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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The obligate predator<jats:named-content content-type="genus-species">Bdellovibrio bacteriovorus</jats:named-content>HD100 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 of<jats:named-content content-type="genus-species">B. bacteriovorus</jats:named-content>HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ<jats:sub>Bd</jats:sub>). The primary structure of PhaZ<jats:sub>Bd</jats:sub>suggests 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. PhaZ<jats:sub>Bd</jats:sub>has been extracellularly produced using different hypersecretor Tol-pal mutants of<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>and<jats:named-content content-type="genus-species">Pseudomonas putida</jats:named-content>as recombinant hosts. The recombinant PhaZ<jats:sub>Bd</jats:sub>has 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. PhaZ<jats:sub>Bd</jats:sub>is 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.</jats:p>

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

“…In both cases, plates were incubated for 48 h at 30°C. On the other hand, extracellular depolymerase activity was measured by the quick and simple qualitative spot test method as previously described (Martínez et al 2012) but in 50 mM Tris-HCl buffer, pH 8. Finally, depolymerase activity of the recombinant purified enzyme was determined by the quantitative standard turbidimetric assay (Martínez et al 2012) measuring the decrease of the turbidity of a homogeneous P(HO-co-HH) suspension with slight modifications.…”

“…On the other hand, extracellular depolymerase activity was measured by the quick and simple qualitative spot test method as previously described (Martínez et al 2012) but in 50 mM Tris-HCl buffer, pH 8. Finally, depolymerase activity of the recombinant purified enzyme was determined by the quantitative standard turbidimetric assay (Martínez et al 2012) measuring the decrease of the turbidity of a homogeneous P(HO-co-HH) suspension with slight modifications. Five hundred microliters of standard reaction mixture contained 15 mM Glycine-NaOH buffer, pH 10, and 1 mg mL −1 of sonicated homogenous suspension of P(HO-co-HH) in deionized water were preincubated at 30°C for 3 min; then, the reaction was started by the addition of 2 μg of pure enzyme and after 30 min of incubation at 30°C, the turbidity decrease at 600 nm was measured.…”

“…Indeed, mcl-PHA degrading microorganisms are less frequently found in the environment and few mcl-PHA depolymerases have been described so far. Most of these mcl-PHA depolymerases belong to Gram-negative bacteria, predominantly Pseudomonas species (Schirmer and Jendrossek 1994;Gangoiti et al 2010;Jendrossek and Handrick 2002), although an extracellularlike mcl-PHA depolymerase has been characterized in the obligated predator Bdellovibrio bacteriovorus HD100 (Martínez et al 2012). Interestingly, a novel subgroup of mcl-PHA depolymerases has been recently identified in Actinobacteria, as those from Streptomyces roseolus SL3 (Gangoiti et al 2012) and Streptomyces venezuelae SO1 (Santos et al 2013).…”

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

“…Several mcl PHA depolymerases have been biochemically characterized. However, only the PhaZ PflGK13 -coding gene and a few other homologous genes have been cloned and sequenced (21,29,37,50), including an mcl PHA depolymerase from the predator Bdellovibrio bacteriovorus (31). Additionally, in a recent work, a significant different gene from a thermophilic bacterium, T. thermophilus HB8, has been identified (36).…”

“…Currently, very few mcl PHA depolymerases have been characterized in comparison to the number of scl PHA depolymerases studied (26). To date, most of the mcl PHA depolymerases reported belong to Gram-negative bacteria, predominantly Pseudomonas species (22,31). The poly(3-hydroxyoctanoate) depolymerase from Pseudomonas fluorescens GK13 (PhaZ PflGK13 ) was the first mcl PHA depolymerase studied in detail at the molecular level (49)(50)(51).…”