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1.
Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined
in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on
the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA
copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting
of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene,
recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications. 相似文献
2.
Yingda Wang Shijun Qian Guangzhen Meng Shuzheng Zhang 《Applied biochemistry and biotechnology》2001,95(2):93-101
The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved
regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed
in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid
and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The
recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data. 相似文献
3.
Huddedar SB Shete AM Tilekar JN Gore SD Dhavale DD Chopade BA 《Applied biochemistry and biotechnology》2002,102(1-6):21-39
Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight
Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to
be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobacter genospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography.
Purified IAA was confirmed by 1H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth
inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of
Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 × 10−5, and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead.
This is the first report of plasmid-encoded IAA production in the genus Acinetobacter. 相似文献
4.
Hong Kui Leung Yun Chung Kwok Sui Yi Law Kin Ho Lo Wai Hung Chua Hong Yu Peter Hoi Fu 《Applied biochemistry and biotechnology》2000,84(1-9):381-390
Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain,
are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ70 promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS−, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively. 相似文献
5.
A recombinant plasmid pFS239 containing the geoe coding for K99 antigen of Escherichia coli and wide-host-range plasmid pKT230 has been cloned in E. coli C600. pFS239 has been transferred to Yersinia enterocolitica strains D29, L15 and L15 (pYV15) through triparental mating. In Y. enterocolitica transconjugants the expression of VW antigens and calcium dependence which represent the propertics associated with the virulence plastmid of Y. enterocolitica remains unchanged. 相似文献
6.
An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of
RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose,
(R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers
by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced. 相似文献
7.
The thermogenic curves of metabolism of the four strains of Escherichia coli pUC19cab/JM109, pUC19cab/XL-IBlue, JM109 and XL-IBlue were determined using the LKB-2277 BioActivity Monitor and the ampoule
method at 37°C. The pUC19cab/JM109 and pUC19cab/XL-IBlue are recombinant E. coli strains bearing the same foreign plasmid pUC19cab, which confers the ability to produce polyhydroxyalkanoates (PHAs). The
yield of PHAs of pUC19cab/XL-IBlue was higher than that of pUC19cab/JM109. XL-IBlue and JM109 were the host bacteria. The
heat flow of these strains was XL-Blue≈JM109>pUC19cab/JM109>pUC19cab/XL-IBlue. These results indicate an obvious interrelation
between the PHAs production and the heat flow rate of E. coli strains. 相似文献
8.
L. N. Yang F. Xu L. X. Sun Z. C. Tan H. D. Tan Z. B. Zhao J. G. Liang 《Journal of Thermal Analysis and Calorimetry》2006,85(3):807-810
A microcalorimetric
technique based on the bacterial heat output was applied to evaluate the influence
of antibiotics PIP (Piperacillin Sodium)
and composite preparation of PIP and SBT (Sulbactam
Sodium) on the growth of E. coli
DH5α. The power–time curves of the growth metabolism of E. coli DH5α were studied using a TAM Air Isothermal
Microcalorimeter at 37°C. By analyzing the power–time curves, the
parameters such as growth rate constants (k),
inhibitory ratio (I), the maximum heat
power (P
m) and the
time of the maximum heat power (t
m)
were obtained. The results show that different concentrations of antibiotics
affect the growth metabolism of E. coli
DH5α. The PIP in the concentration range of 0–0.05 μg mL–1
has a stimulatory effect on the E. coli
DH5α growth, while the PIP of higher concentrations (0.05 –0.25
μg mL–1) can inhibit its growth. It seems
that the composite preparation composed of PIP and SBT cannot improve the
inhibitory effect on E. coli DH5α
as compared with the PIP. 相似文献
9.
Kaijun Xiang Yan Qiao Chi Bun Ching Chang Ming Li 《Electrochemistry communications》2009,11(8):1593-1595
Faster electron transfer between bacteria and electrodes in microbial fuel cells can significantly improve the power density of MFCs for practical applications. A recombinant Escherichia coli (E. coli) strain overexpressing glycerol dehydrogenase (GldA) was engineered as the MFC biocatalyst instead of the natural bacteria. Efficient mediators were produced in the fuel cell with this engineered E. coli resulting in lower polarization and much higher power density than with natural E. coli and E. coli with electro-evolved mediators. For the first time, we demonstrate that engineering E. coli by introduction of appropriate oxidoreductase via gene manipulation can greatly improve the rate of electron transfer. This work provides an efficient and economic approach to biologically engineering bacteria for improving MFC performance. 相似文献
10.
