Molecular Characterization Purification and Immobilization of Bacterial Carbonic Anhydrase for Efficient CO2 Conversion

Abstract

The gradual increase in atmospheric carbon dioxide (CO2) caused by the use of fossil fuels has recently raised environmental concern. To mitigate the CO2 level, novel conversion strategies are urgently needed. Numerous biological, physical, and chemical approaches have already been exploited for CO2 conversion. Among these, the conversion of CO2 using carbonic anhydrase (CA) is an eco-friendly approach that transform millions of CO2 molecules into bicarbonate ions (HCO3-). Furthermore, HCO3- formed during CO2 conversion can be precipitated into calcium carbonates (CaCO3), which is an essential raw material in various industries. Thus, a CA-producing bacterium, Corynebacterium flavescensT5was isolated from cow saliva.The maximum CA production was obtained by the optimizing production parameters.The optimum production parameters were media (nutrient broth), temperature (40°C), inoculum volume 4% (v/v), inoculum age (24 h), and agitation speed 120 rpm. The optimized reaction parameters were reaction pH (7.0), buffer molarity (50mM), reaction time (10 min), and temperature (35°C). All the tested organic solvents and denaturing agents inhibits the enzyme activity. As per our knowledge, C. flavescens was reported first time for the CA production and found to be promising candidate. After that, the keratin particles were used to immobilize C. flavescens cells to improve the CO2 conversion efficacy.The optimum glutaraldehyde concentration, temperature, and incubation period were found to be 0.6 % (v/v), 25 h, and 4°C. After 10 cycles, theproduction of CaCO3 for immobilized cells was found to be 53.46%. The FE-SEM analysis of synthesized CaCO3 showed the main form of crystal was vaterite. The FTIR analysis confirms the functional groups, while the XRD analysis revealed the crystalline structure of CaCO3. The whole cell immobilization onkeratin particles proved tobe effective for CO2conversion. Furthermore, the CA was purified using column chromatographywith 10.4-folds purification having molecular weight approximately 29 kDa on SDS-PAGE. Further, MALDI-TOF-MS analysis confirmed that the purified proteinshowed 58% of the sequence coverage with K. pneumoniae. The purified CA exhibited a slightly basic pH (7.5) and reaction temperature (35°C). To improve the physiochemical properties and stability of the enzyme, the nanoflower of purified CA was synthesized.The flower-like morphology of carbonic anhydrase nanoflowers (CANF) was confirmed by FE-SEM. The CANFshowed 90% of immobilization yield and a good catalytic activity. The optimal reaction temperature of CANF was 40°C, whereas the optimal pH and reaction time were7.5 and 10 min, respectively. The CANF has shown improved thermostability and storage stability in comparison to free CA. The CANF exhibited a lower Km value (4.7 mM) as compared to free CA (5.1 mM), suggesting that CANF has higher accessibility for substrate. Furthermore, the CANF retained 80% of relative activity after nine cycles of reuse. The application of CANF for CO2 conversion showed higher CaCO3 production (1.71-folds) than free CA.This study proved that CANF has a promising future for converting CO2 into CaCO3, thus it can utilize to mitigate the CO2 level in the environment which is one of the major concerns of the 21st century.