Application of medium-chain-length plyhydroxyalkanoate depolymerases for polyester degradation and synthesis

  1. GANGOITI MUÑECAS, JOANA
Supervised by:
  1. Juan Luis Serra Ferrer Director
  2. María Jesús Llama Fontal Director

Defence university: Universidad del País Vasco - Euskal Herriko Unibertsitatea

Fecha de defensa: 22 May 2012

Committee:
  1. Francisco Castillo Rodríguez Chair
  2. Fernando Luis Hernando Echevarría Secretary
  3. María Auxiliadora Prieto Jiménez Committee member
  4. María-José Bonete Committee member
  5. Carmen Gabriela Boeriu Committee member
Department:
  1. Bioquímica y Biología Molecular

Type: Thesis

Teseo: 115728 DIALNET

Abstract

Polyhydroxyalkanoates (PHAs) are environmentally friendly polyesters that are biosynthesized by numerous microorganisms. According to the number of carbon atoms of the side chain of the monomers, PHAs are classified as short-chain-length (SCL) PHAs (3 to 5 carbon atoms) and medium-chain-length (MCL) PHAs (6 to 14 carbon atoms). Extensive studies have demonstrated that PHAs can easily be degraded by microorganisms, by releasing specific PHA depolymerases. Many extracellular SCL-PHA depolymerases have been characterized in depth and more than 20 genes have been identified. In the case of extracellular MCL-PHA depolymerases, only the coding gene phaZGK13 of Pseudomonas fluorescens GK13, and a few homologous genes in other strains, have been cloned and characterized. Depending on the depolymerase, as a result of PHA enzymic degradation, the end products are only monomers, both monomers and dimers, or a mixture of oligomers. Enantiomer pure (R)-3-hydroxyalkanoic acid [(R)-3-HA] monomers are very attractive building blocks of interest not only in the biomedical and pharmaceutical fields, but also for being used as starting materials to obtain other new polyesters. Thus, the development of a cost-effective industrial process for the production of both MCL-PHA depolymerase enzyme and (R)-3-HA monomers is of considerable interest.In this work, based on the strong tendency of the P(3HO) depolymerase of P. fluorescens GK13 to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of P. fluorescens GK13 grown on mineral medium supplemented with P(3HO), as the sole carbon and energy source, has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(R)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (R)-3-HO monomers under the same conditions.On the other hand, the potential of using the P(3HO) depolymerase from Pseudomonas fluorescens GK13 in anhydrous media to catalyze ester-forming reactions has been investigated. Specifically, the MCL-PHA depolymerase catalyzes the ring-opening polymerization of racemic ¿-butyrolactone (¿-BL), L- and D-lactide (LLA, DLA) with high yield resulting in low molecular weight polymers.Additionally, a novel extracellular MCL-PHA depolymerase from Streptomyces roseolus SL3 was purified, characterized and its applicability in (R)-3HA production was studied. The enzyme showed an Mr of 28 kDa, with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified S. roseolus SL3 enzyme hydrolyzed poly(3-hydroxyoctanoic acid) [P(3HO)] and PCL, but not poly(3-hydroxybutyric acid) [P(3HB)], PES and PLA. Moreover, the MCL-PHA depolymerase can hydrolyze various substrates for esterases such as tributyrin and p-nitrophenyl (PNP)-alkanoates, its maximum activity being measured with PNP-octanoate. In contrast, the P(3HO) depolymerase of P. fluorescens GK13 does not show hydrolytic activity toward tributyrin whereas only weak activity was detected toward PCL. Thus, it is likely that the MCL-PHA depolymerase produced from S. roseolus SL3 has a wider range of substrate specificity than the P. fluorescensGK13 enzyme. Interestingly, when P(3HO) was used as substrate the main hydrolysis product was the monomer of (R)-3-hydroxyoctanoate after 72 h of reaction. Considering these valuable attributes, the knowledge of the nature of this enzyme was highly interesting. Thus, the MCL-PHA depolymerase gene from S. roseolus SL3 was partially amplified. The deduced amino acid sequence shared high similarity in amino acid sequence to proteins from several Actinobacteria recently identified a new type of MCL-PHA depolymerases by our group in a parallel work. All these novel MCL-PHA depolymerases did not show homology to already characterized MCL-PHA depolymerases from Pseudomonas.