title: Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions.
abstract: The phaZ                         (                            Sex                         ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T                         (m)) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: García-Hidalgo
authors.forename: Daniel
authors.initials: D
authors.lastname: Hormigo
authors.forename: María Auxiliadora
authors.initials: MA
authors.lastname: Prieto
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D008274:Magnesium / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D044366:Transition Temperature
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase; D008274:Magnesium; D002118:Calcium; D020155:3-Hydroxybutyric Acid