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

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

Abstract

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

Keywords

CA gene Carbonic anhydrase Codon optimization Protein engineering 

Notes

Acknowledgements

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.

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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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