Title: 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 Martinez V, de Santos PG, Garcia-Hidalgo J, Hormigo D, Prieto MA, Arroyo M, de la Mata I Ref: Applied Microbiology & Biotechnology, 99:9605, 2015 : PubMed
Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % alpha-helix, 43.9 % beta-sheet, 19.4 % beta-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the alpha/beta hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 degrees C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.
Title: Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal Martinez V, de la Pena F, Garcia-Hidalgo J, de la Mata I, Garcia JL, Prieto MA Ref: Applied Environmental Microbiology, 78:6017, 2012 : PubMed
The obligate predator Bdellovibrio bacteriovorus 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 B bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate mcl-PHA depolymerase PhaZ(Bd) The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the alpha/beta-hydrolase fold family and has a typical serine hydrolase catalytic triad serine-histidine-aspartic acid in agreement with other PHA depolymerases and lipases PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts The recombinant PhaZ(Bd 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(Bd) 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.
The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) x 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.
Title: The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J Ref: BMC Bioinformatics, 10:89, 2009 : PubMed
BACKGROUND: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases. RESULTS: The PHA Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) has been established as a tool for systematic analysis of this enzyme family. The DED contains sequence entries of 587 PHA depolymerases, which were assigned to 8 superfamilies and 38 homologous families based on their sequence similarity. For each family, multiple sequence alignments and profile hidden Markov models are provided, and functionally relevant residues are annotated. CONCLUSION: The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.
Cholinergic neurons originating from the basal forebrain innervate the entire cortical mantle. Choline-sensitive microelectrodes were used to measure the synaptic release of cortical acetylcholine (ACh) at a subsecond resolution in rats performing a task involving the detection of cues. Cues that were detected, defined behaviorally, evoked transient increases in cholinergic activity (at the scale of seconds) in the medial prefrontal cortex (mPFC), but not in a nonassociational control region (motor cortex). In trials involving missed cues, cholinergic transients were not observed. Cholinergic deafferentation of the mPFC, but not motor cortex, impaired cue detection. Furthermore, decreases and increases in precue cholinergic activity predicted subsequent cue detection or misses, respectively. Finally, cue-evoked cholinergic transients were superimposed over slower (at the timescale of minutes) changes in cholinergic activity. Cortical cholinergic neurotransmission is regulated on multiple timescales to mediate the detection of behaviorally significant cues and to support cognitive performance.
