title: In vitro activation, purification, and characterization of Escherichia coli expressed aryl-alcohol oxidase, a unique H2O2-producing enzyme.
abstract: Aryl-alcohol oxidase (AAO), a flavoenzyme with unique spectral and catalytic properties that provides H2O2 for fungal degradation of lignin, has been successfully activated in vitro after Escherichia coli expression. The recombinant AAO (AAO*) protein was recovered from inclusion bodies of E. coli W3110 transformed with pFLAG1 containing the aao cDNA from Pleurotus eryngii. Optimization of in vitro refolding yielded 75% active enzyme after incubation of AAO* protein (10 microg/ml) for 80 h (at 16 degrees C and pH 9) in the presence of glycerol (35%), urea (0.6 M), glutathione (GSSG/GSH molar ratio of 2), and FAD (0.08 mM). For large-scale production, the refolding volume was 15-fold reduced and over 45 mg of pure active AAO* was obtained per liter of E. coli culture after a single anion-exchange chromatographic step. Correct FAD binding and enzyme conformation were verified by UV-visible spectroscopy and circular dichroism. Although the three enzymes oxidized the same aromatic and aliphatic polyunsaturated primary alcohols, some differences in physicochemical properties, including lower pH and thermal stability, were observed when the activated enzyme was compared with fungal AAO from P. eryngii (wild enzyme) and Emericella nidulans (recombinant enzyme), which are probably related to the absence of glycosylation in the E. coli expressed AAO.
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ruiz-Dueñas
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferreira
authors.forename: María J
authors.initials: MJ
authors.lastname: Martínez
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D032842:Emericella / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000201:enzymology / Q000378:metabolism; D020076:Pleurotus / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase