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KMID : 0364820230590030204
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Korean Journal of Microbiology
2023 Volume.59 No. 3 p.204 ~ p.213
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Recombinant expression and purification of α-carbonic anhydrases from a water bear Ramazzottius varieornatus in Escherichia coli
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Choi Ye-Jin
Jo Byung-Hoon
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Abstract
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Carbonic anhydrase (CA) is an enzyme that catalyzes the reversible hydration of CO2, effectively accelerating carbon capture, utilization, and storage (CCUS) processes to achieve carbon neutrality. The discovery and utilization of stable CA is essential for efficient and cost-effective CCUS. Ramazzottius varieornatus, a species of tardigrade, is resistant to various physical and chemical stresses, and CAs from this species are expected to be stable under these stressful conditions. In this study, for the first time, we attempted the recombinant expression and purification of six α-CAs (RvCAs) from R. varieornatus in Escherichia coli. Additionally, we tested the expression of RvCAs with a fusion of the NEXT tag to enhance the soluble expression. With the exception of RvCA5, all of the recombinant RvCAs showed mostly insoluble expression. Subsequently, RvCA5 and NEXT-RvCA5, which were expressed mostly in soluble forms, were successfully purified. The catalytic activity of RvCA5 was found to be extremely low for both ester hydrolysis and CO2 hydration. Subsequent studies on RvCA5 will be interesting in terms of enzyme activity and stability, and further research on the remaining RvCA enzymes is anticipated to uncover novel stable CAs.
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KEYWORD
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Escherichia coli, Ramazzottius varieornatus, carbonic anhydrase, NEXT tag, recombinant expression
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