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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Protein solubility can limit enzyme performance in industrial applications. This is the case for some carbonic anhydrases (CAs), key enzymes for CO2 capture and utilization. In this study, we investigated an \u03b1-class CA from the thermophilic bacterium Hydrogenimonas thermophila (HtCA), which was predominantly expressed as an insoluble protein in Escherichia coli. Surface analysis using Molecular Operating Environment (MOE) revealed extensive hydrophobic regions, suggesting a basis for its poor solubility. To improve solubility, three C-terminal fusion tags were evaluated (Gb1, ng3-NEXT, and T7B9). All tagged variants showed markedly increased soluble expression as determined by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS-PAGE) analysis. To reduce surface hydrophobicity, selected residues were substituted with charged amino acids. Most variants displayed improved solubility, and V136D showed enhanced thermostability, retaining 76% activity after exposure to 90 \u00b0C for an hour. However, the F177D variant completely lost all enzymatic activity, highlighting the importance of evaluating both solubility and catalytic function during protein engineering. Molecular dynamics simulations supported the experimental findings, revealing that thermostable variants exhibited reduced structural fluctuations and favorable free-energy landscapes, while the inactive F177D mutant sampled a broader conformational space and higher-energy conformations, consistent with decreased structural stability and loss of catalytic activity.<\/jats:p>","DOI":"10.3390\/ijms27167498","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T15:51:05Z","timestamp":1787327465000},"page":"7498","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering of a Carbonic Anhydrase from Hydrogenimonas thermophila Through Fusion Tags and Surface Mutagenesis Enhances Solubility While Revealing Stability\u2013Function Relationships"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8720-9733","authenticated-orcid":false,"given":"Colleen Varaidzo","family":"Manyumwa","sequence":"first","affiliation":[{"name":"Novo Nordisk Foundation Biotechnology Research Institute for the Green Transition, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Carsten","family":"Jers","sequence":"additional","affiliation":[{"name":"Novo Nordisk Foundation Biotechnology Research Institute for the Green Transition, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8860-6853","authenticated-orcid":false,"given":"Ivan","family":"Mijakovic","sequence":"additional","affiliation":[{"name":"Novo Nordisk Foundation Biotechnology Research Institute for the Green Transition, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"},{"name":"Department of Life Sciences, Chalmers University of Technology, SE-41296 Gothenburg, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1907","DOI":"10.1016\/j.bpj.2012.01.060","article-title":"Toward a molecular understanding of protein solubility: Increased negative surface charge correlates with increased solubility","volume":"102","author":"Kramer","year":"2012","journal-title":"Biophys. 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