Isolation of novel medium-chain-length polyhydroxyalkanoate depolymerase producers. Properties and applications of the enzyme

Abstract

Polyhydroxyalkanoates (PHAs) are bacterial storage polyesters currently receiving much attention due to their synthesis from renewable resources and their applicability as biodegradable and biocompatible plastics. The ability to degrade extracellular PHA depends on the release of specific extracellular PHA depolymerases. Most of these identified enzymes are specific for only Short-Chain-Length (SCL)-PHAs (HAs with 3 to 5 C-atoms). In contrast, a limited number of works have evaluated the biochemical characteristics of Medium-Chain-Length (MCL)-PHA depolymerases that are specific to polymers consisting of 3-hydroxyalkanoates with 6 or more carbon atoms. To date most of MCL-PHA depolymerases reported belong to Gram-negative bacteria, predominantly Pseudomonas species. Among Gram-positive bacteria, only the MCL-PHA depolymerases from Streptomyces sp. KJ-72 and Rhodococcus equi P2 have been characterized. Moreover, the interest in the study of PHA hydrolysis catalysed by PHA depolymerases lies not only in their potential use as bioplastics or biomaterials but also in the production of chiral (R)-hydroxyalkanoic acids [(R)-HAs]. Enantiomer pure (R)-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. Depending on the specificity of the depolymerase, as a result of enzymic PHA degradation, the end products can be only monomers, both monomers and dimers, or a mixture of oligomers. On the other hand, enzymic syntheses in vitro have become effective methods for designing and synthesizing environmentally acceptable polymeric materials. The majority of enzymes studied for polymerization reactions were members of the lipase family. As lipases and other serine hydrolases, all PHA depolymerases characterized to date to the molecular level commonly show a catalytic triad (Ser-His-Asp) in their catalytic domains. In particular, Ser is part of a lipase-box pentapeptide Gly-X1-Ser-X2-Gly. Although none of the currently known PHA depolymerases show lipase activity, the fact that catalytic centers of PHA depolymerases resemble those of lipases encouraged different researchers to study the activity of PHA depolymerases in anhydrous media.In this work 19 MCL-PHA degrading microorganisms were isolated from natural sources. Out of those nineteen isolates, eight Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyse other biodegradable plastics such as SCL-PHAs, poly(¿-caprolactone) (PCL), poly(ethylene succinate) (PES) and poly(L-lactide) [P(L-LA)] was studied. Only the strains belonging to Streptomyces genera showed the ability to degrade not only MCL-PHAs, but also SCL-PHAs and PCL. However, none of the isolates were able to hydrolyze PES and P(L-LA).Motivated by the little information available in the literature about the MCL-PHA depolymerase activity of Gram-positive bacteria, the bacterium Streptomyces venezuelae SO1 was selected, and its extracellular depolymerase was purified and characterized. The enzyme showed a molecular mass of 27 kDa, with a pI value of 5.9. Its maximum activity was observed at pH 9 and 50ºC, and retained more than 70% of its initial activity after 8 h at 40 ºC. The purified enzyme hydrolyzed poly(3-hydroxyoctanoic acid) P(3HO) and PCL, but not poly(3-hydroxybutyric acid), PES and P(L-LA). 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. However, the enzyme did not show lipase activity. The enzymic activity was markedly enhanced in the presence of low concentrations of detergents and organic solvents, being inhibited by dithiothreitol and EDTA. The MCL-PHA depolymerase from S. venezuelae SO1 showed higher thermostability and wider substrate specificity than others MCL-PHA depolymerases from Pseudomonas strains already known.To evaluate the biotechnological application of the MCL-PHA depolymerase, its ability to produce (R)-3-hydroxyoctanoate in aqueous media or to catalyze ester-forming reactions in anhydrous media was investigated. In this sense, the enzyme catalyzed the hydrolysis of P(3HO) to monomeric units after 3 days at 30ºC. Moreover, the MCL-PHA depolymerase of S. venezuelae SO1 was identified as a new enzyme for polymer synthesis showing different specificity and other valuable attributes in comparison to the well known lipase biocatalyst Novozyme 435. The enzyme catalyzed the ring-opening polymerization of racemic ¿-butyrolactone, L- and D-lactide with high yield resulting in low molecular weight polymers. On the other hand, ¿-caprolactone and pentadecalactone, which show high polymerizability using Novozyme 435 as catalyst, were not polymerized by the MCL-PHA depolymerase. Apart from this, the enzyme from S. venezuelae SO1 slightly promoted the transacylation of substrates showing an alkyl moiety, such as lauric acid and ethyl-3-hydroxyoctanoate, yielding only 8% and 9% of conversion after 72 and 48 h, respectively.On the other hand, the MCL-PHA depolymerase from S. venezuelae SO1 was subjected to de novo-sequencing and the deduced peptide sequences were used to identify novel MCL-PHA depolymerases from several Actinobacteria. All these enzymes shared high similarity in amino acid sequences but they did not show homology to already characterized MCL-PHA depolymerases. These results suggest that these distinct enzymes might represent a new family of MCL-PHA depolymerases. Additionally, these results offer the possibility of cloning and expressing these distinct enzymes for their possible use in biotechnological processes.