CN107365377A - A kind of preparation method of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection - Google Patents
A kind of preparation method of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection Download PDFInfo
- Publication number
- CN107365377A CN107365377A CN201710645248.7A CN201710645248A CN107365377A CN 107365377 A CN107365377 A CN 107365377A CN 201710645248 A CN201710645248 A CN 201710645248A CN 107365377 A CN107365377 A CN 107365377A
- Authority
- CN
- China
- Prior art keywords
- flaxen fiber
- historical relic
- fiber element
- protein matter
- powder
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000835 fiber Substances 0.000 title claims abstract description 41
- 230000008878 coupling Effects 0.000 title claims abstract description 23
- 238000010168 coupling process Methods 0.000 title claims abstract description 23
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000000843 powder Substances 0.000 claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 229940088598 enzyme Drugs 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000008367 deionised water Substances 0.000 claims abstract description 19
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 19
- 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 16
- 239000008103 glucose Substances 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000000047 product Substances 0.000 claims abstract description 15
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims abstract description 12
- 229920002678 cellulose Polymers 0.000 claims abstract description 10
- 239000001913 cellulose Substances 0.000 claims abstract description 10
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 9
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 9
- 108010059892 Cellulase Proteins 0.000 claims abstract description 8
- 229940106157 cellulase Drugs 0.000 claims abstract description 8
- 238000005238 degreasing Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 27
- 238000001556 precipitation Methods 0.000 claims description 19
- 239000003643 water by type Substances 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000001976 enzyme digestion Methods 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 7
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 7
- 229960002163 hydrogen peroxide Drugs 0.000 claims description 7
- 238000007654 immersion Methods 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000003801 milling Methods 0.000 claims description 2
- 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
- 229920002488 Hemicellulose Polymers 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 3
- 239000004744 fabric Substances 0.000 abstract description 3
- 238000011081 inoculation Methods 0.000 abstract description 3
- FCRIOPQKJNGNNX-UHFFFAOYSA-N formic acid;propan-2-one Chemical compound OC=O.CC(C)=O FCRIOPQKJNGNNX-UHFFFAOYSA-N 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 238000005057 refrigeration Methods 0.000 description 7
- 238000000227 grinding Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 150000002431 hydrogen Chemical class 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 3
- 229910001948 sodium oxide Inorganic materials 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010022355 Fibroins Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- MJFMYEVFESUAQP-UHFFFAOYSA-N formic acid propan-2-one hydrate Chemical compound O.OC=O.CC(C)=O MJFMYEVFESUAQP-UHFFFAOYSA-N 0.000 description 2
- 229920005610 lignin Polymers 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
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing 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
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Emergency Medicine (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to historical relic detection field, disclose a kind of preparation method of the flaxen fiber element enzymolysis product coupling protein matter for historical relic detection, degreasing is carried out to flaxen fiber element using benzene and ethanol, using acetone formic acid water, this mixed system removes delignification, after removing hemicellulose with alkali lye again, obtained cellulose is digested using cellulase and β glucuroides, hyperfiltration technique recovery enzyme is utilized after enzymolysis, filtrate is freeze-dried to obtain glucose powder, glucose powder and ovalbumin is taken to be dissolved in deionized water and potassium bromide solution, and placed in climatic chamber, then freeze-drying obtains glucose ovalbumin conjugate.The coupling macromolecular obtained using method provided by the invention is relatively stable, using this coupling macromolecular as comlete antigen, inoculation is carried out to animal, you can obtain corresponding specific antibody, feasibility is provided for the bast fiber fabrics in identification ancient textiles historical relic.
Description
Technical field
The present invention relates to historical relic detection field, more particularly to a kind of flaxen fiber element enzymolysis product coupling for historical relic detection
The preparation method of protein.
