CN103149198B - Process for rapidly and quantitatively detecting heparosan through enzymatic method - Google Patents
Process for rapidly and quantitatively detecting heparosan through enzymatic method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 63
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 46
- 108010083213 heparitinsulfate lyase Proteins 0.000 claims abstract description 27
- 238000002835 absorbance Methods 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 14
- 239000012488 sample solution Substances 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims description 50
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- 238000001514 detection method Methods 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 11
- 238000013016 damping Methods 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 4
- FNEHAOQZWPHONV-UHFFFAOYSA-N 9h-carbazole;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C3=CC=CC=C3NC2=C1 FNEHAOQZWPHONV-UHFFFAOYSA-N 0.000 abstract description 8
- 239000007853 buffer solution Substances 0.000 abstract 1
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- 238000006243 chemical reaction Methods 0.000 description 20
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a process for rapidly and quantitatively detecting heparosan through an enzymatic method. The process comprises the following steps: 1, taking a sample to be tested, dissolving the sample in a buffer solution having a pH value of 7.0-7.5 to prepare a sample solution, uniformly mixing, balancing at 25-35DEG C for 0.1-10min, adding an enough mount of a Heparinase III enzyme, uniformly mixing, carrying out water bath treatment at 25-35DEG C for 0.1-3h, adding an HCl solution, centrifuging, uniformly mixing, taking the obtained supernatant, detecting by an ultraviolet spectrophotometer through treating a sample not subjected to an enzymatic reaction as a blank contrast, and reading out the absorbance A235nm of the sample; 2, preparing a heparosan standard substance solution having gradient concentrations, reacting and detecting according to the method in step 1, and drawing a standard curve of the absorbance and a substrate concentration; and 3, obtaining the concentration of heparosan in the sample solution through contrasting the standard curve according to the absorbance A235nm, and converting to obtain the content of heparosan in the sample. Compared with a sulfuric acid-carbazole method and a dry weight method, the method provided by the invention has the advantages of strong specificity, high precision, good reappearance and the like.
Description
(1) technical field
The present invention relates to a kind of method of enzyme process Quantitative detection heparosan.
(2) background technology
Heparosan, also known as N-acetylheparosan, (-GlcUA-1,4-GlcNAc-1,4-)
n, be that E.coli O10:K5:H4(is called for short E.coli K5) etc. the disaccharides recurring unit of polysaccharide skeleton in bacterial strain pod membrane, can be used as the biosynthesizing precursor of heparin and Heparan sulfate.The detection method of current heparoan mainly contains sulfate-carbazole, dry weight method, the steps such as dry weight method needs ferment product to carry out to be separated, alcohol precipitation, oven dry, weighing, containing more impurity (protein, other polysaccharide etc.) in the Thick many candies finally obtained, measuring accuracy is poor.And the assay method of sulfuric acid carbazole method a kind of glucuronic acid content that to be the scope of application wider, measurement of the polysaccharide content is also the content by first measuring uronic acid in polysaccharide, be converted into the content of polysaccharide again, the method is widely used in the mensuration of glucuronic acid content in the acidic polysaccharoses such as chondroitin, hyaluronic acid, heparin, Heparan sulfate, cucumber polysaccharide, pectin polysaccharide at present, this assay method is subject to the interference of impurity in reagent or sample (glucose, salt, ferric ion etc.), the shortcoming such as there is complex operation, poor reproducibility, disturbing factor is many, selectivity is poor.
And selected suitable enzyme, carry out enzyme process detect have quick, easy, selectivity strong and the advantage such as high precision, more and more wider in actual applications at present.The bacterium heparinase of Heparinase III enzyme (EC4.2.2.8) for extracting in Flavobacterium (flavobacterium heparinum), can Specific lytic N-sulphation or N-acetyl glucose amine (GlcNSO
3or GlcNAc) and glucuronic acid between glycosidic bond.Current researcher has proved that heparinase III enzyme can be degraded Heparan sulfate, but does not also have report that heparinase III enzyme is applied to heparosan or Heparan sulfate content detection.
(3) summary of the invention
The object of the invention is a kind of method in order to provide enzyme process Quantitative detection heparosan, it is strong that the method has selectivity compared with sulfuric acid carbazole method, dry weight method, and precision is high, high repeatability and other advantages.
The technical solution used in the present invention is:
A method of enzyme process Quantitative detection heparosan, described method comprises:
(1) testing sample is got, be dissolved in the damping fluid of pH7.0 ~ 7.5 and be mixed with sample solution, mixing, at 25 ~ 35 DEG C, balance 0.1 ~ 10min, add enough Heparinase III enzymes, mixing, 25 ~ 35 DEG C of water-bath 0.1 ~ 3h, add HCl solution, centrifugal mixing, get supernatant and carry out the detection of ultraviolet spectrometry light photometer, not carry out the sample of enzyme reaction for blank, read the absorbance A of sample
235nm; Described testing sample can be the heparosan sample after extracting, and also can be crude product containing heparosan or E.coli K5 fermented supernatant fluid etc.;
(2) prepare the heparosan standard solution of gradient concentration, carry out reacting and detecting according to step (1) method, draw the typical curve of absorbance and concentration of substrate;
(3) reference standard curve, per sample absorbance A
235nm, obtain heparosan concentration value in sample solution, convert and obtain heparosan content in sample.
Described step (1) damping fluid is composed as follows: Tris HCl10 ~ 50mM, NaCl40 ~ 80mM, CaCl
21 ~ 4mM, bovine serum albumin 0.005 ~ 0.02%(w/w), solvent is water, pH7.0 ~ 7.5.
Be optimized enzyme reaction system, described method is as follows:
(1) testing sample is got, be dissolved in buffer A be mixed with sample concentration 1 ~ 500g/L sample solution (sample be extract after heparosan sample time, sample concentration is that 1 ~ 10g/L is advisable, when sample is the crude product containing heparosan, sample concentration can be 100 ~ 500g/L, be preferably 300 ~ 500g/L), get 0.6mL sample solution, mixing, 0.1 ~ 10min is balanced at 25 ~ 35 DEG C, add Heparinase III enzyme liquid 0.05mL, mixing, 25 ~ 35 DEG C of water-bath 0.1 ~ 3h, add 50mMHCl solution 2.35mL, centrifugal mixing, get supernatant and carry out the detection of ultraviolet spectrometry light photometer, not carry out the sample of enzyme reaction for blank, read the absorbance A of sample
235nm, described buffer A is composed as follows: Tris HCl20mM, NaCl50mM, CaCl
24mM, bovine serum albumin 0.01%, solvent is water, pH7.5, described Heparinase III enzyme liquid is dissolved in buffer A by Heparinase III enzyme and obtains, and Mei Ye unit enzyme is lived and is greater than 165U/mL,
(2) prepare the heparosan standard solution of gradient concentration, carry out reacting and detecting according to step (1) method, draw the typical curve of absorbance and concentration of substrate;
(3) reference standard curve, per sample absorbance A
235nm, obtain heparosan concentration value in sample solution, convert and obtain heparosan content in sample.
Beneficial effect of the present invention is mainly reflected in: a kind of method that the invention provides enzyme process Quantitative detection heparosan, the method is applicable to the heparosan sample tests after extraction, also can be the mensuration of heparosan content in crude product containing heparosan or E.coli K5 fermented supernatant fluid, have compared with sulfuric acid carbazole method, dry weight method that selectivity is strong, precision is high, high repeatability and other advantages, and simple to operation.
(4) accompanying drawing explanation
Fig. 1 is the depolymerization effect of heparosan under the different enzyme reaction time; Swimming lane 1 ~ 6 be respectively enzyme reaction 0,15,30,60,90,120min sample;
Fig. 2 is that different Heparinase III enzyme amount is on the impact of enzymatic assays heparosan;
Fig. 3 is the typical curve that in reaction system A, Heparinase III enzyme process detects heparosan.
Fig. 4 is the typical curve that in reaction system B, Heparinase III enzyme process detects heparosan.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The mensuration that embodiment 1:Heparinase III enzyme is lived
Valence link between heparinlyase III Specific lytic glucuronic acid and gucosamine, specific as follows:
HeparinaseIII can carry HepC genetic fragment by E.coli BL21() abduction delivering, separation and Extraction obtains, and also can adopt commercial commercial enzyme, and the assay method that its enzyme is lived is with reference to sigma company HeparinaseIII Enzyme activity assay method.As shown in table 1:
Table 1:Heparinase III Enzyme activity assay reaction system A composition and method
Reagent A:20mM Tris HCl, 50mM NaCl, 4mM CaCl
2, 0.01%(w/v) and bovine serum albumin, solvent is water, pH7.5;
Reagent B:1%(w/w) heparosan sterling aqueous solution
Reagent C：50mM HCl
Reagent D:Hep III liquid (mixed liquor that enzyme mixes with 1:89 mass ratio with A liquid)
The computing formula that enzyme is lived:
Sample enzyme (U/ml)=(A alive
235nm sample-A
235nm is blank) × 2 × 10 × 3 × d (5.50 × 0.02)
Note:
2 conversion coefficients that are 30min to 1h;
10 is the conversion coefficient of 1 μm of ole to 0.1 μm of ole;
3 is reaction system cumulative volume (unit is mL);
Df is enzyme liquid extension rate;
5.50 is the micromole extinction coefficient of unsaturated uronic acid at 235nm;
0.02 is enzyme liquid long-pending (unit is mL).
