English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/51018
Share/Impact:
Statistics
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:

Title

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies

AuthorsArroyo, Miguel; García-Hidalgo, Javier; Villalón, M.; Eugenio, Laura I. de; Hormigo, Daniel; Acebal, Carmen; García, José Luis ; Prieto, María Auxiliadora ; Mata, Isabel de la
KeywordsPHA depolymerase
Immobilization
Refolding
Polyhydroxyalkanoate
Issue Date20-Nov-2011
PublisherSpringer
CitationJournal of Industrial Microbiology and Biotechnology, 38: 1203-1209 (2011)
AbstractPurification 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 Ni2+-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
Description7 páginas, 5 figuras, 1 tabla -- PAGS nros. 1203-1209
Publisher version (URL)http://dx.doi.org/10.1007/s10295-010-0898-z
URIhttp://hdl.handle.net/10261/51018
DOI10.1007/s10295-010-0898-z
ISSN1367-5435
E-ISSN1476-5535
Appears in Collections:(CIB) Artículos
Files in This Item:
There are no files associated with this item.
Show full item record
Review this work
 

Related articles:


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.