共查询到20条相似文献,搜索用时 31 毫秒
1.
Yessotoxin (YTX) is a disulfated polyether toxin produced by marine dinoflagellates. Although there is no clear evidence that YTX is toxic to humans, it is a major cause of false positives in DSP toxin detection by mouse bioassay. We developed a new detection and quantification method for yessotoxin using a BiaCore X Surface plasmon resonance (SPR)-based biosensor. The assay is based in the interaction of YTX with phosphodiesterase enzymes (PDE), one of its cellular targets. The injection of several YTX concentrations (3-12 microM) over immobilized PDE I, showed a dose dependent binding signal, which K(obs) (observed rate constant) allowed us to obtain a calibration curve with a linear fit. The detection of yessotoxin using SPR-based biosensor allows the quantification of the toxin with an automated and repetitive method at concentrations in the range of the 1 mg kg(-1) European regulatory limit. 相似文献
2.
An enzyme sensor for the electrochemical detection of the marine toxin okadaic acid (OA) has been developed. The strategy was based on the inhibition of immobilised protein phosphatase (PP2A) by this toxin and the electrochemical measurement of the enzyme activity by the use of appropriate enzyme substrates, electrochemically active after dephosphorylation by the enzyme. Colorimetric inhibition assays have demonstrated the PP2A from human red blood cells to be more sensitive and to provide a wider linear range than the one produced by genetic engineering. Catechyl monophosphate (CMP) and p-aminophenyl phosphate (p-APP) have been tested as enzyme substrates, the former providing higher electrochemical currents at convenient working potentials (+450 mV vs. Ag/AgCl). Biosensors with 19.1 and 5.0 U of immobilised enzyme have been applied to the OA detection. Whereas the 19.1-U biosensor has provided higher electrochemical currents and more reliable determinations, the 5.0-U one has attained a lower 50% inhibition coefficient (IC50) value (22.19 in front of 154.84 μg L−1) and a larger working range (2.69-171.87 in front of 42.97-171.87 μg L−1). The analysis of toxicogenic dinoflagellate extracts with both biosensors and the comparison with the colorimetric assay and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have demonstrated the applicability of the developed electrochemical devices as screening biotools for the assessment of the toxicity of a sample. 相似文献
3.
Putrescine oxidase ([PO]; E.C. 1.4.3.4), which catalyzes the oxidative deamination of putrescine into γ-aminobutyraldehyde,
has been partially purified from Candida guilliermondii. Among the substrates tested, putrescine has the highest reaction rate, followed by spermidine and cadaverine. The K
IN values for putrescine, spermidine, and cadaverine were 20, 200, and 1.1 mM, respectively. The optimum pH and the temperature for PO were 8.0 and 37°C, respectively. Growth of Candida species on putrescine as the solenitrogen source induced the synthesis of PO that converts putrescine into Δ1-pyrroline and γ-aminobutyric acid. These two products were detected and identified from the culture medium. The enzyme was
not activated by divalent cations. Among the species of Candida tested, the highest enzyme activity was found in cell-free extracts of C. guilliermondii. The pathway of putrescine degradation was identified by substrate analysis to be along the nonacetylated pathway in C. guilliermondii. 相似文献
4.
A Simple Colorimetric Enzymatic-Assay for Okadaic Acid Detection Based on the Immobilization of Protein Phosphatase 2A in Sol-Gel 总被引:2,自引:0,他引:2
Okadaic acid (OA), a lipophilic toxin, is produced by Dinophysis and Prorocentrum, and causes diarrheic shellfish poisoning to humans. The mechanism of OA action is based on the reversible inhibition of
protein phosphatase type 2A (PP2A) by the toxin. Therefore, this inhibition could be used to develop assay for OA detection.