Enzyme activity unit defines:
At pH7.0, under 25 DEG C of conditions, it is 1 enzyme activity unit that every 1 hour depolymerization heparosan generates Heparinase III amount required for 0.1 μm of unsaturated uronic acid of ol.
According to formula, the enzyme finally recording the present invention heparinase used III liquid storage is lived as 8.25U/ μ L.
The depolymerization effect of embodiment 2:Heparinase III couple of heparosan
0.02g heparosan sample (sterling of non-desalination) is dissolved in 0.2mL damping fluid (A liquid), HepIII enzyme liquid storage 5 μ L(enzyme 8.25U/ μ alive L is added) after 25 DEG C of balances, 25 DEG C of water-baths, at 15min, 30min, 60min, 90min, 120min takes out 30uL reactant liquor respectively and carries out PAGE electrophoresis, the results are shown in Figure 1.
Mix through the Heparosan sample of HepIII depolymerization and sample-loading buffer 1: 1, loading electrophoresis, electrophoresis apparatus is PROTEAN II xi Cell (BIO-RAD, USA), power supply is Power PacTMUniversal Power Supply (BIO-RAD, USA).The PAGE test of polysaccharide is following to be described:
1) clean glass plate is placed in gel maker, topping-up is hunted leak.
2) separation gel of variable concentrations is prepared: the separation gel damping fluid of 35mL10%, the ammonium persulfate of 500 μ L10%, the TEMED of 50 μ L10%.Slowly join after mixing in glue groove, topping-up condenses under room temperature.
3) the concentrated glue of preparation: the distilled water on separation gel upper strata is blotted as far as possible, comb is inserted in glue groove.The concentrated glue damping fluid of concentrated glue: the 15mL5% of preparation, the ammonium persulfate of 500 μ L10%, the TEMED of 50 μ L10%.Slowly add concentrated glue in abundant mixing backward glue groove, condense under room temperature.
4), after gelling to be concentrated gathers, carefully comb is extracted from glue groove.
5) offset plate is taken out from gel maker be put in electrophoresis main body both sides, add electrode buffer and fill glue hole.
6) after loading, 400V constant voltage electrophoresis.5 DEG C of circulation ethanol are passed in electrophoresis process,
7) when electrophoresis is to offset plate bottommost 2 ~ 4cm, electrophoresis is stopped.Carefully from glass plate, strip gel.
8) gel dyes 30min in 0.5%Alcian Blue, and clear water decolouring carries out gel image scanning after spending the night.
Wherein needed for electrophoresis, solution preparation situation is as follows:
Tris-borate buffer (1L): 6.183g boric acid, 12.11g Tris, 3.72g EDTA-Na
2, add 900mL distilled water, to adjust after pH to 8.3 again constant volume to 1L.
10% separation gel damping fluid (500mL): 48.35g acrylamide, 1.65g methylene diacrylamide, adds Tris-borate buffer constant volume to 500mL.
5% concentrated glue damping fluid (100mL): 4.75g acrylamide, 0.25g methylene diacrylamide, adds 80mL Tris-borate buffer, and after adjusting pH to 6.3, constant volume is to 100mL.
10% ammonium persulfate (1mL): 0.1g ammonium persulfate, adds 1mL distilled water.
10%TEMED(10mL): 1mL ammonium persulfate, 9mL distilled water is added.
0.5% Ai Er Xinlan (250mL): 1.25g Alcian Blue, adds 1.5% acetic acid constant volume to 250mL.
0.1% phenol red: 0.01g phenol red adds 10mL distilled water.
0.1% phenol red of sample-loading buffer: 2mL, 25% sucrose of 8mL.
Electrophoretic buffer (1L): 93g glycocoll, after 24.2g Tris dissolves with 900mL distilled water, constant volume is to 1L.
Heparinase III couple of heparosan has good depolymerization effect as seen from Figure 1.
