CN107266548A - The method that a kind of utilization ionic liquid and protease extract tussah silk fibroin albumen - Google Patents
The method that a kind of utilization ionic liquid and protease extract tussah silk fibroin albumen Download PDFInfo
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- CN107266548A CN107266548A CN201710626825.8A CN201710626825A CN107266548A CN 107266548 A CN107266548 A CN 107266548A CN 201710626825 A CN201710626825 A CN 201710626825A CN 107266548 A CN107266548 A CN 107266548A
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- China
- Prior art keywords
- ionic liquid
- fibroin albumen
- fibroin
- tussah silk
- protease
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- 108010022355 Fibroins Proteins 0.000 title claims abstract description 98
- 239000002608 ionic liquid Substances 0.000 title claims abstract description 70
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 44
- 239000004365 Protease Substances 0.000 title claims abstract description 44
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000000284 extract Substances 0.000 title claims abstract description 15
- 239000003513 alkali Substances 0.000 claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 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
- 239000000843 powder Substances 0.000 claims abstract description 17
- 239000000835 fiber Substances 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 102000035195 Peptidases Human genes 0.000 claims abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims description 31
- 108090000790 Enzymes Proteins 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 238000001035 drying Methods 0.000 claims description 18
- 239000012460 protein solution Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims description 10
- 239000011734 sodium Substances 0.000 claims description 7
- 239000000356 contaminant Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- OSDWBNJEKMUWAV-UHFFFAOYSA-N Allyl chloride Chemical compound ClCC=C OSDWBNJEKMUWAV-UHFFFAOYSA-N 0.000 claims description 5
- 206010013786 Dry skin Diseases 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical class CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 claims description 4
- 229910021538 borax Inorganic materials 0.000 claims description 4
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 4
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000001704 evaporation Methods 0.000 claims 1
- 230000008020 evaporation Effects 0.000 claims 1
- 238000009738 saturating Methods 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 abstract 1
- 239000000460 chlorine Substances 0.000 description 15
- 235000008708 Morus alba Nutrition 0.000 description 9
- 240000000249 Morus alba Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 230000006378 damage Effects 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000006641 stabilisation Effects 0.000 description 3
- 238000011105 stabilization Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000009991 scouring Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000255791 Bombyx Species 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 229920003043 Cellulose fiber Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 108010013296 Sericins Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- QKSIFUGZHOUETI-UHFFFAOYSA-N copper;azane Chemical compound N.N.N.N.[Cu+2] QKSIFUGZHOUETI-UHFFFAOYSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108091005899 fibrous proteins Proteins 0.000 description 1
- 102000034240 fibrous proteins Human genes 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229940059936 lithium bromide Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- XLSZMDLNRCVEIJ-UHFFFAOYSA-N methylimidazole Natural products CC1=CNC=N1 XLSZMDLNRCVEIJ-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000006365 thiocyanation reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Insects & Arthropods (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cosmetics (AREA)
Abstract
Field is extracted the present invention relates to fibroin albumen, a kind of method that utilization ionic liquid and protease extract tussah silk fibroin albumen is disclosed, tussah silk is handled using alkaline process degumming, fibroin fiber is obtained;Fibroin fiber is mixed with previously prepared [AMIM] Cl ionic liquids, the agitating and heating certain time in oil bath, preliminarily solubilised is carried out to tussah silk, PM13 alkali proteases are added to be digested, fibroin albumen is progressively dissolved using ionic liquid and PM13 alkali proteases, by add absolute ethyl alcohol remove ionic liquid, so as to get fibroin albumen dissolve again in deionized water, by dialysing and being freeze-dried to obtain pure silk fibroin powder.The extracting method of this tussah silk fibroin albumen has that reaction condition is gentle, chemical levels are few, ionic liquid is recyclable, and tussah silk fibroin albumen effectively can be dissolved.
Description
Technical field
Field is extracted the present invention relates to fibroin albumen, more particularly to one kind extracts tussah silk using ionic liquid and protease
The method of fibroin albumen.
