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.

Polyester hydrolytic and synthetic activity catalyzed by the medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces venezuelae SO1

“…Thus, the biochemical and catalytic properties of MCL-PHA depolymerases from Actinomycetes are not well-documented. The interest in the study of PHAs hydrolysis catalyzed by PHA depolymerases lies not only in their potential use as bioplastics or biomaterials but also in the production of chiral (R)-3-HAs (Arroyo et al 2011). Depending on the depolymerase, as a result of enzymatic PHA degradation, the end products are only monomers, both monomers and dimers, or a mixture of oligomers (Jendrossek and Handrick 2002).…”

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…Perhaps the most popular domain is the poly-His tag, with high affinity for metal chelates (Figure 5) (e.g. for purification by IMAC) (Porath et al, 1975, Porath and Olin, 1983, Porath, 1992 but the range of these affinity peptides is huge and still growing: domains of affinity for cellulose, chitin binding domain, peptide tags, among 9 others (Arroyo et al, 2011, Bello-Gil et al, 2014, Bergeron et al, 2009, Bolivar and Nidetzky, 2012a, b, Cassimjee et al, 2011, Chern and Chao, 2005, Daunert et al, 2007, Kondo and Teshima, 1995, Kowsari et al, 2014, Kweon et al, 2005, Linder and Teeri, 1997, Martinez et al, 2000, Mateo et al, 2001b, Moldes et al, 2004a, Moldes et al, 2004b, Scaramozzino et al, 2005, Shpigel et al, 1999, Vishwanath et al, 1995, Wang et al, 2013a, Wiesbauer et al, 2011, Zhao et al, 2013. Table 1 shows a summary of the main domains used for this purpose while Table 2 shows some specific examples of uses of these domains.…”