Molecular Biotechnology

, Volume 61, Issue 3, pp 200–208 | Cite as

Isolation and Expression Analysis of Three Types of α-Carbonic Anhydrases from the Antarctic Alga Chlamydomonas sp. ICE-L under Different Light Stress Treatments

  • Chongli Shi
  • Meiling An
  • ·Jinlai MiaoEmail author
  • Yingying He
  • Zhou Zheng
  • Changfeng Qu
  • Xixi Wang
  • Huan Lin
  • Junhong Liu
original paper


Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that can reversibly catalyse the hydration reaction of carbon dioxide. Antarctic algae are the most critical component of the Antarctic ecosystem; algae can enter the carbon cycle food chain by fixing carbon dioxide from the air. In this study, the complete open reading frames (ORFs) of CA1 (GenBank ID KY826431), CA2 (GenBank ID KY826432), and CA3 (GenBank ID KY826433), encoding CAs in the Antarctic ice microalga Chlamydomonas. sp. ICE-L, were successfully cloned using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression patterns of CAs under blue light, under UV light, and in the dark were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The CA1, CA2, and CA3 ORFs encode proteins of 376, 430, and 419 amino acids, respectively. Phylogenetic analysis revealed that all amino acid sequences showed high homology with those of C. sp. ICE-L. There are six types of algal CAs; we hypothesised that the CAs studied here are most likely α-CAs. Expression analysis showed that the transcription level of the CAs was influenced by both UV light and blue light. These findings provide additional insight into the molecular mechanisms of CAs and will accelerate the development of CAs for applications in agriculture and environmental governance.


Chlamydomonas sp. ICE-L qRT-PCR Bioinformatic analysis Carbonic anhydrase 



This work was supported by the National Key Research and Development Program of China (Grant No. 2018YFD0900705; 2018YFD0901103), the Natural Science Foundation of China (Grant Nos. 41576187, No. 41776203, No. 21576145), Key Research and Development Program of Shandong Province (Grant Nos. 2016YYSP017, No. 2016ZDJS06A03, No. 2017GHY15112, No. 2018YYSP024, Grant No. 2018GHY115034), Public Science and Technology Research Funds Projects of Ocean (Grant No. 201405015), Deep Sea Biological Resources Plan of China (Grant No. DY135-B2-14), Qingdao Entrepreneurship & Innovation Pioneers Program (Grant No. 15-10-3-15-(44)-zch), and Ningbo Public Service Platform for High-Value Utilization of Marine Biological Resources (Grant No. NBHY-2017-P2).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain studies with animals.

Supplementary material

12033_2018_152_MOESM1_ESM.emf (151 kb)
Supplementary material 1 (EMF 150 KB)
12033_2018_152_MOESM2_ESM.emf (168 kb)
Supplementary material 2 (EMF 168 KB)
12033_2018_152_MOESM3_ESM.emf (152 kb)
Supplementary material 3 (EMF 152 KB)