The determination of enzyme amount in embodiment 3:Heparinase III Enzyme activity assay reaction system
With 10g/L heparosan sterling solution for substrate, with the D liquid containing different Heparinase III enzyme activity unit and its reaction, reaction system is with table 1.Fig. 2 is the graph of a relation of different enzyme amount and absorbance, and horizontal ordinate represents enzyme activity unit in D liquid, and ordinate is absorbance.
As can be seen from Figure 2, for the heparosan solution of 10g/L, along with the increase of Heparinase in reaction system III enzyme amount, absorbance is progressively increasing, and when the consumption of enzyme is more than 8.25U, absorbance is constant, keeps OD
235nm0.981, represent that enzyme reaction is complete, substrate degradation product no longer increases.Thus determine that the heparosan solution of complete depolymerization 10g/L needs enzyme amount to be 8.25U, the enzyme amount therefore added for following experiment is all greater than 8.25U.Under this enzyme dosage, obtain Heparinase III enzyme process in reaction system A and detect the typical curve (Fig. 3) of heparosan.
Embodiment 4: enzyme process and sulfuric acid carbazole method comparing heparosan detection method of content in crude product
(1) enzyme process
Take a certain amount of heparosan crude product respectively to react, reaction system is identical with method and table 1.When crude product concentration is 500g/L, add the OD of reactant liquor after excessive enzyme reaction 0.5h
235with the OD of reactant liquor after reaction 1h
235identical, illustrate that the substrate heparosan depolymerization under this enzyme amount in 500g/L crude product solution is complete, the like, lower than the substrate heparosan in the crude product solution of this concentration also depolymerization completely.So, adopt 0.5h just can ensure in the reaction time to react completely in detection system.According to the equation of Fig. 3, can convert and obtain the testing result (table 2) of heparosan content in each concentration crude product.
Table 2: enzyme process detects the content of heparosan in crude product
The average of heparosan content percentage under calculating variable concentrations, known application enzyme process, in this sample, heparosan content is about 1.62%.
(2) sulfuric acid carbazole method
Table 3: sulfuric acid carbazole method detects the content of crude product heparosan
Known application carbazole method, in this sample, heparosan content is about 1.89%.By more known, the measured value of application enzyme process to unknown sample heparosan content is slightly less than measures income value with carbazole method, shows that enzymatic assays interference is less, can be applied to the mensuration to crude product heparosan content.But this system detectable concentration limit is too high, sterling typical curve lowest detection lower limit is about 1.6g/L.
Embodiment 5: fermentation liquor detects the impact of heparosan content to enzyme process
Supernatant heparosan crude product being dissolved in respectively pure water and E.coli K5 fermentation liquor is made into series of samples concentration (table 4), and it is identical with table 1 to add reaction system A(method) detect heparosan content.The results are shown in Table 4, when crude product concentration is 500g/L, add excessive enzyme reaction 0.5h and 1h, OD
235nmremain unchanged, substrate complete reaction is described.As can be known from the above table under identical content in crude product, the absorbance recorded in fermentation liquor, a little more than content in crude product, should be the impact containing a small amount of heparosan in fermentation liquor.But when heparosan crude product concentration is higher than 300g/L, in fermentation liquor, the mensuration of heparosan content is compared with the heparosan content in crude product aqueous solution, and difference is not remarkable.Show that being also applicable to being applied to heparosan content in directly to fermentation liquor at heparosan enzyme process detects.
Table 4: fermentation liquor detects the impact of heparosan content to enzyme process
| Crude product heparosan content (g/L) | OD 235nm aqueous solution | OD 235nm fermentation liquor |
| 100 | 0.118 | 0.197 |
| 200 | 0.250 | 0.330 |
| 300 | 0.483 | 0.487 |
| 400 | 0.715 | 0.735 |
| 500 | 0.918 | 0.925 |
Embodiment 6: the optimization of enzyme detection system
Apply above-mentioned enzyme system to measure heparosan content and need the content of heparosan in sample liquid to be greater than 1.6g/L(and heparosan crude product to be greater than 100g/L), the concentration of substrate of needs is larger.Because the output of Escherichia coli K5 sweat is sometimes lower than 1.6g/L, heparosan content monitoring in Escherichia coli K5 fermentation liquor directly can not be applied in.Therefore need adjustment enzyme reaction system, to reduce the concentration limit of heparosan in liquid to be measured, improve the detection threshold of enzyme detection system.Table 4 is system after adjustment.
