Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

doi:10.1016/j.cjche.2020.01.003

Chinese Journal of Chemical Engineering ›› 2020, Vol. 28 ›› Issue (8): 2167-2172.DOI: 10.1016/j.cjche.2020.01.003

• Biotechnology and Bioengineering • Previous Articles Next Articles

Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

Chen Ma, Jing Wang, Xuelin Wang, Dandan Mai, Yuqi Jin, Kequan Chen, Xin Wang, Pingkai Ouyang

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China

Received:2019-10-10 Revised:2019-11-26 Online:2020-09-19 Published:2020-08-28
Contact: Xin Wang
Supported by:
The National Natural Science Foundation of China (Grant No. 21576134,Grant No. 21606127, Grant No. 21390200, Grant No. 21706126), the National Key Research and Development Program of China (Grant No. 2016YFA0204300) and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

Chen Ma, Jing Wang, Xuelin Wang, Dandan Mai, Yuqi Jin, Kequan Chen, Xin Wang, Pingkai Ouyang

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China

通讯作者: Xin Wang
基金资助:
The National Natural Science Foundation of China (Grant No. 21576134,Grant No. 21606127, Grant No. 21390200, Grant No. 21706126), the National Key Research and Development Program of China (Grant No. 2016YFA0204300) and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

Abstract

Abstract: In this study, we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). First, adenylate cyclase from Escherichia coli MG1655 (EAC) and Bordetella Pertussis (BAC) were expressed in E. coli BL21 (DE3) and comparatively analyzed for their activities. As a result, EAC from E. coli MG1655 exhibited a higher activity. However, amount of EAC were obtained in an insoluble form. Therefore, we expressed the first 446 amino acids of EAC (EAC446) to avoid the inclusion body. The effects of induction temperature, incubation time, and incubation pH were further evaluated to improve the expression of EAC446. Subsequently, the reaction process for the production of cAMP with ATP as a starting material was investigated. As none of cAMP was detected in the whole-cell based biocatalytic process, the reaction catalyzed by the crude enzyme was determined for cAMP production. What's more, the reaction temperature, reaction pH, metal ion additives and substrate concentration was optimized, and the maximum cAMP production of 18.45 g·L^-1 was achieved with a yield of 95.4% after bioconversion of 6 h.

Key words: Adenylate cyclase, Cyclic adenosine monophosphate (cAMP), Adenosine triphosphate (ATP), Bioconversion

摘要： In this study, we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). First, adenylate cyclase from Escherichia coli MG1655 (EAC) and Bordetella Pertussis (BAC) were expressed in E. coli BL21 (DE3) and comparatively analyzed for their activities. As a result, EAC from E. coli MG1655 exhibited a higher activity. However, amount of EAC were obtained in an insoluble form. Therefore, we expressed the first 446 amino acids of EAC (EAC446) to avoid the inclusion body. The effects of induction temperature, incubation time, and incubation pH were further evaluated to improve the expression of EAC446. Subsequently, the reaction process for the production of cAMP with ATP as a starting material was investigated. As none of cAMP was detected in the whole-cell based biocatalytic process, the reaction catalyzed by the crude enzyme was determined for cAMP production. What's more, the reaction temperature, reaction pH, metal ion additives and substrate concentration was optimized, and the maximum cAMP production of 18.45 g·L^-1 was achieved with a yield of 95.4% after bioconversion of 6 h.

关键词: Adenylate cyclase, Cyclic adenosine monophosphate (cAMP), Adenosine triphosphate (ATP), Bioconversion

Chen Ma, Jing Wang, Xuelin Wang, Dandan Mai, Yuqi Jin, Kequan Chen, Xin Wang, Pingkai Ouyang. Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli[J]. Chinese Journal of Chemical Engineering, 2020, 28(8): 2167-2172.

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Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

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