4'-去甲基表鬼臼毒素生物转化为4'-去甲基表鬼臼酸
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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。
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.
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。
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.
引文
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2.Giri A,Narasu ML.Production of podophyllotoxin from Podophyllum hexandrum:a potential natural product for clinically useful anticancer drugs.Cytotechnology,2000,34:17-26.
3.Imbert TF.Discovery of podophyHotoxins.Biochimie,1998,80:207-222.
4.Gordaliza M,Garcia PA,Corral JMM,Castro MA,Gomez-Zurita MA.Podophyllotoxin:distribution,sources,applications and new cytotoxic derivatives.Toxin,2004,44:441-459.
5.Pez-Perez JL,Olmo E,PascuaI-Teresa B,Abad A,Feliciano AS.Synthesis and cytotoxicity of hydrophobic esters of podophyllotoxins.Bioor Med Chem Lett,2004,14:1283-1286.
6.Castro MA,Corral JM,Gordaliza M,Go'mez-Zurita MA,Puente ML,Betancur-Galvis LA,Sierra J,Feliciano AS.Synthesis,cytotoxicity and antiviral activity of podophyliotoxin analogues modified in the E-ring.Eur J Med Chem,2003,38:899-911.
7.许重远,贾江滨,陈振德.4′-去甲表鬼臼毒素衍生物4β-取代结构修饰研究进展.中草药,2000,31(5):389-392.
8.汪世龙,姚思德,王玫,李义久,张志成,张曼维.鬼臼毒素及其衍生物的化学活性研究.辐射研究与辐射工艺学报,1997,15(2):86-91.
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