Background technology
Silk is mainly made up of fibroin albumen and sericin, and wherein fibroin albumen accounts for 70%, be silk it is main into
Point.Silk gum is globular preteins, contains a large amount of polar hydrophilic side bases in composition amino acid, stability is poor, dissolves in acid, alkali, albumen
In the solution such as enzyme and low concentration sodium carbonate;And fibroin is fibrous protein, natural fibroin fiber contains a large amount of intramoleculars and divided
Hydrogen bond between son, crystallinity is dissolved in common solvent compared with Gao Ernan.
Relatively common in life is mulberry silk and tussah silk, mainly by glycine(Gly), alanine(Ala), serine
(Ser)And tyrosine(Tyr)Composition, but amino acid content and distributional difference are larger.In tussah silk peptide, with reactive group
Amino acid(Such as arginine, aspartic acid)More than mulberry silk, tryptophan, the content of histidine are 5 times of mulberry silk, tussah silk
Alanine content be 40%, mulberry silk is 30%.Therefore the bending of peptide chain and entanglement degree ratio mulberry silk are big in tussah silk, this just makes
Rigidity, the solvent resistant ability of tussah silk are higher than mulberry silk.
At present, in research process, frequently with strong acid, highly basic, high concentration salting liquid such as high concentration CaCl2Solution, copper
Ammonia solution, lithium-bromide solution, thiocyanation lithium solution etc. dissolve to Bombyx silk albumen, but these solvents are present to albumen
Matter molecular degradation is serious, toxic, unstable, the shortcomings of be not easily recycled, at the same to the extraction effect of tussah silk fibroin albumen compared with
Difference.
Ionic liquid (IL) is the green solvent of the great application prospect of a class of rising in recent years, is widely used in electrochemistry
, organic synthesis, chemical separating, material prepare etc. field.So-called ionic liquid is exactly in room temperature(Or a little higher than room temperature
Temperature)Under the ionic system that is in a liquid state, it is different from common are machine solvent, there is powerful electrostatic phase interaction in ionic liquid
With, therefore show unusual feature:Non-volatile, high stability, good conduction and thermal conductivity, selective dissolution power with
Designability, while having characteristic environmentally friendly, can be recycled.
The content of the invention
In order to solve the above-mentioned technical problem, tussah silk silk is extracted using ionic liquid and protease the invention provides one kind
The method of fibroin.The extracting method of the present invention is more gentle, damages small to fibroin albumen, nonhazardous environmentally friendly using raw material, and
Extraction efficiency is higher.
The present invention concrete technical scheme be:A kind of utilization ionic liquid and protease extract the side of tussah silk fibroin albumen
Method, in terms of g and mL, comprises the following steps:
1)3.5-4.5g tussah silks are weighed, are cleaned with deionized water, surface contaminant, drying is removed.
2)By the tussah silk of drying with 1:95-105 bath raio is in the Na containing 0.4-0.6wt%2PO4With 0.8-1.2wt%'s
C17H3525-35min is boiled in COONa mixed solution, degumming process is carried out, altogether degumming four times.
In scouring processes, with the hydrolysis of silk gum, the concentration of amino acid gradually increases in solution, pH reductions, degumming efficiency
Weaken, so alkali need to be added to maintain the stabilization of degumming liquid pH value, but alkali concn crosses conference damage fibroin, so using
C17H35COONa is used as buffer.
3)After degumming, tussah silk is washed by rubbing with the hands more than 4 times with deionized water, 55-65 DEG C of baking oven is put into, obtain the silk of drying
Cellulose fiber.
4)The fibroin fiber of 1.8-2.2g dryings is weighed, with 1:In 45-55 bath raio immersion [AMIM] Cl ionic liquids, control
Reaction temperature processed stirs 5-7h in the range of 80 ~ 100 DEG C in oil bath, after the completion of reaction, is cooled to room temperature.
5)To step 4)PM is added in intermediate ion liquid13- basic protein enzyme powder 0.25-0.35g, in 40 ~ 55 DEG C of oil
5-7h is stirred in bath, is obtained after fibroin/ionic liquid solution, the enzyme that goes out is carried out.
Biology enzyme is a kind of nontoxic and environment-friendly catalyst, possesses efficient specificity;And consumption is few, water
Solution condition(Temperature, pH)Gently, pollution is reduced, is economized on resources.Ionic liquid (IL) is the great application of a class of rising in recent years
The green solvent of prospect, certain dissolubility is shown to fibroin albumen.
The present invention uses PM13Alkali protease is modified, to keep stabilization of the alkali protease in ionic liquid
Property and activity.Alkali protease is also a kind of protein in itself, and ionic liquid has certain destruction, PM to its structure13
Place is a kind of comb copolymer, can be covered in alkali protease surface, prevent alkali protease from being destroyed by solion, simultaneously
By compatibility of the PM13 chains to IL, the stability of enzyme can be not only improved, it is ensured that activity of the enzyme in ionic liquid, and make
Enzyme is uniformly dispersed in IL.
In addition, the amino acid composition of tussah silk and mulberry silk has significant differences, although alanine and sweet ammonia in two kinds of silks
The content of content alanine all in 75% or so, but tussah silk of acid is more than glycine, and mulberry silk is then on the contrary, this causes
On peptide chain structure, tussah silk is based on the peptide chain of propyl- third, and the methyl in propyl- the third peptide chain structure side base so that the shape between side base
Into intensive hydrophobic effect, tussah silk is set to be difficult to dissolve.Simultaneously as the adhesion of tussah silk crystal region is more than mulberry silk, lead
Cause its dissolubility poor.Therefore, during being dissolved to it, first with solvability of the ionic liquid to fibroin albumen, in height
Preliminarily solubilised is carried out to fibroin albumen under the conditions of temperature, PM is added13- alkali protease, while using ion liquid dissolving
Hydrolysis and efficiency of the enzyme to fibroin albumen can also be improved.
The present invention uses PM13Alkali protease is modified, to keep stabilization of the alkali protease in ionic liquid
Property and activity.Alkali protease is also a kind of protein in itself, and ionic liquid has certain destruction, PM to its structure13
Place is a kind of comb copolymer, can be covered in alkali protease surface, prevent alkali protease from being destroyed by solion, simultaneously
By compatibility of the PM13 chains to IL, the stability of enzyme can be not only improved, it is ensured that activity of the enzyme in ionic liquid, and make
Enzyme is uniformly dispersed in IL.
6)Treat that fibroin/ionic liquid solution is cooled to room temperature, add absolute ethyl alcohol, soak repeatedly, separate out fibroin albumen,
Mixture is filtered by vacuum, deionized water is added into the fibroin albumen filtered out, is filtered after soaking repeatedly, then solution is filled
Enter the 20-28h that dialysed in bag filter, obtain pure silk fibroin protein solution.
7)Obtained silk fibroin protein solution is freeze-dried, you can obtain silk fibroin powder.
Preferably, step 4)In, the preparation method of [AMIM] the Cl ionic liquids is:By 1.0-1.5:1 mole
Than adding allyl chloride and N- methylimidazoles into reaction vessel, 6 ~ 8h is stirred at reflux in 55-65 DEG C of oil bath, reaction is finished
Afterwards, excessive 2- methallyl chlorides are removed using rotary evaporation, light yellow clear liquid are obtained, i.e. [AMIM] Cl ionic liquids
After body, freeze-drying 20-28h, thaw stand-by.
In the above-mentioned methods, it is necessary to which obtained product is freeze-dried, water that may be present in ionic liquid is removed
Point.Water is polar solvent, brings part hydrogen bond into, and ionic liquid is had an effect, so that ionic liquid can be reduced to fibroin albumen
Dissolution.
Preferably, step 5)In, the PM13The preparation method of-alkali protease is:To 90-110mL 0.1M boron
0.10-0.20g alkali proteases and 3.5-4.0g PM are added in sour sodium cushioning liquid13, it is slowly stirred under the conditions of 1-5 DEG C
0.5-1.5h, ultrafiltration removes unreacted PM under conditions of additional 45-55mL 1mmol/L HCl13, in triplicate, by filtrate
It is freeze-dried, obtains PM13- basic protein enzyme powder, it is stand-by.
