Guimarães, José R.Carballares Navarro, DiegoRocha Martín, JavierTardioli, Paulo W.Fernandez Lafuente, Roberto2025-09-162025-09-162022-10-24Guimarães, J. R., Carballares, D., Rocha-Martin, J., Tardioli, P. W., & Fernandez-Lafuente, R. (2022). Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization. International Journal of Molecular Sciences, 23(21). https://doi.org/10.3390/IJMS2321128081661-659610.3390/ijms232112808https://hdl.handle.net/20.500.14352/123975This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES, Finance Code 001; CAPES-PRINT, number 88887.571985/2020-00), MCIN/AEI/10.13039/501100011033 (PID2021-122398OB-I00). DC thanks to Ministerio de Ciencia e Innovación-Spanish Government by a FPI.Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article1422-0067https://doi.org/10.3390/ijms232112808https://www.mdpi.com/1422-0067/23/21/12808open access66544.478577.15Solid phase enzyme modificationImmobilized lipase physicochemical modificationImmobilized lipase mineralizationEnzyme features tuningIngeniería químicaBioquímica (Biología)3303 Ingeniería y Tecnología Químicas2403 Bioquímica2302.09 Enzimología2210.01 Catálisis