NDLI: Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions

Content Provider	Springer Nature Link
Author	García Hidalgo, Javier Hormigo, Daniel Prieto, María Auxiliadora Arroyo, Miguel Mata, Isabel
Copyright Year	2011
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.
Starting Page	1975
Ending Page	1988
Page Count	14
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	93
Issue Number	5
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-08-16
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Extracellular PHB depolymerase Streptomyces exfoliatus Rhodococcus sp. T104 Cloning Biotechnology Microbiology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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