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Currently, Parkinson\u2019s disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography\u2014IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 \u00b0C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 \u00b0C and \u221280 \u00b0C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.<\/jats:p>","DOI":"10.3390\/ijms232012298","type":"journal-article","created":{"date-parts":[[2022,10,17]],"date-time":"2022-10-17T02:29:46Z","timestamp":1665973786000},"page":"12298","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase"],"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, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Augusto Q.","family":"Pedro","sequence":"additional","affiliation":[{"name":"Chemistry Department, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universit\u00e1rio de Santiago, 3810-193 Aveiro, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Diana M.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adriana E.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8205-1041","authenticated-orcid":false,"given":"Marino F. A.","family":"Santos","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M\u00e1rcia A. S.","family":"Correia","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3096-8325","authenticated-orcid":false,"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3004-0543","authenticated-orcid":false,"given":"Maria J.","family":"Rom\u00e3o","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"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, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, 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"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2212","DOI":"10.1038\/nprot.2007.321","article-title":"The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability","volume":"2","author":"Niesen","year":"2007","journal-title":"Nat. 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