CN113862250A - Immobilization method of chitosanase - Google Patents
Immobilization method of chitosanase Download PDFInfo
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- CN113862250A CN113862250A CN202111200437.6A CN202111200437A CN113862250A CN 113862250 A CN113862250 A CN 113862250A CN 202111200437 A CN202111200437 A CN 202111200437A CN 113862250 A CN113862250 A CN 113862250A
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- chitosanase
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- 108010089807 chitosanase Proteins 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 37
- 108090000790 Enzymes Proteins 0.000 claims abstract description 82
- 102000004190 Enzymes Human genes 0.000 claims abstract description 82
- 229920001661 Chitosan Polymers 0.000 claims abstract description 46
- 229920005989 resin Polymers 0.000 claims abstract description 45
- 239000011347 resin Substances 0.000 claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 16
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 14
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 14
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 12
- 239000011591 potassium Substances 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 59
- 230000000694 effects Effects 0.000 claims description 55
- 239000007853 buffer solution Substances 0.000 claims description 36
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 238000005406 washing Methods 0.000 claims description 21
- 238000001914 filtration Methods 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 11
- 230000003100 immobilizing effect Effects 0.000 claims description 11
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 2
- FUKWVANJMZUZNA-UHFFFAOYSA-M [OH-].[Na+].B([O-])(O)O.[Na+] Chemical compound [OH-].[Na+].B([O-])(O)O.[Na+] FUKWVANJMZUZNA-UHFFFAOYSA-M 0.000 claims description 2
- ZLFRJHOBQVVTOJ-UHFFFAOYSA-N dimethyl hexanediimidate Chemical compound COC(=N)CCCCC(=N)OC ZLFRJHOBQVVTOJ-UHFFFAOYSA-N 0.000 claims description 2
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 claims description 2
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims 1
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 abstract description 20
- 238000011084 recovery Methods 0.000 abstract description 10
- 238000003860 storage Methods 0.000 abstract description 7
- 239000003638 chemical reducing agent Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 73
- 238000006460 hydrolysis reaction Methods 0.000 description 16
- 230000007062 hydrolysis Effects 0.000 description 12
- 239000000047 product Substances 0.000 description 10
- 239000012295 chemical reaction liquid Substances 0.000 description 8
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000413 hydrolysate Substances 0.000 description 3
- 150000003141 primary amines Chemical group 0.000 description 3
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 239000003822 epoxy resin Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 229920000647 polyepoxide Polymers 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010059892 Cellulase Proteins 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
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- 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
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- 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
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01132—Chitosanase (3.2.1.132)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the technical field of enzyme engineering, and particularly relates to a chitosan enzyme immobilization method, wherein ESR-3 is selected as carrier resin, the carrier resin ESR-3 is activated by using a cross-linking agent glutaraldehyde, then chitosan enzyme liquid is added, the two carry out covalent bonding reaction, and finally a reducing agent sodium borohydride/potassium borohydride is added for treatment to obtain immobilized chitosan enzyme; the obtained immobilized chitosanase can be used for hydrolyzing a chitosan solution to produce water-soluble chitosan oligosaccharide with low molecular weight; the method has the advantages of high storage and operation stability after the chitosan enzyme is immobilized, easy separation of the enzyme from a substrate and a product, contribution to recovery and reutilization of the enzyme and the like, and is suitable for industrial application.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and provides a chitosanase immobilization method.
Background
Chitosan (EC.3.2.1.132) is a specific enzyme for degrading chitosan, and acts on beta-1, 4-glycosidic bond of chitosan to decompose into low molecular weight chitosan oligosaccharide. The chitosan oligosaccharide has unique biological functions, has wide application in the fields of medicine, agriculture, food and the like, and is a hot spot for research and development at home and abroad at present.