Background technology
Be unearthed a large amount of textile historical relics in China in recent years, identifies that its affiliated species has to the history culture for studying China
Important meaning.Has there are several more accurate sensitive methods at present in identification for silk goods, wherein exempted from enzyme-linked again
Epidemic disease determining adsorption, Western Blotting are representative, and they are established 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, detect in ancient textiles whether contain silk using these antibody cans.But the main component of flaxen fiber is fiber
Element, animal immune can not be carried out directly as comlete antigen, therefore, it is difficult to utilize enzyme linked immunosorbent assay (ELISA), Western
The methods of Blotting, is identified the linen 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 flaxen fiber element enzymolysis product for historical relic detection is even
Join the preparation method of protein.The present invention is handled the enzymolysis product glucose of flaxen fiber element 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
Flaxen fiber kind fabric provide feasibility.
The present invention concrete technical scheme be：A kind of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection
Preparation method, in terms of g, mL, comprise the following steps：
1）9-11 g flaxen fibers are taken, are pulverized, 45-55 mL benzene is added, 20-30 mL ethanol immersion treatments, fiber is carried out
Ungrease treatment, processing mixed solution are evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the flaxen fiber after degreasing spend from
Sub- water is rinsed, and flaxen fiber powder is obtained after drying.
2）The flaxen fiber powder after 4-6g drying is taken, adds 95-105 mL acetone and 45-55 mL deionized waters, stirring is mixed
Close uniformly after be added dropwise 8-12mL formic acid, after immersion to solution centrifugal processing, take bottom precipitation continue with previous solu handle and from
The heart, repeat and repeatedly finally precipitated, with deionized water rinsing, add 180-220mL 7-9wt% sodium hydroxide,
Filtered after heating 10-14h in 70-80 DEG C of water-bath, by precipitation deionized water rinsing.
Using acetone-formic acid-water, this mixed system can substantially completely remove lignin the present invention, recycle alkali soluble
Liquid removes the cellulose purity lifting that hemicellulose makes to obtain.
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
Filtered after hot 4-6h, by precipitation deionized water rinsing, cellulose powder is obtained after drying.
4）2-3g cellulose powders are taken, add 70-80mL deionized waters, weigh 0.4-0.6 g cellulase and 0.1-
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 handles cellulose the speed that can accelerate enzymolysis using complex enzyme, shortens technological process, simultaneously
Also the degree of enzymolysis can be improved, increases yield.
5）After enzyme digestion reaction terminates, ultrafiltration is carried out to enzymolysis liquid using milipore filter, and filtrate is stirred into processing and mixed, collects filter
Liquid, vacuum freeze drying obtain glucose powder.
6）Milipore filter is removed, it cleaned so that the enzyme on film to be eluted, vacuum refrigeration is carried out to eluent
Dry, the enzyme being recycled.
The present invention is reclaimed using hyperfiltration technique to enzyme, and the enzymatic activity for reclaiming to obtain has been saved and has been produced into more than 92%
This.
7）Take 0.15-0.25 g ovalbumins and 0.8-1.2 g glucose powder to be dissolved in 18-22mL deionized waters, stir
Vacuum freeze drying is carried out after mixing uniformly, lyophilized rear gained powder is dissolved in 25-35 mL saturations potassium bromide solutions and at 55-65 DEG C,
Placed 6-8 days in relative humidity 60-70% climatic chamber.
The enzymolysis product glucose of cellulose can be produced into new coupling with ovalbumin coupling by the method to divide greatly
Son, and obtained coupling macromolecular is relatively stable.
8）To step 7）Middle resulting solution carries out vacuum freeze drying, finally obtains glucose-ovalbumin conjugate.
Preferably, step 1）In, milling time 3-5min, soak time 6-10h, drying temperature are 45-55 DEG C.
Preferably, step 2）In, soak time 1.5-2.5h, centrifugation rate is 900-1100 r/min.
Preferably, step 3）In, the feed liquid mass ratio of precipitation and hydrogenperoxide steam generator is 1:25-35, drying temperature are
45-55℃。
Preferably, step 4）In, the enzyme activity of cellulase and beta-glucosidase is 10000 u/g.
