CN107266561B - Preparation method of cotton cellulose enzymatic hydrolysate coupled protein for cultural relic detection - Google Patents
Preparation method of cotton cellulose enzymatic hydrolysate coupled protein for cultural relic detection Download PDFInfo
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- 229920000742 Cotton Polymers 0.000 title claims abstract description 37
- 229920002678 cellulose Polymers 0.000 title claims abstract description 19
- 239000001913 cellulose Substances 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 title claims abstract description 12
- 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
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 7
- 239000000413 hydrolysate Substances 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 44
- 239000008367 deionised water Substances 0.000 claims description 35
- 229910021641 deionized water Inorganic materials 0.000 claims description 35
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 30
- 239000000843 powder Substances 0.000 claims description 24
- 239000002244 precipitate Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 17
- 229940088598 enzyme Drugs 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000010438 heat treatment Methods 0.000 claims description 15
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 15
- 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 description 14
- 238000004108 freeze drying Methods 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 238000000967 suction filtration Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 229940098773 bovine serum albumin Drugs 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 10
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 108010059892 Cellulase Proteins 0.000 claims description 9
- 229940106157 cellulase Drugs 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000000227 grinding Methods 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical class [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 230000008020 evaporation Effects 0.000 claims description 4
- 238000005238 degreasing Methods 0.000 claims description 3
- 229940068918 polyethylene glycol 400 Drugs 0.000 claims description 2
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 229920002521 macromolecule Polymers 0.000 description 6
- 239000004753 textile Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000004744 fabric Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010022355 Fibroins Proteins 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009870 specific binding Effects 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
Abstract
The invention relates to the field of cultural relics detection, and discloses a preparation method of cotton cellulose enzymatic hydrolysate coupled protein for cultural relics detection.
Description
Technical Field
The invention relates to the field of cultural relic detection, in particular to a preparation method of a cotton cellulose enzymatic hydrolysate conjugate protein for cultural relic detection.
Background
In recent years, China unearthes a large number of textile cultural relics, and the identification of the varieties of the textile cultural relics has great significance for the research of the historical culture of China. Several relatively precise and sensitive methods have been developed for the identification of silk fabrics, and these methods are represented by enzyme-linked immunosorbent assay, Western Blotting, and most of them are based on the basic immunological knowledge that specific binding occurs between antigen and corresponding antibody. Since silk contains a large amount of protein, the silk fibroin is used as a complete antigen to carry out immune injection on animals, antibodies capable of being specifically combined with the silk fibroin can be obtained through corresponding treatment, and whether the ancient textiles contain the silk or not can be detected by using the antibodies. However, the main component of cotton fiber is cellulose, and the cotton fiber cannot be directly used as a complete antigen for animal immunization, so that the identification of cotton fabrics in ancient textile cultural relics by using methods such as enzyme-linked immunosorbent assay, WesternBlotting and the like is difficult.
Disclosure of Invention
In order to solve the technical problem, the invention provides a preparation method of a cotton cellulose enzymolysis product coupled protein for cultural relic detection. The invention couples the glucose of the enzymolysis product of the cotton cellulose and the biomacromolecule to form the coupled macromolecule through a series of treatments. The invention couples the cellulose product with protein creatively, which provides feasibility for identifying cotton fabrics in ancient textile cultural relics.
The specific technical scheme of the invention is that the preparation method of the cotton cellulose enzymatic hydrolysate conjugate protein for cultural relic detection comprises the following steps in g and m L:
1) grinding 9-11 g of cotton fibers into powder, adding 45-55 m L benzene and 20-30m L ethanol for soaking, degreasing the fibers, evaporating the mixed solution under reduced pressure to recover benzene and ethanol, washing the degreased cotton fibers with deionized water, and drying to obtain cotton fiber powder.
2) Taking 4-6g of dried cotton fiber powder, adding 4-6wt% of acetic acid of 180-class 220m L, heating in a water bath at 70-80 ℃ for 10-14 h, then carrying out suction filtration, taking the precipitate, washing with deionized water, adding 7-9wt% of sodium hydroxide of 180-class 220m L, heating in a water bath at 70-80 ℃ for 10-14 h, then carrying out suction filtration, and washing the precipitate with deionized water.
3) Taking the precipitate obtained in the step 2), adding 8-12wt% hydrogen peroxide solution, heating in 40-50 ℃ water bath for 4-6h, filtering, washing the precipitate with deionized water, and drying to obtain cellulose powder.
