Bolívar Bolívar, Juan ManuelNidetzky, Bernd2024-01-122024-01-122012Bolivar, J. M., & Nidetzky, B. (2012). Positively charged mini-protein Z basic2 as a highly efficient silica binding module: Opportunities for enzyme immobilization on unmodified silica supports. Langmuir, 28(26), 10040-10049. https://doi.org/10.1021/LA30123480743-746310.1021/la3012348https://hdl.handle.net/20.500.14352/92775Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Zbasic2 (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Zbasic2 protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Zbasic2 proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Zbasic2 proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Zbasic2 and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal article1520-5827https://doi.org/10.1021/la3012348open access66.0577.1Immobilized enzymeamino acid oxidaseGlassGlucosyltransferasesProtein StructureL-Amino Acid OxidaseIngeniería químicaBiotecnologíaQuímica industrialBioquímica (Química)2302 Bioquímica3302 Tecnología Bioquímica3303 Ingeniería y Tecnología Químicas