  1. 1.
    Kupriyanova, E., Pronina, N., & Los, D. (2017). Carbonic anhydrase—A universal enzyme of the carbon-based life. Photosynthetica, 55(1), 3–19.Google Scholar
  2. 2.
    NOAA ESRL (2012). National Oceanic and Atmospheric Administration, Earth System Research Laborary. link
  3. 3.
    Firth, P., Fisher, S. G. (1992). Global Climate Change and Freshwater Ecosystems (pp. 234–249). New York: Springer-Verlag.Google Scholar
  4. 4.
    Khalifah, R. G. (1971). The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. Journal of Biological Chemistry, 246(8), 2561–2573.Google Scholar
  5. 5.
    Stewart, C., & Hessami, M. A. (2005). A study of methods of carbon dioxide capture and sequestration––the sustainability of a photosynthetic bioreactor approach. Energy Conversion and Management, 46(3), 403–420.Google Scholar
  6. 6.
    Badger, M. (2003). The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms. Photosynthesis Research, 77(2–3), 83.Google Scholar
  7. 7.
    Zhang, Y. T., Zhang, L., Chen, H. L., & Zhang, H. M. (2010). Selective separation of low concentration CO2 using hydrogel immobilized CA enzyme based hollow fiber membrane reactors. Chemical Engineering Science, 65(10), 3199–3207.Google Scholar
  8. 8.
    van Hille, R., Fagan, M., Bromfield, L., & Pott, R. (2014). A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae. Journal of Applied Phycology, 26(1), 377–385.Google Scholar
  9. 9.
    Moroney, J. V., & Somanchi, A. (1999). How do Algae concentrate CO2 to increase the efficiency of photosynthetic carbon fixation? Plant Physiology, 119(1), 9–16.Google Scholar
  10. 10.
    Tsai, D. D.-W., Chen, P. H., & Ramaraj, R. (2017). The potential of carbon dioxide capture and sequestration with algae. Ecological Engineering, 98, 17–23.Google Scholar
  11. 11.
    Dimario, R. J., Clayton, H., Mukherjee, A., Ludwig, M., & Moroney, J. V. (2017). Plant carbonic anhydrases—structures, locations, evolution and physiological roles. Molecular Plant, 10(1), 30–46.Google Scholar
  12. 12.
    Rudenko, N., Ignatova, L., Fedorchuk, T., & Ivanov, B. (2015). Carbonic anhydrases in photosynthetic cells of higher plants. Biochemistry, 80(6), 674–687.Google Scholar
  13. 13.
    So, A. K.-C., Espie, G. S., Williams, E. B., Shively, J. M., Heinhorst, S., & Cannon, G. C. (2004). A novel evolutionary lineage of carbonic anhydrase (ε class) is a component of the carboxysome shell. Journal of Bacteriology, 186(3), 623–630.Google Scholar
  14. 14.
    Poole, J. H., Tyack, P. L., Stoeger-Horwath, A. S., & Watwood, S. (2005). Animal behaviour: Elephants are capable of vocal learning. Nature, 434(7032), 455.Google Scholar
  15. 15.
    Suzuki, K., Yang, S.-Y., Shimizu, S., Morishita, E. C., Jiang, J., Zhang, F., et al. (2011). The unique structure of carbonic anhydrase αCA1 from Chlamydomonas reinhardtii. Acta Crystallographica Section D, 67(10), 894–901.Google Scholar
  16. 16.
    De Luca, V., Vullo, D., Del Prete, S., Carginale, V., Osman, S. M., AlOthman, Z., Supuran, C. T., et al. (2016). Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the antarctic bacterium Colwellia psychrerythraea. Bioorganic and Medicinal Chemistry, 24(4), 835–840.Google Scholar
  17. 17.
    Sage, R. F., & Coleman, J. R. (2001). Effects of low atmospheric CO2 on plants: More than a thing of the past. Trends in Plant Science, 6(1), 18–24.Google Scholar
  18. 18.
    Capasso, C., & Supuran, C. T. (2015). Bacterial, fungal and protozoan carbonic anhydrases as drug targets. Expert Opinion on Therapeutic Targets, 19(12), 1689–1704.Google Scholar
  19. 19.
    Capasso, C., & Supuran, C. T. (2015). An overview of the alpha-, beta-and gamma-carbonic anhydrases from bacteria: Can bacterial carbonic anhydrases shed new light on evolution of bacteria? Journal of Enzyme Inhibition and Medicinal Chemistry, 30(2), 325–332.Google Scholar
  20. 20.
    Ozensoy Guler, O., Capasso, C., & Supuran, C. T. (2016). A magnificent enzyme superfamily: Carbonic anhydrases, their purification and characterization. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(5), 689–694.Google Scholar
  21. 21.
    Lehtonen, J., Parkkila, S., Vullo, D., Casini, A., Scozzafava, A., & Supuran, C. (2004). Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: A novel target for the drug design. Bioorganic and Medicinal Chemistry Letters, 14(14), 3757–3762.Google Scholar
  22. 22.
    Lindskog, S. (1960). Purification and properties of bovine erythrocyte carbonic anhydrase. Biochimica Et Biophysica Acta, 39(2), 218–226.Google Scholar
  23. 23.
    Lane, T. W., Saito, M. A., George, G. N., Pickering, I. J., Prince, R. C., & Morel, F. M. (2005). Biochemistry: A cadmium enzyme from a marine diatom. Nature, 435(7038), 42.Google Scholar
