Biotechnology and Bioprocess Engineering

, Volume 14, Issue 1, pp 32–37

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

  • Rishiram Ramanan
  • Krishnamurthi Kannan
  • Nadimuthu Vinayagamoorthy
  • Kunga Mohan Ramkumar
  • Saravana Devi Sivanesan
  • Tapan Chakrabarti
Article

DOI: 10.1007/s12257-008-0099-z

Cite this article as:
Ramanan, R., Kannan, K., Vinayagamoorthy, N. et al. Biotechnol Bioproc E (2009) 14: 32. doi:10.1007/s12257-008-0099-z

Abstract

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn2+, Co2+, Cu2+, Fe3+, Mg2+, and anion SO4 increased enzyme activity while strong inhibition was observed in the presence of Hg2+, Cl, HCO3, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the Vmax and Km values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The Ki value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.

Keywords

carbonic anhydrase Bacillus subtilis purification N-terminal amino acids enzyme characterization 

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg GmbH 2009

Authors and Affiliations

  • Rishiram Ramanan
    • 1
  • Krishnamurthi Kannan
    • 1
  • Nadimuthu Vinayagamoorthy
    • 1
  • Kunga Mohan Ramkumar
    • 2
  • Saravana Devi Sivanesan
    • 1
  • Tapan Chakrabarti
    • 1
  1. 1.Environmental Biotechnology DivisionNational Environmental Engineering Research Institute (NEERI)Nehru Marg, NagpurIndia
  2. 2.Department of Biotechnology, Bharathidasan Institute of TechnologyBharathidasan UniversityTrichy, Tamil NaduIndia

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