CN111286789B - Complex enzyme degumming agent and degumming method - Google Patents
Complex enzyme degumming agent and degumming method Download PDFInfo
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 52
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 42
- 108010029541 Laccase Proteins 0.000 claims abstract description 89
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 66
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 45
- 108010059820 Polygalacturonase Proteins 0.000 claims abstract description 41
- 239000011259 mixed solution Substances 0.000 claims abstract description 34
- 240000008564 Boehmeria nivea Species 0.000 claims abstract description 28
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 24
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- 108020004410 pectinesterase Proteins 0.000 claims abstract description 15
- 108010070456 protopectinase Proteins 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 15
- 238000003756 stirring Methods 0.000 claims abstract description 11
- 238000002791 soaking Methods 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims description 30
- XPOLVIIHTDKJRY-UHFFFAOYSA-N acetic acid;methanimidamide Chemical compound NC=N.CC(O)=O XPOLVIIHTDKJRY-UHFFFAOYSA-N 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 25
- 230000035484 reaction time Effects 0.000 claims description 14
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 13
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 12
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 12
- 108090000856 Lyases Proteins 0.000 claims description 12
- 102000004317 Lyases Human genes 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 12
- 238000005470 impregnation Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 claims description 9
- PIPNXYUYZWKFLP-UHFFFAOYSA-N C(C)(=O)O.C(C)(=O)O.C(=N)N Chemical compound C(C)(=O)O.C(C)(=O)O.C(=N)N PIPNXYUYZWKFLP-UHFFFAOYSA-N 0.000 claims description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 229960001051 dimercaprol Drugs 0.000 claims description 3
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 20
- 239000000835 fiber Substances 0.000 abstract description 14
- 239000006227 byproduct Substances 0.000 abstract description 5
- 108010087558 pectate lyase Proteins 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 description 45
- 229920005610 lignin Polymers 0.000 description 13
- 239000000084 colloidal system Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 229920002488 Hemicellulose Polymers 0.000 description 4
- 229920001277 pectin Polymers 0.000 description 4
- 239000001814 pectin Substances 0.000 description 4
- 235000010987 pectin Nutrition 0.000 description 4
- 229920002230 Pectic acid Polymers 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 238000007598 dipping method Methods 0.000 description 3
- 108010062085 ligninase Proteins 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000003607 modifier Substances 0.000 description 3
- 239000010318 polygalacturonic acid Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001699 photocatalysis Effects 0.000 description 2
- HQFLTUZKIRYQSP-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole-6-sulfonic acid Chemical compound OS(=O)(=O)C1=CC=C2N(CC)CSC2=C1 HQFLTUZKIRYQSP-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 description 1
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000009120 camo Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 235000005607 chanvre indien Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 239000011487 hemp Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 108010039928 polymethylgalacturonase Proteins 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01C—CHEMICAL OR BIOLOGICAL TREATMENT OF NATURAL FILAMENTARY OR FIBROUS MATERIAL TO OBTAIN FILAMENTS OR FIBRES FOR SPINNING; CARBONISING RAGS TO RECOVER ANIMAL FIBRES
- D01C1/00—Treatment of vegetable material
- D01C1/02—Treatment of vegetable material by chemical methods to obtain bast fibres
Landscapes
- Chemical & Material Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Textile Engineering (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Chemical Or Physical Treatment Of Fibers (AREA)
Abstract
The invention belongs to the technical field of ramie degumming, and provides a compound enzyme degumming agent and a degumming method. The degumming agent comprises the following raw materials: protopectinase, polygalacturonase, polygalacturonate lyase, pectin esterase and modified laccase. The complex enzyme is stable and has excellent degumming performance. The degumming method comprises adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain mixed solution; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and soaking the raw ramie in the mixed solution and stirring. The degumming effect is excellent by adopting the method, the degummed ramie has excellent mechanical property, the fiber yield is high, and the byproducts are few.
