Biological approach of CO2 sequestration: Production and application of Carbonic anhydrase.

Abstract

In past few decades, green-house gases, mainly CO2 has been growing concern. To handle this problem, an efficient CO2- sequestration technology is needed. Anthropogenic activities and evolving industries are two of the major contributors in the elevation of atmospheric CO2. Chemical and physical methods have been used for on-site CO2-reduction form industrial outlets. The overall processes are costly and environmentally unfriendly. Biocatalyst based CO2-reduction has been much popular because of its high rate of conversion and efficiency. The overall production cost can be minimized by reusability of enzyme and thermostability. Carbonic anhydrase from Sulfurihydrogenibium azorense (SazCA), the most active CA ever tested, was expressed, and purified from Nicotiana benthamiana by transient expression and Cellulose-CBM3 mediated one-step purification respectively giving a yield of 350mg/kg of fresh weight. Immobilized SazCA on microcrystalline cellulose retained 70% of its CO2-hydration activity after 24h at 70 ℃ while free form could only retain 45% under same conditions. Thus, immobilization not only enhanced the heat stability but reusability of enzyme in multiple rounds of CO2-hydration reaction. To further enhance the existing model, we adopted protein engineering approach and shuffled the protein domains between different alpha-CAs to produce a novel and ultra-stable recombinant carbonic anhydrase which can withstand under high temperature, alkaline pH, and high saline conditions. Domain swapping between SazCA and PmCA (CA from Persephonella marina EX-H1), two variants, SPS and PSP were generated, and both were found structurally stable, but SPS was the most stable when compared to its parental design while PSP was similar liek PmCA. Furthermore, addition of two loop sequences from halotolerant dCAII gave rise to SPS_1 and SPS_2 variants. Comparing with wild type (SazCA and PmCA), thermal stability of chimeric CAs (SPS, SPS_1 and SPS_2) not only increased under standard conditions but also at high salt and alkaline conditions demonstrating variants’ ultrastability. We also tested CO2 reduction by SPS using synthetic and real sea water. Addition of 100 µg of SPS in real seawater accelerated the CO2 reaction rate much faster. It took only 10 minutes to drop pH from 9.1 to 8.14 in real sea water. For the first time, we applied a unique approach to generate ultra-stable CAs having broad range of stability under different harsh conditions making them good candidates for industrial bio- catalyst as it profitably able to capture indoor air CO2 using seawater.