Compared with the traditional method for producing chitosan oligosaccharide by acid degradation or oxidative degradation, the method for producing chitosan oligosaccharide by using the biological enzyme method has the advantages of simple process, small environmental pollution, small molecular weight of the product, good water solubility, high biological activity and the like, so that more and more attention is paid and great development is brought in recent years. Many manufacturers degrade chitosan by using non-specific enzymes such as cellulase, protease, lipase and the like, but have the defects of long production period, low yield and the like; the chitosan enzyme can specifically hydrolyze chitosan, can specifically act on GlcN-GlcN, GlcNAc-GlcN and GlcNAc-GlcNAc glycosidic bonds, can hydrolyze chitosan into chitosan oligosaccharide with low molecular weight, and has the advantages of mild reaction condition, no side reaction, high chitosan oligosaccharide yield, no environmental pollution and the like.
The use of free chitosanase in practical applications has the following disadvantages: the stability of the enzyme is poor, and the enzyme is easily denatured and inactivated by the influence of pH value and temperature; the enzyme is water-soluble, and cannot be separated and recycled for reuse after reaction, so that the production process often has a step of raising the temperature to inactivate the enzyme, which not only causes the increase of cost and the waste of energy, but also increases the chance of polluting products, thereby limiting the application of the products. The immobilized chitosanase can be used for well solving the problems, not only can the problems of enzyme recovery and reuse, enzyme pollution and the like be solved, but also the stability of the enzyme is improved, the tolerance of the enzyme to acid, alkali, high temperature and other conditions is greatly improved, and meanwhile, the immobilized chitosanase is used for catalyzing, the reaction operation can be simplified, the enzymatic reaction can be realized continuously and automatically, and therefore, the research and development of the immobilized enzyme have extremely important significance.
At present, methods for immobilizing chitosanase are more applied, such as an embedding method, an adsorption method, a cross-linking method and the like, however, the quantity of the adsorption, cross-linking or embedding immobilized enzyme is very small, the activity loss of the enzyme is serious, even if the recovery rate of the activity of the enzyme reaches 80%, the consumption cost is too high, in addition, the contact between a substrate and an enzyme activity site is influenced after embedding, and the steric hindrance is increased; the half-life period and survival time of the enzyme are short, and the stability is low, so that the enzyme is difficult to apply to industrial production.
Therefore, whether to provide a more suitable method for immobilizing the chitosan enzyme becomes a difficult point and a key point of research.
Disclosure of Invention
Aiming at the defects of the technology, the invention provides a chitosan enzyme immobilization method, which comprises the steps of selecting ESR-3 as carrier resin, activating the carrier resin ESR-3 by using a cross-linking agent glutaraldehyde, then adding chitosan enzyme liquid, carrying out covalent bonding reaction on the two, and finally adding a reducing agent sodium borohydride/potassium borohydride for treatment to obtain immobilized chitosan enzyme; the obtained immobilized chitosanase can be used for hydrolyzing a chitosan solution to produce water-soluble chitosan oligosaccharide with low molecular weight; the method has the advantages of high storage and operation stability after the chitosan enzyme is immobilized, easy separation of the enzyme from a substrate and a product, contribution to recovery and reutilization of the enzyme and the like, and is suitable for industrial application.
The ESR-3 carrier resin selected by the invention is the best carrier resin selected by the inventor, and the inventor simultaneously tests immobilized enzymes of macroporous resin, epoxy resin, ESR-2, ESR-3 and ESR-5 resin to find that the immobilized chitosanase after the ESR-3 is immobilized has high enzyme activity recovery rate and high enzyme activity, the resin is purchased from Tianjin Nankai and science and technology Limited company, the resin is provided with primary amine functional groups, and the stability and the enzyme activity can be improved by using the resin as a chitosanase carrier, and on the basis of the resin, the following specific immobilization method is provided:
a chitosanase immobilization method comprises the following steps:
(1) activation of the support
Weighing a certain amount of resin ESR-3 carrier, adding the carrier into a buffer solution, simultaneously adding a cross-linking agent with a certain concentration, and placing the carrier on a shaking table for oscillation to react for 2-5 h; taking out the carrier, washing the resin by buffer solution, and filtering the resin to dryness for later use by taking the eluent as the standard without the cross-linking agent;
(2) immobilization of chitosanase
Weighing a proper amount of activated resin carrier, adding the buffer solution, wherein the buffer solution can be used for immersing all the carriers; adding a proper amount of chitosanase enzyme liquid with activity, uniformly mixing, and then placing on a shaking table to oscillate until the chitosanase is immobilized; filtering out the carrier immobilized with the chitosanase, respectively washing with pure water and sodium chloride solution, filtering out the carrier after washing, adding sodium borohydride/potassium solution, stirring and filtering, collecting the carrier, and detecting the enzyme activity to obtain the immobilized chitosanase.