4) Taking 2-3g of cellulose powder, adding 70-80m L deionized water, weighing 0.4-0.6 g of cellulase and 0.1-0.3 g of β -glucosidase, adding into the solution, adjusting the pH to 4-5, and carrying out enzymolysis reaction for 7-9 h.
The invention can accelerate enzymolysis speed, shorten process flow, improve enzymolysis degree and increase yield by using the compound enzyme to treat cellulose.
5) Transferring 8-12m L polyethylene glycol-400 into a beaker, adding 4-6 m L deionized water, finally adding 8-12m L prepared 30wt% ammonium sulfate solution, stirring uniformly, and storing at 1-5 deg.C to obtain extract.
6) After the enzymolysis reaction is finished, adding 20-30m L extract into the residual solution, stirring and mixing uniformly, placing in an environment with the temperature of 1-5 ℃ for 7-9h, then carrying out centrifugal treatment on the solution, layering the solution after centrifugation, respectively collecting upper and lower liquid phases, carrying out vacuum freeze drying, freeze-drying the upper organic phase to obtain enzyme, recycling, and freeze-drying the lower solution to obtain glucose powder.
The product after the cellulose hydrolysis is micromolecular glucose, and the cellulose and β -glucosidase are biomacromolecules.
7) Dissolving 0.15-0.25 g bovine serum albumin and 0.8-1.2 g glucose powder in 18-22m L deionized water, stirring, vacuum freeze drying, dissolving the powder in 25-35 m L saturated potassium bromide solution, and standing at 55-65 deg.C and relative humidity of 60-70% in a constant temperature and humidity cabinet for 6-8 days.
By the method, the enzymolysis product glucose of cellulose and bovine serum albumin can be coupled to generate new coupling macromolecules, and the obtained coupling macromolecules are stable.
8) And (3) carrying out vacuum freeze drying on the solution obtained in the step 7) to finally obtain the glucose-bovine serum albumin conjugate.
Preferably, in the step 1), the grinding time is 3-5min, the soaking time is 6-10h, and the drying temperature is 45-55 ℃.
Preferably, in the step 3), the mass ratio of the precipitate to the feed liquid of the hydrogen peroxide solution is 1:25-35, and the drying temperature is 45-55 ℃.
Preferably, in the step 4), the enzyme activities of the cellulase and the β -glucosidase are 10000 u/g.
Preferably, in step 6), the centrifugation rate is 10000-14000 r/min.
Compared with the prior art, the invention has the beneficial effects that:
1. the method provided by the invention can be used for coupling and producing new coupling macromolecules by the enzymolysis product glucose of the cellulose and the bovine serum albumin, the obtained coupling macromolecules are relatively stable, the coupling macromolecules are taken as complete antigens, and the corresponding specific antibodies can be obtained by carrying out immune injection on animals, thereby providing feasibility for identifying cotton fabrics in ancient textile cultural relics.
2. The method can accelerate the enzymolysis by using the complex enzyme to treat the cellulose, shorten the experimental process, improve the enzymolysis degree and increase the yield, and then recycle the enzyme by using extraction, so that the activity of the enzyme obtained by recycling is more than 90 percent, thereby saving the production cost to a great extent.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
1) Grinding 10 g of cotton fiber for 3 minutes, placing the cotton fiber in a beaker, adding 45 m of L benzene and 20m of L ethanol, soaking for 6 hours to degrease the fiber, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased cotton fiber with deionized water for 5 times, and drying the degreased cotton fiber in an oven at 50 ℃.
2) Taking 5 g of dried cotton fiber powder, adding 200 m of L% acetic acid, heating in 75 ℃ water bath for 12 h, performing suction filtration, taking precipitate, washing with deionized water for 5 times, adding 200 m of L% sodium hydroxide, heating in 75 ℃ water bath for 12 h, performing suction filtration, and washing the precipitate with deionized water for 5 times.
3) And (3) adding 8% hydrogen peroxide with the material liquid mass ratio of 1:30 into the finally obtained precipitate in the step (2), heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4) And (3) adding 75 m of L deionized water into 2.5 g of the cellulose powder obtained in the step (3), accurately weighing 0.4 g of cellulase with the enzyme activity of 10000 u/g and 0.1 g of β -glucosidase with the enzyme activity of 10000 u/g, adding the cellulase and the β -glucosidase into the solution, adjusting the pH value to 4, and carrying out enzymolysis reaction for 8 hours.