  24. 24.
    Sinetova, M. A., Kupriyanova, E. V., Markelova, A. G., Allakhverdiev, S. I., & Pronina, N. A. (2012). Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1817(8), 1248–1255.Google Scholar
  25. 25.
    Moroney, J., Bartlett, S., & Samuelsson, G. (2001). Carbonic anhydrases in plants and algae. Plant, Cell & Environment, 24(2), 141–153.Google Scholar
  26. 26.
    Dionisio, M. L., Tsuzuki, M., & Miyachi, S. (1989). Blue light induction of carbonic anhydrase activity in Chlamydomonas reinhardtii. Plant and Cell Physiology, 30(2), 215–219.Google Scholar
  27. 27.
    Gao, S., Zhao, W., Li, X., You, Q., Shen, X., Guo, W., Wang, S., et al. (2017). Identification and characterization of miRNAs in two closely related C 4 and C 3 species of Cleome by high-throughput sequencing. Scientific Reports, 7, 46552.Google Scholar
  28. 28.
    Ynalvez, R., Xiao, Y., Ward, A., Cunnusamy, K., & Moroney, J. (2010). Identification and characterization of two closely related beta-carbonic anhydrases from Chlamydomonas reinhardtii. Physiologia Plantarum, 133(1), 15–26.Google Scholar
  29. 29.
    An, M., Mou, S., Zhang, X., Ye, N., Zhou, Z., Cao, S., et al. (2013). Temperature regulates fatty acid desaturases at a transcriptional level and modulates the fatty acid profile in the Antarctic microalga Chlamydomonas sp. ICE-L. Bioresource Technology, 134(2), 151–157.Google Scholar
  30. 30.
    He, Y., Wang, Y., Zhou, Z., Liu, F., An, M., He, X., et al. (2017) Cloning and stress-induced expression analysis of calmodulin in the antarctic alga Chlamydomonas sp. ICE-L. Current Microbiology, 1–9.Google Scholar
  31. 31.
    Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Higgins, D. G. (1997) The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(25), 4876–4882.Google Scholar
  32. 32.
    Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783–791.Google Scholar
  33. 33.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2– ∆∆CT method. Method, 25(4), 402–408.Google Scholar
  34. 34.
    Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., et al. (2018) SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research.Google Scholar
  35. 35.
    Sumi, K. R., Nou, I.-S., & Kho, K. H. (2016). Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis. Gene, 588(2), 173–179.Google Scholar
  36. 36.
    Coviello, V., Marchi, B., Sartini, S., Quattrini, L., Marini, A. M., Simorini, F., Taliani, S., et al. (2016). 1, 2-Benzisothiazole derivatives bearing 4-, 5-, or 6-alkyl/arylcarboxamide moieties inhibit carbonic anhydrase isoform IX (CAIX) and cell proliferation under hypoxic conditions. Journal of Medicinal Chemistry, 59(13), 6547–6552.Google Scholar
  37. 37.
    Engel, B. D., Schaffer, M., Cuellar, L. K., Villa, E., Plitzko, J. M., & Baumeister, W. (2015). Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife, 4, e04889.Google Scholar
  38. 38.
    Qu, C., He, Y., Zheng, Z., An, M., Li, L., Wang, X., He, X., et al. (2018). Cloning, expression analysis and enzyme activity assays of the α-carbonic anhydrase gene from Chlamydomonas sp. ICE-L. Molecular Biotechnology, 60(1), 21–30.Google Scholar
  39. 39.
    Benlloch, R., Shevela, D., Hainzl, T., Grundström, C., Shutova, T., Messinger, J., Samuelsson, G., et al. (2015) Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii. Plant Physiology, 167(3):950–962.Google Scholar
  40. 40.
    Dedeoglu, N., De Luca, V., Isik, S., Yildirim, H., Kockar, F., Capasso, C., & Supuran, C. T. (2015). Cloning, characterization and anion inhibition study of a β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans. Bioorganic and medicinal chemistry, 23(13), 2995–3001.Google Scholar
  41. 41.
    Supuran, C. T. (2017). Advances in structure-based drug discovery of carbonic anhydrase inhibitors. Expert Opinion on Drug Discovery, 12(1), 61–88.Google Scholar
  42. 42.
    Supuran, C. T. (2008). Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nature Reviews Drug Discovery, 7(2), 168–181.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chongli Shi
    • 1
    • 2
  • Meiling An
    • 2
    • 3
    • 4
  • ·Jinlai Miao
    • 2
    • 3
    • 4
    Email author
  • Yingying He
    • 2
    • 4
  • Zhou Zheng
    • 2
    • 3
    • 4
  • Changfeng Qu
    • 2
  • Xixi Wang
    • 2
  • Huan Lin
    • 1
  • Junhong Liu
    • 1
  1. 1.College of Chemical EngineeringQingdao University of Science and TechnologyQingdaoChina
  2. 2.Key Laboratory of Marine Bioactive Substances, First Institute of OceanographyState Oceanic AdministrationQingdaoChina
  3. 3.Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and TechnologyQingdaoChina
  4. 4.Medical CollegeQingdao UniversityQingdaoChina

Personalised recommendations