Dien Bruce S. Nichols Nancy N. O'Bryan Patricia J. Bothast Rodney J. 《Applied biochemistry and biotechnology》2000,84(1-9):181-196
Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLO1297, which contains the genes
from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically.
Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but noselective
antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLO1297 in anaerobic culture. The
fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from
stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures.
Fermentations were completed within 60 h and ethanol yields were 86–92% of theoretical. Maximal ethanol concentrations were
3.9–4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v)
total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with
FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable.
Author to whom all correspondence and reprint requests should be addressed.
Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard
of the product, and the use of the name by USDA im plies no approval of the product to the exclusion of others that may also
be suitable. 相似文献
11.
《Analytical letters》2012,45(9):2155-2166
Abstract A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 106?108 cells·cm?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 105 cells · cm?3. 相似文献
12.
Gagik G. Gurzadyan Helmut Gorner Dietrich Schulte-Frohlinde 《Photochemistry and photobiology》1993,58(4):477-485
Plasmid pTZ18R and calf thymus DNA in aerated neutral aqueous solution were irradiated by continuous 254 nm light. The quantum yields are φssb= 4.0 × 10-5 and φdsb= 1.4 × 10-6 for single- and double-strand break formation, respectively, φbr= 2.3 × 10-5 for base release, φdn= 2.1 × 10-3 for destruction of nucleotides, and φicl×φlds× 1 × 10-6 for interstrand cross-links and locally denatured sites, respectively. The presence of Tris-HCI/ ethylenediaminetetraacetic acid (10:1, pH 7.5) buffer strongly reduces φssb, The corresponding φ values, obtained on employing pulsed 193 nm laser irradiation, are much larger than those using λirr, = 254 nm. This is ascribed to a contribution of chemical reactions induced by photoionization, which is absent for 254 nm irradiation. The quantum yields of inactivation of plasmid DNA (λirr= 254 nm) were measured by transformation of the Escherichia coli strains AB1157 (wild type), φina(1157) = 1.6 × 10-4, AB1886 (uvr-), φina(1886) = 4.2 × 10-4, AB2463 (rec-), φina(2463) = 4.1 × 10-4 and AB2480 (uvr- rec-), φina(2480) = 3.1 × 10-3. The quantum yields of inactivation of plasmid DNA are compared with those of the four E. coli strains (denoted as chromosomal DNA inactivation) obtained from the literature. The results for E. coli strain AB2480 show that the chromosomal DNA and the plasmid DNA are both inactivated by a single pyrimidine photodimer per genome. With the E. coli strain AB2463 inactivation of plasmid and chromosomal DNA is the same for the same total damage per genome and is ~ 10 times smaller than for AB2480. This is explained by photodimer repair in chromosomal and plasmid DNA and by the absence of dsb repair in both cases. In the repair wild-type strain AB1157, inactivation of the plasmid DNA is roughly 100 times higher than that of the chromosomal DNA. We postulate that a portion of this difference is due to repair of dsb by the recA system in chromosomal DNA and that such repair does not take place in the plasmid DNA. The biological results from 254 nm irradiation are compared with those from 193 nm laser irradiation. 相似文献
13.
Byoung Chul Park Sukhoon Koh Changsoo Chang Se Won Suh Dae-Sil Lee Si Myung Byun 《Applied biochemistry and biotechnology》1997,62(1):15-27
The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped
in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified
enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native
condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability
tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation
is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than
those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations
such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity. 相似文献
14.
化学需氧量(CODCr)与总有机碳(TOC)指标均可用来表征废水的有机物污染程度.不过,以CODCr表征有机物污染程度一般只能反映水中部分有机物的量,而TOC能更全面地反映废水中有机物的含量.以丙烯酸生产外排废水为研究对象,分析结果表明:在一定范围内,废水中CODCr与TOC满足关系式y=1.147 4x+74.75(R2=0.955 26),回收率范围98%~115%,TOC测定结果的精密度高、可靠、准确,在一定条件下,可使用TOC来间接换算CODCr. 相似文献
15.