Description
Technical Field
The invention belongs to the technical field of ramie degumming, and particularly relates to a compound enzyme degumming agent and a degumming method.
Background
Degumming ramie refers to a process for preparing ramie fibers by removing the gum of raw ramie. The length, fineness, strength and the like of ramie single fibers are suitable for spinning requirements, but the fibers are adhered together by colloid in raw ramie and full degumming is required. The colloid in ramie mainly comprises pectin, hemicellulose and lignin. In recent years, the research of adopting compound enzyme degumming is more fierce. The complex enzyme mostly comprises pectinase, hemicellulase and ligninase.
The ligninase can degrade a solid lignin barrier in an intercellular layer, so that the fibers are resolved, and pectin and hemicellulose can be better degraded only by resolving the fibers. Laccase is one of the ligninases. Laccase is popular because of the mild catalytic oxidation reaction conditions and the water as a byproduct. However, instability of laccase is easily caused by changes in external factors such as temperature, pH or enzyme system. Thus easily causing its deactivation and loss of action.
The ramie degumming process in the prior art mainly comprises chemical degumming, biological degumming and biological-chemical combined degumming. The biological degumming process is relatively expensive; chemical degumming and biological-chemical combined degumming both use a large amount of chemical reagents, and are easy to cause chemical pollution.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the first objective of the present invention is to provide a compound enzyme degumming agent, which is stable and has excellent degumming performance.
In view of the above-mentioned deficiencies in the prior art, a second object of the present invention is to provide a degumming method, which has an excellent degumming effect, and the degummed ramie has excellent mechanical properties, a high fiber yield and few byproducts.
In order to achieve the purpose, the invention adopts the following solution:
the compound enzyme degumming agent comprises the following raw materials in parts by weight: 25-30 parts of polymethyl galacturonase, 30-37 parts of polygalacturonic acid lyase, 16-22 parts of polygalacturonase and 10 parts of modified laccase; the preparation of the modified laccase comprises the following steps: (1) Adding 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into a laccase aqueous solution in sequence for reaction and then dialyzing to obtain carboxyl modified laccase; (2) And sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into the carboxyl modified laccase to react and dialyzing to obtain the modified laccase.
A degumming method comprises adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:1.5-2.5; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and soaking the raw ramie in the mixed solution and stirring.
The compound enzyme degumming agent and the degumming method provided by the invention have the beneficial effects that:
(1) The complex enzyme degumming agent provided by the invention is prepared from protopectinase, polygalacturonase, polygalacturonate lyase, pectinesterase and polygalacturonase in the proportion. The compound enzyme degumming agent can be suitable for complex components of ramie colloid and can carry out degumming in a targeted manner. The compound enzyme degumming agent particularly adopts the modified laccase to degrade the solid lignin in the intercellular layer, so that the fiber is resolved out to degrade the colloid by other enzymes. The modified laccase obtained by the preparation method of the modified laccase can improve the activity and stability of the modified laccase at the same time, so that the phenomena of inactivation and the like cannot occur in the process of degrading lignin, and the degradation of colloid is further assisted. The complex enzyme degumming agent provided by the invention has excellent degumming performance.
(2) The degumming method provided by the invention comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the compound enzyme degumming agent to prepare a mixed solution, placing the mixed solution into a sealing box, and performing impregnation degumming on raw ramie under the irradiation of ultraviolet light. In the application, the photocatalytic oxidation is combined with biological catalysis, and the lignin is degraded under the combined action, so that the lignin in a solid intercellular layer is further degraded, and the fibers are resolved. The reaction conditions of the process are mild. The degumming effect is excellent by adopting the method, the degummed ramie has excellent mechanical property, the fiber yield is high, and the byproducts are few.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The complex enzyme degumming agent and the degumming method provided by the embodiment of the invention are specifically explained below.
