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1.
Arief Budi Witarto Takafumi Ohtera Koji Sode 《Applied biochemistry and biotechnology》1999,77(1-3):159-168
We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made
up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried
out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser 771, of a chimeric PQQGDHs on
their thermal stability. A series of Ser 771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction
governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97 A3 acquired stability
at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed.
These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining
the overall structure of β-propeller proteins. 相似文献
2.
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant,
and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have
been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone
synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein
in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at
OD600 induction time of 0.5 was found to be the best condition for chalcone synthase secretion. 相似文献
3.
The biological activity of a kind of hetero-bimetallic Schiff-base complex was studied using Escherichia coli (E. coli) cell as the target. By microcalorimetry, the difference of anti-bacterial activity between the binuclear Schiff-base and
the ligand was determined and analyzed. To analyze the inhibition of the bacterial growth internally, the E. coli cells grown in the presence of hetero-bimetallic Schiff-base complex were observed by scanning electron microscopy. The images
in high resolution revealed the damage of outer cell membrane caused the inhibitory effect on E. coli. Inductively coupled plasma-mass spectrometry results proved the absorption of the complex by cells, which confirmed the
interaction between the Schiff-base and biological macromolecule. 相似文献
4.
Sunhee Lee Yeontae Jung Seunghan Lee Jinwon Lee 《Applied biochemistry and biotechnology》2013,169(5):1606-1619
Microorganisms have been used for biodiesel (fatty acid methyl ester) production due to their significant environmental and economic benefits. The aim of the present research was to develop new strains of Escherichia coli K-12 MG1655 and to increase the content of long-chain fatty acids by overexpressing essential enzymes that are involved in the fatty acid synthase elongation cycle. In addition, the relationship of β-ketoacyl-acyl carrier protein (ACP) synthase (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydrase (fabZ), and β-enoyl-ACP reductase (fabI) with respect to fatty acid production was investigated. The four enzymes play a unique role in fatty acid biosynthesis and elongation processes. We report the generation of recombinant E. coli strains that produced long-chain fatty acids to amounts twofold over wild type. To verify the results, NAD+/NADH ratios and glucose analyses were performed. We also confirmed that FabZ plays an important role in producing unsaturated fatty acids (UFAs) as E. coli SGJS25 (overexpressing the fabZ gene) produced the highest percentage of UFAs (35 % of total long-chain fatty acids), over wild type and other recombinants. Indeed, cis-9-hexadecenoic acid, a major UFA in E. coli SGJS25, was produced at levels 20-fold higher than in wild type after 20 h in culture. The biochemically engineered E. coli presented in this study is expected to be more economical for producing long-chain fatty acids in quality biodiesel production processes. 相似文献
5.
Fabrication of an Electrochemical E. coli Biosensor in Biowells Using Bimetallic Nanoparticle‐Labelled Antibodies 下载免费PDF全文
Haslet Eksi Remziye Güzel Burcu Güven Ismail Hakki Boyaci Ali Osman Solak 《Electroanalysis》2015,27(2):343-352
An electrochemical biosensor was developed for the determination of Escherichia coli (E. coli) in water. For this purpose, silver‐gold core‐shell (Ag@Au) bioconjugates and anti‐E. coli modified PS‐microwells were designed in a sandwich‐type format in order to obtain higher sensitivity and selectivity. Ag@Au bimetallic nanoparticles were synthesized by co‐reduction method. The core‐shell formation was analyzed by using UV‐Vis spectroscopy and transmission electron microscopy. Biotin labeled anti‐E. coli antibodies were coupled with Ag@Au nanoparticles to form bioconjugates. The electrochemical immunosensor was prepared by immobilizing anti‐E. coli on polystyrene (PS)‐microwells via chemical bonding. These modified microwells were identified with X‐ray photoelectron spectroscopy and surface enhanced Raman spectroscopy. E. coli was sandwiched between Ag@Au bioconjugates and anti‐E. coli on PS‐microwells at different concentrations. The relationship between the E. coli concentration and stripping current of gold ions (Au3+) were investigated by square wave anodic stripping voltammetry at pencil graphite electrode. The proposed method can provide some advantages such as lower detection limit and shorter detection time. The electrochemical response for the immunosensor was linear with the concentration of the E. coli in the range of 101 and 105 cfu/mL with a limit of detection 3 cfu/mL. The procedure maintains good sensitivity and repeatability and also offers utility in the fields of environmental monitoring and clinical diagnosis. 相似文献
6.