The roles of the rel+ gene and of 4-thiouridine in killing and photoprotection of Escherichia coli by near-ultraviolet radiation 总被引:3,自引:0,他引:3
Abstract— Photoprotection is a reduction in response to far-UV (190–300. nm) radiation in cells that have been previously exposed to longer wavelengths. It has been proposed that photoprotection operates by means of a growth delay that permits more time for dark repair. Growth delay in Escherichia coli utilizes 4-thiouridine (4Srd) in transfer RNA as a chromophore and it requires the rel+ gene, which exerts a stringent control upon RNA synthesis. Mutants that were either rel or 4Srd? were isolated from E. coli B, utilizing a near-UV-induced growth-delay selection technique. The rel mutants, which undergo little growth delay after near-UV irradiation, show only 50% as much photoprotection as wild types, while 4Srd? mutants show no photoprotection at all. Thus, photoprotection appears to utilize 4Srd as its sole chromophore in E. coli B and B/r, and no more than 50% of photoprotection in these strains can be a result of near-UV-induced growth delay. 相似文献
16.
A. Abderrahmane L. Yi G. Wen-Ying S. Ping Q. Song-Sheng 《Journal of Thermal Analysis and Calorimetry》2002,68(3):909-916
In this paper, a promoter-probe plasmid pKK232-8 was used as a vector, which functioned in Escherichia coli TG1 host. The plasmid DNA fragments from Pseudomonas maltophilia AT18 chromosome DNA active as promoter inEscherichia coli TG1, the promoter function was studied by means of microcalorimetry, the promoter is about 800 bp DNA, it can promote the chloramphenicol
(Cm) gene in plasmid pKK232-8, the Cm resistance level is about 80 μg mL–1, the promoter activity is high. It implicates that there are probably many promoters in Pseudomonas maltophilia AT18 chromosome. All these information is readily obtained by an LKB 2277-204 heat conduction microcalorimeter. Microcalorimetry
is a quantitative, inexpensive, and versatile method for microbiological genetic research.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
17.
Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed
by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted
in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa.
The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up. 相似文献
18.
Radiocomplexation of fleroxacin (FXN) with technetium-99m and its characterization in terms of in vitro stability in saline
and serum solutions, in vitro binding with live and heat-killed Escherichia coli, and biodistribution in male Wistar rats (MWR) artificially infected with live and heat-killed E. coli was studied. The 99mTc-FXN complex showed a radiochemical purity (RCP) yield of 98.10 ± 0.24% at 30 min using 125 μg of stannous fluoride, 74 MBq
of sodium pertechnetate, and 2 mg of FXN. The complex was found to be more than 90% stable up to 4 h after constitution in
normal saline. In serum, the emergence of 16.50% undesirable species was observed within 16 h of incubation at 37 °C. The
99mTc-FXN complex showed saturated in vitro binding with E. coli with a maximum value of 65.00% at 90 min. A fivefold increase in uptake of the complex was noted in the infected when compared
with the inflamed and normal muscle of the MWR infected with live E. coli. The stable radiochemical profile in saline and serum, saturated in vitro binding with E. coli and increased uptake in the infected muscle, confirmed the potential of the 99mTc-FXN complex as an E. coli infection imaging agent. 相似文献
19.
Han Seung Chae Kong-Hwan Kim Sun Chang Kim Pyung Cheon Lee 《Applied biochemistry and biotechnology》2010,162(8):2333-2344
Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order
to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene
and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene
formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids,
also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer
for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest
volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion,
we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information
can serve as the basis for the subsequent development of microorganisms for carotenoids of interest. 相似文献
20.
Kin-Ho Law Pui-Ling Chan Wai-Sum Lau Yin-Chung Cheng Yun-Chung Leung Wai-Hung Lo Hugh Lawford Hoi-Fu Yu 《Applied biochemistry and biotechnology》2004,114(1-3):361-372
Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One
of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable
properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry
weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor
culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed
by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated
clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior
E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry
and medicine. 相似文献