title: Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of recombinant poly-3-hydroxybutyrate depolymerase from Streptomyces exfoliatus.
abstract: Cross-linked enzyme aggregates of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces exfoliatus (PhaZ(Sex)-CLEAs) have been prepared. Acetone was used as the precipitating agent, while addition of bovine serum albumin (BSA) facilitated CLEAs formation. Conditions for enzyme precipitation and cross-linking have been optimized, and confocal scanning microscopy showed a homogeneous enzyme distribution in the biocatalyst. Obtained PhaZ(Sex)-CLEAs presented an average size of 50-300 μm, showing a high PHB depolymerase activity of 255 U/g wet biocatalyst at 40°C and pH 7.0. Temperature-activity profile of PhaZ(Sex)-CLEAs at pH 8.0 showed that the highest activity for pNPB hydrolysis was achieved at 60°C, whereas pH-activity profile at 40°C indicated that highest activity for PHB hydrolysis was achieved at pH 7.0. Additionally, immobilized biocatalyst could be recycled at least for 20 consecutive batch reactions without loss of catalytic activity, and showed higher pH and temperature stability, and better tolerance to several organic solvents than its soluble counterpart.
authors.affiliation: Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hormigo
authors.forename: Javier
authors.initials: J
authors.lastname: García-Hidalgo
authors.forename: Carmen
authors.initials: C
authors.lastname: Acebal
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D001709:Biotechnology / Q000379:methods*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D018613:Microscopy, Confocal; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D059027:Recycling; D012995:Solubility / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
chemical_list: D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D012997:Solvents; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase