CN102559643B - Method for preparing fructose lysine enzyme and application of preparing fructose lysine enzyme - Google Patents
Method for preparing fructose lysine enzyme and application of preparing fructose lysine enzyme Download PDFInfo
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Abstract
The invention provides a method for preparing fructose lysine enzyme and a glycated albumin detection kit, wherein the glycated albumin detection kit is prepared by utilizing the fructose lysine enzyme that is prepared by adopting the method. The method comprises the operation steps as follows: strains are screened so as to obtain 11 to 82 aspergillus strains of the high-activity fructose lysine enzyme; the 11 to 82 aspergillus strains are cultivated, and mycelia are collected; the mycelia are suspended in buffer solution, and then are processed through cell disruption and centrifugation, and supernatant fluid is collected; the supernatant fluid is processed through fractional precipitation by adopting ammonium sulfate solution, and deposits are collected so as to obtain crude extracts; the crude extracts are processed through hydrophobic chromatography, and eluant is collected; in addition, the collected eluant is processed through affinity chromatography, and then eluant is collected, that is, fructose lysine enzyme solution is obtained. The method has the advantages as follows: the preparation steps are simple, and the obtained fructose lysine enzyme achieves high purity, high activity and high reaction specificity.
Description
Technical field
The present invention relates to medical test technical field, be specifically related to a kind of preparation method and application thereof of the fructose lysyl oxidase using in medical test.
Background technology
Diabetes are class metabolic pattern diseases that occur in hyperglycemia population, can cause the most systems of body to be subject to serious infringement, especially nerve and blood vessel.Estimate according to the World Health Organization, diabetes mellitus in China number has occupied second place of the world.Diabetes will become the most serious public health problem of future 50 years China.
Glycosylated albumin (Glycated albumin, GA) refer to that non-enzymatic saccharification react occurs for glucose and albumin N-terminal in human serum and the product that forms, wherein 90% with albumin chain in Methionin ε-NH2 residue react, its reaction principle is for first both form unsettled glycosylamine (Glycosylamine) or Schiff base (Schiff Base), and the latter forms stable keto-amine (ketoamine) through irreversible glycosamine (Amadori) rearrangement reaction again.Because the albuminous transformation period is approximately 20 days, so detecting, glycosylated albumin can be used to detect 2-3 week average blood sugar level in the past.At present, glycosylated albumin has become a requisite test item of diabetic subject, and compared with glycolated hemoglobin, it is more suitable for the index as assessment diabetes dialysis patients hospital care and mortality risk.
So the glycosylated albumin amount in human serum of how accurately determining becomes the key of clinical detection glycosylated albumin.Existing market mainly adopts enzyme process for detection of the glycosylated albumin in human serum.Its reaction principle, for first using proteolytic enzyme that glycated protein is digested to low-molecular-weight saccharification polypeptide, is used fructosyl amino acid enzyme catalysis saccharification polypeptide generation oxidizing reaction to generate polypeptide (or amino acid), arabino-hexosone and H subsequently
2o
2.The H discharging
2o
2measure by end reaction colorimetry, it is proportional in the absorption value at 600nm place and the concentration of glycosylated albumin.Concrete reaction process is as follows:
From above-mentioned reaction principle and reactions steps, fructosyl amino acid enzyme is the key enzyme during glycosylated albumin is measured, and can fructosyl amino acid enzyme become the key that accurately measure glycosylated albumin in human serum.The fructosyl amino acid enzyme that is applicable to glycosylated albumin mensuration need have following characteristics:
1, specificity is high: fructose Methionin and fructose Methionin polypeptide are had to high vigor, to other fructosyl amino acid low vitalities except fructose Methionin or there is no vigor;
2, vigor is high;
3, Heat stability is good;
4, pH sphere of action is wide, pH good stability.
And therefore the shortcoming such as the import reagent box existence and stability of existing mensuration glycosylated albumin poor (being dry powder), required enzyme amount are large in the market, specificity is not high requires the highly active fructosyl amino acid enzyme of exploitation to solve above-mentioned drawback.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of fructose lysyl oxidase, and the method preparation process is simple, and the fructose lysyl oxidase purity obtaining is high, activity is high, atopic is high.
The technical solution adopted in the present invention is:
A preparation method for fructose lysyl oxidase, comprises following operation steps:
(1) bacterium: many strains Aspergillus bacterial strain is fermented, collect mycelia and obtain the crude extract containing fructose lysyl oxidase, take fructose Methionin and fructosyl Methionin polypeptide as substrate, utilize quinone method to measure the activity of fructose lysyl oxidase, the Aspergillus strain 11-82 of high reactivity fructose lysyl oxidase is produced in screening;
(2) cultivate: with culture medium culturing 11-82 Aspergillus strain, collect mycelia and clean;
(3) just carry: get the mycelia of having cleaned and be suspended in damping fluid, through cytoclasis, centrifugal, collect supernatant liquor; The weightmeasurement ratio of mycelia and damping fluid is 1: 3～5;
(4) ammonium sulfate precipitation: use ammoniumsulphate soln to carry out fractionation precipitation to step (3) gained supernatant liquor, be dissolved in buffer A finally collecting the precipitation obtaining, obtain crude extract;
(5) hydrophobic chromatography: with buffer A balance hydrophobic chromatography post, then the crude extract loading that step (4) is obtained absorption, after absorption finishes, carry out gradient elution with ammoniumsulphate soln, collect elutriant;
(6) affinity chromatography: the fructosyl amino acid except fructose Methionin is coupled on immobilization carrier, with after bufferA balance chromatography column, by the elutriant loading after hydrophobic chromatography post wash-out, carry out affine absorption, after absorption, carry out gradient elution with ammoniumsulphate soln, collect elutriant, obtain fructose lysyl oxidase solution;
Wherein: buffer A is 15%～30% the saturated ammonium sulphate of preparing as end liquid using the Tris-Hcl damping fluid of 5～15mM, pH8～8.5.
In described step (1), 11-82 Aspergillus strain is the bacterial classification from row filter.11-82 Aspergillus strain Classification And Nomenclature is aspergillus niger, Latin name is Aspergillus niger, be preserved in and be positioned at China Committee for Culture Collection of Microorganisms of Pekinese of China common micro-organisms center (CGMCC), the address of depositary institution is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on October 21st, 2011, and deposit number is CGMCC No.5364.
The present invention is fermented to 250 strain Aspergillus bacterial strains, after cleaning, centrifugal collection mycelia, be resuspended in damping fluid, after ultrasonic disruption lysing cell, centrifugal collection supernatant liquor, utilize quinone method to measure fructose lysyl oxidase activity, and screening obtain active higher fructose lysyl oxidase and bacterium producing multi enzyme preparation thereof.
The present invention adopts " two-step approach " to screen the fructose lysyl oxidase activity that originates from different aspergillus tubigensis.First round screening is using fructose Methionin as substrate, measures fructose lysyl oxidase to its action activity, wherein 153 strain non-activities, and 41 strain vigor are low, and 56 strain vigor performances are higher.Then 56 strain aspergillus tubigensis are carried out to second takes turns screening, adopts fructosyl Methionin polypeptide as substrate (being the stand-in of glycosylated albumin hydrolysate), wherein 40 strain non-activities, and residue 16 strain vigor performances differ.In addition, the fructose lysyl oxidase that this 16 strain is produced using fructose glycine and fructose α-amino-isovaleric acid as substrate, is measured its activity respectively.Finally, the 16 strain bacterial strains that screening is obtained carry out Physiology and biochemistry, Molecular Identification, determine that it belongs to 6 kinds of aspergillus, in table 1.
Table 1
| Numbering | Bacterial classification | Fru-lys | Fru-lys-his | Fru-Gly | Fru-Val |
| Aspergillus sp.11-08 | A.niger | 108.5 | 65.8 | 3.6 | 6.3 |
| Aspergillus sp.11-115 | A.niger | 94.9 | 78.4 | 2.7 | 8.1 |
| Aspergillus sp.11-82 | A.niger | 88.2 | 71.6 | 1.9 | 3.7 |
| Aspergillus sp.11-245 | A.niger | 29.8 | 18.9 | 0.9 | 9.6 |
| Aspergillus sp.11-117 | A.niger | 15.7 | 9.6 | 2.6 | 6.8 |
| Aspergillus sp.11-216 | A.niger | 19.8 | 14.6 | 3.2 | 11.2 |
| Aspergillus sp.11-75 | A.fumigatus | 24.3 | 19.3 | 5.9 | 9.7 |
| Aspergillus sp.11-01 | A.fumigatus | 11.3 | 3.2 | 1.6 | 7.6 |
| Aspergillus sp.11-33 | A.fumigatus | 8.9 | 5.4 | 0.4 | 15.8 |
| Aspergillus sp.11-25 | A.terreus | 16.7 | 6.8 | 6.6 | 12.1 |
| Aspergillus sp.11-98 | A.terreus | 21.1 | 12.9 | 1.2 | 8.9 |
| Aspergillus sp.11-46 | A.glaucus | 17.8 | 8.6 | 0.9 | 14.7 |
| Aspergillus sp.11-26 | A.glaucus | 13.4 | 7.8 | 1.9 | 4.3 |
| Aspergillus sp.11-10 | A.glaucus | 22.8 | 13.5 | 0.8 | 7.9 |
| Aspergillus sp.11-150 | A.flavus | 15.7 | 2.6 | 2.4 | 10.4 |
| Aspergillus sp.11-235 | A.versicolor | 14.8 | 4.6 | 1.3 | 8.1 |
Wherein: Fru-lys is fructose Methionin, Fru-lys-his is the two peptides of fructosyl Methionin Histidine, and Fru-Gly is fructose glycine, and Fru-Val is fructose α-amino-isovaleric acid.
As can be seen from Table 1, in the 16 strain aspergillus that screen, wherein Aspergillus niger totally 6 strains, and no matter to fructose Methionin, or fructosyl Methionin polypeptide all shows the enzyme higher compared with other bacterial strains and lives, therefore select enzyme three the highest strains alive, numbering is respectively 11-08,11-115,11-82.Also can be found out by table 1 in addition, the fructose lysyl oxidase that this three strains bacterial strain produces is higher to the activity of fructose Methionin and the two peptides of fructose Methionin Histidine, and it is lower to the activity of fructose glycine and fructose α-amino-isovaleric acid, therefore present higher specificity, thereby the fructose Methionin that this three strains bacterium is produced is further purified, so that for further study to zymologic property.
Although the bacterial strain fermenting enzyme work of 11-82 is slightly low compared with 11-08,11-11, but its zymologic property excellence, 60 ℃ of insulation half hours, still can preserve 50% activity, and within the scope of this very wide pH of pH4-10, enzyme is lived all very high, thus elect our final bacterial strain as, for carrying out the preparation of fructose lysyl oxidase.
As preferably, in described step (2), the composition of substratum comprises fructose Methionin 5～10g/L, glucose 5～15g/L, dipotassium hydrogen phosphate 0.5～1.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.5～1.5g/L, magnesium sulfate 0.2～0.8g/L, calcium chloride 0.05～0.2g/L, VITAMIN mixing solutions 0.5～2 ‰, metallic ion mixed liquor 0.5～2 ‰, the pH of substratum is 4.5～6.5, and wherein the composition of VITAMIN mixing solutions comprises V
b1hcl 0.5～1.5g/dL, riboflavin 1.5～2.5mg/dL, calcium pantothenate 1～3mg/dL, V
b6hcl 1～3mg/dL, vitamin H 0.05～0.2mg/dL, benzaminic acid 0.5～2mg/dL, niacin 1～3mg/dL, folic acid 0.05～0.15mg/dL, the composition of metal mixed solution comprises magnesium sulfate 1～3g/L, zinc sulfate 1.5～3g/L, copper sulfate 0.1～1g/L, cobalt chloride 0.1～0.5g/L, Sodium orthomolybdate 0.2～0.3g/L, boric acid 0.2～0.6g/L, potassiumiodide 0.01～0.15g/L.
As preferably, in described step (3), damping fluid is phosphoric acid buffer or Tris-Hcl damping fluid.
As preferably, in described step (6), immobilization carrier is NHS-activated Sepharose 4 fast Flow or CNBr-activated Sepharose 4 fast Flow.
The present invention also provides a kind of glycosylated albumin detection kit, as the application of the fructose lysyl oxidase of above-mentioned preparation.This test kit is made up of following two kinds of reagent, wherein:
Reagent 1:
Reagent 2:
Described damping fluid is the one in Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, acetic acid-sodium acetate buffer, phthalic acid-hydrochloride buffer or glycine-hydrochloride buffer.
Described sanitas is the one in MIT, proclin300.
Described stablizer is the one in glycerine, tween 20.
The present invention is based on wild-type Aspergillus is fermented, the Aspergillus strain of high reactivity fructose lysyl oxidase is produced in screening, the aspergillus tubigensis obtaining from screening again, extract fructose lysyl oxidase, the extraction process adopting is separation and Extraction fructose lysyl oxidase simply and rapidly, extracting method gentleness, make enzyme keep native conformation, thereby obtain compared with the enzyme product of higher specific activity and more excellent stability.
The method of current existing document, patent report purifying fructosyl amino acid enzyme, mainly adopt ammonium sulfate precipitation, cross ion exchange column, drainage column, sieve chromatography etc., some steps also will repeat 2-3 time, not only process is loaded down with trivial details, expense is large, and final yield is also very low, only be only applicable to the research in laboratory, can not meet the demand of preparing in enormous quantities test kit.The present invention only adopts three steps (ammonium sulfate precipitation, hydrophobic chromatography, affinity chromatography) just can obtain the purification schemes of pure enzyme, not only greatly simplified purge process, and yield reaches 47%.
The preparation method of fructose lysyl oxidase provided by the invention, not only method is easy and simple to handle, and final yield and purity all higher, purification effect excellence.The fructose lysyl oxidase of separation and purification gained, active high, detect for glycosylated albumin test kit, enzyme amount used reduces, and atopic improves, and is suitable for preparation and the application of test kit, can be widely used in clinical detection.
Classification And Nomenclature from the bacterial classification 11-82 of row filter Aspergillus strain is aspergillus niger, Latin name is Aspergillus niger, preservation date is on October 21st, 2011, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCC No.5364.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, but is not limited to this.
A preparation method for fructose lysyl oxidase, comprises following operation steps:
(1) 250 strain Aspergillus bacterial strains are fermented, collect mycelia and obtain the crude extract containing fructose lysyl oxidase, utilize quinone method to measure the activity of fructose lysyl oxidase, the Aspergillus strain 11-82 of high reactivity fructose lysyl oxidase is produced in screening.
(2) with culture medium culturing 11-82 Aspergillus strain, collect mycelia and clean.
(3) just carry: get in the Tris-Hcl damping fluid that mycelia that 180g (weight in wet base) cleaned is suspended in 10mM, pH8.5 (containing 2mM DTT), ultrasonic disruption lysing cell, by lysate centrifugal 30min under 20000g rotating speed, collect supernatant liquor, sampling records the ratio enzyme of fructose lysyl oxidase and lives as 0.196U/mg.
(4) ammonium sulfate precipitation: using ammoniumsulphate soln (concentration respectively is 45%, 70%) is that enzyme extract carries out fractionation precipitation to supernatant liquor, be dissolved in buffer A finally collecting the precipitation obtaining, obtain crude extract, sampling records the ratio enzyme of fructose lysyl oxidase and lives as 0.402U/mg.
(5) hydrophobic chromatography: select adsorption column (2.2cm × 30cm), dress phenyl-sepharose 1500mL, with after buffer A balance pillar, the crude extract loading absorption that step (4) is obtained, after absorption finishes, carry out gradient elution with the ammoniumsulphate soln of 25-0%, collect elutriant, sampling records the ratio enzyme of fructose lysyl oxidase and lives as 18.6U/mg.
(6) affinity chromatography: fructose α-amino-isovaleric acid is coupled on CNBr-activated Sepharose, with after buffer A balance chromatography column, by the elutriant loading after hydrophobic chromatography post wash-out, carry out affine absorption, after absorption, carry out gradient elution with the ammoniumsulphate soln of 25-0%, collect elutriant, sampling records the ratio enzyme of fructose lysyl oxidase and lives as 94.7U/mg.
Wherein, buffer A is 25% the saturated ammonium sulphate (containing 2mM DTT) of preparing as end liquid using the Tris-Hcl damping fluid of 10mM, pH8.5
Above-mentioned steps (3)～step (6) finishes fructose lysyl oxidase to carry out the detection of correlation parameter, and result is as shown in table 2.
Table 2
The measuring method of above-mentioned fructose lysyl oxidase activity is as follows:
1, preparation detects the cell pyrolysis liquid that enzyme is lived: get 50mL bacterium liquid, the centrifugal 15min of 5500rpm collects mycelia, after ultrapure water is clean, centrifugal, Eddy diffusion is in 30mM Tris-Hcl damping fluid (pH8.0), adopt ultrasonic disruption lysing cell, by lysate under 20000g rotating speed centrifugal 30 minutes, collect supernatant liquor.
2, adopt Xylene Brilliant Cyanine G method to measure protein content.
3, the mensuration of enzymic activity: get 50uL lysate and be added in reaction mixture, 37 ℃ of temperature are bathed 5-10 minute, measure the light absorption value at 505 places.The composition of reaction mixture is: 100umol Tris-Hcl (pH8.0), and the amino Sedatine of 4.5umol 4-, 6.0umol phenol, 6.0unit/mL peroxidase, 5.0umol substrate, cumulative volume is 3mL.Wherein, a unit of enzyme activity is defined as: at 37 ℃, per minute catalysis produces the needed enzyme amount of 0.5umol quinone dyestuff.The umol product that actual enzyme activity forms by the every mg albumen of per minute is measured.
The present invention selects fructose Methionin or the fructose Methionin polypeptide substrate as the activation analysis of fructose lysyl oxidase.
The synthesis step of fructose Methionin is as follows: selected Methionin and glucose (mol ratio is 1: 10), take anhydrous methanol as solvent, 4h refluxes the in the situation that of boiling, after having reacted, by methyl alcohol evaporate to dryness, with phosphate buffered saline buffer (10mM, pH7.4,) dissolved solids resistates, then carry out separation and purification through reversed-phase HPLC, finally obtain purity and be 98% fructose Methionin.
The synthesis step of fructosyl Methionin polypeptide is as follows: choose dissolved state containing Methionin polypeptide (as: lys-phe, lys-ser, lys-arg, lys-ile, lys-leu, lys-val, lys-glu, lys-tyr, lys-ala, lys-his, lys-leu-val, lys-cys-cys) and glucose (mol ratio is 1: 4), take anhydrous methanol as solvent, heating in water bath 2h at 64 ℃, the product obtaining carries out separation and purification through silicagel column and TLC, then carries out phenetic analysis through mass spectrum (LC-MS).Result shows, the final yield of above-mentioned fructosyl Methionin polypeptide is between 8～67%, and wherein Gly-lys-ser yield is the highest, is 67%.Respectively take above-mentioned fructosyl Methionin polypeptide as substrate, measure fructose lysyl oxidase to its activity, result shows, the enzymic activity take Gly-lys-his as substrate is the highest, reaches 70.7U/L, therefore select the substrate of Gly-lys-his as the activation analysis of fructose lysyl oxidase.
The above embodiment of the present invention is can not be used for limiting the present invention to explanation of the present invention, and any change in implication and the scope suitable with claims of the present invention, all should think to be included in the scope of claims.
Claims (2)
1. a preparation method for fructose lysyl oxidase, is characterized in that comprising following operation steps:
(1) cultivate: with culture medium culturing 11-82 Aspergillus strain, collect mycelia and clean;
(2) just carry: get the mycelia of having cleaned and be suspended in damping fluid, through cytoclasis, centrifugal, collect supernatant liquor; The weightmeasurement ratio of mycelia and damping fluid is 1 ︰ 3～5;
(3) ammonium sulfate precipitation: use ammoniumsulphate soln to carry out fractionation precipitation to step (2) gained supernatant liquor, be dissolved in buffer A finally collecting the precipitation obtaining, obtain crude extract;
(4) hydrophobic chromatography: with buffer A balance hydrophobic chromatography post, then the crude extract loading that step (3) is obtained absorption, after absorption finishes, carry out gradient elution with ammoniumsulphate soln, collect elutriant;
(5) affinity chromatography: the fructosyl amino acid except fructose Methionin is coupled on immobilization carrier, with after buffer A balance chromatography column, by the elutriant loading after hydrophobic chromatography post wash-out, carry out affine absorption, after absorption, carry out gradient elution with ammoniumsulphate soln, collect elutriant, obtain fructose lysyl oxidase solution;
Wherein: buffer A is 15%～30% the saturated ammonium sulphate of preparing as end liquid using the Tris-Hcl damping fluid of 5～15mM, pH8～8.5;
In described step (1), 11-82 Aspergillus strain is the bacterial classification from row filter, Classification And Nomenclature is aspergillus niger, Latin name is Aspergillus niger, on October 21st, 2011, in the center preservation of China Committee for Culture Collection of Microorganisms of Pekinese common micro-organisms, deposit number was CGMCC No.5364;
In described step (1), the composition of substratum comprises fructose Methionin 5～10g/L, glucose 5～15g/L, dipotassium hydrogen phosphate 0.5～1.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.5～1.5g/L, magnesium sulfate 0.2～0.8g/L, calcium chloride 0.05～0.2g/L, VITAMIN mixing solutions 0.5～2 ‰, metallic ion mixed liquor 0.5～2 ‰, the pH of substratum is 4.5～6.5, and wherein the composition of VITAMIN mixing solutions comprises V
b1hcl0.5～1.5g/dL, riboflavin 1.5～2.5mg/dL, calcium pantothenate 1～3mg/dL, V
b6hcl1～3mg/dL, vitamin H 0.05～0.2mg/dL, benzaminic acid 0.5～2mg/dL, niacin 1～3mg/dL, folic acid 0.05～0.15mg/dL, the composition of metallic ion mixed liquor comprises magnesium sulfate 1～3g/L, zinc sulfate 1.5～3g/L, copper sulfate 0.1～1g/L, cobalt chloride 0.1～0.5g/L, Sodium orthomolybdate 0.2～0.3g/L, boric acid 0.2～0.6g/L, potassiumiodide 0.01～0.15g/L;
In described step (2), damping fluid is phosphoric acid buffer or Tris-Hcl damping fluid;
In described step (5), immobilization carrier is NHS-activated Sepharose 4 fast Flow or CNBr-activated Sepharose 4 fast Flow.
2. a glycosylated albumin detection kit, is characterized in that: formed by following two kinds of reagent, wherein:
Reagent 1:
Reagent 2:
Described damping fluid is the one in Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, acetic acid-sodium acetate buffer, phthalic acid-hydrochloride buffer or glycine-hydrochloride buffer;
Described sanitas is the one in MIT or proclin300;
Described stablizer is the one in glycerine, tween 20.
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| CN104164473A (en) * | 2013-05-16 | 2014-11-26 | 北京豪迈生物工程有限公司 | Glycated albumin enzymatic detection kit and detection method thereof |
| CN103695380B (en) * | 2013-12-27 | 2016-05-25 | 宁波美康生物科技股份有限公司 | Fructosyl amino acid oxidase, preparation method and the glycosylated albumin detection kit containing this enzyme |
| CN111676236A (en) * | 2020-03-12 | 2020-09-18 | 北京达成生物科技有限公司 | Escherichia coli expression method of recombinant FLOD protein |
| CN117286037B (en) * | 2023-11-20 | 2024-03-01 | 西北农林科技大学深圳研究院 | Filamentous fungi and application thereof in gas metabolism |
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| CN102559643A (en) | 2012-07-11 |
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