Heterofunctional supports for the one-step purification,immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports |
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| Institution: | 1. Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain;2. Departamento de Microbiología, Instituto de Fermentaciones Industriales-CSIC, C/Juan de la Cierva 3, 2006 CSIC, Madrid, Spain;1. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria;2. Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria;1. College of Pharmaceutical Science, Hebei University, Baoding 071002, China;2. Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Science, Hebei University, Baoding 071002, China;1. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12/l, A-8010 Graz, Austria;2. Austrian Centre of Industrial Biotechnology (acib), Petersgasse 14, A-8010 Graz, Austria;1. Departamento de Biologia – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – Universidade de São Paulo, Brazil;2. Departamento de Bioquímica e Imunologia – Faculdade de Medicina de Ribeirão Preto – Universidade de São Paulo, Brazil;3. Departamento de Biotecnología y Microbiologia de los Alimentos, Instituto de Ciencias de la Alimentación, CIAL-CSIC, Calle Nicolás Cabrera 9, Campus UAM, Cantoblanco, 28049, Spain;4. Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica – CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain;1. Departamento de Bioquímica e Imunologia – Faculdade de Medicina de Ribeirão Preto – Universidade de São Paulo, Brazil;2. Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil;3. Department of Chemical and Biomolecular Engineering – Johns Hopkins University, USA;4. Departamento de Química – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – Universidade de São Paulo, Brazil;5. Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil;6. Centro de Ciências Médicas e Farmacêuticas – UNIOESTE – Cascavel, Paraná, Brazil;7. Departamento de Biologia – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – Universidade de São Paulo, Brazil |
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| Abstract: | For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme. |
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