CN107266561A - A kind of preparation method of the gossypin enzymolysis product coupling protein matter detected for historical relic - Google Patents
A kind of preparation method of the gossypin enzymolysis product coupling protein matter detected for historical relic Download PDFInfo
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- CN107266561A CN107266561A CN201710645257.6A CN201710645257A CN107266561A CN 107266561 A CN107266561 A CN 107266561A CN 201710645257 A CN201710645257 A CN 201710645257A CN 107266561 A CN107266561 A CN 107266561A
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- 230000008878 coupling Effects 0.000 title claims abstract description 22
- 238000010168 coupling process Methods 0.000 title claims abstract description 22
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 22
- SJRXVLUZMMDCNG-UHFFFAOYSA-N Gossypin Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=C(O)C2=O SJRXVLUZMMDCNG-UHFFFAOYSA-N 0.000 title claims abstract description 13
- SJRXVLUZMMDCNG-KKPQBLLMSA-N gossypin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=C(O)C2=O SJRXVLUZMMDCNG-KKPQBLLMSA-N 0.000 title claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 26
- 229920000742 Cotton Polymers 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 229940088598 enzyme Drugs 0.000 claims abstract description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000008367 deionised water Substances 0.000 claims abstract description 18
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 17
- 239000008103 glucose Substances 0.000 claims abstract description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 12
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims abstract description 12
- 108010059892 Cellulase Proteins 0.000 claims abstract description 10
- 229940106157 cellulase Drugs 0.000 claims abstract description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 9
- 229920002678 cellulose Polymers 0.000 claims abstract description 8
- 239000001913 cellulose Substances 0.000 claims abstract description 8
- 239000000284 extract Substances 0.000 claims abstract description 8
- 238000011282 treatment Methods 0.000 claims abstract description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 6
- 238000005238 degreasing Methods 0.000 claims abstract description 6
- 239000007791 liquid phase Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 38
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000001556 precipitation Methods 0.000 claims description 19
- 238000000967 suction filtration Methods 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 14
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 13
- 239000003643 water by type Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000001976 enzyme digestion Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 8
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 8
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 229960002163 hydrogen peroxide Drugs 0.000 claims description 7
- 239000000835 fiber Substances 0.000 claims description 6
- 108010088751 Albumins Proteins 0.000 claims description 5
- 102000009027 Albumins Human genes 0.000 claims description 5
- 239000012888 bovine serum Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000007654 immersion Methods 0.000 claims description 2
- 238000003801 milling Methods 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims 1
- 238000005057 refrigeration Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 6
- 102000036639 antigens Human genes 0.000 abstract description 6
- 108091007433 antigens Proteins 0.000 abstract description 6
- 239000004753 textile Substances 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 3
- 239000004744 fabric Substances 0.000 abstract description 3
- 238000011081 inoculation Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 229920002488 Hemicellulose Polymers 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 abstract 1
- 238000000227 grinding Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010022355 Fibroins Proteins 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
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- Biotechnology (AREA)
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- General Chemical & Material Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Emergency Medicine (AREA)
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- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to historical relic detection field, disclose a kind of preparation method of the gossypin enzymolysis product coupling protein matter detected for historical relic, first to gossypin degreasing, after delignification and hemicellulose being removed through acetic acid and naoh treatment, cellulose is digested using cellulase and β glucuroides, deionized water ammonium sulfate polyethylene glycol 400 extract is prepared again, levels liquid phase after extraction is freeze-dried respectively, the enzyme that upper strata is recycled, lower floor obtains glucose powder, glucose powder and bovine serum albumin are dissolved in deionized water and potassium bromide solution, and placed in climatic chamber, then freeze-drying obtains glucose bovine serum albumin conjugate.The coupling macromolecular that the method provided using the present invention is obtained relatively is stablized, using this coupling macromolecular as comlete antigen, inoculation is carried out to animal, you can obtain corresponding specific antibody, feasibility is provided for the cotton fabric category in identification ancient textiles historical relic.
Description
Technical field
The present invention relates to historical relic detection field, more particularly to a kind of gossypin enzymolysis product coupling detected for historical relic
The preparation method of protein.
Background technology
China has been unearthed a large amount of textile historical relics in recent years, identifies that its affiliated species has to the history culture for studying China
Important meaning.Identification for silk goods has occurred in that several more accurate sensitive methods at present, wherein exempted from enzyme-linked again
Epidemic disease determining adsorption, Western Blotting are representative, and they set up mostly can occur spy between antigen and corresponding antibody
The opposite sex is combined in this immunology ABC.Because silk contains a large amount of protein in itself, so utilizing silk fibroin
Inoculation is carried out to animal as comlete antigen, can be obtained by respective handling can be anti-with fibroin albumen specific binding
Body, just can detect in ancient textiles whether contain silk using these antibody.But the main component of cotton fiber is fiber
Element, it is impossible to carry out animal immune directly as comlete antigen, therefore, it is difficult to utilize enzyme linked immunosorbent assay (ELISA), Western
The methods such as Blotting are identified the bafta in ancient textiles historical relic.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of gossypin enzymolysis product idol detected for historical relic
Join the preparation method of protein.The present invention is handled the enzymolysis product glucose of gossypin and large biological molecule by a series of
Coupling forms coupling macromolecular.Using this coupling macromolecular as comlete antigen, inoculation is carried out to animal, you can obtain relative
The specific antibody answered, the present invention is with a creative way by cellulose product and albumen coupling, in identification ancient textiles historical relic
Cotton fabric category provide feasibility.
The present invention concrete technical scheme be：A kind of gossypin enzymolysis product coupling protein matter detected for historical relic
Preparation method, in terms of g, mL, comprises the following steps：
1）9-11 g cotton fibers are taken, are pulverized, 45-55 mL benzene is added, 20-30 mL ethanol immersion treatments are carried out to fiber
Ungrease treatment, processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the cotton fiber after degreasing spend from
Sub- water is rinsed, and cotton fiber powder is obtained after drying.
2）The cotton fiber powder after 4-6g drying is taken, 180-220 mL 4-6wt% acetic acid is added, in 70-80 DEG C of water
Suction filtration after 10-14 h is heated in bath, precipitation is taken, 180-220mL 7-9wt% sodium hydroxide is added after being washed with deionized,
Suction filtration after 10-14 h is heated in 70-80 DEG C of water-bath, by precipitation deionized water rinsing.
3）Take step 2）Middle gained precipitation, addition concentration is 8-12wt% hydrogenperoxide steam generators, is added in 40-50 DEG C of water-bath
Suction filtration after hot 4-6h, by precipitation deionized water rinsing, cellulose powder is obtained after drying.
4）2-3g cellulose powders are taken, 70-80mL deionized waters are added, 0.4-0.6 g cellulase and 0.1- is weighed
Regulation pH is 4-5 after 0.3 g beta-glucosidase is added into solution, carries out enzyme digestion reaction 7-9h.
The present invention is handled cellulose progress using complex enzyme can accelerate the speed of enzymolysis, shorten technological process, simultaneously
Also the degree of enzymolysis can be improved, increases yield.
5）8-12 mL PEG-4000s are pipetted into beaker, 4-6 mL deionized waters is subsequently added into, is eventually adding 8-12
The 30wt% ammonium sulfates that mL is prepared in advance, stir after being stored at 1-5 DEG C, extract are made.
6）After enzyme digestion reaction terminates, 20-30mL extracts are added in surplus solution, 1-5 is placed in after being uniformly mixed
7-9h in DEG C environment, then carries out centrifugal treating to solution, and solution is layered after centrifugation, and levels liquid phase is collected respectively and carries out vacuum
Freeze-drying, obtains enzyme after upper organic phase is lyophilized, can reuse, and glucose powder is obtained after lower floor's solution is lyophilized.
The later product of cellulase hydrolysis is small molecule glucose, and cellulase and beta-glucosidase are biological big point
Son, the present invention is recycled using extraction to enzyme, is reclaimed obtained enzymatic activity more than 90%, is largely saved
Production cost.
7）0.15-0.25 g bovine serum albumins and 0.8-1.2 g glucose powder is taken to be dissolved in 18-22mL deionized waters,
Vacuum freeze drying is carried out after stirring, lyophilized rear gained powder is dissolved in 25-35 mL saturations potassium bromide solutions and in 55-65
DEG C, placed 6-8 days in relative humidity 60-70% climatic chamber.
The enzymolysis product glucose of cellulose and bovine serum albumin can be coupled by the new coupling of generation by the method big
Molecule, and obtained coupling macromolecular is relatively stable.
8）To step 7）Middle resulting solution carries out vacuum freeze drying, finally obtains glucose-bovine serum albumin conjugate.
Preferably, step 1）In, milling time is 3-5min, and soak time is 6-10h, and drying temperature is 45-55 DEG C.
Preferably, step 3）In, the feed liquid mass ratio of precipitation and hydrogenperoxide steam generator is 1:25-35, drying temperature is
45-55℃。
Preferably, step 4）In, the enzyme activity of cellulase and beta-glucosidase is 10000 u/g.
Preferably, step 6）In, centrifugation rate is 10000-14000 r/min.
It is compared with the prior art, the beneficial effects of the invention are as follows：
1st, the method that the present invention is provided can be new by the way that enzymolysis product glucose and the bovine serum albumin coupling of cellulose are produced
Coupling macromolecular, and obtained coupling macromolecular relatively stablize, using it is this coupling macromolecular as comlete antigen, animal is exempted from
Epidemic disease is injected, you can obtain corresponding specific antibody, is provided for the cotton fabric category in identification ancient textiles historical relic feasible
Property.
2nd, the present invention is handled cellulose progress using complex enzyme can accelerate the speed of enzymolysis, shorten experiment flow, together
When can also improve the degree of enzymolysis, increase yield, then enzyme recycled using extraction, the enzymatic activity that recovery is obtained exists
More than 90%, largely save production cost.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1
1）10 g cotton fibers are taken, grinding is placed in beaker after 3 minutes, add 45 mL benzene, 20 mL ethanol soak 6 h with to fibre
Dimension carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the cotton fiber after degreasing
With deionized water rinsing 5 times, dried in 50 DEG C of baking oven.
2）The cotton fiber powder after 5 g drying is taken, 200 mL 4% acetic acid is added, 12 h is heated in 75 DEG C of water-bath
Suction filtration, takes precipitation afterwards, and 7% sodium hydroxide that 200 mL are added after 5 times is washed with deionized, and is heated in 75 DEG C of water-bath
Suction filtration after 12 h, by precipitation deionized water rinsing 5 times.
3）Last gained in step 2 is taken to precipitate, it is 1 to add feed liquid mass ratio:30 8% hydrogen peroxide, in 45 DEG C of water-baths
Suction filtration after 5 h of middle heating, by precipitation deionized water rinsing 5 times, dries in 50 DEG C of baking ovens, obtains cellulose powder.
4）Gained cellulose powder in 2.5 g steps 3 is taken, 75 mL deionized waters are added, the g enzyme activity of precise 0.4 is
Regulation pH is after the beta-glucosidase that 10000 u/g cellulase and 0.1 g enzyme activity are 10000 u/g is added into solution
4, carry out the h of enzyme digestion reaction 8.
5）A 100 mL beakers are taken, 8 mL PEG-4000s are pipetted with pipette into beaker, 5 mL are subsequently added into
Deionized water, is eventually adding 30% ammonium sulfate that 8 mL are prepared in advance, stirs after storage at 4 DEG C.
6）After enzyme digestion reaction terminates, added in surplus solution in 25 mL steps 5 and match somebody with somebody extract, be uniformly mixed
After be placed in 8 h in 4 DEG C of environment, then by solution under 12000 r/min rotating speed from, after centrifugation solution be layered, collect respectively
Levels liquid phase carries out vacuum freeze drying, obtains enzyme after upper organic phase is lyophilized, can reuse, after lower floor's solution is lyophilized
Obtain glucose powder.
7）Take gained glucose powder in 0.2 g bovine serum albumins and 1 g steps 6 to be dissolved in 20 mL deionized waters, stir
Vacuum freeze drying is carried out after uniform to solution, lyophilized rear gained powder is dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C,
Placed 7 days in the climatic chamber of relative humidity 65%.
8）Vacuum freeze drying is carried out to resulting solution in step 7, glucose-bovine serum albumin conjugate is finally obtained.
Embodiment 2
1）10 g cotton fibers are taken, grinding is placed in beaker after 4 minutes, add 50 mL benzene, 25 mL ethanol soak 8 h with to fibre
Dimension carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the cotton fiber after degreasing
With deionized water rinsing 5 times, dried in 50 DEG C of baking oven.
2）The cotton fiber powder after 5 g drying is taken, 200 mL 5% acetic acid is added, 12 h is heated in 75 DEG C of water-bath
Suction filtration, takes precipitation afterwards, and 8% sodium hydroxide that 200 mL are added after 5 times is washed with deionized, and is heated in 75 DEG C of water-bath
Suction filtration after 12 h, by precipitation deionized water rinsing 5 times.
