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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 hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30\u201335 \u00b0C and significant enzymatic activity below 20 \u00b0C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca2+ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca2+ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca2+ binding site, while Rho13 and I3C6 both maintained one stably bound Ca2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as 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":"10.3390\/biom15030415","type":"journal-article","created":{"date-parts":[[2025,3,14]],"date-time":"2025-03-14T07:02:16Z","timestamp":1741935736000},"page":"415","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Cold-Active Starch-Degrading Enzymes from a Cold and Alkaline Greenland Environment: Role of Ca2+ Ions and Conformational Dynamics in Psychrophilicity"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-8505-9994","authenticated-orcid":false,"given":"Malthe Kj\u00e6r","family":"Bendtsen","sequence":"first","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"}]},{"given":"Jan Stanislaw","family":"Nowak","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7003-6055","authenticated-orcid":false,"given":"Pedro","family":"Paiva","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto, Rua do Campo Alegre, s\/n, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7843-7374","authenticated-orcid":false,"given":"Marcos","family":"L\u00f3pez Hern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"},{"name":"Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7015-5460","authenticated-orcid":false,"given":"Pedro","family":"Ferreira","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto, Rua do Campo Alegre, s\/n, 4169-007 Porto, Portugal"}]},{"given":"Jan Skov","family":"Pedersen","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"},{"name":"Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"}]},{"given":"Nicolai Sundgaard","family":"Bekker","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark"}]},{"given":"Elia","family":"Viezzi","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5375-6264","authenticated-orcid":false,"given":"Francesco","family":"Bisiak","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5413-4667","authenticated-orcid":false,"given":"Ditlev E.","family":"Brodersen","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus, Denmark"}]},{"given":"Lars Haastrup","family":"Pedersen","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4706-4023","authenticated-orcid":false,"given":"Athanasios","family":"Zervas","sequence":"additional","affiliation":[{"name":"Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2748-4722","authenticated-orcid":false,"given":"Pedro A.","family":"Fernandes","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto, Rua do Campo Alegre, s\/n, 4169-007 Porto, Portugal"}]},{"given":"Maria Joao","family":"Ramos","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto, Rua do Campo Alegre, s\/n, 4169-007 Porto, Portugal"}]},{"given":"Peter","family":"Stougaard","sequence":"additional","affiliation":[{"name":"Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0799-6894","authenticated-orcid":false,"given":"Mariane Schmidt","family":"Th\u00f8gersen","sequence":"additional","affiliation":[{"name":"Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2918-8989","authenticated-orcid":false,"given":"Daniel E.","family":"Otzen","sequence":"additional","affiliation":[{"name":"Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, 8000 Aarhus, Denmark"},{"name":"Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus, Denmark"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Liu, Y., Zhang, N., Ma, J., Zhou, Y., Wei, Q., Tian, C., Fang, Y., Zhong, R., Chen, G., and Zhang, S. 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