title: Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.
abstract: Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % β-sheet, 19.4 % β-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/β hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Virginia
authors.initials: V
authors.lastname: Martínez
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.|Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain.
authors.forename: Patricia Gómez
authors.initials: PG
authors.lastname: de Santos
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: García-Hidalgo
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.|Department of Pharmacy and Biotechnology, Biomedical Sciences Faculty, European University of Madrid, Madrid, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hormigo
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: M Auxiliadora
authors.initials: MA
authors.lastname: Prieto
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Arroyo
authors.affiliation: Department of Biochemistry and Molecular Biology, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain. idlmata@ucm.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: de la Mata
mesh_terms: D001711:Biotransformation; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D008970:Molecular Weight; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase
keywords: 3-Hydroxyalkanoic acids; Polyhydroxyalkanoate depolymerase; Streptomyces exfoliatus