3）Last gained in step 2 is taken to precipitate, it is 1 to add feed liquid mass ratio:30 10% hydrogen peroxide, in 45 DEG C of water-baths
Suction filtration after 5 h of middle heating, by precipitation deionized water rinsing 5 times, dries in 50 DEG C of baking ovens, obtains cellulose powder.
4.）Gained cellulose powder in 2.5 g steps 3 is taken, 75 mL deionized waters are added, the g enzyme activity of precise 0.5 is
Regulation pH is after the beta-glucosidase that 10000 u/g cellulase and 0.2 g enzyme activity are 10000 u/g is added into solution
4.5, carry out the h of enzyme digestion reaction 8.
5）A 100 mL beakers are taken, 10 mL PEG-4000s are pipetted with pipette into beaker, 5 mL are subsequently added into
Deionized water, is eventually adding 30% ammonium sulfate that 10 mL are prepared in advance, stirs after storage at 4 DEG C.
6）After enzyme digestion reaction terminates, added in surplus solution in 25 mL steps 5 and match somebody with somebody extract, be uniformly mixed
After be placed in 8 h in 4 DEG C of environment, then by solution under 12000 r/min rotating speed from, after centrifugation solution be layered, collect respectively
Levels liquid phase carries out vacuum freeze drying, obtains enzyme after upper organic phase is lyophilized, can reuse, after lower floor's solution is lyophilized
Obtain glucose powder.
7）Take gained glucose powder in 0.2 g bovine serum albumins and 1 g steps 6 to be dissolved in 20 mL deionized waters, stir
Vacuum freeze drying is carried out after uniform to solution, lyophilized rear gained powder is dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C,
Placed 7 days in the climatic chamber of relative humidity 65%.
8）Vacuum freeze drying is carried out to resulting solution in step 7, glucose-bovine serum albumin conjugate is finally obtained.
Embodiment 3
1）10 g cotton fibers are taken, grinding is placed in beaker after 5 minutes, add 55 mL benzene, 30 mL ethanol soak 10 h with right
Fiber carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, and the cotton after degreasing is fine
Wesy's deionized water rinsing 5 times, is dried in 50 DEG C of baking oven.
2）The cotton fiber powder after 5 g drying is taken, 200 mL 6% acetic acid is added, 12 h is heated in 75 DEG C of water-bath
Suction filtration, takes precipitation afterwards, and 9% sodium hydroxide that 200 mL are added after 5 times is washed with deionized, and is heated in 75 DEG C of water-bath
Suction filtration after 12 h, by precipitation deionized water rinsing 5 times.
3）Last gained in step 2 is taken to precipitate, it is 1 to add feed liquid mass ratio:30 12% hydrogen peroxide, in 45 DEG C of water-baths
Suction filtration after 5 h of middle heating, by precipitation deionized water rinsing 5 times, dries in 50 DEG C of baking ovens, obtains cellulose powder.
4）Gained cellulose powder in 2.5 g steps 3 is taken, 75 mL deionized waters are added, the g enzyme activity of precise 0.6 is
Regulation pH is after the beta-glucosidase that 10000 u/g cellulase and 0.3 g enzyme activity are 10000 u/g is added into solution
5, carry out the h of enzyme digestion reaction 8.
5）A 100 mL beakers are taken, 12 mL PEG-4000s are pipetted with pipette into beaker, 5 mL are subsequently added into
Deionized water, is eventually adding 30% ammonium sulfate that 12 mL are prepared in advance, stirs after storage at 4 DEG C.
6）After enzyme digestion reaction terminates, added in surplus solution in 25 mL steps 5 and match somebody with somebody extract, be uniformly mixed
After be placed in 8 h in 4 DEG C of environment, then by solution under 12000 r/min rotating speed from, after centrifugation solution be layered, collect respectively
Levels liquid phase carries out vacuum freeze drying, obtains enzyme after upper organic phase is lyophilized, can reuse, after lower floor's solution is lyophilized
Obtain glucose powder.
7）Take gained glucose powder in 0.2 g bovine serum albumins and 1 g steps 6 to be dissolved in 20 mL deionized waters, stir
Vacuum freeze drying is carried out after uniform to solution, lyophilized rear gained powder is dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C,
Placed 7 days in the climatic chamber of relative humidity 65%.
