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

2012 ◽  
Vol 78 (17) ◽  
pp. 6017-6026 ◽  
Author(s):  
Virginia Martínez ◽  
Fernando de la Peña ◽  
Javier García-Hidalgo ◽  
Isabel de la Mata ◽  
José Luis García ◽  
...  
Keyword(s):  
Chain Length ◽  
Large Set ◽  
Serine Hydrolase ◽  
Bdellovibrio Bacteriovorus ◽  
Short Chain Length ◽  
Content Type ◽  
Pha Depolymerase ◽  
Biochemical Evidence ◽  
Medium Chain ◽  
Medium Chain Length

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.

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

2012 ◽  
Vol 78 (20) ◽  
pp. 7229-7237 ◽  
Author(s):  
Joana Gangoiti ◽  
Marta Santos ◽  
María Auxiliadora Prieto ◽  
Isabel de la Mata ◽  
Juan L. Serra ◽  
...  
Keyword(s):  
Chain Length ◽  
De Novo ◽  
Hydrolysis Product ◽  
Maximum Activity ◽  
Short Chain Length ◽  
Great Ability ◽  
Content Type ◽  
Pha Depolymerase ◽  
Medium Chain ◽  
Medium Chain Length

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.

scholarly journals Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442

2006 ◽  
Vol 282 (7) ◽  
pp. 4951-4962 ◽  
Author(s):  
Laura I. de Eugenio ◽  
Pedro García ◽  
José M. Luengo ◽  
Jesús M. Sanz ◽  
Julio San Román ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Chain Length ◽  
Biochemical Characterization ◽  
Current Knowledge ◽  
Dimensional Structure ◽  
Serine Hydrolase ◽  
Short Chain Length ◽  
Intracellular Enzyme ◽  
Medium Chain ◽  
Medium Chain Length

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a coreα/β hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.

scholarly journals Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum

2016 ◽  
Vol 82 (20) ◽  
pp. 6132-6140 ◽  
Author(s):  
Daniel Heinrich ◽  
Matthias Raberg ◽  
Philipp Fricke ◽  
Shane T. Kenny ◽  
Laura Morales-Gamez ◽  
...  
Keyword(s):  
Chain Length ◽  
Synthesis Gas ◽  
Rhodospirillum Rubrum ◽  
Molar Fraction ◽  
Human Nutrition ◽  
Production Costs ◽  
Short Chain Length ◽  
Content Type ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTThe purple nonsulfur alphaproteobacteriumRhodospirillum rubrumS1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes fromPseudomonas putidaKT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooFfromR. rubrumS1 in a PHA-negative mutant ofR. rubrum. P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooFpromoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Placpromoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion ofphaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCLaccumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition.IMPORTANCEPolyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacteriumRhodospirillum rubrumwas genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.

scholarly journals Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

2013 ◽  
Vol 79 (12) ◽  
pp. 3813-3821 ◽  
Author(s):  
Jo-Ann Chuah ◽  
Satoshi Tomizawa ◽  
Miwa Yamada ◽  
Takeharu Tsuge ◽  
Yoshiharu Doi ◽  
...  
Keyword(s):  
Chain Length ◽  
Fold Increase ◽  
Short Chain ◽  
Pha Synthase ◽  
Wild Type ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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