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However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs\u2019 design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation\/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation\/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at \u221280 \u00b0C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.<\/jats:p>","DOI":"10.3390\/ijms23137264","type":"journal-article","created":{"date-parts":[[2022,7,1]],"date-time":"2022-07-01T00:21:50Z","timestamp":1656634910000},"page":"7264","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation"],"prefix":"10.3390","volume":"23","author":[{"given":"Ana M.","family":"Gon\u00e7alves","sequence":"first","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9155-7581","authenticated-orcid":false,"given":"\u00c2ngela","family":"Sousa","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]},{"given":"Augusto Q.","family":"Pedro","sequence":"additional","affiliation":[{"name":"CICECO-Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Campus Universit\u00e1rio de Santiago, 3810-193 Aveiro, Portugal"}]},{"given":"Maria J.","family":"Rom\u00e3o","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}]},{"given":"Jo\u00e3o A.","family":"Queiroz","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1802-8998","authenticated-orcid":false,"given":"Eug\u00e9nia","family":"Gallardo","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Laborat\u00f3rio de F\u00e1rmaco-Toxicologia, UBI Medical, Universidade da Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6910-7576","authenticated-orcid":false,"given":"Lu\u00eds A.","family":"Passarinha","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"},{"name":"Laborat\u00f3rio de F\u00e1rmaco-Toxicologia, UBI Medical, Universidade da Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2147\/NDT.S2404","article-title":"COMT inhibition with tolcapone in the treatment algorithm of patients with Parkinson\u2019s disease (PD): Relevance for motor and non-motor features","volume":"4","author":"Antonini","year":"2008","journal-title":"Neuropsychiatr. 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