In this work, a colorimetric test based on the PP2A inhibition was developed for OA detection. PP2A from GTP and Millipore
was immobilized on silica sol-gel, and the detection was performed. A limit of detection of 0.29 and 1.14 μg/L was respectively
observed for enzyme from GTP and Millipore. The immobilization technique provided a tool to preserve the enzymatic activity,
which is very unstable in solution. The PP2A immobilized sol-gel exhibited a storage stability of near 5 months, when microtiter
plate with enzyme-immobilized polymer was kept at −18C°. The combination of the simplicity of the colorimetric method, along
with long storage stability achieved by sol-gel immobilization, demonstrated the potentiality of this technique to be used
for commercial purpose. 相似文献
5.
Søren N. Olsen Erik Lumby Kc McFarland Kim Borch Peter Westh 《Applied biochemistry and biotechnology》2011,163(5):626-635
Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end,
development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis.
In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis
of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about
500 pmol glucose s−1. Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the
complexity of the substrate. Dose–response experiments with a typical cellulase cocktail enabled a multidimensional analysis
of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis
rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion)
but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile
is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due
to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced
hydrolysis rate over time. 相似文献
6.
A thermostable cyclodextrinase (EC 3.2.1.54) fromBacillus stearothermophilus HY-1 was purified to homogeneity by disc-electrophoresis after sonication disruption, ammonium sulfate fractionation, DEAE-cellulose(DE32)
column chromatography, hydroxyapatite chromatography, Sephadex G150 gel-filtration, and α-cyclodextrin-AH-Sepharose 4B affinity
chromatography. The enzyme was purified 230-fold with 21.2% of activity recovery. The optimal substrates of the enzyme were
α-, Β-, and γ-cyclodextrins and linear maltooligosaccharides, and the final product was mainly maltose. The enzyme could hydrolyze
pullulan to produce panose. It could also hydrolyze soluble starch, amylose, and amylopectin, but not glycogen. The Km and
Vmax for α-, Β-, and γ-cyclodextrins were 1.79, 1.67, and 2.50 mg/mL, and 336, 185, and 208 Μmol/mg/min, respectively. The molecular
weight of the enzyme was 61,000 by SDS-gel-electrophoresis. The isoelectric point was pH 5.0. The enzyme was most active at
pH 6.2 and 55‡C, and it was strongly inhibited by Cu2+, Hg2+, Zn2+, Pb2+, and slightly by Fe2+. The effect of some protein modification reagents on the activity of the enzyme suggested that tryptophan and histidine residue(s)
may be located at the active site. The amino acid composition of the enzyme was also determined. 相似文献
7.
Xiumei Feng Yuying Liu Qingcheng Kong Jianshan Ye Xiaohua Chen Jianqiang Hu Zhiwu Chen 《Journal of Solid State Electrochemistry》2010,14(6):923-930
Rod-constructed zinc oxide (ZnO) microspheres (RZnOMs), consisting of hundreds of needle-like ZnO nanorods, were utilized
to explore a novel biosensor through coupling with myoglobin (Mb) in the presence of chitosan (Chi). Biocompatibility and
electrochemical properties of the resulting ZnO-Chi-Mb composite film were studied by Fourier-transform infrared spectroscopy
and cyclic voltammetry. The results revealed that the RZnOMs-based composite was a satisfying matrix for proteins to effectively
retain their native structure and bioactivity. With advantages of the unique inorganic material, facilitated direct electron
transfer of the metalloenzymes was acquired on the RZnOMs-based enzyme electrode. Moreover, the RZnOMs-based biosensor also
displayed significant electrocatalytic activity for the reduction of hydrogen peroxide with an apparent Michaelis–Menten constant
(32 μM), wide linear range (2–490 μM), and low detection limit (0.21 μM, S/N = 3). These indicated that the RZnOMs were one of the ideal candidate materials for direct electrochemistry of redox proteins
and related biosensor construction. 相似文献
8.
Ah-Reum Park Hye-Jung Kim Jung-Kul Lee Deok-Kun Oh 《Applied biochemistry and biotechnology》2010,160(8):2236-2247
We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave
a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular
mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 oC. The half-lives of
the enzyme at 70, 80, and 90 oC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity
with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity
was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with
lactose. 相似文献
9.
