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1.
Feiwei Zhang Dairong Qiao Hui Xu Chong Liao Shilin Li Yi Cao 《Journal of microbiology (Seoul, Korea)》2009,47(3):351-357
Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order
to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni2+-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 μM and 161.9 μM, respectively, which demonstrated that this XR had dual coenzyme specificity.
Moreover, this XR showed the highest catalytic efficiency (kcat=1.44×l04 min−1) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L · h xylitol productivity. This XR coupled
with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production. 相似文献
2.
We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be
thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K
m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation;
however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered
near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active
site. A homology three-dimensional model was developed and the structural information was combined with activity and stability
data to give functional clues about the asparaginase. 相似文献
3.
Ching-Nan Lin Wan-Jr Syu Wei-Sheng W Sun Jenn-Wei Chen Tai-Hung Chen Ming-Jaw Don Shao-Hung Wang 《Journal of biomedical science》2010,17(1):84
Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin
treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying
plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide
stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance
when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for
the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished
apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture
supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation. 相似文献
4.
A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing
238 mg/liter culture. With increase in culture cell density to A
600 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was
in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed
enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of
the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic
analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K
m and k
cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec−1, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase
from B. halodurans. 相似文献
5.
6.
A rod-shaped, gram-negative bacterium Stenotrophomonas sp. SMSP-1 was isolated from the sludge of a wastewater treating system of a pesticide manufacturer. Strain SMSP-1 could
hydrolyze methyl parathion to p-nitrophenol (PNP) and dimethyl phosphorothioate but could not degrade PNP further. Strain SMSP-1 was able to hydrolyze other
organophosphate pesticides, including fenitrothion, ethyl parathion, fenthion, and phoxim, but not chlorpyrifos. A 4395-bp
DNA fragment, including an organophosphorus hydrolase encoding gene ophc2, was cloned from the chromosome of strain SMSP-1 using the shotgun technique. Its sequence analysis showed that ophc2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase), and orf1 (encoding a CDF family heavy metal/H+ antiporter). The ophc2 gene was effectively expressed in E. coli. This is the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas. 相似文献
7.
N. Yu. Markelova 《Microbiology》2010,79(6):777-779
Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached
to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection
and public health is discussed. 相似文献
8.
Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
9.
10.
Z. B. Liu Q. Wan Y. F. Yue H. W. Yang J. M. Wang X. F. Li Y. Yang 《Russian Journal of Plant Physiology》2011,58(3):478-483
Ascorbate peroxidase (APX) is an important enzyme to scavenge the reactive oxygen species (ROS), which are often caused by
the salt stress. Here, APX cDNA from Brassica napus was amplified by RT-PCR and cloned into the prokaryotic expression vector pGEX-6p-1 to express BnAPX as a glutathione S-transferase (GST) fusion protein. The recombinant expression plasmid was then transformed into Escherichia coli BL21 (DE3) and induced with 0.2 mM IPTG at 28°C. The enzyme activity analysis of the induced protein showed the GST-APX protein
had the similar enzyme activity with the other found APXs, which decompose H2O2. Moreover, the GST-APX fusion protein was purified by affinity chromatography using the glutathione-Sepharose 4B column.
The purified GST-APX protein was then used to immunize rabbits to obtain the anti-BnAPX serum, which was suitable to recognize
both the recombinant exogenous BnAPX and the endogenous BnAPX in vivo by western blotting and the immunohistochemical experiment.
Furthermore, the immuno-fluorescent microscopy observation revealed that BnAPX was expressed in the chloroplasts. Finally,
the bacteria expressing BnAPX grew much faster in the presence of 3% NaCl than the control cells, indicating that the transformant expressing BnAPX acquired resistance to salt stress. 相似文献
11.
The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions
for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to
be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their
concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions
selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times
higher than that using the non-optimized culture conditions (2 U/mL). 相似文献
12.
Shuyu Wu Eirini Chouliara Lars Bogø Jensen Anders Dalsgaard 《Acta veterinaria Scandinavica》2008,50(1):38
Background
Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli. 相似文献13.
Atsuko Uzura Fumiki Nomoto Akiko Sakoda Yukifumi Nishimoto Michihiko Kataoka Sakayu Shimizu 《Applied microbiology and biotechnology》2009,83(4):617-626
A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated
from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed
to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme.
The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several
short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone
reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM
3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction. 相似文献
14.