Preferably, step 5）In, the interception of milipore filter is 30kDa, and it is 18-22 DEG C to control temperature during ultrafiltration, pressure
For 0.15-0.25Mpa, filtrate stir speed (S.S.) is 180-220 r/min.
It is compared with the prior art, the beneficial effects of the invention are as follows：
1st, method provided by the invention can be by the way that the enzymolysis product glucose of cellulose and ovalbumin coupling be produced newly
Macromolecular is coupled, and obtained coupling macromolecular is relatively stable, and using this coupling macromolecular as comlete antigen, animal is immunized
Injection, you can obtain corresponding specific antibody, feasibility is provided for the bast fiber fabrics in identification ancient textiles historical relic.
2nd, using acetone-formic acid-water, this mixed system can substantially completely remove lignin the present invention, recycle alkali
Solution removes the cellulose purity lifting that hemicellulose makes to obtain.
3rd, the present invention handles cellulose the speed that can accelerate enzymolysis using complex enzyme, shortens experiment flow, together
When can also improve the degree of enzymolysis, increase yield, then enzyme reclaimed using hyperfiltration technique, the enzymatic activity for reclaiming to obtain exists
More than 92%, save production cost.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1
1）10 g flaxen fibers are taken, grinding is placed in beaker after 3 minutes, adds 45 mL benzene, 20 mL ethanol, 6 h are with to fibre for immersion
Dimension carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the flaxen fiber after degreasing
With deionized water rinsing 5 times, dried in 50 DEG C of baking oven.
2）The flaxen fiber powder after 5 g drying is taken, 100 mL acetone and 50 mL deionized waters is added, is uniformly mixed
10 mL formic acid are added dropwise later, solution is centrifuged under 1000 r/min rotating speed after soaking 2 h, takes bottom precipitation to continue before using
State solution to handle and centrifuge, repeat 4 times and finally precipitated, with deionized water rinsing 5 times, add 200 mL 7% hydrogen
Sodium oxide molybdena, filtered after 12 h are heated in 75 DEG C of water-bath, 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
Filtered after 5 h are heated in bath, by precipitation deionized water rinsing 5 times, dried in 50 DEG C of baking ovens, obtain cellulose powder.
4）Gained cellulose powder in 2.5 g steps 3 is taken, adds 75 mL deionized waters, 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.）After enzyme digestion reaction terminates, ultrafiltration is carried out to enzymolysis liquid, uses milipore filter of the interception for 30kDa, ultrafiltration time control
Temperature processed is 20 DEG C, and pressure is 0.2 Mpa, and filtrate is stirred under 200 r/min rotating speed, collects filtrate, vacuum refrigeration
It is dried to obtain glucose powder.
6.）Milipore filter is removed, it cleaned so that the enzyme on film to be eluted, vacuum refrigeration is carried out to eluent
Dry, the enzyme being recycled.
7）Gained glucose powder in 0.2 g ovalbumins and 1 g steps 5 is taken to be dissolved in 20 mL deionized waters, stirring is equal
Carry out vacuum freeze drying after even to solution, it is lyophilized after gained powder be dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C, phase
To being placed 7 days in the climatic chamber of humidity 65%.
8 ）Vacuum freeze drying is carried out to resulting solution in step 7, finally obtains glucose-ovalbumin conjugate.
Embodiment 2
1）10 g flaxen fibers are taken, grinding is placed in beaker after 4 minutes, adds 50 mL benzene, 25 mL ethanol, 8 h are with to fibre for immersion
Dimension carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the flaxen fiber after degreasing
With deionized water rinsing 5 times, dried in 50 DEG C of baking oven.
