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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (7): 50-57    DOI: 10.13523/j.cb.2103021
技术与方法     
氨茶碱与柠檬酸盐协同作用促进环磷酸腺苷发酵生产
李志刚1,2,顾阳2,谭海2,张中华2,常景玲1,2,*()
1 现代生物育种河南省协同创新中心 新乡 453003
2 河南科技学院生命科技学院 新乡 453003
Enhanced Cyclic Adenosine Monophosphate Fermentation Production by Aminophylline and Citrate Coupling Addition
LI Zhi-gang1,2,GU Yang2,TAN Hai2,ZHANG Zhong-hua2,CHANG Jing-ling1,2,*()
1 Collaborative Innovation Center of Modem Biological Breeding of Henan Province, Xinxiang 453003, China
2 School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China
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摘要：

目的: 探究通过抑制磷酸二酯酶活性促进cAMP发酵合成的工艺方法。方法: 在7 L发酵罐上进行添加氨茶碱的发酵实验,通过对发酵主要参数、关键酶活性、能量代谢水平等进行分析,针对性提出了氨茶碱与柠檬酸盐协同作用促进cAMP合成的发酵工艺。结果: 与对照相比,添加5 mg/L氨茶碱批次的cAMP产量提高25.9%,副产物腺苷浓度减少41.6%,两批次中腺苷酸环化酶和琥珀腺苷酸脱氢酶活性无显著改变,而磷酸二酯酶和5'-核苷酸酶活性明显下降。能量代谢分析结果表明,两批次的胞内ATP/AMP相比于对照批次明显降低,而AMP水平却显著上升,ATP合成水平成为限制产物积累的主要因素。氨茶碱与柠檬酸盐协同添加的cAMP发酵工艺中,cAMP产量达到4.48 g/L,比单独添加柠檬酸钠和氨茶碱分别提高22.1%和13.8%,副产物腺苷浓度仅为0.98 g/L,分别降低51.7%和25.3%。结论: 氨茶碱抑制了磷酸二酯酶和5'-核苷酸酶活性,减少cAMP分解和副产物合成,显著提高cAMP产量,然而ATP合成水平成为产物积累的限制因素。氨茶碱与柠檬酸盐协同添加工艺将抑制磷酸二酯酶活性和提高能量代谢水平相结合,进一步促进了产物发酵合成。
关键词： cAMP;  磷酸二酯酶;  氨茶碱;  腺苷;  柠檬酸钠    
Abstract:

Objective: The aim of this study is to develop a novel cAMP fermentation process via inhibiting phosphodiesterase activity.Methods: cAMP fermentations with aminophylline addition were conducted in a 7 L fermenter and corresponding fermentation kinetics, key enzymes activities and energy metabolism levels were analyzed. Finally, a fermentation process with aminophylline and citrate coupling added was proposed and conducted for improving cAMP content.Results: Due to 5 mg/L aminophylline added, cAMP concentration was increased by 25.9% with a decrease of 41.6% for coproduct adenosine when compared with those of a control group. There were no obvious deviation for adenylate cyclase and succinyladenylate dehydrogenase activities in the two fermentation batches whereas the activities of phosphodiesterase and 5'-nucleotide enzyme were obvious lower than those of the control group. The energy metabolism analysis results showed that intracellular ATP/AMP were lower than control with an improved AMP level, indicating that ATP synthesis had been the main limited factor for product accumulation. Using the fermentation process with aminophylline and citrate coupling addition, cAMP content achieved 4.48 g/L with 22.1% and 13.8% increasement than those of aminophylline and citrate individual added, and adenosine content was 0.98 g/L with 51.7% and 25.3% lower than individual added.Conclusion: With aminophylline added, the activities of phosphodiesterase and 5'-nucleotide enzyme were inhibited and cAMP content was also improved obviously due to decreased cAMP decomposition and adenosine synthesis. However, the ATP synthesis level had been the main limiting factor for improving cAMP production. The fermentation process with aminophylline and citrate coupling addition could decrease cAMP decomposition and improve energy metabolism simultaneously, which further promoted cAMP fermentation synthesis.
Key words: Cyclic adenosine monophosphate    Phosphodiesterase    Aminophylline    Adenosine    Citrate
收稿日期: 2021-03-11 出版日期: 2021-08-03
ZTFLH:  Q819  
通讯作者: 常景玲     E-mail: changjl001@126.com
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引用本文:

李志刚,顾阳,谭海,张中华,常景玲. 氨茶碱与柠檬酸盐协同作用促进环磷酸腺苷发酵生产[J]. 中国生物工程杂志, 2021, 41(7): 50-57.

LI Zhi-gang,GU Yang,TAN Hai,ZHANG Zhong-hua,CHANG Jing-ling. Enhanced Cyclic Adenosine Monophosphate Fermentation Production by Aminophylline and Citrate Coupling Addition. China Biotechnology, 2021, 41(7): 50-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103021    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I7/50

图1  氨茶碱添加量对cAMP生物合成的影响
图2  添加氨茶碱对7 L发酵罐中cAMP发酵性能的影响
图3  氨茶碱对产物合成代谢中关键酶活性的影响
图4  添加氨茶碱对ATP合成及呼吸链脱氢酶活性的影响
图5  氨茶碱与柠檬酸钠协同作用促进cAMP发酵合成
图6  氨茶碱与柠檬酸钠偶合添加对细胞生理状态的影响
图7  氨茶碱与柠檬酸盐协同作用条件下cAMP合成代谢
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