲基表鬼臼毒素结构修饰研究提供了新途径,促进了生物转化技术在化学成分结构修饰研究的应用,为其他化合物结构修饰研究提供借鉴意义。更多还原

【Abstract】 For the first time,the biotransformation process of 4’-demethylepipodophyllotoxin to demethylepipodophyllic acid was developed in this work.The performance of the biotransformation process was improved by the study on its kinetics and optimization.Firstly,based on the capability of modifying 4’-demethylepipodophyllotoxin structure,4 trains(i.e,Bacillus fusiformis CICC 20463,Bacillus subtilis CCTCC AB93174,Pseudomonas aeruginosa CCTCC AB 9306 and Pseudomonas oleovorans CGMCC 1.1641) were screened out from 8 tested strains.Among them,B.fusiformis was selected for the following study because of its high substrate conversion. Biotransformation product was separated from the biotransformation borth by macroporous resin D312,and identified as 4’-demethylepipodophyllic acid by the comparison analysis of EI-MS and NMR spectrums with 4’-demethylepipodophyllotoxin.So,the biotransformation process from 4’-demethylepipodophyllotoxin to 4’-demethylepipodophyllic acid was set up.Secondly,dynamics and optimization of biotransformation process were investigated.The results show that maximum 4’-demethylepipodophyllic acid concentration(2.81±0.21 mg/L) was obtained at 5.0 g/L yeast extract and 10.0 g/L peptone;4’-demethylepipodophyllic acid concentration was enhanced as sucrose increasing in this study,and 2.94±0.17 mg/L 4’-demethylepipodophyllic acid was obtained at 40.0 g/L sucrose;3 g/L sodium chloride is harmful for cell growth but help to the production of 4’-demethylepipodophyllic acid(4.10±0.18 mg/L);100 mg/L 4’-demethylepipodophyllotoxin is suitable for the production of 4’-demethylepipodophyllic acid(6.47±0.35 mg/L).It is notable that 4’-demethylepipodophyllic acid concentration was influenced by pH obviously, maximum 4’-demethylepipodophyllic acid concentration was increased to 38.78 mg/L at pH 9.0.The biotransformation technology was firstly applied in the structure modification of 4’-demethylepipodophyllotoxin,and more solulable biotransformation product (4’-demethylepipodophyllic acid) was produced.The biotransformation process was developed and optimized,and increased 4’-demethylepipodophyllic acid concentration obviously.This work gave a new way to the structure modification of 4’-demethylepipodophyllotoxin,promoted the application of biotransformation technology in the modification of chemicals and provided references to modification of other chemicals.更多还原