RESUMO
Bacteria isolated from marine sediments were screened for their ability to accumulate polyhydroxyalkanoates. Among the isolates, four Vibrio spp. (strain M11, M14, M20 and M31) were studied in detail. All synthesized intracellular lipid inclusions during growth on diverse carbon sources including acetate, glycerol, succinate, glucose and sucrose. The inclusions were identified to be poly-beta-hydroxybutyrate (PHB) using gas chromatography and nuclear magnetic resonance analysis. No other type of polyhydroxyalkanoates (PHAs) was found to be accumulated by these marine isolates, suggesting that the diversity of PHAs produced in marine environments may be not as versatile as found in other environments. Strain M11 accumulated PHB in concentrations as high as 41% of cell dry weight when grown in medium containing 4% of sodium chloride. One of the Vibrio spp. was identified to be closely related to Vibrio natriegens (98% identity) by partial 16S rDNA sequence homology. V. natriegens has the shortest generation time (9.8 min) of any bacterium and this characteristic may be an exploitable trait for the industrial production of PHB.
Assuntos
Sedimentos Geológicos/microbiologia , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Vibrio/isolamento & purificação , Vibrio/metabolismo , Carbono/farmacologia , Meios de Cultura , Hidroxibutiratos/isolamento & purificação , Microscopia Eletrônica de Transmissão , Filogenia , Poliésteres/isolamento & purificação , Sais/farmacologia , Vibrio/crescimento & desenvolvimento , Vibrio/ultraestruturaRESUMO
The lectin receptors concanavalin A (ConA) and wheat germ lectin (WGL) were successfully conjugated to a thermo-responsive hydrogel polymer (poly-N-isopropylacrylamide) (PNIPAAm) as an affinity ligand. The coupling efficiencies of the ConA and WGL to PNIPAAm were 5.1% and 44%, respectively. These lectin receptor-polymer conjugates were then tested for their efficiency in purification of various polysaccharides or polysaccharide-containing compounds such as beta-glucan. Results indicated that these conjugates separated various polysaccharides from a complex mixture. The use of a thermo-responsive polymer in low-temperature purification of potentially heat-labile glycoproteins is advantageous. Additionally, other affinity ligands could be coupled to the polymer for separation of the respective bioactive molecules.
Assuntos
Polímeros/química , Polissacarídeos/química , Resinas Acrílicas/química , Concanavalina A/química , Glucanos/química , Hidrogel de Polietilenoglicol-Dimetacrilato , Lectinas/química , Ligantes , Oligossacarídeos , Ligação Proteica , Temperatura , Fatores de Tempo , Aglutininas do Germe de Trigo/químicaRESUMO
Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.
Assuntos
Proteínas de Bactérias/genética , Coenzima A Ligases/genética , Cupriavidus necator/genética , Proteínas de Membrana Transportadoras/genética , Ácidos Pentanoicos/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Coenzima A Ligases/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Cupriavidus necator/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucose/metabolismo , Hidroxibutiratos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Óperon , Poliésteres/metabolismo , Propionatos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66~70% of total cell material of these recombinant strains.