2）The flaxen fiber powder after 5 g drying is taken, 100 mL acetone and 50 mL deionized waters is added, is uniformly mixed
10 mL formic acid are added dropwise later, solution is centrifuged under 1000 r/min rotating speed after soaking 2 h, takes bottom precipitation to continue before using
State solution to handle and centrifuge, repeat 4 times and finally precipitated, with deionized water rinsing 5 times, add 200 mL 8% hydrogen
Sodium oxide molybdena, filtered after 12 h are heated in 75 DEG C of water-bath, 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
Filtered after 5 h of middle heating, by precipitation deionized water rinsing 5 times, dried in 50 DEG C of baking ovens, obtain cellulose powder.
4）Gained cellulose powder in 2.5 g steps 3 is taken, adds 75 mL deionized waters, 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）After enzyme digestion reaction terminates, ultrafiltration is carried out to enzymolysis liquid, uses milipore filter of the interception for 30kDa, ultrafiltration time control
Temperature processed is 20 DEG C, and pressure is 0.2 Mpa, and filtrate is stirred under 200 r/min rotating speed, collects filtrate, vacuum refrigeration
It is dried to obtain glucose powder.
6）Milipore filter is removed, it cleaned so that the enzyme on film to be eluted, vacuum refrigeration is carried out to eluent
Dry, the enzyme being recycled.
7）Gained glucose powder in 0.2 g ovalbumins and 1 g steps 5 is taken to be dissolved in 20 mL deionized waters, stirring is equal
Carry out vacuum freeze drying after even to solution, it is lyophilized after gained powder be dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C, phase
To being placed 7 days in the climatic chamber of humidity 65%.
8）Vacuum freeze drying is carried out to resulting solution in step 7, finally obtains glucose-ovalbumin conjugate.
Embodiment 3
1）10 g flaxen fibers are taken, grinding is placed in beaker after 5 minutes, adds 55 mL benzene, 30 mL ethanol, 10 h are with to fibre for immersion
Dimension carries out ungrease treatment, and processing mixed solution is evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the flaxen fiber after degreasing
With deionized water rinsing 5 times, dried in 50 DEG C of baking oven.
2）The flaxen fiber powder after 5 g drying is taken, 100 mL acetone and 50 mL deionized waters is added, is uniformly mixed
10 mL formic acid are added dropwise later, solution is centrifuged under 1000 r/min rotating speed after soaking 2 h, takes bottom precipitation to continue before using
State solution to handle and centrifuge, repeat 4 times and finally precipitated, with deionized water rinsing 5 times, add 200 mL 9% hydrogen
Sodium oxide molybdena, filtered after 12 h are heated in 75 DEG C of water-bath, 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
Filtered after 5 h of middle heating, by precipitation deionized water rinsing 5 times, dried in 50 DEG C of baking ovens, obtain cellulose powder.
4）Gained cellulose powder in 2.5 g steps 3 is taken, adds 75 mL deionized waters, 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）After enzyme digestion reaction terminates, ultrafiltration is carried out to enzymolysis liquid, uses milipore filter of the interception for 30kDa, ultrafiltration time control
Temperature processed is 20 DEG C, and pressure is 0.2 Mpa, and filtrate is stirred under 200 r/min rotating speed, collects filtrate, vacuum refrigeration
It is dried to obtain glucose powder.
6）Milipore filter is removed, it cleaned so that the enzyme on film to be eluted, vacuum refrigeration is carried out to eluent
Dry, the enzyme being recycled.
7）Gained glucose powder in 0.2 g ovalbumins and 1 g steps 5 is taken to be dissolved in 20 mL deionized waters, stirring is equal
Carry out vacuum freeze drying after even to solution, it is lyophilized after gained powder be dissolved in 30 mL saturations potassium bromide solutions and at 60 DEG C, phase
To being placed 7 days in the climatic chamber of humidity 65%.
8）Vacuum freeze drying is carried out to resulting solution in step 7, finally obtains glucose-ovalbumin conjugate.