5) A100 m L beaker was taken, 8 m L PEG-400 was pipetted into the beaker, followed by addition of 5m L deionized water and finally 8 m L of a pre-prepared 30% ammonium sulfate solution, stirred well and stored at 4 ℃.
6) And after the enzymolysis reaction is finished, adding 25 m L of the extract prepared in the step 5 into the residual solution, stirring and mixing uniformly, placing in an environment at 4 ℃ for 8 h, then separating the solution at the rotating speed of 12000 r/min, layering the solution after centrifugation, respectively collecting upper and lower liquid phases, performing vacuum freeze drying, freeze-drying the upper organic phase to obtain the enzyme which can be recycled, and freeze-drying the lower solution to obtain glucose powder.
7) And (3) dissolving 0.2 g of bovine serum albumin and 1 g of glucose powder obtained in the step (6) in 20m L deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30m L saturated potassium bromide solution, and placing in a constant temperature and humidity box with the relative humidity of 60 ℃ for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-bovine serum albumin conjugate.
Example 2
1) Grinding 10 g of cotton fiber for 4 minutes, placing the cotton fiber in a beaker, adding 50m L benzene and 25 m L ethanol, soaking for 8 hours to degrease the fiber, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased cotton fiber with deionized water for 5 times, and drying in an oven at 50 ℃.
2) Taking 5 g of dried cotton fiber powder, adding 200 m of L5% acetic acid, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, taking the precipitate, washing with deionized water for 5 times, adding 200 m of L% sodium hydroxide, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, and washing the precipitate with deionized water for 5 times.
3) And (3) taking the precipitate finally obtained in the step (2), adding 10% hydrogen peroxide with the material liquid mass ratio of 1:30, heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4.) taking 2.5 g of the cellulose powder obtained in the step 3, adding 75 m L deionized water, accurately weighing 0.5 g of cellulase with the enzyme activity of 10000 u/g and 0.2 g of β -glucosidase with the enzyme activity of 10000 u/g into the solution, adjusting the pH value to 4.5, and carrying out enzymolysis reaction for 8 hours.
5) A100 m L beaker was taken, 10 m L PEG-400 was pipetted into the beaker, followed by 5m L of deionized water and finally 10 m L of a pre-prepared 30% ammonium sulfate solution, stirred well and stored at 4 ℃.
6) And after the enzymolysis reaction is finished, adding 25 m L of the extract prepared in the step 5 into the residual solution, stirring and mixing uniformly, placing in an environment at 4 ℃ for 8 h, then separating the solution at the rotating speed of 12000 r/min, layering the solution after centrifugation, respectively collecting upper and lower liquid phases, performing vacuum freeze drying, freeze-drying the upper organic phase to obtain the enzyme which can be recycled, and freeze-drying the lower solution to obtain glucose powder.
7) And (3) dissolving 0.2 g of bovine serum albumin and 1 g of glucose powder obtained in the step (6) in 20m L deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30m L saturated potassium bromide solution, and placing in a constant temperature and humidity box with the relative humidity of 60 ℃ for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-bovine serum albumin conjugate.
Example 3
1) Grinding 10 g of cotton fiber for 5 minutes, placing the cotton fiber in a beaker, adding 55 m L benzene and 30m L ethanol, soaking for 10 hours to degrease the fiber, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased cotton fiber with deionized water for 5 times, and drying in an oven at 50 ℃.
2) Taking 5 g of dried cotton fiber powder, adding 200 m of L6% acetic acid, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, taking the precipitate, washing with deionized water for 5 times, adding 200 m of L% sodium hydroxide, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, and washing the precipitate with deionized water for 5 times.
3) And (3) taking the precipitate finally obtained in the step (2), adding 12% hydrogen peroxide with the material liquid mass ratio of 1:30, heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4) And (3) adding 75 m L deionized water into 2.5 g of the cellulose powder obtained in the step (3), accurately weighing 0.6 g of cellulase with the enzyme activity of 10000 u/g and 0.3 g of β -glucosidase with the enzyme activity of 10000 u/g, adding the cellulase and the β -glucosidase into the solution, adjusting the pH value to 5, and performing enzymolysis reaction for 8 hours.
5) A100 m L beaker was taken, 12m L PEG-400 was pipetted into the beaker, followed by 5m L of deionized water and finally 12m L of a pre-prepared 30% ammonium sulfate solution, stirred well and stored at 4 ℃.