Furthermore, the specific operation steps provided by the inventor are as follows:
(1) activation of the support
Weighing a certain amount of resin ESR-3 carrier, adding the carrier into a buffer solution, simultaneously adding a cross-linking agent with a certain concentration, and placing the carrier on a shaking table for oscillation to react for 2-5 h; taking out the carrier, washing the resin by buffer solution, and filtering the resin to dryness for later use by taking the eluent as the standard without the cross-linking agent;
the buffer solution is an alkaline buffer solution with the pH value of more than 7.0, and the buffer solution can be used for immersing all carriers, more specifically, the use amount of the buffer solution can be limited to that the mass-to-volume ratio of the resin carrier to the buffer solution is 1g:0.8mL-1g:10 mL;
can be selected from phosphate buffer, Tris buffer, sodium borate-sodium hydroxide buffer;
the cross-linking agent is one of glutaraldehyde, hexamethylene diamine, maleic anhydride, double nitrogen benzidine-2, 2' -disulfuric acid, hexamethylene isocyanate, 1, 5-difluoro-2, 4-nitro-benzenesulfone and dimethyl adipimidate, and the dosage of the cross-linking agent is 0.2-5% of the volume dosage of the buffer solution; preferably glutaraldehyde;
the cross-linking agent can form a covalent bond with primary amine groups on ESR-3 resin, and then is combined with chitosanase in a covalent bond mode, so that the chitosanase is immobilized;
(2) immobilization of chitosanase
Weighing a proper amount of activated resin carrier, adding the buffer solution, wherein the buffer solution can be used for immersing all the carriers;
adding a proper amount of chitosanase enzyme liquid with activity (the activity of the chitosanase enzyme liquid is 10-300U/mL), uniformly mixing, and placing on a shaking bed to oscillate for more than 20h, wherein the mass-volume ratio of the resin carrier mass to the chitosanase enzyme liquid is 1g:5mL-1g:30 mL;
filtering out the carrier immobilized with the chitosanase, and washing with pure water until the effluent liquid of ninhydrin detection is negative; and finally, washing with 1mol/L sodium chloride solution until ninhydrin test shows negative, filtering out the carrier, adding sodium borohydride/potassium borohydride solution, stirring for 15min, filtering, collecting the carrier, and detecting enzyme activity to obtain the immobilized chitosanase.
The dosage of the buffer solution in the step is that the mass-volume ratio of the resin carrier to the buffer solution is 1g:0.8mL-1g:10 mL;
the concentration of the sodium borohydride/potassium borohydride solution adopted in the step (2) is 0.2mM-0.5mM, and the dosage is 1g:2mL-1g:10mL of the mass-volume ratio of the resin carrier mass to the sodium borohydride/potassium borohydride solution;
in the prior art, distilled water is generally adopted for cleaning in the enzyme immobilization operation, and a sodium borohydride/potassium borohydride solution is adopted for cleaning in the application, so that the immobilized chitosanase is more stable and cannot easily fall off mainly because the sodium borohydride/potassium borohydride solution has a strong reduction effect.
The invention also provides the immobilized chitosanase prepared by the scheme.
After obtaining the immobilized chitosanase, the inventors carried out the following performance tests:
(1) enzyme activity detection
And (3) measuring the activity of free chitosan enzyme: 10mL of 2% chitosan-acetic acid solution was added with 1mL of the enzyme solution of chitosan, and reacted in a thermostatic water bath at 55 ℃ for 10 min. And (3) after the degradation reaction is finished, inactivating at 80 ℃ for 10min, taking out 2mL of the solution, adding the solution into 2mL of 2% NaOH solution, mixing uniformly, centrifuging, and removing unreacted chitosan oligosaccharide. 1mL of the supernatant was subjected to a 3, 5-dinitrosalicylic acid (DNS) colorimetric method to determine the amount of reducing sugar, and the activity of the free enzyme was calculated.
