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Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L

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Abstract

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

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Acknowledgements

This work was funded by Natural Science Foundation of China (No. 41576187), Basic Scientific Fund for National Public Research Institutes of China (No. 2016Q10), Postdoctoral Applied Research Projects of Qingdao (No. QDBSH2015015), Key Research and Development Program of Shandong Province (Nos. 2016YYSP017, 2016ZDJS06A03, 2017GHY15112), Natural Science Foundation of China-Shandong Joint Fund (No. U1606403), Public Science and Technology Research Funds Projects of Ocean (No. 201405015), Qingdao Entrepreneurship and Innovation Pioneers Program (No. 15-10-3-15-(44)-zch), Shandong Provincial Natural Science Foundation (No. ZR2017QD008), and Polar Strategic Foundation of China (No. 20150303).

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  1. Changfeng Qu, Yingying He and Zhou Zheng contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Marine Bioactive Substances, First Institute of Oceanography, State Oceanic Administration, Qingdao, 266061, China

    Changfeng Qu, Yingying He, Zhou Zheng, Meiling An, Lulu Li, Xixi Wang, Xiaodong He, Yibin Wang, Fangming Liu & Jinlai Miao

  2. Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China

    Changfeng Qu, Zhou Zheng & Jinlai Miao

  3. Medical College, Qingdao University, Qingdao, 266071, China

    Meiling An & Jinlai Miao

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  1. Changfeng Qu
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Qu, C., He, Y., Zheng, Z. et al. Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L. Mol Biotechnol 60, 21–30 (2018). https://doi.org/10.1007/s12033-017-0040-9

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