Raw materials used in the present invention, equipment, it is the conventional raw material, equipment of this area unless otherwise noted；In the present invention
Method therefor, it 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 the 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 (6)
1. it is a kind of for historical relic detection flaxen fiber element 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 flaxen fibers are taken, are pulverized, 45-55 mL benzene is added, 20-30 mL ethanol immersion treatments, fiber is carried out
Ungrease treatment, processing mixed solution are evaporated under reduced pressure after terminating to reclaim benzene and ethanol, the flaxen fiber after degreasing spend from
Sub- water is rinsed, and flaxen fiber powder is obtained after drying；
2）The flaxen fiber powder after 4-6g drying is taken, adds 95-105 mL acetone and 45-55 mL deionized waters, is stirred
8-12mL formic acid is added dropwise after even, to solution centrifugal processing after immersion, takes bottom precipitation to continue to be handled and centrifuged with previous solu, weight
Multiple operation is repeatedly finally precipitated, and with deionized water rinsing, 180-220mL 7-9wt% sodium hydroxide is added, in 70-80
DEG C water-bath in heat 10-14h after filter, 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
Filtered after 6h, by precipitation deionized water rinsing, cellulose powder is obtained after drying；
4）2-3g cellulose powders are taken, add 70-80mL deionized waters, weigh 0.4-0.6 g cellulase and 0.1-0.3 g
Beta-glucosidase add into solution after regulation pH be 4-5, progress enzyme digestion reaction 7-9h；
5）After enzyme digestion reaction terminates, ultrafiltration is carried out to enzymolysis liquid using milipore filter, and filtrate is stirred into processing and mixed, collects filtrate, very
Vacuum freecing-dry obtains glucose powder；
6）Milipore filter is removed, it cleaned so that the enzyme on film to be eluted, vacuum freeze drying is carried out to eluent,
The enzyme being recycled；
7）0.15-0.25 g ovalbumins and 0.8-1.2 g glucose powder is taken to be dissolved in 18-22mL deionized waters, stirring is equal
Vacuum freeze drying is carried out after even, lyophilized rear gained powder is dissolved in 25-35 mL saturations potassium bromide solutions and at 55-65 DEG C, relatively
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-ovalbumin conjugate.
A kind of 2. preparation side of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection as claimed in claim 1
Method, it is characterised in that step 1）In, milling time 3-5min, soak time 6-10h, drying temperature are 45-55 DEG C.
A kind of 3. preparation side of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection as claimed in claim 1
Method, it is characterised in that step 2）In, soak time 1.5-2.5h, centrifugation rate is 900-1100 r/min.
A kind of 4. preparation side of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection 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 45-
55℃。
A kind of 5. preparation side of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection 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.
A kind of 6. preparation side of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection as claimed in claim 1
Method, it is characterised in that step 5）In, the interception of milipore filter is 30kDa, and it is 18-22 DEG C to control temperature during ultrafiltration, and pressure is
0.15-0.25Mpa, filtrate stir speed (S.S.) are 180-220 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710645248.7A CN107365377B (en) | 2017-08-01 | 2017-08-01 | Preparation method of hemp cellulose hydrolysate conjugate protein for cultural relic detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710645248.7A CN107365377B (en) | 2017-08-01 | 2017-08-01 | Preparation method of hemp cellulose hydrolysate conjugate protein for cultural relic detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107365377A true CN107365377A (en) | 2017-11-21 |
| CN107365377B CN107365377B (en) | 2020-07-14 |
Family
ID=60310177
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710645248.7A Active CN107365377B (en) | 2017-08-01 | 2017-08-01 | Preparation method of hemp cellulose hydrolysate conjugate protein for cultural relic detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107365377B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104406992A (en) * | 2014-12-31 | 2015-03-11 | 浙江理工大学 | Method for detecting ancient silk fabrics by transmission electron microscope |