Luciana C. Costa Mônica R. C. Marques Rodrigo B. Tiosso Jefferson P. Cantarim Fábio Merçon 《Macromolecular Symposia》2012,319(1):121-128
Summary: In this study we report both the development of dithiocarbamate resins from the commercial hypercrosslinked resin MN-250 and the evaluation of the biocidal capacity of this material against E. coli ATCC25922 suspensions. The preparation of dithiocarbamate resins followed a synthetic pathway based on nitration of resins, reduction of nitro groups and reaction with CS2 in an alkaline medium. The biocidal capacity was evaluated by means of elution of E. coli suspensions (103–107 cells/mL) through columns containing the resin and plating on LB nutrient medium solidified with Bacto agar. We can conclude that hypercrosslinked resins with dithiocarbamate groups have potential biocidal action. 相似文献
7.
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
8.
Bennani M Badri S Baibai T Oubrim N Hassar M Cohen N Amarouch H 《Applied biochemistry and biotechnology》2011,165(1):290-299
Shiga toxin Escherichia coli (STEC), also called verotoxin-producing E. coli, is a major cause of food-borne illness, capable of causing hemorrhagic colitis and hemolytic–uremic syndrome (HUS). This
study was carried out to evaluate the presence of (STEC) and E. coli O157:H7 in shellfish and Mediterranean coastal environments of Morocco. The contamination of shellfish and marine environment
with Shiga toxin-producing E. coli (STEC) and E. coli O157:H7, was investigated during 2007 and 2008. A total of 619 samples were analyzed and 151 strains of E. coli were isolated. The presence of the stx1, stx2, and eae genes was tested in E. coli isolates strains using a triplex polymerase chain reaction. STEC was detected in three positives samples (1.9%), corresponding
to the serotype O157:H7, the others Shiga toxin-producing E. coli non-O157 were also detected. 相似文献
9.
Rodica Elena Ionescu 《Electroanalysis》2023,35(8):e202300003
A rapid and cost-effective method to specifically identify and quantify pathogenic Escherichia coli (E. coli) bacteria in aqueous samples and food products is highly recommended to avoid the degradation of human health that can unfortunately lead to fatal cases. To overcome these borderline situations, portable and easy-to-use screening devices are needed for the non-expert public and confirmed by medical personnel/physicians who can quickly guide/prescribe antibiotic treatments. In such a context, nanotechnologies are very promising and useful tools due to the remarkable optical, chemical and physical properties of biocompatible nanomaterials deposited or synthesized on traditional solid electrodes that greatly improve the detection limit and the selectivity of nanostructured-based biosensors. With this in mind, this review summarizes the latest advances in the bioelectrochemical detection of E. coli and its related products using different biosensor configurations in saline buffers and spiked real samples, namely food products (milk, fruits, vegetables), body fluids (blood, urine, swine feces) and river water. 相似文献
10.
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
11.
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
12.
Shaochi Wang Alexandra P. Breslawec Crystal Li Prof. Myles B. Poulin 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(47):10719-10723
Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function-driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para-nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related β-hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries. 相似文献
13.
1,3-Propanediol (1,3-PD) is an intermediate in chemical and polymer synthesis. We have previously expressed the genes of a
biochemical pathway responsible for 1,3-PD production, thedha regulon ofKlebsiella pneumoniae, inEscherichia coli. An analysis of the maximum theoretical yield of 1,3-PD from glycerol indicates that the yield can be improved by the cofermentation
of sugars, provided that kinetic constraints are overcome. The yield of 1,3-PD from glycerol was improved from 0.46 mol/mol
with glycerol alone to 0.63 mol/mol with glucose cofermentation and 0.55 mol/mol with xylose cofermentation. The engineeredE. coli also provides a model system for the study of metabolic pathway engineering. 相似文献
14.
Comparative Study of Substrate- and Stereospecificity of Penicillin G Amidases from Different Sources and Hybrid Isoenzymes 总被引:1,自引:0,他引:1
Boris Galunsky Karsten Lummer Volker Kasche 《Monatshefte für Chemie / Chemical Monthly》2000,131(6):623-632
Summary. Four natural pencillin G amidase variants from different sources and two genetically constructed hybrid enzymes were produced
and purified to homogeneity. The specificity constants of one enzyme (E. coli) were found to differ six orders of magnitude for hydrolytic transformations within a wide range of substrates. The substrate
specificity of the homologous penicillin amidases was found to differ less than one order of magnitude for hydrolysis of the
most specific and up to two orders of magnitude for the less specific substrates. The -substrate specificity in hydrolytic and transfer reactions (studied mainly with the E. coli enzyme) varied more than three orders of magnitude for the different substrates. The penicillin amidases were found to be
R-specific in the S
1-binding site and S-specific in the -binding site. The S
1-stereoselectivity differs less than one order of magnitude for the different variants. The -stereoselectivity is more pronounced, increases with nucleophile specificity, and was found to differ up to three orders
of magnitude in transfer reactions for the enzyme from E. coli. The observed variation of enatioselectivity for different penicillin amidases and one substrate can also be achieved by changes
in temperature. Comparison of substrate- and stereospecificity of penicillin amidases from different sources and hybrid isoenzymes
suggests that similar changes can be expected for enzyme variants derived by rational protein design or directed evolution.
