![]() However, the examined disulfide bonds were unfavorable, or the resultant stabilizing effects were not significant. The study showed the capability to predict potential residue pairs for disulfide engineering. developed a computational algorithm to stabilize human CA II (hCAII) 10. Disulfide bonds, including those found in naturally occurring proteins, are believed to decrease conformational entropy of unfolded state of protein, thereby providing folded state of protein with increased stability 17.Ī few studies have engineered CAs with disulfide bonds. Various proteins have been successfully engineered by introducing disulfide bridges 15, 17, 18, 19, 20, 21, 22. In particular, disulfide engineering is a powerful strategy for enhancing protein thermostability. Although information on protein structure is an essential prerequisite for rational engineering, protein structure databases are fast-growing and design technologies are becoming more robust for better estimation 16. In contrast, rational design provides a fast, universal, and technically easier way to manipulate enzymes for desired traits. Furthermore, the method requires a specific high-throughput selection/screening tool for each type of enzyme, whose implementation is greatly challenging and at times not feasible 14, 15. Although the directed evolution has proven effective for the given purpose 3, it is generally a time-consuming and labor intensive process. CAs have been engineered for use as stable biosensors or catalysts 3, 8, 9, 10, 11, 12, 13. Protein engineering has been widely applied to enzymes to increase thermostability. Thus, an improved thermostability of CA is a highly sought goal for successful industrial application. However, the application of CA has been limited due to the unstable nature of proteins, because CO 2 reduction facilities generally operate under harsh environments such as high temperature conditions 6, 7. Due to the potential cost effectiveness and environmentally friendly properties, the use of CA in biomimetic CO 2 sequestration is considered a promising alternative for reducing anthropogenic CO 2, one of the most urgent environmental issues 3, 4, 5, 6. CAs are ubiquitous metalloenzymes that play various physiological functions in diverse forms of life and are classified into five different classes (i.e., α, β, γ, δ, and ζ) 2. Similar content being viewed by othersĬarbonic anhydrase (CA) is an enzyme that rapidly catalyzes the hydration of carbon dioxide (CO 2) into bicarbonate (HCO 3 −) and proton (H +) 1. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 ☌. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. ![]() One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. ![]() In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. ![]()
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