Preferably, the PM13For MW15000 pectinations PEG.
Preferably, in PM13It is cold under the conditions of -20 DEG C in advance before freeze-drying in the preparation process of-alkali protease
Freeze 4h.
Preferably, step 5)In, go out enzyme when, at least 30min is incubated in the oil bath more than 80 DEG C.
Preferably, step 6)In, the molecular cut off of the bag filter is 3500.
It is compared with the prior art, the beneficial effects of the invention are as follows:
(1)The present invention utilizes PM13Alkali protease is modified, PM is utilized13To the affinity interaction of ionic liquid, not only carry
The high stability and reactivity of enzyme, while adding dispersing uniformity of the enzyme in ionic liquid, is conducive to it to fibroin
The hydrolysis of albumen.
(2)The present invention adds C in scouring processes17H35COONa maintains alkaline environment as buffer, improves degumming
Efficiency, while reducing the injury to fibroin fiber.
(3)The present invention is progressively handled fibroin albumen, carried using the dual dissolution effect of ionic liquid and biology enzyme
The high solubility of fibroin albumen.
(4)In the present invention, biology enzyme is nontoxic, with it is environment-friendly, while consumption is few, economize on resources;It is specific high, make
With mild condition, the destruction to fibroin albumen is small.Environmentally friendly and ionic liquid is " green solvent ", group can be designed, and
It is easily recycled, can be recycled.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1:
The method that a kind of utilization ionic liquid and protease extract tussah silk fibroin albumen, comprises the following steps:
1)4g tussah silks are weighed as sample, are cleaned with deionized water, surface contaminant, drying is removed.
2)By the sample of drying with 1:100 bath raio is containing 0.5% Na2PO4And 1%C17H35In COONa mixed solution
30min is boiled, degumming process is carried out, altogether degumming four times.
3)After degumming, sample is washed by rubbing with the hands more than 4 times with deionized water, 60 DEG C of baking ovens are put into, dry fibroin is obtained fine
Dimension.
4)The fibroin fiber of 2g dryings is weighed, with 1:In 50 bath raio immersion [AMIM] Cl ionic liquids, control reaction temperature
Degree stirs 6h in the range of 80 DEG C in oil bath.After the completion of reaction, room temperature is cooled to.
5)To 4)PM is added in intermediate ion liquid13- basic protein enzyme powder 0.3g, stirs 6h in 40 DEG C of oil bath, obtains
To after fibroin/ionic liquid solution, the enzyme that goes out is carried out.
6)Treat that fibroin/ionic liquid solution is cooled to room temperature, add absolute ethyl alcohol, soak repeatedly, have fibroin albumen analysis
Go out.Mixture is filtered by vacuum, deionized water is added into the fibroin albumen filtered out, is filtered after soaking repeatedly, then will be molten
Liquid is fitted into the bag filter of molecular cut off 3500 24h that dialyses, and obtains pure silk fibroin protein solution.
7)Obtained silk fibroin protein solution is freeze-dried, you can obtain silk fibroin powder.
Wherein, PM13The preparation of-alkali protease:0.15g alkali is added into 100mL 0.1M sodium borate buffer solution
Property protease and 3.75g PM13(Pectination PEG MW15000), 1h is slowly stirred under the conditions of 4 DEG C, in additional 50mL 1mmol/L
Ultrafiltration removes unreacted PM under conditions of HCl13, in triplicate.Filtrate is freeze-dried, PM is obtained13- basic protein
Enzyme powder, it is stand-by.
The preparation of [AMIM] Cl ionic liquids:According to 1.25:1 mol ratio added into three-necked flask allyl chloride and
N- methylimidazoles, are stirred at reflux 6h in 60 DEG C of oil baths, after completion of the reaction, and excessive 2- methallyls are removed using rotary evaporation
Base chlorine, obtains light yellow clear liquid, i.e. [AMIM] Cl ionic liquids, after freeze-drying 24h, thaws stand-by.
Embodiment 2:
The method that a kind of utilization ionic liquid and protease extract tussah silk fibroin albumen, comprises the following steps:
1)4g tussah silks are weighed as sample, are cleaned with deionized water, surface contaminant, drying is removed.
2)By the sample of drying with 1:100 bath raio is containing 0.5% Na2PO4And 1%C17H35In COONa mixed solution
30min is boiled, degumming process is carried out, altogether degumming four times.
3)After degumming, sample is washed by rubbing with the hands more than 4 times with deionized water, 60 DEG C of baking ovens are put into, dry fibroin is obtained fine
Dimension.
4)The fibroin fiber of 2g dryings is weighed, with 1:In 50 bath raio immersion [AMIM] Cl ionic liquids, control reaction temperature
Degree stirs 6h in the range of 90 DEG C in oil bath.After the completion of reaction, room temperature is cooled to.
5)To 4)PM is added in intermediate ion liquid13- basic protein enzyme powder 0.3g, stirs 6h in 47 DEG C of oil bath, obtains
To after fibroin/ionic liquid solution, the enzyme that goes out is carried out.
6)Treat that fibroin/ionic liquid solution is cooled to room temperature, add absolute ethyl alcohol, soak repeatedly, have fibroin albumen analysis
Go out.Mixture is filtered by vacuum, deionized water is added into the fibroin albumen filtered out, is filtered after soaking repeatedly, then will be molten
Liquid is fitted into the bag filter of molecular cut off 3500 24h that dialyses, and obtains pure silk fibroin protein solution.
7)Obtained silk fibroin protein solution is freeze-dried, you can obtain silk fibroin powder.
Wherein, PM13The preparation of-alkali protease:0.15g alkali is added into 100mL 0.1M sodium borate buffer solution
Property protease and 3.75g PM13(Pectination PEG MW15000), 1h is slowly stirred under the conditions of 4 DEG C, in additional 50mL 1mmol/L
Ultrafiltration removes unreacted PM under conditions of HCl13, in triplicate.Filtrate is freeze-dried, PM is obtained13- basic protein
Enzyme powder, it is stand-by.
The preparation of [AMIM] Cl ionic liquids:According to 1.25:1 mol ratio adds allyl chloride and N- into three-necked flask
Methylimidazole, is stirred at reflux 7h in 60 DEG C of oil baths, after completion of the reaction, and excessive 2- methacrylics are removed using rotary evaporation
Chlorine, obtains light yellow clear liquid, i.e. [AMIM] Cl ionic liquids, after freeze-drying 24h, thaws stand-by.
Embodiment 3:
The method that a kind of utilization ionic liquid and protease extract tussah silk fibroin albumen, comprises the following steps:
1)4g tussah silks are weighed as sample, are cleaned with deionized water, surface contaminant, drying is removed.
2)By the sample of drying with 1:100 bath raio is containing 0.5% Na2PO4And 1%C17H35In COONa mixed solution
30min is boiled, degumming process is carried out, altogether degumming four times.
3)After degumming, sample is washed by rubbing with the hands more than 4 times with deionized water, 60 DEG C of baking ovens are put into, dry fibroin is obtained fine
Dimension.
4)The fibroin fiber of 2g dryings is weighed, with 1:In 50 bath raio immersion [AMIM] Cl ionic liquids, control reaction temperature
Degree stirs 6h in the range of 100 DEG C in oil bath.After the completion of reaction, room temperature is cooled to.
5)To 4)PM is added in intermediate ion liquid13- basic protein enzyme powder 0.3g, stirs 6h in 55 DEG C of oil bath, obtains
To after fibroin/ionic liquid solution, the enzyme that goes out is carried out.
6)Treat that fibroin/ionic liquid solution is cooled to room temperature, add absolute ethyl alcohol, soak repeatedly, have fibroin albumen analysis
Go out.Mixture is filtered by vacuum, deionized water is added into the fibroin albumen filtered out, is filtered after soaking repeatedly, then will be molten
Liquid is fitted into the bag filter of molecular cut off 3500 24h that dialyses, and obtains pure silk fibroin protein solution.
7)Obtained silk fibroin protein solution is freeze-dried, you can obtain silk fibroin powder.