8）Vacuum freeze drying is carried out to resulting solution in step 7, glucose-bovine serum albumin conjugate is finally obtained.
Raw materials used in the present invention, equipment, is the conventional raw material, equipment of this area unless otherwise noted；Side used in the present invention
Method, is the conventional method of this area unless otherwise noted.
It is described above, only it is presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, it is every according to the present invention
Any simple modification, change and equivalent transformation that technical spirit is made to above example, still fall within the technology of the present invention side
The protection domain of case.
Claims (5)
1. it is a kind of for historical relic detect gossypin enzymolysis product coupling protein matter preparation method, it is characterised in that with g,
ML is counted, and is comprised the following steps：
1）9-11 g cotton fibers are taken, are pulverized, 45-55 mL benzene is added, 20-30 mL ethanol immersion treatments are carried out to fiber
Ungrease treatment, processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the cotton fiber after degreasing spend from
Sub- water is rinsed, and cotton fiber powder is obtained after drying；
2）The cotton fiber powder after 4-6g drying is taken, 180-220 mL 4-6wt% acetic acid is added, in 70-80 DEG C of water-bath
Suction filtration after 10-14 h is heated, precipitation is taken, 180-220mL 7-9wt% sodium hydroxide is added after being washed with deionized,
Suction filtration after 10-14 h is heated in 70-80 DEG C of water-bath, by precipitation deionized water rinsing；
3）Take step 2）Middle gained precipitation, addition concentration is 8-12wt% hydrogenperoxide steam generators, and 4- is heated in 40-50 DEG C of water-bath
Suction filtration after 6h, by precipitation deionized water rinsing, cellulose powder is obtained after drying；
4）2-3g cellulose powders are taken, 70-80mL deionized waters are added, 0.4-0.6 g cellulase and 0.1-0.3 g is weighed
Beta-glucosidase add into solution after regulation pH be 4-5, carry out enzyme digestion reaction 7-9h；
5）8-12 mL PEG-4000s are pipetted into beaker, 4-6 mL deionized waters is subsequently added into, is eventually adding 8-12 mL
The 30wt% ammonium sulfates prepared in advance, stir after being stored at 1-5 DEG C, extract are made；
6）After enzyme digestion reaction terminates, 20-30mL extracts are added in surplus solution, 1-5 DEG C of ring is placed in after being uniformly mixed
7-9h in border, then carries out centrifugal treating to solution, and solution is layered after centrifugation, and levels liquid phase is collected respectively and carries out vacuum refrigeration
Dry, obtain enzyme after upper organic phase is lyophilized, can reuse, glucose powder is obtained after lower floor's solution is lyophilized；
7）Take 0.15-0.25 g bovine serum albumins and 0.8-1.2 g glucose powder to be dissolved in 18-22mL deionized waters, stir
Vacuum freeze drying is carried out after uniform, lyophilized rear gained powder is dissolved in 25-35 mL saturations potassium bromide solutions and in 55-65 DEG C, phase
To being placed 6-8 days in humidity 60-70% climatic chamber；
8）To step 7）Middle resulting solution carries out vacuum freeze drying, finally obtains glucose-bovine serum albumin conjugate.
2. a kind of preparation side of gossypin enzymolysis product coupling protein matter detected for historical relic as claimed in claim 1
Method, it is characterised in that step 1）In, milling time is 3-5min, and soak time is 6-10h, and drying temperature is 45-55 DEG C.
3. a kind of preparation side of gossypin enzymolysis product coupling protein matter detected for historical relic as claimed in claim 1
Method, it is characterised in that step 3）In, the feed liquid mass ratio of precipitation and hydrogenperoxide steam generator is 1:25-35, drying temperature is 45-
55℃。
4. a kind of preparation side of gossypin enzymolysis product coupling protein matter detected for historical relic as claimed in claim 1
Method, it is characterised in that step 4）In, the enzyme activity of cellulase and beta-glucosidase is 10000 u/g.
5. a kind of preparation side of gossypin enzymolysis product coupling protein matter detected for historical relic as claimed in claim 1
Method, it is characterised in that step 6）In, centrifugation rate is 10000-14000 r/min.
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| CA2310616C (en) * | 1997-11-26 | 2009-02-03 | David R. Schneider | Method and apparatus for preserving human saliva for testing |
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| CA2310616C (en) * | 1997-11-26 | 2009-02-03 | David R. Schneider | Method and apparatus for preserving human saliva for testing |
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