title: Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies.
abstract: Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni(2+)-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Arroyo
authors.forename: J
authors.initials: J
authors.lastname: García-Hidalgo
authors.forename: M
authors.initials: M
authors.lastname: Villalón
authors.forename: L
authors.initials: L
authors.lastname: de Eugenio
authors.forename: D
authors.initials: D
authors.lastname: Hormigo
authors.forename: C
authors.initials: C
authors.lastname: Acebal
authors.forename: J L
authors.initials: JL
authors.lastname: García
authors.forename: M A
authors.initials: MA
authors.lastname: Prieto
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
mesh_terms: D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000201:enzymology*; D058849:Protein Refolding; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C080782:poly(3-hydroxyoctanoic acid) depolymerase