Expression of an Environmentally Friendly Enzyme, Engineered Carbonic Anhydrase, in Escherichia coli

  • Mohaddeseh Mohsenpour
  • Zahra Noormohammadi
  • Shiva Irani
  • Nour AmirmozafariEmail author
Research paper


Carbon dioxide (CO2) is one of the main causes for global warming. The most important enzyme that converts CO2 into bicarbonate and prevents CO2 emissions in the environment is carbonic anhydrase (CA). The aim of this study was to clone the heat-stable CA gene in Escherichia coli host. The CA gene coding frame of Caminibacter mediatlanticus was optimized based on the more frequently used codons by E. coli. Accordingly, nine codons were inserted in this gene fragment. After CA gene insertion in cloning vector (pGH), it was sub-cloned in pBI121 expression vector and transformed into E. coli XL1-Blue. The accuracy of gene cloning was confirmed by PCR, restriction enzyme digestion and sequencing. Cell extract was prepared from recombinant bacteria. Culture supernatants were assayed for CA activity by the color change of bromothymol blue as indicator. The time for the indicator color changes were recorded and the assays were repeated five times. The average of color change time for cell extract of recombinant, non-recombinant E. coli and control reaction were 1.5, 16, and 48 min, respectively. The amount of active enzyme was calculated to be 92.8 units/ml and the enzyme retained its activity after of incubation at 60 °C, 65 °C, and 70 °C. Due to the positive properties of CA from C. mediatlanticus, codon optimization, modification, and expression of this gene, using a simple and inexpensive method, may be used to obtain CA with enhanced properties.

Graphical Abstract


CA gene Carbonic anhydrase Codon optimization Protein engineering 



We thank the Islamic Azad University personnel for their assistance and cooperation in the use of equipment.

Compliance with Ethical Standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.