Received December 20, 1999. Accepted (revised) February 4, 2000 相似文献
15.
Boris Galunsky Karsten Lummer Volker Kasche 《Monatshefte für Chemie / Chemical Monthly》2000,26(4):623-632
Four natural pencillin G amidase variants from different sources and two genetically constructed hybrid enzymes were produced and purified to homogeneity. The specificity constants of one enzyme (E. coli) were found to differ six orders of magnitude for hydrolytic transformations within a wide range of substrates. The substrate specificity of the homologous penicillin amidases was found to differ less than one order of magnitude for hydrolysis of the most specific and up to two orders of magnitude for the less specific substrates. The -substrate specificity in hydrolytic and transfer reactions (studied mainly with the E. coli enzyme) varied more than three orders of magnitude for the different substrates. The penicillin amidases were found to be R-specific in the S 1-binding site and S-specific in the -binding site. The S 1-stereoselectivity differs less than one order of magnitude for the different variants. The -stereoselectivity is more pronounced, increases with nucleophile specificity, and was found to differ up to three orders of magnitude in transfer reactions for the enzyme from E. coli. The observed variation of enatioselectivity for different penicillin amidases and one substrate can also be achieved by changes in temperature. Comparison of substrate- and stereospecificity of penicillin amidases from different sources and hybrid isoenzymes suggests that similar changes can be expected for enzyme variants derived by rational protein design or directed evolution. 相似文献
16.
Absence of repair replication following ultraviolet irradiation of an excision-deficient mutant of Escherichia coli 总被引:2,自引:0,他引:2
The excision -repair of damaged DNA in bacteria and other systems probably requires at least three enzymes to carry out the following steps in sequence: (1) Recognition of a structural distortion in the DNA and the production of an endonucleolytic cleavage of the damaged strand near the lesion. (2) The simultaneous peeling back of the damaged strand and resynthesis of the excised region, with eventual cleavage of the damaged segment from the DNA. (3) The rejoining of the newly synthesized strand to contiguous parental DNA. Evidence for all three steps has been obtained from in vivo studies. The E. coli DNA polymerase has been shown to carry out step # 2 in vitro [1] and the polynucleotide ligase has the required specificity for step # 3[2–4]. An enzyme responsible for step # 1 has been purified from Micrococcus lysodeikticus [5,6] but not from E. coli, although a class of u.v. sensitive mutants in E. coli has been shown to be defective in this step in the repair sequence. In such mutants the release of pyrimidine dimers from the damaged DNA is not observed during post-irradiation growth of u.v. irradiated cultures [7]. It would be predicted, as a consequence, that the next step, non-conservative repair replication, would not be seen in these mutants. Hanawalt and Petti-john showed this to be true for the double mutant E. coli B8-1 that includes a deficiency in dimer excision [8]. In the present study we have looked more closely at an E. coli K-12 strain that has only the uvrA6 deficiency that results in inability to excise pyrimidine dimers. 相似文献
17.
《Analytical letters》2012,45(16):2559-2570
A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. The NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1 × 10?12 M DNA target and 50 cfu/μL E. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation. 相似文献
18.
Counting of Escherichia coli by a microflow cytometer based on a photonic–microfluidic integrated device 下载免费PDF全文
Tianyi Guo Yin Wei Changqing Xu Benjamin R. Watts Zhiyi Zhang Qiyin Fang Haiying Zhang P. Ravi Selvaganapathy M. Jamal Deen 《Electrophoresis》2015,36(2):298-304
Counting of Escherichia coli DH5α‐cell suspensions in PBS is performed using a microflow cytometer based on a photonic–microfluidic integrated device. Side‐scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a hemocytometer. The detection efficiency is correlated to the ratio of sample to sheath flow rates. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2–4 μm) as their scattering intensities are different. 相似文献
19.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was optimized to discriminate
between wild-type and ampicillin-resistant Escherichia coli. Only ampicillin-resistant E. coli displayed an m/z ≈ 29,000 peak, which was confirmed as β-lactamase by in-gel digestion followed by peptide mass fingerprinting. Rapid MALDI-TOF
MS detection of antibiotic-resistance could fulfill an important clinical need, providing critical phenotypic information
beyond genus–species identification. 相似文献
20.
M. Sc. Marian J. Menke Dr. Pascal Schneider Dr. Christoffel P. S. Badenhorst Dr. Andreas Kunzendorf B. Sc. Florian Heinz Dr. Mark Dörr Dr. Martin A. Hayes Prof. Uwe T. Bornscheuer 《Angewandte Chemie (International ed. in English)》2023,62(51):e202313912
Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l -methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l -homocysteine into H2S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates. 相似文献