{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T02:54:48Z","timestamp":1784256888306,"version":"3.55.0"},"reference-count":78,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T00:00:00Z","timestamp":1764115200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T00:00:00Z","timestamp":1764115200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The 16S microbial community profiling of a metagenomics library from geothermal spring at Lisvori (Lesvos island, Greece) enabled the identification of a putative sequence exhibiting 95% identity to the \u03b3\u2010type carbonic anhydrase (\u03b3\u2010CA) from\n                    <jats:italic>Caloramator australicus<\/jats:italic>\n                    (\u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CA). The sequence of \u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CA was amplified by PCR, cloned, and expressed in\n                    <jats:italic>E. coli<\/jats:italic>\n                    . Activity assays showed that \u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CA possesses very low, but detectable, anhydrase activity, while exhibiting no measurable esterase activity. Differential scanning fluorimetry (DSF) revealed that the enzyme shows high thermal stability with a melting temperature (\n                    <jats:italic>T<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>m<\/jats:italic>\n                    <\/jats:sub>\n                    ) approximately 65\u201375\u00b0C in the pH range between 5.5 and 9.0. The structure of \u03b3\n                    <jats:italic>\u2010Ca<\/jats:italic>\n                    CA was determined by X\u2010ray crystallography at 1.11\u2009\u00c5 resolution, the highest resolution reported so far for a \u03b3\n                    <jats:italic>\u2010<\/jats:italic>\n                    CA. The enzyme was crystallized as a trimer in the crystallographic asymmetric unit and contains three zinc\u2010binding sites, one at each interface of neighboring subunits of the trimer. Structure\u2010based rational design enabled the design and creation of a mutant enzyme (\u03b3\n                    <jats:italic>\u2010Ca<\/jats:italic>\n                    CAmut) which possessed a heptapeptide insertion at the active\u2010site loop and two\u2010point mutations. Kinetic analysis demonstrated that \u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure\u2010guided engineering. The thermostability of \u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X\u2010ray crystallographic analysis of the \u03b3\u2010\n                    <jats:italic>Ca<\/jats:italic>\n                    CAmut structure at 2.1\u2009\u00c5 resolution, provided detailed structural insights into how the mutations impact the overall enzyme structure, function, and thermostability. These findings provide valuable structural and functional insights into \u03b3\u2010CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design.\n                  <\/jats:p>","DOI":"10.1002\/pro.70396","type":"journal-article","created":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T13:59:00Z","timestamp":1764165540000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Structural and functional characterization of a metagenomically derived \u03b3\u2010type carbonic anhydrase and its engineering into a hyperthermostable esterase"],"prefix":"10.1002","volume":"34","author":[{"given":"Charoutioun S.","family":"Bodourian","sequence":"first","affiliation":[{"name":"Laboratory of Enzyme Technology, Department of Biotechnology School of Applied Biology and Biotechnology, Agricultural University of Athens  Athens 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E.","family":"Labrou","sequence":"additional","affiliation":[{"name":"Laboratory of Enzyme Technology, Department of Biotechnology School of Applied Biology and Biotechnology, Agricultural University of Athens  Athens Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,11,26]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909052925"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/md13041688"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(05)80360-2"},{"key":"e_1_2_11_5_1","first-page":"93","volume-title":"Enzymes","author":"Angeli 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