6) And after the enzymolysis reaction is finished, adding 25 m L of the extract prepared in the step 5 into the residual solution, stirring and mixing uniformly, placing in an environment at 4 ℃ for 8 h, then separating the solution at the rotating speed of 12000 r/min, layering the solution after centrifugation, respectively collecting upper and lower liquid phases, performing vacuum freeze drying, freeze-drying the upper organic phase to obtain the enzyme which can be recycled, and freeze-drying the lower solution to obtain glucose powder.
7) And (3) dissolving 0.2 g of bovine serum albumin and 1 g of glucose powder obtained in the step (6) in 20m L deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30m L saturated potassium bromide solution, and placing in a constant temperature and humidity box with the relative humidity of 60 ℃ for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-bovine serum albumin conjugate. The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (5)
1. A preparation method of a cotton cellulose enzymolysis product coupled protein for cultural relic detection is characterized by comprising the following steps of (g and m L):
1) grinding 9-11 g of cotton fibers into powder, adding 45-55 m L benzene and 20-30m L ethanol for soaking, degreasing the fibers, performing reduced pressure evaporation on the mixed solution after the degreasing to recover benzene and ethanol, washing the degreased cotton fibers with deionized water, and drying to obtain cotton fiber powder;
2) taking 4-6g of the cotton fiber powder dried in the step 1), adding 4-6wt% of acetic acid of 180-class 220m L, heating in a water bath at 70-80 ℃ for 10-14 h, then carrying out suction filtration, taking the precipitate, washing with deionized water, adding 7-9wt% of sodium hydroxide of 180-class 220m L, heating in a water bath at 70-80 ℃ for 10-14 h, then carrying out suction filtration, and washing the precipitate with deionized water;
3) taking the precipitate obtained in the step 2), adding 8-12wt% hydrogen peroxide solution, heating in a water bath at 40-50 ℃ for 4-6h, then carrying out suction filtration, washing the precipitate with deionized water, and drying to obtain cellulose powder;
4) taking 2-3g of the cellulose powder obtained in the step 3), adding 70-80m L deionized water, weighing 0.4-0.6 g of cellulase and 0.1-0.3 g of β -glucosidase, adding into the solution, adjusting the pH to 4-5, and carrying out enzymolysis reaction for 7-9 h;
5) transferring 8-12m L polyethylene glycol-400 into a beaker, adding 4-6 m L deionized water, finally adding 8-12m L prepared 30wt% ammonium sulfate solution, stirring uniformly, and storing at 1-5 deg.C to obtain extract;
6) after the enzymolysis reaction is finished, adding 20-30m L extract into the residual solution, stirring and mixing uniformly, placing in an environment at 1-5 ℃ for 7-9h, then carrying out centrifugal treatment on the solution, layering the solution after centrifugation, respectively collecting upper and lower liquid phases, carrying out vacuum freeze drying, freeze-drying the upper organic phase to obtain enzyme which can be recycled, and freeze-drying the lower solution to obtain glucose powder;
7) dissolving 0.15-0.25 g of bovine serum albumin and 0.8-1.2 g of glucose powder obtained in the step 6) in 18-22m L deionized water, uniformly stirring, performing vacuum freeze drying, dissolving the powder obtained after freeze drying in 25-35 m L saturated potassium bromide solution, and placing in a constant temperature and humidity box with the relative humidity of 60-70% for 6-8 days at 55-65 ℃;
8) and (3) carrying out vacuum freeze drying on the solution obtained in the step 7) to finally obtain the glucose-bovine serum albumin conjugate.
2. The method for preparing the cotton cellulose enzymolysis product coupled protein for cultural relic detection according to claim 1, wherein in the step 1), the grinding time is 3-5min, the soaking time is 6-10h, and the drying temperature is 45-55 ℃.
3. The method for preparing the cotton cellulose enzymatic hydrolysate coupled protein for cultural relic detection according to claim 1, wherein in the step 3), the mass ratio of the precipitate to the hydrogen peroxide solution is 1:25-35, and the drying temperature is 45-55 ℃.
4. The method for preparing the cotton cellulose enzymatic hydrolysate conjugate protein for cultural relic detection according to claim 1, wherein in the step 4), the enzyme activities of the cellulase and the β -glucosidase are 10000 u/g.
5. The method as claimed in claim 1, wherein the centrifugation rate in step 6) is 10000-14000 r/min.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2310616C (en) * | 1997-11-26 | 2009-02-03 | David R. Schneider | Method and apparatus for preserving human saliva for testing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2310616C (en) * | 1997-11-26 | 2009-02-03 | David R. Schneider | Method and apparatus for preserving human saliva for testing |
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