{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T22:23:28Z","timestamp":1791411808519,"version":"4.3.3"},"reference-count":70,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T00:00:00Z","timestamp":1755648000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T00:00:00Z","timestamp":1755648000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 GM57498"],"award-info":[{"award-number":["R01 GM57498"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000015","name":"U.S. Department of Energy","doi-asserted-by":"publisher","award":["DE-FG02-05ER15650"],"award-info":[{"award-number":["DE-FG02-05ER15650"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2025,8,20]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Extremophilic microorganisms are promising candidates for industrial and analytical biocatalysis.\n                      <jats:italic toggle=\"yes\">Haloferax volcanii<\/jats:italic>\n                      , a halophilic archaeon that prefers glycerol over glucose, channels this substrate into central metabolism through glycerol kinase (GK). Here, we report the biochemical properties of\n                      <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                      GK and its potential for biotechnological applications. An N-terminal His-tagged GK was functional\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      and yielded 3 mg\/L culture\u20144.5 times more enzyme than a C-terminal StrepII-tagged version. Size exclusion chromatography revealed a glycerol-induced oligomeric shift from homodimer to a dimer-dominant state with detectable tetramer. The purified enzyme showed robust activity across broad pH and salinity ranges, with optimal activity at 100 mM NaCl and 50\u00b0C\u201360\u00b0C. It retained catalytic activity in 5%\u201310% dimethyl sulfoxide (DMSO) and crude glycerol containing methanol. His-GK was freeze-thaw stable and thermotolerant in 2\u202fM NaCl buffers. In the absence of ligands, the enzyme\u2019s melting temperature (T\n                      <jats:sub>m<\/jats:sub>\n                      ) was 80\u00b0C. Glycerol increased the T\n                      <jats:sub>m<\/jats:sub>\n                      to 85\u00b0C, and combinations with MgCl\u2082 (84\u00b0C) or ATP (88\u00b0C) provided further stabilization. The highest T\n                      <jats:sub>m<\/jats:sub>\n                      (89\u00b0C) occurred with all three ligands, suggesting a cumulative stabilizing effect. Kinetic analyses revealed positive cooperativity for glycerol, ATP, and Mg\u00b2\n                      <jats:sup>+<\/jats:sup>\n                      ; Mn\u00b2\n                      <jats:sup>+<\/jats:sup>\n                      and Co\u00b2\n                      <jats:sup>+<\/jats:sup>\n                      also supported the activity.\n                      <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                      GK is the first known GK to exhibit positive cooperativity with glycerol and ATP. Its high stability and substrate flexibility support its use in biodiesel waste valorization,\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      biocatalysis, and biosensor development\u2014applications demanding robust, specific, and stable enzymes.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        This study reveals that\n                        <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                        GK exhibits positive cooperativity for glycerol, ATP, and Mg\u00b2\n                        <jats:sup>+<\/jats:sup>\n                        , a kinetic feature not previously reported for glycerol kinases. This behavior enables steep, switch-like responses to small substrate changes, offering unique advantages for biosensor design. Importantly,\n                        <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                        GK also maintains high activity under extreme salinity, temperature, broad pH, and solvent conditions that typically limit enzyme use in industrial and environmental applications. These traits make this GK an ideal candidate for enzyme-based biosensors, which often suffer from poor tolerance to pH, solvent, and thermal stress. Its robustness supports its use in cross-linked enzyme crystals, an immobilization method that enhances enzyme stability and reusability under harsh conditions. Moreover, GKs are already employed in Mg\u00b2\n                        <jats:sup>+<\/jats:sup>\n                        detection kits; however,\n                        <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                        GK\u2019s ability to tolerate and respond to diverse divalent cations (e.g., Co\u00b2\n                        <jats:sup>+<\/jats:sup>\n                        , Mn\u00b2\n                        <jats:sup>+<\/jats:sup>\n                        ) broadens their potential for pollutant detection and environmental monitoring. These features collectively position\n                        <jats:italic toggle=\"yes\">H. volcanii<\/jats:italic>\n                        GK as a valuable biocatalyst for biosensing,\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        diagnostics, and biotechnological applications requiring both precision and durability.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00886-25","type":"journal-article","created":{"date-parts":[[2025,7,8]],"date-time":"2025-07-08T08:29:08Z","timestamp":1751963348000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Biochemical properties of glycerol kinase from the hypersaline-adapted archaeon\n                    <i>Haloferax volcanii<\/i>"],"prefix":"10.1128","volume":"91","author":[{"given":"Karol M.","family":"Sanchez","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02y3ad647","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida","place":["Gainesville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6105-0923","authenticated-orcid":true,"given":"Julie A.","family":"Maupin-Furlow","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02y3ad647","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida","place":["Gainesville, USA"]},{"id":[{"id":"https:\/\/ror.org\/02y3ad647","id-type":"ROR","asserted-by":"publisher"}],"name":"Genetics Institute, University of Florida","place":["Gainesville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1039\/c0gc00307g"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1186\/1754-6834-5-13"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.rser.2020.110501"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.3390\/en15093381"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1007\/s43393-021-00036-w"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.3390\/diagnostics15081037"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1007\/s40843-017-9069-3"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac202701c"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.3390\/bios14030143"},{"key":"e_1_3_3_11_2","volume-title":"Enzymes and their biotechnological applications","author":"Rampelotto P","year":"2015","unstructured":"Rampelotto P. 2015. 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