And (3) measuring the activity of the immobilized chitosan enzyme: taking 10mL of 2% chitosan-acetic acid solution, adding 0.1g of immobilized chitosanase, and placing in a constant temperature water bath kettle at 55 ℃ for reaction for 10 min. After the reaction, 2mL of the solution was immediately taken out and added to 2mL of 2% NaOH solution, and the mixture was mixed and centrifuged to remove unreacted chitosan. And (3) taking 1mL of supernatant, determining the amount of reducing sugar according to a 3, 5-dinitrosalicylic acid (DNS) colorimetric method, and calculating the activity of the immobilized enzyme.
Definition of enzyme activity unit: under certain conditions, the amount of enzyme required to hydrolyze chitosan per minute to release 1. mu. mol of reducing sugar is defined as 1 enzyme activity unit (U).
The activity recovery rate (%) of the immobilized chitosanase is equal to the enzyme activity (U) fixed by the resin/the enzyme activity (U) added initially before the fixation multiplied by 100%;
the chitosan hydrolysis ratio (%) — reducing sugar content/initial chitosan content × 100%;
through the detection of the method, the activity of the free chitosan detected in the invention is 30.62U/mL, and the detection is carried out after the ESR-3 resin and the chitosanase liquid are immobilized according to the ratio of 1g to 15mL, so that the activity of the immobilized enzyme is 455.9U/g, and the activity recovery rate of the immobilized enzyme is 99.26%.
(2) Stability of immobilized chitosanase
a. Storage stability of immobilized chitosanase
The immobilized chitosanase was stored at 4 ℃ and after 30 days the residual activity of the enzyme after storage was determined under optimal hydrolysis conditions. The result shows that the immobilized chitosanase keeps more than 90% of enzyme activity after being stored for 30 days.
b. Operational stability of immobilized chitosanase
The stability is a main index for measuring the practical value of the immobilized enzyme, and the operation stability is represented by a repeated hydrolysis substrate experiment of the immobilized enzyme:
adding immobilized enzyme into 2% chitosan-acetic acid solution, and hydrolyzing the immobilized enzyme once every 30min under the optimal hydrolysis condition. After the primary hydrolysis reaction is finished, washing the immobilized enzyme by deionized water, removing residual degradation substrates and degradation products on the immobilized enzyme, then carrying out the hydrolysis reaction again, and so on; the concentration of reducing sugar is determined for the enzymolysis product by referring to a DNS colorimetric sugar determination method, and the residual activity of the immobilized enzyme is calculated, and the result shows that the enzyme activity of the immobilized enzyme obtained by the invention is still kept above 80% after the chitosan is repeatedly hydrolyzed for 10 times.
The inventors also carried out the relevant experiments for hydrolyzing chitosan using the immobilized chitosanase obtained above as follows:
preparing chitosan substrate with concentration of 1-6 wt%, adding 1-3 vt% acetic acid solution, swelling, adjusting pH value to 4.0-6.0, performing enzymolysis reaction at 50-60 deg.C for 20-130min according to certain mass of immobilized enzyme, sampling and detecting: the reaction is stopped when the reaction solution is dropped into 2 percent NaOH solution and no white flocculent precipitate is generated;
wherein the dosage of the immobilized enzyme is as follows: the total enzyme activity of the immobilized chitosanase required to be added for 1g of chitosan is 8-50U.
Analysis of hydrolysate chitosan oligosaccharide shows that the viscosity average molecular weight is 1-3KDa, and the water solubility is good.
The technical scheme of the invention has the following advantages:
the primary amine resin carrier material and the cross-linking agent used by the immobilized chitosanase are easily obtained from the market, and the cost is low; glutaraldehyde is used as a cross-linking agent, the glutaraldehyde reacts with amino residues on a resin carrier and chitosanase to form a covalent bond, and the covalent bond is reduced from a double bond to a single bond under the action of a reducing agent sodium/potassium borohydride, so that the stability of the immobilized enzyme is improved, the immobilization reaction condition is mild, the process flow is simple, and the operation is easy.
When free chitosanase is adopted, the system needs to be inactivated after enzymolysis, so that the color of the product chitosan oligosaccharide is darkened, the chroma of the product is increased, and the improvement of the product quality is not utilized; the immobilized chitosanase has high stability, long operation life and long storage life, and the enzyme may be reused and has obviously lowered cost and is suitable for large scale production.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention are within the scope of the present invention, and in the following examples, the percentages are percentages by weight unless otherwise specified.
Example 1
A chitosanase immobilization method comprises the following specific steps:
activation of chitosanase vectors
5g of carrier resin ESR-3 was weighed into an Erlenmeyer flask, and NaH was added2PO4-Na2HPO4100mL of buffer (50mM, pH8.0), 2mL of 25% glutaraldehyde solution was added, and the mixture was shaken at 150rpm on a shaker at 25 ℃ for 2 hours. The carrier is filtered off, washed 5 times with 30mL of the same buffer solution, and filtered to dryness for later use based on the washing of residual glutaraldehyde.
Immobilization of chitosanase
Adding NaH into 1g of the activated carrier2PO4-Na2HPO45mL of buffer solution (50mM, pH8.0), 5mL of enzyme solution containing 300U of chitosanase (the enzyme activity of the enzyme solution is 60U/mL) is added, after uniform mixing, the bottle mouth is sealed, and the mixture is placed on a shaking table at 25 ℃ and is oscillated for 20 hours at 150 rpm;
filtering out the resin carrier, washing with pure water until the ninhydrin detection effluent is negative, washing with 1mol/L NaCl solution until ninhydrin detection is negative, and collecting the carrier.
Adding 100mM sodium borohydride solution with the final volume percentage of 3% into the resin carrier of the cross-linked chitosanase, continuously stirring, standing for 3 hours at the temperature of 2-8 ℃, and further reducing covalent bonds; collecting the carrier and detecting the enzyme activity; the enzyme activity of the immobilized enzyme is 297.23U, the activity recovery rate of the immobilized enzyme is 99.08 percent, and the obtained immobilized chitosanase is stored at 4 ℃ for later use.
Example 2
A chitosanase immobilization method comprises the following specific steps:
5g of carrier resin ESR-3 was weighed into an Erlenmeyer flask, and NaH was added2PO4-Na2HPO4100mL of a buffer (50mM, pH8.0), 2mL of a 25% glutaraldehyde solution were added, and the mixture was shaken at 150rpm on a shaker at 25 ℃ for 3 hours. The carrier is filtered off, washed 5 times with 30mL of the same buffer solution, and filtered to dryness for later use based on the washing of residual glutaraldehyde.
Immobilization of chitosanase
Adding NaH into 1g of the activated carrier2PO4-Na2HPO45mL of buffer solution (50mM, pH8.0), 10mL of enzyme solution containing 486U of chitosanase (the enzyme activity of the enzyme solution is 48.6U/mL) is added, after uniform mixing, the bottle mouth is sealed, and the mixture is placed on a shaking table at 25 ℃ and under 150rpm and oscillated for 21 h;
filtering out the resin carrier, washing with pure water until the ninhydrin detection effluent is negative, washing with 1mol/L NaCl solution until ninhydrin detection is negative, and collecting the carrier.
Adding 100mM sodium borohydride solution with the final volume percentage of 3% into the resin carrier of the cross-linked chitosanase, continuously stirring, standing for 3 hours at the temperature of 2-8 ℃, and further reducing covalent bonds; and collecting the carrier, detecting the enzyme activity, wherein the enzyme activity of the immobilized enzyme is 482.03U, the activity recovery rate of the immobilized enzyme is 99.24%, and the obtained immobilized chitosanase is stored at 4 ℃ for later use.
Example 3
Hydrolyzed chitosan using immobilized chitosanase
Preparing a chitosan substrate with the concentration of 2%, adding 1.5% acetic acid solution, fully swelling in a water bath with the temperature of 55 ℃, adjusting the pH value to be 5.5, adding 0.2g of the immobilized chitosan enzyme in the embodiment 1, performing enzymolysis for 20min at the temperature of 55 ℃, taking reaction liquid, dropping 2% NaOH solution until no white flocculent precipitate is generated, stopping the reaction, and filtering the immobilized chitosan enzyme for the next use; the reaction solution is detected by an acetylacetone method, the viscosity average molecular weight of the chitosan oligosaccharide generated by hydrolysis is 473.23Da, the chroma is 429.65 once, and the water solubility is good.
