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。
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.
作者
李志刚
顾阳
谭海
张中华
常景玲
LI Zhi-gang;GU Yang;TAN Hai;ZHANG Zhong-hua;CHANG Jing-ling(Collaborative Innovation Center of Modem Biological Breeding of Henan Province,Xinxiang 453003,China;School of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2021年第7期50-57,共8页
China Biotechnology
基金
河南省科技攻关项目(192102210193)
河南科技学院高层次人才科研启动项目(2015006)对本研究的资助
关键词
CAMP
磷酸二酯酶
氨茶碱
腺苷
柠檬酸钠
Cyclic adenosine monophosphate
Phosphodiesterase
Aminophylline
Adenosine
Citrate