In this example, the preparation of the modified laccase includes: (1) Adding 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into the laccase aqueous solution in sequence for reaction and then dialyzing to obtain the carboxyl modified laccase. The 2,3-bis-mercaptopropanol serving as the laccase inhibitor can protect the active points of the laccase backbone and avoid the influence on the active points of the backbone in the modification process. 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is used as a hydroxyl active agent and can activate amino acid at the side chain and the tail end of an amino acid residue in the laccase, and the first formamidine acetate aqueous solution is used as a modifier and can provide a crosslinking reaction between an amino group and the activated laccase to form a peptide bond.
In this example, the mass ratio of laccase, 2,3-dimercaprol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and formamidine acetate in the first aqueous formamidine acetate solution in the aqueous laccase solution is: 1:0.02-0.04:0.05-0.08:0.13-0.15. The reaction time is controlled to be 62-84min, and the reaction temperature is controlled to be 45-52 ℃. To achieve the above effects.
In this example, the dialysis solution used for dialysis was a dipotassium phosphate/citric acid buffer. So as to terminate the reaction and remove substances that are not completely reacted in the crosslinking reaction.
(2) And sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into the carboxyl modified laccase to react and dialyzing to obtain the modified laccase. Wherein N, N' -carboxydiimidazole is used as an amino modifier to activate the amino groups on the side chains and the terminal amino acids of the amino acid residues in the laccase modified by the hydroxyl groups, and a second formamidine diacetate aqueous solution is used as a modifier to further perform a guanylation reaction with the modified amino groups.
In this example, the mass ratio of formamidine acetate in the aqueous solution of N, N' -carboxydiimidazole and second formamidine diacetate was: 1:0.07-0.09:0.13-0.15. The reaction time is controlled to be 70-88min, and the reaction temperature is controlled to be 40-50 ℃. To achieve the above effects.
The inventor finds that the method can increase the activity and stability of the laccase and avoid the inactivation and the loss of the effect of the laccase due to the influence of external factors in the process of degrading the lignin. The laccase with increased activity and stability can be more fully used for degrading lignin so that the fiber is analyzed, and other substances in the colloid are more favorably and fully contacted with the complex enzyme degumming agent so as to be degraded.
The embodiment of the invention provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 25-30 parts of protopectinase, 15-20 parts of polygalacturonase, 14-18 parts of polygalacturonase lyase, 8-14 parts of pectinesterase, 16-22 parts of polygalacturonase and 10 parts of modified laccase. Further, in this example, protopectinase is 26-28 parts, polygalacturonase is 16-19 parts, polygalacturonate lyase is 15-17 parts, pectin esterase is 9-13 parts, polygalacturonase is 17-21 parts, and modified laccase is 10 parts. Further, 27 parts of protopectinase, 17 parts of polygalacturonase, 16 parts of polygalacturonase lyase, 10 parts of pectinesterase, 20 parts of polygalacturonase and 10 parts of modified laccase. The compound enzyme degumming obtained by the raw materials and the mixture ratio thereof can degrade pectin and hemicellulose more fully after the modified laccase degrades lignin fully, and the raw materials are mutually cooperated and cooperated to degrade pectin, hemicellulose and lignin fully in the colloid.
The embodiment of the invention also provides a degumming method, which comprises the step of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain mixed solution. The concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:1.5-2.5. Placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and soaking the raw ramie in the mixed solution and stirring. Wherein the temperature in the closed box is controlled at 55-60 deg.C, pH is 5.0-5.5, the reaction time is 24h, and ultraviolet irradiation intensity is 5.00 + -0.10 μ Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
in the present application, the inventors combine photocatalysis and biocatalysis to achieve further lignin degradation under the premise of environmental friendliness and mild reaction conditions. The titanium dioxide is cheap, stable and antioxidant, and in the application, the titanium dioxide absorbs ultraviolet rays and can degrade organic matters in lignin under the action of a catalyst hydrogen peroxide. Meanwhile, the hydrogen peroxide can be used as a supplier of the laccase to provide oxygen for assisting the laccase to degrade the lignin better, so that an excellent degradation effect is achieved.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a modified laccase: (1) Adding 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into a laccase aqueous solution in sequence for reaction and then dialyzing to obtain carboxyl modified laccase; wherein the mass ratio of the laccase, 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and formamidine acetate in the first formamidine acetate aqueous solution is as follows: 1:0.02:0.05:0.13; controlling the reaction time to be 62min and the reaction temperature to be 45 ℃; (2) Sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into carboxyl modified laccase to react and dialyze to obtain modified laccase; wherein the mass ratio of the N, N' -carboxyl diimidazole to the formamidine acetate in the second formamidine acetate aqueous solution is as follows: 1:0.07:0.13; the reaction time was controlled at 70min and the reaction temperature was controlled at 40 ℃.