Two β-glucosidase/xylosidase genes, Rubg3A and Rubg3B, were cloned from yak rumen uncultured microorganisms by metagenome method and function-based screening. Recombinant RuBG3A
and RuBG3B purified from Escherichia coli were characterized for enzymatic properties, and they exhibited activity against 4-nitrophenyl-β-d-glucopyranoside and 4-nitrophenyl-β-d-xylopyranoside, suggesting bifunctional β-glucosidase/xylosidase activity. Chromatography analysis showed that they could
effectively hydrolyze cellooligosaccharide substrates, indicating the facilitation in saccharification of cellulose. RuBG3A
and RuBG3B can also increase the reducing sugar released in xylan hydrolysis to 218% and 169%, respectively, through synergism
with xylanase, suggesting their application in hemicellulose saccharification. Molecular modeling and substrate docking showed
that there should be one active center responsible for the bifunctional activity in each enzyme, since the active site pocket
is substantially wide to allow the entry of both β-glucosidic or β-xylosidic substrates, which elucidated the structure–function
relationship in substrate specificities. Therefore, the enzymatic properties, the participation in hydrolysis of cellooligosaccharides,
and the synergism with xylanase make RuBG3A and RuBG3B very interesting candidates for saccharification of both cellulose
and hemicellulose. 相似文献
10.
Ramadan A. Abuknesha Fiona Jeganathan Rens DeGroot Dirk Wildeboer Robert G. Price 《Analytical and bioanalytical chemistry》2010,396(7):2547-2558
A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection
step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for
proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates
and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP
and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The
detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from
Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 μunits mL−1 for chymotrypsin, 0–12 μunits mL−1 for type IX, 0–35 μunits mL−1 for type XIV and 0–100 μunits mL−1 for type XXIV. The detection limits of the proteases studied were 89 μunits mL−1 for chymotrypsin, 0.26 μunits mL−1 for type IX, 5.8 μunits mL−1 for type XIV and 6.5 μunits mL−1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase
enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format. 相似文献
11.
Fanjul-Bolado P González-García MB Costa-García A 《Analytical and bioanalytical chemistry》2006,385(7):1202-1208
p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble
product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare
screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the
detection limit achieved for p-nitrophenol was 2×10−8 mol L−1, almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical
detection method, measurement of 7×10−14 mol L−1 alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed
carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin
produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry. 相似文献
12.
The purified α-amylase of Geobacillus thermoleovorans had a molecular mass of 26 kDa with a pI of 5.4, and it was optimally active at 100 °C and pH 8.0. The T
1/2 of α-amylase at 100 °C increased from 3.6 to 5.6 h in the presence of cholic acid. The activation energy and temperature
quotient (Q
10) of the enzyme were 84.10 kJ/mol and 1.31, respectively. The activity of the enzyme was enhanced strongly by Co2+ and Fe2+; enhanced slightly by Ba2+, Mn2+, Ni2+, and Mg2+; inhibited strongly by Sn2+, Hg2+, and Pb2+, and inhibited slightly by EDTA, phenyl methyl sulfonyl fluoride, N-ethylmaleimide, and dithiothreitol. The enzyme activity was not affected by Ca2+ and ethylene glycol-bis (β-amino ethyl ether)-N,N,N,N-tetra acetic acid. Among different additives and detergents, polyethylene glycol 8000 and Tween 20, 40, and 80 stabilized
the enzyme activity, whereas Triton X-100, glycerol, glycine, dextrin, and sodium dodecyl sulfate inhibited to a varied extent.
α-Amylase exhibited activity on several starch substrates and their derivatives. The K
m and K
cat values (soluble starch) were 1.10 mg/ml and 5.9 × 103 /min, respectively. The enzyme hydrolyzed raw starch of pearl millet (Pennisetum typhoides) efficiently. 相似文献
13.
