Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

doi:10.1016/j.cjche.2022.01.020

中国化学工程学报 ›› 2023, Vol. 53 ›› Issue (1): 56-62.DOI: 10.1016/j.cjche.2022.01.020

• Full Length Article • 上一篇下一篇

Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

Dahai Jiang, Zhidi Min, Jing Leng, Huanqing Niu, Yong Chen, Dong Liu, Chenjie Zhu, Ming Li, Wei Zhuang, Hanjie Ying

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

收稿日期:2021-09-01 修回日期:2022-01-22 出版日期:2023-01-28 发布日期:2023-04-08
通讯作者: Huanqing Niu,E-mail:huanqingniu@njtech.edu.cn
基金资助:
This work was supported by Jiangsu Province Natural Science Foundation for Distinguished Young Scholars (BK20190035), Jiangsu Government Scholarship for Overseas Studies (JS-2019-053), Key Research & Development plan of Jiangsu Province (BE2019001), the National Natural Science Foundation of China (2217080044 and 22008119), the Natural Science Foundation of Jiangsu Province (BK20202002), the National Key Research and Development Program of China (2021YFC2101204), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R28), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.

Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

Dahai Jiang, Zhidi Min, Jing Leng, Huanqing Niu, Yong Chen, Dong Liu, Chenjie Zhu, Ming Li, Wei Zhuang, Hanjie Ying

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

Received:2021-09-01 Revised:2022-01-22 Online:2023-01-28 Published:2023-04-08
Contact: Huanqing Niu,E-mail:huanqingniu@njtech.edu.cn
Supported by:
This work was supported by Jiangsu Province Natural Science Foundation for Distinguished Young Scholars (BK20190035), Jiangsu Government Scholarship for Overseas Studies (JS-2019-053), Key Research & Development plan of Jiangsu Province (BE2019001), the National Natural Science Foundation of China (2217080044 and 22008119), the Natural Science Foundation of Jiangsu Province (BK20202002), the National Key Research and Development Program of China (2021YFC2101204), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R28), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.

摘要/Abstract

摘要： Adenylate cyclase (AC) is the key enzyme that catalyzes the formation of cAMP from ATP. In this study, we discovered two novel class III ACs with a halophilic property from Thermobifida halotolerans DSM 44931 (ThAC) and Haloactinopolyspora alba DSM 45211 (HaAC), respectively. The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively. The presence of 2500 and 2200 mmol·L^-1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC, with 22-fold and 7.4-fold higher activities compared to those without NaCl, respectively. Several divalent metal ions were found to activate the recombinant ACs to different extents, and the optimal metal ion was Mg²⁺ for both ThAC and HaAC with concentrations of 80 mmol·L^-1 and 40 mmol·L^-1 respectively. Purified ThAC and HaAC had the optimal specific activities ((4.59 ±0.35)×10⁴ and (7.76 ±0.52)×10⁴ U·mg^-1) and catalytic efficiency (4.47 and 5.30 L·mmol^-1·s^-1) at 45 ℃ and 40 ℃ respectively, while the optimum pH of both two recombinant ACs was 10.0. This is the first report of the halophilic Class III ACs, which could make new contributions to explore and study ACs for further associated investigations.

关键词: Enzyme, Protein stability, Biotechnology, Halophilic adenylate cyclase, cAMP

Abstract: Adenylate cyclase (AC) is the key enzyme that catalyzes the formation of cAMP from ATP. In this study, we discovered two novel class III ACs with a halophilic property from Thermobifida halotolerans DSM 44931 (ThAC) and Haloactinopolyspora alba DSM 45211 (HaAC), respectively. The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively. The presence of 2500 and 2200 mmol·L^-1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC, with 22-fold and 7.4-fold higher activities compared to those without NaCl, respectively. Several divalent metal ions were found to activate the recombinant ACs to different extents, and the optimal metal ion was Mg²⁺ for both ThAC and HaAC with concentrations of 80 mmol·L^-1 and 40 mmol·L^-1 respectively. Purified ThAC and HaAC had the optimal specific activities ((4.59 ±0.35)×10⁴ and (7.76 ±0.52)×10⁴ U·mg^-1) and catalytic efficiency (4.47 and 5.30 L·mmol^-1·s^-1) at 45 ℃ and 40 ℃ respectively, while the optimum pH of both two recombinant ACs was 10.0. This is the first report of the halophilic Class III ACs, which could make new contributions to explore and study ACs for further associated investigations.

Key words: Enzyme, Protein stability, Biotechnology, Halophilic adenylate cyclase, cAMP

Dahai Jiang, Zhidi Min, Jing Leng, Huanqing Niu, Yong Chen, Dong Liu, Chenjie Zhu, Ming Li, Wei Zhuang, Hanjie Ying. Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba[J]. 中国化学工程学报, 2023, 53(1): 56-62.

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Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

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