  1. Achal V, Mukherjee A (2015) Review of microbial precipitation for sustainable construction. Constr Build Mater 93:1224–1235. CrossRefGoogle Scholar
  2. Alvizo O, Nguyen LJ, Savile CK, Bresson JA, Lakhapatri SL, Solis EO, Fox RJ, Broering JM, Benoit MR, Zimmerman SA, Novick SJ, Liang J, Lalonde JJ (2014) Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas. Proc Natl Acad Sci USA 111:16436–16441. CrossRefGoogle Scholar
  3. Bian Y, Rong Z, Chang TMS (2011) Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase: a novel biotechnology-based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant. Artif Cells Blood Substit Immobil Biotechnol 39:127–136. CrossRefGoogle Scholar
  4. Borchert M, Saunders P (2013) Heat-stable carbonic anhydrases and their use. Patent US 8697428 B2Google Scholar
  5. Bose H, Satyanarayana T (2017) Microbial carbonic anhydrases in biomimetic carbon sequestration for mitigating global warming: prospects and perspectives. Front Microbiol 8:1615. CrossRefGoogle Scholar
  6. Faridi S, Satyanarayana T (2016) Novel alkalistable α-carbonic anhydrase from the polyextremophilic bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration. Environ Sci Pollut Res 23:15236–15249. CrossRefGoogle Scholar
  7. Jo BH, Park TY, Park HJ, Yeon YJ, Yoo YJ, Cha HJ (2016) Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration. Sci Rep 6:29322. CrossRefGoogle Scholar
  8. Kaar JL, Oh HI, Russell AJ, Federspiel WJ (2007) Towards improved artificial lungs through biocatalysis. Biomaterials 28:3131–3139. CrossRefGoogle Scholar
  9. Kanth BK, Min K, Kumari S, Jeon H, Jin ES, Lee J (2012) Expression and characterization of codon-optimized carbonic anhydrase from Dunaliella species for CO2 sequestration application. Appl Biochem Biotechnol 167:2341–2356. CrossRefGoogle Scholar
  10. Kanth BK, Jun SY, Kumari S (2014) Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: its expression and characterization for CO2 sequestration Applications. Proc Biochem 49:2114–2121. CrossRefGoogle Scholar
  11. Ki MR, Min K, Kanth BK, Lee J, Pack SP (2013) Expression, reconstruction and characterization of codon-optimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application. Bioprocess Biosyst Eng 36:375–381. CrossRefGoogle Scholar
  12. Kumar V, Satyanarayana T (2014) Production of thermo-alkali-stable xylanase by a novel polyextremophilic Bacillus halodurans TSEV1 in cane molasses mediumand its applicability in making whole wheat bread. Bioprocess Biosyst Eng 37:1043–1053. CrossRefGoogle Scholar
  13. Lee JH, Kwak NS, Lee IY, Jang KR, Lee DW, Jang SG, Kim BK, Shim JG (2015) Performance and economic analysis of commercial-scale coal-fired power plant with post-combustion CO2 capture. Korean J Chem Eng 32:800–807. CrossRefGoogle Scholar
  14. McCranor BJ, Bozym RA, Vitolo MI, Fierke CA, Bambrick L, Polster BM (2012) Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J Bioenergy Biomembr 44:253–263. CrossRefGoogle Scholar
  15. Mesbah NM, Wiegel J (2012) Life under multiple extreme conditions: diversity and physiology of the halophilic alkalithermophiles. Appl Environ Microbiol 78:4074–4082. CrossRefGoogle Scholar
  16. Mitchell AC, Dideriksen K, Spangler LH, Cunningham AB, Gerlach R (2010) Microbially enhanced carbon capture and storage by mineral-trapping and solubility-trapping. Environ Sci Technol 44:5270–5276. CrossRefGoogle Scholar
  17. Mohsenpour M, Tohidfar M, Jelodar NB, Jouzani GS (2015) Designing a new marker-free and tissue-specific platform for molecular farming applications. J Plant Biochem Biotechnol 24:433–440. CrossRefGoogle Scholar
  18. Muyssen BT, De Schamphelaere KA, Janssen CR (2006) Mechanisms of chronic waterborne Zn toxicity in Daphnia magna. Aquat Toxicol 77:393–401. CrossRefGoogle Scholar
  19. Prasad S, Khadatare PB, Roy I (2011) Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli. Appl Environ Microbiol 77:4603–4609. CrossRefGoogle Scholar
  20. Sambrook J, Rusell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  21. Satav SS, Bhat S, Thayumanavan S (2010) Feedback regulated drug delivery vehicles: carbon dioxide responsive cationic hydrogels for antidote release. Biomacromol 11:1735–1740. CrossRefGoogle Scholar
  22. Savile CK, Lalonde JJ (2011) Biotechnology for the acceleration of carbon dioxide capture and sequestration. Curr Opin Biotechnol 22:818–823. CrossRefGoogle Scholar
  23. Voordeckers JW, Starovoytov V, Vetriani C (2005) Caminibacter mediatlanticus sp. nov., a thermophilic, chemolithoautotrophic, nitrate-ammonifying bacterium isolated from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge. Int J Syst Evol Microbiol 55:773–779. CrossRefGoogle Scholar
  24. Wang D, Hurst TK, Thompson RB, Fierke CA (2011) Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli. J Biomed Opt 16:087011. CrossRefGoogle Scholar
  25. Warrier R, Lalitha S, Chellappan S (2014) A modified assay of carbonic anhydrase activity in tree species. BBR Biochem Biotechnol Rep 3:48–55. CrossRefGoogle Scholar
  26. Zhang Z, Lian B, Hou W, Chen M, Li X, Shen W (2011) Optimization of nutritional constituents for carbonic anhydrase production by Bacillus mucilaginosus K02. Afr J Biotechnol 10:8403–8413. CrossRefGoogle Scholar

Copyright information

© University of Tehran 2019

Authors and Affiliations

  1. 1.Department of Biology, Science and Research BranchIslamic Azad UniversityTehranIran
  2. 2.Department of Microbiology, Faculty of MedicineIran University of Medical Science and Health ServicesTehranIran

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