IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO₂ SEQUESTERER

Abstract

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.

Keywords:

• Bacillus subtilis
• carbonic anhydrase
• characterization
• CO₂ sequestration
• immobilization

ACKNOWLEDGMENTS

The authors are thankful to Periyar Maniammai University for providing infrastructure facilities to carry out the present study. M. Oviya gratefully acknowledges the Jawaharlal Nehru Memorial Fund, New Delhi, for providing fellowship for doctoral studies. S. S. Giri is thankful to the Department of Science and Technology (DST), Government of India, for providing an INSPIRE fellowship (IF 10575) for doctoral research.

Notes

Note. All the steps were carried out at 4°C.

Note. Enzyme activities were determined by incubating the free and immobilized CA in the presence of various metal ions (1 mM) and inhibitors (1 mM) for 2 hr at 37°C. All the values are represented as mean ± SEM.