| CN104865218A (en) * | 2015-05-06 | 2015-08-26 | 江西出入境检验检疫局检验检疫综合技术中心 | Method for measuring cotton and hemp blend fiber content |
| CN105353118A (en) * | 2015-11-12 | 2016-02-24 | 浙江理工大学 | Detection method of keratin in ancient wool fabrics |
| CN105699660A (en) * | 2016-02-24 | 2016-06-22 | 浙江理工大学 | Specific immunity detection method of ancient wool |
-
2017
- 2017-08-01 CN CN201710645248.7A patent/CN107365377B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104406992A (en) * | 2014-12-31 | 2015-03-11 | 浙江理工大学 | Method for detecting ancient silk fabrics by transmission electron microscope |
| CN104865218A (en) * | 2015-05-06 | 2015-08-26 | 江西出入境检验检疫局检验检疫综合技术中心 | Method for measuring cotton and hemp blend fiber content |
| CN105353118A (en) * | 2015-11-12 | 2016-02-24 | 浙江理工大学 | Detection method of keratin in ancient wool fabrics |
| CN105699660A (en) * | 2016-02-24 | 2016-06-22 | 浙江理工大学 | Specific immunity detection method of ancient wool |
Non-Patent Citations (1)
| Title |
|---|
| 郑秦: "利用丝素蛋白抗体鉴定古代丝织品", 《蚕业科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107365377B (en) | 2020-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103422379B (en) | A kind of preparation method of bagasse cellulose nanofiber membrane | |
| Periyasamy et al. | Carrier-free co-immobilization of xylanase, cellulase and β-1, 3-glucanase as combined cross-linked enzyme aggregates (combi-CLEAs) for one-pot saccharification of sugarcane bagasse | |
| CN101787382B (en) | Method for recovering coin protein sugar dregs and preparing protein nitrogen sources and nitrogen-containing syrup | |
| CN101589153A (en) | Method for producing glucose by enzymatic hydrolysis of cellulose that can be pretreated with an ionic liquid containing a polyatomic anion | |
| CN101550606B (en) | Ramie degumming method utilizing a complex enzyme preparation | |
| CN108385421B (en) | lignocellulose supercritical CO2Blasting and component separation method thereof | |
| CN107829300B (en) | Method for synthesizing grafted lysine viscose fiber | |
| CN110499350B (en) | Sericin hydrolysis method, product and application thereof | |
| CN108316036A (en) | A kind of method that bean dregs prepare fento cellulose | |
| CN108486752B (en) | Preparation method of nano knitted woolen fabric with self-cleaning function | |
| CN106894271B (en) | The quick-fried method with enzymatic hydrogen peroxide oxidation combined pretreatment lignocellulose-like biomass of sulfur trioxide low-grade fever | |
| CN103669006B (en) | A kind of method utilizing Snailase that cotton fabrics is carried out Bio-Pretreatment | |
| US11959112B2 (en) | Process for production of sugar from a cotton-containing textile | |
| CN108048297A (en) | A kind of device applied to oxygenation pretreatment lignocellulosic enzymolysis saccharification water-saving and emission-reducing technique | |
| CN107365377A (en) | A kind of preparation method of flaxen fiber element enzymolysis product coupling protein matter for historical relic detection | |
| Yu et al. | DLC (sa) and DLCA (sa) pretreatments boost the efficiency of microbial lipid production from rice straw via Trichosporon dermatis | |
| CN102168381A (en) | Method for pretreating cotton fabric by using biological enzyme/mediator system and bleaching cotton fabric by using hydrogen peroxide | |
| CN107266561A (en) | A kind of preparation method of the gossypin enzymolysis product coupling protein matter detected for historical relic | |
| CN109090336A (en) | A kind of rice protein powder extracting method increasing protein content | |
| CN112962334A (en) | Natural dyeing auxiliary for dyeing sorghum husk pigment plants and dyeing method | |
| CN107287895B (en) | A method of wrinkle proofing being carried out to cotton fabric using biopolishing waste liquid | |
| CN111270534B (en) | Method for dyeing high-quality bulk fiber plant indigo | |
| CN102174765A (en) | Technological process for making Lian-si paper by using moso bamboo | |
| CN109320631A (en) | A method of extracting hemicellulose | |
| CN107653686A (en) | A kind of preparation method of desizing refining complex enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |