Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

ABSTRACTNineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters,Streptomyces roseolusSL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin andp-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured withpNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of severalActinobacteriastrains, includingS. roseolusSL3, were identified on the basis of the peptidede novosequencing of theStreptomyces venezuelaeSO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.

Identification and Biochemical Evidence of a Medium-Chain-Length Polyhydroxyalkanoate Depolymerase in the Bdellovibrio bacteriovorus Predatory Hydrolytic Arsenal

ABSTRACTThe obligate predatorBdellovibrio bacteriovorusHD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 ofB. bacteriovorusHD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBdsuggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBdhas been extracellularly produced using different hypersecretor Tol-pal mutants ofEscherichia coliandPseudomonas putidaas recombinant hosts. The recombinant PhaZBdhas been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBdis an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

Enzymatic processes for biodegradation of poly(hydroxyalkanoate)s crystals

Poly(hydroxyalkanoate)s (PHAs) have attracted much attention as environmentally compatible polymeric materials that can be produced from renewable carbon resources. Biodegradation of PHA materials occurs by the function of extracellular PHA depolymerase secreted from microorganisms. Thus, elucidation of the enzymatic degradation mechanism for PHA materials is important to design PHA materials with desirable properties and controlled biodegradability. The solid PHA polymer is a water-insoluble substrate but PHA depolymerases are soluble in water. Therefore, the enzymatic degradation of PHA materials is a heterogeneous reaction on the material’s surface. Two distinct processes are involved during the degradation, namely, adsorption of the enzyme on the surface of PHA material and the subsequent hydrolysis of polymer chains. Atomic force microscopy (AFM) is a powerful tool that has been used for the quantitative analysis of PHA crystal degradation. AFM enables the characterization of the crystal surface nanostructure in a buffer solution. By using in-situ (real-time) AFM observations, we recently succeeded in observing the degradation processes of PHA crystals. Subsequently, we were also able to investigate the degradation rates of PHA crystals using the same technique. In this review, we have attempted to give an overview concerning the direct visualization of the adsorption, as well as the hydrolysis reactions of PHA depolymerases at the nanometer scale. In addition, we present other analytical techniques besides AFM as a complimentary approach to analyze the effect of enzyme adsorption on PHA crystals.Key words: poly(hydroxyalkanoate) (PHA), enzymatic degradation, lamellar crystal, PHA depolymerase.