Wherein, PM13The preparation of-alkali protease:0.15g alkali is added into 100mL 0.1M sodium borate buffer solution
Property protease and 3.75g PM13(Pectination PEG MW15000), 1h is slowly stirred under the conditions of 4 DEG C, in additional 50mL 1mmol/L
Ultrafiltration removes unreacted PM under conditions of HCl13, in triplicate.Filtrate is freeze-dried, PM is obtained13- basic protein
Enzyme powder, it is stand-by.
The preparation of [AMIM] Cl ionic liquids:According to 1.25:1 mol ratio added into three-necked flask allyl chloride and
N- methylimidazoles, are stirred at reflux 8h in 60 DEG C of oil baths, after completion of the reaction, and excessive 2- methallyls are removed using rotary evaporation
Base chlorine, obtains light yellow clear liquid, i.e. [AMIM] Cl ionic liquids, after freeze-drying 24h, thaws stand-by.
Contrast experiment:
A kind of method of enzymolysis and extraction fibroin albumen single in aqueous solvent, step is as follows:
1)4g tussah silks are weighed as sample, are cleaned with deionized water, surface contaminant, drying is removed.
2)By the sample of drying with 1:100 bath raio is containing 0.5% Na2PO4And 1%C17H35In COONa mixed solution
30min is boiled, degumming process is carried out, altogether degumming four times.
3)After degumming, sample is washed by rubbing with the hands more than 4 times with deionized water, 60 DEG C of baking ovens are put into, dry fibroin is obtained fine
Dimension.
4)2g fibroin fibers are weighed, with 1:50 bath raio immersion CB buffer solutions(Na2CO3/NaHCO3Buffer solution)In, regulation
PH value of solution is slowly stirred 24h under the conditions of 40 ~ 55 DEG C of oil bath, after the completion of reaction, need to carry out destroy the enzyme treatment between 9 ~ 10.
5)Remaining fibroin fiber is filtered out, with the bag filter of molecular cut off 3500 to silk fibroin protein solution dialysis 48h,
Obtain pure silk fibroin protein solution.
6)Silk fibroin protein solution is freeze-dried 48h, silk fibroin powder is obtained.
Although protease is with higher specificity, single enzymatic isolation method only has 10% or so, dissolving to the solubility of fibroin albumen
Efficiency is very low, and longer to the processing time of silk.So the present invention is first at relatively high temperatures using ionic liquid to fibroin
Fiber is tentatively swelled and dissolved, and adds alkali protease and fibroin albumen is dissolved, and is shortening the same of action time
When, the solubility to fibroin albumen can also be improved, its solubility is up to more than 20%.
Raw materials used in the present invention, equipment, is the conventional raw material, equipment of this area unless otherwise noted;In the present invention
Method therefor, 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 (7)
1. a kind of method that utilization ionic liquid and protease extract tussah silk fibroin albumen, it is characterised in that in terms of g and mL,
Comprise the following steps:
1)3.5-4.5g tussah silks are weighed, are cleaned with deionized water, surface contaminant, drying is removed;
2)By the tussah silk of drying with 1:95-105 bath raio is in the Na containing 0.4-0.6wt%2PO4With 0.8-1.2wt%'s
C17H3525-35min is boiled in COONa mixed solution, degumming process is carried out, altogether degumming four times;
3)After degumming, tussah silk is washed by rubbing with the hands more than 4 times with deionized water, 55-65 DEG C of baking oven is put into, obtain dry fibroin fine
Dimension;
4)The fibroin fiber of 1.8-2.2g dryings is weighed, with 1:In 45-55 bath raio immersion [AMIM] Cl ionic liquids, control is anti-
Answer temperature in the range of 80 ~ 100 DEG C, 5-7h is stirred in oil bath, after the completion of reaction, be cooled to room temperature;
5)To step 4)PM is added in intermediate ion liquid13- basic protein enzyme powder 0.25-0.35g, in 40 ~ 55 DEG C of oil bath
5-7h is stirred, is obtained after fibroin/ionic liquid solution, the enzyme that goes out is carried out;
6)Treat that fibroin/ionic liquid solution is cooled to room temperature, add absolute ethyl alcohol, soak repeatedly, separate out fibroin albumen, to mixed
Compound is filtered by vacuum, and deionized water is added into the fibroin albumen filtered out, is filtered after soaking repeatedly, then solution is loaded into saturating
Dialyse 20-28h in analysis bag, obtains pure silk fibroin protein solution;
7)Obtained silk fibroin protein solution is freeze-dried, you can obtain silk fibroin powder.
