{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,3,20]],"date-time":"2025-03-20T02:10:10Z","timestamp":1742436610902,"version":"3.40.1"},"posted":{"date-parts":[[2025,2,17]]},"group-title":"Biology and Life Sciences","reference-count":0,"publisher":"MDPI AG","license":[{"start":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T00:00:00Z","timestamp":1739750400000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,2,15]]},"abstract":"<jats:p>Cold-active enzymes hold promise in energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolysed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolysed cyclodextrins and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal temperatures around 30-35\u2103 and significant activity below 20\u2103, but Rho13 and I3C6 had an alkaline pH optimum while Ika2 was markedly acidophilic. They show complex dependence on Ca2+ concentration, with activity of Rho13 and I3C6 following a bell-shaped dependence and Ika2 being unaffected; however, removal of Ca2+ reduces stability of all 3 enzymes. Loss of structure occurs well above the optimal activity, showing the characteristic psychrophilic divorce be-tween activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca2+ binding site, while Rho13 and I3C6 both maintain one stably bound Ca2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, founded in a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation is also found in regions outside the active site and may provide easier substrate access and ac-commodation and faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.<\/jats:p>","DOI":"10.20944\/preprints202502.1231.v1","type":"posted-content","created":{"date-parts":[[2025,2,19]],"date-time":"2025-02-19T00:18:17Z","timestamp":1739924297000},"source":"Crossref","is-referenced-by-count":0,"title":["Cold-Active Starch-Degrading Enzymes from a Cold and Alkaline Greenland Environment: Role of Ca&lt;sup&gt;2+&lt;\/sup&gt; Ions and Conformational Dynamics in Psychrophilicity"],"prefix":"10.20944","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-8505-9994","authenticated-orcid":false,"given":"Malthe Kj\u00e6r","family":"Bendtsen","sequence":"first","affiliation":[]},{"given":"Jan Stanislaw","family":"Nowak","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7003-6055","authenticated-orcid":false,"given":"Pedro","family":"Paiva","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7843-7374","authenticated-orcid":false,"given":"Marcos L\u00f3rez","family":"Hern\u00e1ndez","sequence":"additional","affiliation":[]},{"given":"Pedro","family":"Ferreira","sequence":"additional","affiliation":[]},{"given":"Jan Skov","family":"Pedersen","sequence":"additional","affiliation":[]},{"given":"Nicolai Sundgaard","family":"Bekker","sequence":"additional","affiliation":[]},{"given":"Elia","family":"Viezzi","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5375-6264","authenticated-orcid":false,"given":"Francesco","family":"Bisiak","sequence":"additional","affiliation":[]},{"given":"Lars Haastrup","family":"Pedersen","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5413-4667","authenticated-orcid":false,"given":"Ditlev E","family":"Brodersen","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4706-4023","authenticated-orcid":false,"given":"Athanasios","family":"Zervas","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2748-4722","authenticated-orcid":false,"given":"Pedro A.","family":"Fernandes","sequence":"additional","affiliation":[]},{"given":"Maria Joao","family":"Ramos","sequence":"additional","affiliation":[]},{"given":"Peter","family":"Stougaard","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0799-6894","authenticated-orcid":false,"given":"Mariane Schmidt","family":"Th\u00f8gersen","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2918-8989","authenticated-orcid":false,"given":"Daniel E. E.","family":"Otzen","sequence":"additional","affiliation":[]}],"member":"1968","container-title":[],"original-title":[],"deposited":{"date-parts":[[2025,3,20]],"date-time":"2025-03-20T01:31:52Z","timestamp":1742434312000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.preprints.org\/manuscript\/202502.1231\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,17]]},"references-count":0,"URL":"https:\/\/doi.org\/10.20944\/preprints202502.1231.v1","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.3390\/biom15030415","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,2,17]]},"subtype":"preprint"}}