Table 4: the enzyme process detection system B improving hepaerosan detection threshold
Reagent E is the heparosan standard solution (being dissolved in A liquid) of different gradient concentration;
Reagent A:20mM Tris HCl, 50mM NaCl, 4mM CaCl
2, 0.01%(w/v) and bovine serum albumin, solvent is water, pH7.5;
Reagent C:50mM HCl solution;
Reagent D:Hep III liquid (mixed liquor that enzyme liquid storage mixes with 1:3 ratio with A liquid);
Fig. 4 is the typical curve that in reaction system B, Heparinase III enzyme process detects heparosan.
Can extrapolate from Fig. 2 when the heparosan in reaction system is 0.5mg, add Heparinase III 8.25u, substrate is complete reaction.After adjustment system, when heparosan liquid concentration is 0.7g/L, in reaction system, heparosan quality is 0.42mg, be less than 0.5mg, the enzyme activity unit added is greater than 8.25u can ensure substrate complete reaction, therefore the consumption of Reagent D suitably can dilute according to required enzyme activity unit, and total enzyme is lived and is greater than 8.25U, and namely the enzyme of Reagent D is lived and is greater than 165U/mL.
After system adjustment, heparosan after the concentration limit surveyed of heparosan reaches purifying in liquid to be measured is that 0.1g/L(is when to work as concentration be 0.1g/L, OD235 is about 0.06, for can measured value, lower absorption values scene photometer cannot predict or inaccurate), it is 5 ~ 10g/L that crude product heparosan can survey minimum, improves detection threshold (Fig. 4).
The heparosan crude product that the system after above-mentioned optimization of applying is different to two content detects, and the results are shown in Table 5.
Table 5: heparosan detection example in the enzyme reaction system B after optimization
In table 5, the heparosan content average measurement result of sample 1 and sample 2 is respectively 3.53% and 1.55%, and difference is remarkable, shows that the detection that the enzyme reaction system B after optimizing application carries out heparosan is accurately feasible.
Claims (3)
1. a method of enzyme process Quantitative detection heparosan, described method comprises:
(1) testing sample is got, be dissolved in the damping fluid of pH7.0 ~ 7.5 and be mixed with sample solution, mixing, at 25 ~ 35 DEG C, balance 0.1 ~ 10min, add enough Heparinase III enzymes, mixing, 20 ~ 35 DEG C of water-bath 0.1 ~ 3h, add HCl solution, centrifugal mixing, get supernatant and carry out the detection of ultraviolet spectrometry light photometer, not carry out the sample of enzyme reaction for blank, read the absorbance A of sample
235nm; The content that described enzyme system measures heparosan in the sample solution of heparosan content is 1.6g/L-10g/L, and the enzyme amount added is greater than 8.25U/ μ L;
(2) prepare the heparosan standard solution of gradient concentration, carry out reacting and detecting according to step (1) method, draw the typical curve of absorbance and concentration of substrate;
(3) reference standard curve, per sample absorbance A
235nm, obtain heparosan concentration value in sample solution, convert and obtain heparosan content in sample.
2. the method for claim 1, is characterized in that step (1) damping fluid is composed as follows: TrisHCl 10 ~ 50mM, NaCl 40 ~ 80mM, CaCl
21 ~ 4mM, bovine serum albumin 0.005 ~ 0.02%, solvent is water, pH 7.0 ~ 7.5.
3. the method for claim 1, is characterized in that described method is as follows:
(1) get testing sample, be dissolved in buffer A the sample solution being mixed with sample concentration 1 ~ 500g/L, get 0.6mL sample solution, mixing, balance 0.1 ~ 10min at 25 ~ 35 DEG C, add Heparinase III enzyme liquid 0.05mL, mixing, 25 ~ 35 DEG C of water-bath 0.5 ~ 3h, add 50mM HCl solution 2.35mL, centrifugal mixing, get supernatant and carry out the detection of ultraviolet spectrometry light photometer, not carry out the sample of enzyme reaction for blank, read the absorbance A of sample
235nm; Described buffer A is composed as follows: Tris HCl 20mM, NaCl 50mM, CaCl
24mM, bovine serum albumin 0.01%, solvent is water, pH 7.5; Described Heparinase III enzyme liquid is dissolved in buffer A by HeparinaseIII enzyme and obtains, and Mei Ye unit enzyme is lived and is greater than 165U/mL;
(2) prepare the heparosan standard solution of gradient concentration between 0.1 ~ 10g/L, carry out reacting and detecting according to step (1) method, draw the typical curve of absorbance and concentration of substrate;
(3) reference standard curve, per sample absorbance A
235nm, obtain heparosan concentration value in sample solution, convert and obtain heparosan content in sample.
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