Example 2
The embodiment provides a modified laccase: (1) Adding 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into a laccase aqueous solution in sequence for reaction and then dialyzing to obtain carboxyl modified laccase; wherein the mass ratio of the laccase, 2,3-dimercaprol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and formamidine acetate in the first formamidine acetate aqueous solution is as follows: 1:0.04:0.08:0.15; controlling the reaction time to be 84min and the reaction temperature to be 52 ℃; (2) Sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into carboxyl modified laccase to react and dialyze to obtain modified laccase; wherein the mass ratio of the N, N' -carboxyl diimidazole to the formamidine acetate in the second formamidine acetate aqueous solution is as follows: 1:0.09:0.15; the reaction time was controlled at 88min and the reaction temperature was controlled at 50 ℃.
Example 3
The embodiment provides a modified laccase: (1) Adding 2,3-bis-mercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into a laccase aqueous solution in sequence, reacting, and dialyzing to obtain carboxyl modified laccase; wherein the mass ratio of the laccase, 2,3-dimercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and formamidine acetate in the first formamidine acetate aqueous solution is as follows: 1:0.03:0.07:0.14; controlling the reaction time to be 78min and the reaction temperature to be 48 ℃; (2) Sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into carboxyl modified laccase to react and dialyze to obtain modified laccase; wherein the mass ratio of the N, N' -carboxyl diimidazole to the formamidine acetate in the second formamidine acetate aqueous solution is as follows: 1:0.08:0.14; the reaction time was controlled to 80min and the reaction temperature was controlled to 45 ℃.
Example 4
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 25 parts of protopectinase, 20 parts of polygalacturonase, 14 parts of polygalacturonase lyase, 8 parts of pectinesterase, 22 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:2; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, soaking the raw ramie in the mixed solution, and stirring; wherein the temperature in the closed box is controlled at 58 ℃, the pH is 5.3, the reaction is carried out for 24 hours, and the ultraviolet irradiation intensity is 5.00 mu Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
example 5
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 30 parts of protopectinase, 15 parts of polygalacturonase, 18 parts of polygalacturonase lyase, 14 parts of pectinesterase, 16 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:2; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and dipping the raw ramie in the mixed solution and stirring; wherein the temperature in the closed box is controlled at 58 ℃, the pH is 5.3, the reaction is carried out for 24 hours, and the ultraviolet irradiation intensity is 5.00 mu Ecm -2 s -1 Solid-liquid impregnation ratio 1:20.
example 6
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 26 parts of protopectinase, 19 parts of polygalacturonase, 15 parts of polygalacturonic acid lyase, 13 parts of pectinesterase, 17 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:2; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and dipping the raw ramie in the mixed solution and stirring; wherein the temperature in the closed box is controlled at 58 deg.C, pH is 5.3, the reaction time is 24h, and the ultraviolet irradiation intensity is 5.00 μ Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
example 7
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 28 parts of protopectinase, 16 parts of polygalacturonase, 17 parts of polygalacturonase lyase, 9 parts of pectinesterase, 21 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degumming agent to the titanium dioxide in the acetic acid buffer solution is 10:2; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, soaking the raw ramie in the mixed solution, and stirring; wherein the temperature in the closed box is controlled at 58 ℃, the pH is 5.3, the reaction is carried out for 24 hours, and the ultraviolet irradiation intensity is 5.00 mu Ecm -2 s -1 Solid-liquid impregnation ratio 1:20.