2. the method that a kind of utilization ionic liquid and protease as claimed in claim 1 extract tussah silk fibroin albumen, it is special
Levy and be, step 4)In, the preparation method of [AMIM] the Cl ionic liquids is:By 1.0-1.5:1 mol ratio is held to reaction
Allyl chloride and N- methylimidazoles are added in device, 6 ~ 8h is stirred at reflux in 55-65 DEG C of oil bath, after completion of the reaction, using rotation
Evaporation removes excessive 2- methallyl chlorides, obtains light yellow clear liquid, i.e. [AMIM] Cl ionic liquids, is freeze-dried
After 20-28h, thaw stand-by.
3. the method that a kind of utilization ionic liquid and protease as claimed in claim 1 extract tussah silk fibroin albumen, it is special
Levy and be, step 5)In, the PM13The preparation method of-alkali protease is:Sodium borate buffer to 90-110mL 0.1M is molten
0.10-0.20g alkali proteases and 3.5-4.0g PM are added in liquid13, 0.5-1.5h is slowly stirred under the conditions of 1-5 DEG C, outside
Plus ultrafiltration removes unreacted PM under conditions of 45-55mL 1mmol/L HCl13, in triplicate, filtrate is freeze-dried,
Obtain PM13- basic protein enzyme powder, it is stand-by.
4. the method that a kind of utilization ionic liquid and protease as claimed in claim 3 extract tussah silk fibroin albumen, it is special
Levy and be, the PM13For MW15000 pectinations PEG.
5. the method that a kind of utilization ionic liquid and protease as claimed in claim 4 extract tussah silk fibroin albumen, it is special
Levy and be, in PM13In the preparation process of-alkali protease, before freeze-drying, 4h is freezed under the conditions of -20 DEG C in advance.
6. the method that a kind of utilization ionic liquid and protease as claimed in claim 1 extract tussah silk fibroin albumen, it is special
Levy and be, step 5)In, go out enzyme when, at least 30min is incubated in the oil bath more than 80 DEG C.
7. the method that a kind of utilization ionic liquid and protease as claimed in claim 1 extract tussah silk fibroin albumen, it is special
Levy and be, step 6)In, the molecular cut off of the bag filter is 3500.
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CN109295140A (en) * | 2018-10-22 | 2019-02-01 | 浙江海洋大学 | A kind of preparation method of Japanese croaker air bladder collagen protein source dipeptidyl peptidase-IV peptide for inhibiting |
CN110759969A (en) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
CN113603903A (en) * | 2021-08-04 | 2021-11-05 | 上海曜爱生物科技有限公司 | Preparation and application of fillable silk fibroin gel |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109295140A (en) * | 2018-10-22 | 2019-02-01 | 浙江海洋大学 | A kind of preparation method of Japanese croaker air bladder collagen protein source dipeptidyl peptidase-IV peptide for inhibiting |
CN109295140B (en) * | 2018-10-22 | 2022-01-18 | 浙江海洋大学 | Preparation method of collagen-derived dipeptidyl peptidase-IV (DPP-IV) inhibitory peptide of Nibea japonica swim bladder |
CN110759969A (en) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
CN110759969B (en) * | 2019-10-14 | 2021-10-22 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
CN113603903A (en) * | 2021-08-04 | 2021-11-05 | 上海曜爱生物科技有限公司 | Preparation and application of fillable silk fibroin gel |
CN113603903B (en) * | 2021-08-04 | 2023-10-10 | 上海曜爱生物科技有限公司 | Preparation and application of silk fibroin gel capable of being filled |
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