Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions |
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Authors: | Javier?García-Hidalgo Daniel?Hormigo María?Auxiliadora?Prieto Miguel?Arroyo Email author" target="_blank">Isabel?de la?MataEmail author |
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Institution: | 1.Department of Biochemistry and Molecular Biology I, Faculty of Biology,Universidad Complutense de Madrid,Madrid,Spain;2.Department of Environmental Biology,Centro de Investigaciones Biológicas, CSIC,Madrid,Spain |
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Abstract: | The phaZ
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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. |
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