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1.
Nutritional flexibility of oligotrophic and copiotrophic Antarctic bacteria with respect to organic substrates 总被引:1,自引:0,他引:1
Abstract Alcaligenes eutrophus can accumulate poly-3-hydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) containing only 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) units. Granule-associated PHB-synthase was active with d (−)-3-hydroxybutyryl-CoA and d (−)-3-hydroxyvaleryl-CoA of the range of d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C4 –C10 ). In carbon-limited cultures, PHB-synthase was predominantly soluble, becoming granule-associated on transition to nitrogen limitation. Granule-associated PHB-synthase increased in activity at least up to pH 10.0 and K m values of 0.68 mM and 1.63 mM were determined for the C4 and C5 substrates, respectively, at pH 8.5. The soluble PHB-synthase, which was unstable, showed equal activity in the range pH 8.0–10.0, had a K m value for d (−)-3-hydroxybutyryl-CoA of 0.72 mM and an M r of 160,000. PHB does not measurably turn over under steady-state polymer-accumulating conditions. 相似文献
2.
A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP(+)-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of gamma-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD(+)-dependent succinate semialdehyde dehydrogenase in R. eutropha. 相似文献
3.
A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin. 相似文献
4.
A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample.
This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular
MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and
gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7.
The maximum activity was observed at pH 8.7 and 50 °C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain.
The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of
the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
5.
Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight 总被引:7,自引:0,他引:7
Bohmert K Balbo I Kopka J Mittendorf V Nawrath C Poirier Y Tischendorf G Trethewey RN Willmitzer L 《Planta》2000,211(6):841-845
Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described.
These plants accumulated more than 4% of their fresh weight (≈40% of their dry weight) in the form of PHB in leaf chloroplasts.
These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of
a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis
in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating
the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe
effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing
lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that
the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount
of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and
sugar alcohols.
Received: 2 February 2000 / Accepted: 31 March 2000 相似文献
6.
Jieun Kim Jeong Ho Chang Eun-Jung Kim Kyung-Jin Kim 《Biochemical and biophysical research communications》2014
(R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha H16 (RePhaB) is an enzyme that catalyzes the NADPH-dependent reduction of acetoacetyl-CoA, an intermediate of polyhydroxyalkanoates (PHA) synthetic pathways. Polymeric PHA is used to make bioplastics, implant biomaterials, and biofuels. Here, we report the crystal structures of RePhaB apoenzyme and in complex with either NADP+ or acetoacetyl-CoA, which provide the catalytic mechanism of the protein. RePhaB contains a Rossmann fold and a Clamp domain for binding of NADP+ and acetoacetyl-CoA, respectively. The NADP+-bound form of RePhaB structure reveals that the protein has a unique cofactor binding mode. Interestingly, in the RePhaB structure in complex with acetoacetyl-CoA, the conformation of the Clamp domain, especially the Clamp-lid, undergoes a large structural change about 4.6 Å leading to formation of the substrate pocket. These structural observations, along with the biochemical experiments, suggest that movement of the Clamp-lid enables the substrate binding and ensures the acetoacetyl moiety is located near to the nicotinamide ring of NADP+. 相似文献
7.
Porwal S Kumar T Lal S Rani A Kumar S Cheema S Purohit HJ Sharma R Singh Patel SK Kalia VC 《Bioresource technology》2008,99(13):5444-5451
Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H2) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H2 constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H2 producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H2 – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium. 相似文献
8.
Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K2HPO4 (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K2HPO4/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K2HPO4 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K2HPO4 1.0 g/l and inoculum 150.58 ml/l. 相似文献
9.
Chitwadee Phithakrotchanakoon Ratama Daduang Arinthip Thamchaipenet Thidarat Wangkam Toemsak Srikhirin Lily Eurwilaichitr Verawat Champreda 《Applied microbiology and biotechnology》2009,82(1):131-140
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase
containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights
of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V
max and K
m of 3.63 ± 0.16 μmol min−1 mg−1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that
PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm−2 h−1 for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films
was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique
will be of utility for further study of bioplastic degradation. 相似文献
10.
Tom Defoirdt Nico Boon Patrick Sorgeloos Willy Verstraete Peter Bossier 《Biotechnology advances》2009,27(6):680-685
Because of the risk of antibiotic resistance development, there is a growing awareness that antibiotics should be used more carefully in animal production. However, a decreased use of antibiotics could result in a higher frequency of pathogenic bacteria, which in its turn could lead to a higher incidence of infections. Short-chain fatty acids (SCFAs) have long been known to exhibit bacteriostatic activity. These compounds also specifically downregulate virulence factor expression and positively influence the gastrointestinal health of the host. As a consequence, there is currently considerable interest in SCFAs as biocontrol agents in animal production. Polyhydroxyalkanoates (PHAs) are polymers of β-hydroxy short-chain fatty acids. Currently, PHAs are applied as replacements for synthetic polymers. These biopolymers can be depolymerised by many different microorganisms that produce extracellular PHA depolymerases. Interestingly, different studies provided some evidence that PHAs can also be degraded upon passage through the gastrointestinal tract of animals and consequently, adding these compounds to the feed might result in biocontrol effects similar to those described for SCFAs. 相似文献