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

• Published in: Biotechnology and bioprocess engineering 2009, 14(1), , pp.32-37
• Main Authors: Ramanan, Rishiram, Kannan, Krishnamurthi, Vinayagamoorthy, Nadimuthu, Ramkumar, Kunga Mohan, Sivanesan, Saravana Devi, Chakrabarti, Tapan
• Format: Article
• Language: English
• Subjects: Amino acids; Bacillus subtilis; Biotechnology; Carbon dioxide; Cellulose; Chemistry; Chemistry and Materials Science; Chromatography; Cobalt; Enzymatic activity; Enzyme kinetics; Enzymes; Industrial and Production Engineering; Microorganisms; Molecular weight; Morphology; Physiology; Proteins; Studies; 생물공학
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-008-0099-z

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 Zn 2+ , Co 2+ , Cu 2+ , Fe 3+ , Mg 2+ , and anion SO 4 − increased enzyme activity while strong inhibition was observed in the presence of Hg 2+ , Cl − , HCO 3 − , 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 V max and K m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K i 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.