Pengjun Shi Ning Li Peilong Yang Yaru Wang Huiying Luo Yingguo Bai Bin Yao 《Applied biochemistry and biotechnology》2010,162(3):707-718
An α-l-arabinofuranosidase gene, abf51S9, was cloned from Streptomyces sp. S9 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene consisted of 1,506 bp and encoded 501 amino acids with a calculated mass of 55.2 kDa. The
deduced amino acid sequence was highly homologous with the α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic
homogeneity by Ni-NTA affinity chromatography and subsequently characterized. The optimal pH and temperature for the recombinant
enzyme were 6.0 and 60∼65 °C, respectively. The enzyme showed a broad pH range of stability, retaining over 75% of the maximum
activity at pH 5.0 to 11.0. The specific activity, K
m, and V
max with p-nitrophenyl-α-l-arabinofuranoside as substrate were 60.0 U mg−1, 1.45 mM, and 221 μmol min−1 mg−1, respectively. Abf51S9 showed a mild but significant synergistic effect in combination with xylanase on the degradation of
oat-spelt xylan and soluble wheat arabinoxylan substrates with a 1.19- and 1.21-fold increase in the amount of reducing sugar
released, respectively. These favorable properties make Abf51S9 a good candidate in various industrial applications. 相似文献
14.
Swetha Mahalaxmi Colin R. Jackson Clint Williford Charles L. Burandt 《Applied biochemistry and biotechnology》2010,162(5):1414-1422
Biochemical conversion of lignocellulosic biomass to ethanol involves size reduction, preprocessing, pretreatment, enzyme
hydrolysis, and fermentation. In recent years, microbial preprocessing has been gaining attention as a means to produce labile
biomass for lessening the requirement of pretreatment severity. However, loss of sugars due to microbial consumption is a
major consequence, suggesting its minimization through optimization of nutrients, temperature, and preprocessing time. In
this work, we emphasized estimation of fungal preprocessing time, at which higher sugar yields can be achieved after preprocessing
and enzyme hydrolysis. The estimation is based on the enzymatic activity profile obtained by treating switchgrass with Phanerochaete chrysosporium for 28 days. Enzyme assays were conducted once in every 7 days for 28 days, for activities of phenol oxidase, peroxidase,
β-glucosidase, β-xylosidase, and cellobiohydrolase. We found no activity for phenol oxidase and peroxidase, but the greatest
activities for cellulases on the seventh day. We then treated switchgrass for 7 days with P. chrysosporium and observed that the preprocessed switchgrass had higher glucan (39%), xylan (17.5%), and total sugar yields (25.5%) than
the unpreprocessed switchgrass (34%, 37.5%, and 20.5%, respectively, p < 0.05). This verifies the utility of using enzyme assays for initial estimation of preprocessing time to enhance sugar yields. 相似文献
15.
A. Khalyfa S. Kermasha P. Marsot M. Goetghebeur 《Applied biochemistry and biotechnology》1995,53(1):11-27
The partially purified chlorophyllase, obtained from the algaPhaeodactylum tricornutum, was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large
amounts of a single purified chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction
under denaturing conditions demonstrated the presence of two bands with mol wt of 43 ±3 and 46 ±3 kDa. The purification procedure
resulted in 2-and 195-fold increases in chlorophyllase activity compared to that of the partially purified and crude enzymatic
extracts, respectively. The optimum pH for chlorophyllase hydrolytic activity was found to be 8.0. The optimum incubation
time and temperature for the hydrolytic activity of the purified chlorophyllase were found to be 2 h and 31°C, respectively.
The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The
addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and β-carotene, to the
reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward
selective substrates with an order of activity as follows: purified chlorophyllb > purified chlorophylla > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive
and competitive inhibitory effects on chlorophyllase activity with Ki, values of 0.78 mM and 3.75μM, respectively. 相似文献
16.
Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods 总被引:1,自引:0,他引:1
A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using
its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers,
AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed
to have high sensitivity and specificity to ampicillin (K
d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was
used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection
for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in
distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive
enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be
a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry. 相似文献
17.
D. T. Souza A. S. R. Bispo E. P. S. Bon R. R. R. Coelho R. P. Nascimento 《Applied biochemistry and biotechnology》2012,166(6):1575-1585
In the present paper, endo-β-1,4-xylanase production by Aspergillus fumigatus was evaluated in solid-state fermentation using low-cost substrates such as sugarcane bagasse (SCB), brewer’s spent grain
(BSG), and wheat bran (WB). The partial characterization of the crude enzyme was also performed. In the experimental conditions,
the highest levels of endo-β-1,4-xylanase production by A. fumigatus FBSPE-05 occurred within 8 days incubation when using SCB/liquid medium at 1:2 ratio (219.5 U g−1) and 4 days incubation when using WB/liquid medium at 1:1 ratio (215.6 U g−1). Crude enzyme from this last condition was used to enzyme characterization, showing best enzyme activity at 60 °C and pH 6.0,
which suggests a thermophilic endoxylanase. The crude enzyme retained 73% of its activity after 1 h at 60 °C, and zymogram
has shown three bands of endo-β-1,4-xylanase activity, with different molecular masses. A. fumigatus FBSPE-05 was able to grow and produce good levels of endo-β-1,4-xylanase using agro-industrial by-products, making this strain
worthy for further investigation. To our knowledge, this is the first study reporting the use of SCB and/or BSG as sole substrates
for endoxylanase production by solid-state fermentation using A. fumigatus. 相似文献
18.
Marla D. Swain George P. Anderson Dan Zabetakis Rachael D. Bernstein Jinny L. Liu Laura J. Sherwood Andrew Hayhurst Ellen R. Goldman 《Analytical and bioanalytical chemistry》2010,398(1):339-348
Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage
display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using
two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on
microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical
sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating
specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding
assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated
selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT
A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high
degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity
was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on
subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array
assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K
D ) in the range 2.2 × 10−11 to 1.6 × 10−10 M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of
BoNT A, particularly in field screening and monitoring applications. 相似文献
19.
Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification
procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity
chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was
purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight
of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was
found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75
M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K
M and V
max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme. 相似文献
20.
An electrochemical biosensor for the detection of microcystin has been developed based on the inhibition of the protein phosphatase 2A (PP2A) by this cyanobacterial toxin. The enzyme has been immobilised by entrapment using a poly(vinyl alcohol) azide-unit pendant water-soluble photopolymer (PVA-AWP). Electrode supports and immobilisation conditions have been optimised by colorimetric assays, the highest immobilisation yields being obtained with screen-printed graphite electrodes and the 1:2 PP2A:PVA ratio. Catechyl monophosphate (CMP), α-naphthyl phosphate (α-NP) and 4-methylumbelliferyl phosphate (4-MUP) have been used as phosphorylated substrates to monitor the protein phosphatase activity by electrochemical methods, the former providing the highest chronoamperometric currents at appropriate working potentials (+450 mV versus Ag/AgCl). Incubation with standard microcystin solutions has demonstrated the inhibition of the immobilised enzyme, proportional to the toxin concentration. The standard inhibition curve has provided a 50% inhibition coefficient (IC50) of 83 μg L−1, a limit of detection (LOD; 35% inhibition) of 37 μg L−1, and 100% inhibition at about 1000 μg L−1. Real samples of cyanobacterial blooms from the Tarn River (Midi-Pyrénées, France) have been analysed using the developed amperometric biosensor and the toxin contents have been compared to those obtained by a conventional colorimetric protein phosphatase inhibition (PPI) assay and high-performance liquid chromatography (HPLC). The results clearly justify the use of the developed amperometric biosensor as screening method for microcystin detection. 相似文献