{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T14:00:06Z","timestamp":1790172006760,"version":"4.1.0"},"reference-count":53,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32400107"],"award-info":[{"award-number":["32400107"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32500106"],"award-info":[{"award-number":["32500106"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007129","name":"Natural Science Foundation of Shandong Province","doi-asserted-by":"publisher","award":["ZR2024QD143"],"award-info":[{"award-number":["ZR2024QD143"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]},{"name":"The Marine S&T Fund of Shandong Province for Qingdao Marine Science and Technology Center","award":["grant 2022QNLM030004-3"],"award-info":[{"award-number":["grant 2022QNLM030004-3"]}]},{"name":"The SKLMT Frontiers and Challenges Project","award":["grant SKLMTFCP-2023-06"],"award-info":[{"award-number":["grant SKLMTFCP-2023-06"]}]},{"name":"Taishan Scholars Program of Shandong Province, China","award":["tsqn202306092"],"award-info":[{"award-number":["tsqn202306092"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2026,9,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      \u03b1-Amylases (EC 3.2.1.1) are among the most important industrial enzymes in starch saccharification, detergent, paper, and textile industries. Canonical \u03b1-amylases typically contain Ca\n                      <jats:sup>2+<\/jats:sup>\n                      in their structure, which is not directly involved in catalysis but essential in maintaining the structural integrity of \u03b1-amylases. Ca\n                      <jats:sup>2+<\/jats:sup>\n                      ions are easily removed by chelating reagents, which may affect the activity and stability of \u03b1-amylases and thereby hinder their applications in some industries. Here, we identified a metal-free \u03b1-amylase, AmyY, from the bacterium\n                      <jats:italic toggle=\"yes\">Alkalimonas<\/jats:italic>\n                      sp. NCh-2 isolated from an alkaline hot spring. AmyY contains a catalytic module of glycoside hydrolase family 13 (GH13) and a carbohydrate-binding module of family 20 (CBM20). AmyY exhibits high activity under alkaline conditions (pH 8.0\u201311.0) and tolerance to high salinity, chelating reagents, and surfactants. We solved the structures of AmyY and its complex with acarbose. Although the overall topology and active sites of AmyY resemble those of reported GH13 \u03b1-amylases, the structure of AmyY is devoid of metal ions. The inability of AmyY to bind metal ions results from replacements of acidic amino acid residues by neutral ones. Combined with biochemical, structural, and bioinformatic data, we demonstrated that AmyY and its homologs represent a class of metal-free \u03b1-amylases in GH13. Moreover, structural analyses revealed that the CBM20 of AmyY is highly flexible, and we obtained a mutant with enhanced thermostability by truncating the CBM20. This mutant demonstrates remarkable wash performance and desizing capability at alkaline pH, which underscores its application potential in the detergent and textile industries.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        While canonical GH13 \u03b1-amylases typically contain Ca\n                        <jats:sup>2+<\/jats:sup>\n                        , our biochemical and structural data reveal that AmyY has evolved a metal-free architecture. This is achieved through the replacement of acidic metal-coordinating residues by neutral ones, abolishing metal binding without compromising catalytic efficiency. This work expands the structural and functional diversity of GH13 by defining a class of metal-free \u03b1-amylases, with AmyY and its homologs as representatives. Furthermore, the engineered AmyY with enhanced thermostability offers a candidate biocatalyst for industrial applications, such as the detergent industry. The structure of AmyY also offers a blueprint for engineering other \u03b1-amylases into metal-independent forms.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00660-26","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T12:58:38Z","timestamp":1786539518000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Biochemical and structural characterization of AmyY from\n                    <i>Alkalimonas<\/i>\n                    sp. NCh-2 reveals a class of metal-free \u03b1-amylases in glycoside hydrolase family 13"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1523-3475","authenticated-orcid":false,"given":"Fang","family":"Zhao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04rdtx186","id-type":"ROR","asserted-by":"publisher"}],"name":"Ministry of Education Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Shandong Provincial Key Laboratory of Microbial Resource Exploration and Innovative Utilization & College of Marine Life Sciences, Ocean University of China","place":["Qingdao, China"]},{"name":"Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center","place":["Qingdao, 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