example 8
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 27 parts of protopectinase, 17 parts of polygalacturonase, 16 parts of polygalacturonase lyase, 10 parts of pectinesterase, 20 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:2; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, and dipping the raw ramie in the mixed solution and stirring; wherein the temperature in the closed box is controlled at 58 ℃, the pH is 5.3, the reaction is carried out for 24 hours, and the ultraviolet irradiation intensity is 5.00 mu Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
example 9
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 27 parts of protopectinase, 17 parts of polygalacturonase, 16 parts of polygalacturonase lyase, 10 parts of pectinesterase, 20 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:1.5; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, soaking the raw ramie in the mixed solution, and stirring; wherein the temperature in the closed box is controlled at 55 deg.C, pH is 5.0, the reaction time is 24h, and the ultraviolet irradiation intensity is 4.90 μ Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
example 10
The embodiment provides a compound enzyme degumming agent, which comprises the following raw materials in parts by weight: 27 parts of protopectinase, 17 parts of polygalacturonase, 16 parts of polygalacturonic acid lyase, 10 parts of pectinesterase, 20 parts of polygalacturonase and 10 parts of the modified laccase provided by the embodiment 3;
the embodiment provides a degumming method, which comprises the steps of adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degumming agent to the titanium dioxide in the acetic acid buffer solution is 10:2.5; placing the mixture in a closed box, providing ultraviolet radiation, and making the ramieImmersing hemp in the mixed solution and stirring; wherein the temperature in the closed box is controlled at 60 deg.C, pH is 5.5, the reaction time is 24h, and the ultraviolet irradiation intensity is 5.10 μ Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
experimental example 1
The experimental method comprises the following steps: example 1-3 laccase Activity assay: by measuring O 2 The method (3-ethylbenzothiazole-6-sulfonic Acid (ABTS) is used as a substrate) is used for measuring the laccase activity; (2) half-life analysis of laccase of examples 1-3: taking a certain amount of laccase liquid, keeping the temperature at 50 ℃ for a certain time (10 min, 20min, 30min, 40min, 50min, 60min, 90min, 120min …), and rapidly cooling. 0.5mL of the treated laccase solution was removed, the residual activity was determined according to the laccase activity assay procedure in (1), and the half-life was obtained by using a logarithmic method for the data obtained. Common laccase as control group; the test results are shown in table 1:
TABLE 1 laccase Activity and thermostability
| Sample (I) | Relative Activity (%) | Relative half-life (%) |
| Example 1 | 285.4 | 202.5 |
| Example 2 | 290.5 | 190.4 |
| Example 3 | 380.8 | 180.2 |
| Laccase enzymes | 100 | 100 |
Note: the relative activity of the unmodified laccase is defined as 100%, and the activity of the modified laccase is based on the laccase; the relative half-life of the unmodified laccase is defined as 100%, and the relative half-life of the modified laccase is based on the laccase.
As can be seen from the results in Table 1, the modified laccases obtained by the methods of examples 1-3 have increased activity and stability.
Experimental example 2
The degumming methods provided in examples 4-10 were used to prepare dry refined ramie according to the prior art by the subsequent steps of impregnation (washing, oiling, drying), and the residual gum rate, fiber strength, appearance and yield of the dry refined ramie provided in examples 4-10 were determined, and the results are shown in table 2:
TABLE 2
As can be seen from the data in Table 2, the dry refined ramie prepared by the degumming methods provided in the embodiments 4 to 10 of the present invention has low residual gum ratio, strong fiber strength, high yield and good appearance.
In conclusion, the compound enzyme degumming agent and the degumming method provided by the invention are adopted. The complex enzyme is stable and has excellent degumming performance. The degumming effect is excellent by adopting the method, the degummed ramie has excellent mechanical property, the fiber yield is high, and the byproducts are few.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A degumming method, which is characterized in that: adding titanium dioxide, 30% hydrogen peroxide and acetic acid buffer solution into the complex enzyme degumming agent to obtain a mixed solution; the concentration ratio of the complex enzyme degelling agent to the titanium dioxide in the acetic acid buffer solution is 10:1.5-2.5; placing the mixed solution in a closed box, providing irradiation ultraviolet rays, soaking the raw ramie in the mixed solution, and stirring;
the compound enzyme degumming agent comprises the following raw materials in parts by weight: 25-30 parts of protopectinase, 15-20 parts of polygalacturonase, 14-18 parts of polygalacturonase lyase, 8-14 parts of pectinesterase, 16-22 parts of polygalacturonase and 10 parts of modified laccase;
the preparation of the modified laccase comprises the following steps: (1) Adding 2,3-bis-mercaptopropanol, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and a first formamidine acetate aqueous solution into a laccase aqueous solution in sequence, reacting, and dialyzing to obtain carboxyl modified laccase; (2) Sequentially adding N, N' -carboxyl diimidazole and formamidine diacetate aqueous solution into the carboxyl modified laccase to react and dialyzing to obtain the modified laccase;
the mass ratio of the laccase in the laccase aqueous solution, the 2,3-dimercaprol, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the formamidine acetate in the first formamidine acetate aqueous solution is as follows: 1:0.02-0.04:0.05-0.08:0.13-0.15;
the mass ratio of the carboxyl modified laccase to the N, N' -carboxyl diimidazole to the formamidine acetate in the second formamidine acetate aqueous solution is as follows: 1:0.07-0.09:0.13-0.15.
2. The degumming process according to claim 1, characterized in that: the dialyzate used for dialysis was a dipotassium hydrogen phosphate/citric acid buffer.
3. The degumming process according to claim 2, characterized in that: in the step (1) of preparing the modified laccase, the reaction time is 62-84min, and the reaction temperature is controlled to be 45-52 ℃.
4. The degumming process according to claim 3, characterized in that: in the step (2) of preparing the modified laccase, the reaction time is 70-88min, and the reaction temperature is controlled to be 40-50 ℃.
5. The degumming process according to claim 1, characterized in that: the raw materials comprise the following components in parts by weight: 26-28 parts of protopectinase, 16-19 parts of polygalacturonase, 15-17 parts of polygalacturonase lyase, 9-13 parts of pectin esterase, 17-21 parts of polygalacturonase and 10 parts of modified laccase.
6. The degumming process according to claim 5, characterized in that: the raw materials comprise the following components in parts by weight: 27 parts of protopectinase, 17 parts of polygalacturonase, 16 parts of polygalacturonase, 10 parts of pectinesterase, 20 parts of polygalacturonase and 10 parts of modified laccase.
7. The degumming process according to claim 1, characterized in that: the temperature in the closed box is controlled to be 55-60 ℃, the pH is 5.0-5.5, the reaction is carried out for 24 hours, and the ultraviolet irradiation intensity is 5.00 +/-0.10 mu Ecm -2 s -1 Solid-liquid impregnation ratio of 1:20.
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| CN101649494A (en) * | 2009-09-04 | 2010-02-17 | 吴鹏 | Novel biological degumming agent for ramie raw materials |
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| JP2006094811A (en) * | 2004-09-30 | 2006-04-13 | Tokyo Institute Of Technology | Modified laccase and process for treating aromatic compounds |
| CN101649494A (en) * | 2009-09-04 | 2010-02-17 | 吴鹏 | Novel biological degumming agent for ramie raw materials |
| CN107385523A (en) * | 2017-07-19 | 2017-11-24 | 苏州凯邦生物技术有限公司 | A kind of biological enzyme jute fiber degumming agent |
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