The purpose of this work was to characterize the cellular and proteomic responses of Escherichia coli JK-17 exposed to the rose flower extract (Rosa hybrida). The bacterial isolate was enriched and isolated from contaminated food. 16S rRNA sequence analyses revealed that the strain
was 99% similar to the E. coli species cluster; therefore, this strain was designated E. coli JK-17. The rose flower extract showed a dose-dependent antibacterial effect on E. coli JK-17. Treatment of E. coli JK-17 with 50 and 100 mg/mL of the rose flower extract completely inhibited growth within 12 and 6 h of incubation. The stress
shock proteins (SSPs) were induced with different concentrations of rose flower extract. The proteins were identified as 70-kDa
DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using anti-DnaK and anti-GroEL monoclonal antibodies. The levels of SSPs
induced by the rose flower extract increased when the exposure time to the rose flower extract was increased. SDS-PAGE with
silver staining revealed that the amount of lipopolysaccharide (LPS) in E. coli JK-17 increased or decreased with different concentrations and exposure times of the rose flower extract. To identify proteins
induced by the rose flower extract, 2-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of E. coli JK-17 cultures. In the pH range of 4 ∼ 7, more than 250 spots were detected on the silver stained gels. Notably, 15 protein
spots were increased or decreased after treatment with the rose flower extract. Twelve up-regulated proteins were identified
as chaperones (DnaK and GroEL) and porin proteins (PhoE, RfaI, RfaG, MdoH, and WzzE) by MALDITOF mass spectrometry, and three
down-regulated proteins were identified, including proteins involved in energy and DNA metabolism (SdhA and GyrB), and amino
acid biosynthesis (GltK). Using scanning electron microscopic analysis, some cells were shown to adopt irregular rod shapes
and wrinkled surfaces after treatment with the rose flower extract. These results provide clues for better understanding the
mechanism of rose flower extract-induced stress and cytotoxicity in E. coli JK-17. 相似文献
15.
Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK,
DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously
expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into
the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression
of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip–HlyA
fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA. 相似文献
16.
Khomaini Hasan Wangsa Tirta Ismaya Idar Kardi Yandi Andiyana Susanti Kusumawidjaya Safri Ishmayana Toto Subroto Soetijoso Soemitro 《Biologia》2008,63(6):1044-1050
α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size
exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE
analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble
the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex
G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme.
However, it was found that the K
m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies
showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain,
while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested
enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested
enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A. 相似文献
17.
Aspergillus niger is widely used as an enzyme source in industries. Considering its enzymic potential, A. niger was studied for its acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase), and invertase (EC 3.2.1.26,
β-fructofuranoside fructohydrolase) activity in defined media supplemented with 1%, 3%, or 5% sucrose concentrations. Both
these enzymes play a key role in phosphate and carbon metabolism in plants, animals, and microorganisms and hence are interesting
from the standpoint of biotechnological applications. Ontogenic changes in extracellular, cytoplasmic, and wall-bound enzyme
activities of A. niger were studied. Growth in terms of fresh weight showed inverse correlation with pH. At higher pH values, both enzyme activities
were higher in the medium supplemented with low sucrose concentration. It was observed that the more the fresh weight of fungi
decreased, the greater was the enzyme activity observed. It is suggested that these enzymes may participate in autolysis of
fungi and, on the other hand, could prove to be a potential source of industrial application and exploitation. 相似文献
18.
Lena Jansson Jonas Ångström Michael Lebens Susann Teneberg 《Glycoconjugate journal》2010,27(1):171-179
A novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits (termed LCTBK) has previously been described, and also the native heat-labile enterotoxin bind to some extent to blood group A/B terminated
glycoconjugates. The blood group antigen binding site is located at the interface of the B-subunits. Interestingly, the same
area of the B-subunits has been proposed to be involved in binding of the heat-labile enterotoxin to lipopolysaccharides on
the bacterial cell surface. Binding of the toxin to lipopolysaccharides does not affect the GM1 binding capacity. The present
study aimed at characterizing the relationship between the blood group A/B antigen binding site and the lipopolysaccharide
binding site. However, no binding of the B-subunits to E. coli lipopolysaccharides in microtiter wells or on thin-layer chromatograms was obtained. Incubation with lipopolysaccharides
did not affect the binding of the B-subunits of heat-labile enterotoxin of human isolates to blood group A-carrying glycosphingolipids,
indicating that the blood group antigen site is not involved in LPS binding. However, the saccharide competition experiments
showed that GM1 binding reduced the affinity for blood group A determinants and vice versa, suggesting that a concurrent occupancy of the two binding sites does not occur. The latter finding is related to a connection
between the blood group antigen binding site and the GM1 binding site through residues interacting with both ligands. 相似文献
19.
M. Yu. Kordyukova O. V. Zatsepina M. A. Polzikov 《Applied Biochemistry and Microbiology》2011,47(2):123-127
cDNA of human gene Surf-6 (hSutf-6) was amplified and cloned into vector pGEX-2T for the expression in the bacterial system of protein hSURF-6 translationally
fused to glutathione-S-transferase. The resulting vector is named as pGEX-2T-GST-hSurf-6. Superproducer of chimeric protein
GST-hSURF-6 was obtained on the basis of Escherichia coli strain BL21-CodonPlus(DE3)-RIL. Its purification was performed by the affinity chromatography on L-glatathione-sepharose.
The proportion of recombinant protein GST-hSURF-6 in the optimized conditions was not less than 15% of the total bacterial
protein, and up to 7 mg of the protein was isolated from 1 liter of culture of the producer strain. The final fraction of
eluate contained approximately 80% of GST-hSURF-6. The amount and the purity of the isolated protein were sufficient to immunize
animals and obtain antibodies. Protein GST-hSURF-6 can also be used as an affinity ligand for revealing protein partners of
hSURF-6 in human cells. 相似文献
20.
L. E. Petrovskaya A. A. Shulga O. V. Bocharova Ya. S. Ermolyuk E. A. Kryukova V. V. Chupin M. J. J. Blommers A. S. Arseniev M. P. Kirpichnikov 《Biochemistry. Biokhimii?a》2010,75(7):881-891
To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and
hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most
of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output
(up to 50 mg/liter). 相似文献