title: Coating of soluble and immobilized enzymes with ionic polymers: full stabilization of the quaternary structure of multimeric enzymes.
abstract: This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).
authors.affiliation: Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Bolivar
authors.forename: Javier
authors.initials: J
authors.lastname: Rocha-Martin
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.forename: Jose
authors.initials: J
authors.lastname: Berenguer
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez-Lafuente
authors.forename: Jose M
authors.initials: JM
authors.lastname: Guisan
mesh_terms: D002411:Cation Exchange Resins / Q000378:metabolism; D020099:Coated Materials, Biocompatible; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D011549:Pseudomonas / Q000201:enzymology; D012965:Sodium Chloride / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D002411:Cation Exchange Resins; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D011108:Polymers; D012965:Sodium Chloride; D011094:Polyethyleneimine; D005560:Formate Dehydrogenases; D005969:Glutamate Dehydrogenase; D005976:Glutaral