Example 4
Hydrolyzed chitosan using immobilized chitosanase
Preparing a chitosan substrate with the concentration of 6%, adding 2.5% acetic acid solution, fully swelling in 55 ℃ water bath, adjusting the pH value to be 5.5, adding 0.2g of the immobilized chitosan enzyme in the embodiment 1, performing enzymolysis for 20min at the temperature of 55 ℃, taking reaction liquid, dropping 2% NaOH solution until no white flocculent precipitate is generated, stopping the reaction, and filtering the immobilized chitosan enzyme for the next use; the reaction solution is detected by an acetylacetone method, the viscosity average molecular weight of the chitosan oligosaccharide generated by hydrolysis is 569.62Da, the chroma is 1835.69 once, and the water solubility is good.
Experimental example 5
Stability of the immobilized chitosanase prepared in example 1
1) Storage stability of immobilized chitosanase
10.0g of the immobilized chitosanase was weighed out and stored at 4 ℃ after 30 days, and the residual activity of the enzyme after storage was determined at 0.5g under the conditions of optimal hydrolysis of the enzyme. The result shows that the immobilized chitosanase keeps more than 90% of enzyme activity after being stored for 30 days.
2) Operational stability of immobilized chitosanase
0.2g of immobilized chitosanase was added to a 2% chitosan solution, and one enzymatic hydrolysis process was performed every 20min under optimal hydrolysis conditions. After the primary hydrolysis reaction is finished, the immobilized enzyme is washed by deionized water, and then the chitosan substrate is added again for hydrolysis reaction. And (4) determining the concentration of reducing sugar by referring to a DNS colorimetric method, and calculating the residual activity of the immobilized chitosanase. The result shows that the enzyme activity can still be maintained at 80 percent after the chitosan is repeatedly hydrolyzed by the immobilized enzyme for 10 times.
Comparative example 1
Preparing a chitosan substrate with the concentration of 2%, adding 1.5% acetic acid solution, fully swelling in a water bath with the temperature of 55 ℃, adjusting the pH value to be 5.5, adding 100U of free chitosanase, carrying out enzymolysis at the temperature of 55 ℃ for 20min, taking reaction liquid, dripping 2% NaOH solution into the reaction liquid, stopping the reaction when no white flocculent precipitate is generated, and putting the reaction liquid into a boiling water bath for inactivation for 15min to obtain a hydrolysate of chitosan oligosaccharide; the viscosity average molecular weight of the chitosan oligosaccharide produced by hydrolysis is 3000Da, the water solubility is good, but the chroma is 1969.89 ever, because the enzyme activity is still needed to be inactivated at high temperature after the hydrolysis of the free enzyme is finished, generally the condition of boiling water bath for 10-20min is adopted, the chroma of the chitosan oligosaccharide of the hydrolysis product is high under the high temperature environment, and the appearance of the product is affected, and it can be seen that compared with the example 3, the viscosity average molecular weight and the chroma of the chitosan oligosaccharide obtained in the example 3 are far lower than those of the comparative example.
Comparative example 2
Preparing a chitosan substrate with the concentration of 6%, adding 2.5% acetic acid solution, fully swelling in 55 ℃ water bath, adjusting the pH value to be 5.5, adding 300U of free chitosanase, carrying out enzymolysis at the temperature of 55 ℃ for 20min, taking reaction liquid, dripping 2% NaOH solution into the reaction liquid, stopping the reaction when no white flocculent precipitate is generated, and putting the reaction liquid into boiling water bath for inactivation for 15min to obtain a hydrolysate of chitosan oligosaccharide; the reaction solution was examined by acetylacetone method, and the chitosan oligosaccharide produced by hydrolysis had a viscosity average molecular weight of 3069Da and good water solubility, but had a higher color value of 6983.59, and it was found that the viscosity average molecular weight and color value of the chitosan oligosaccharide obtained in example 4 were much lower than those of the comparative example, as compared with example 4.
Comparative example 3
Weighing 5g of carrier epoxy resin into an erlenmeyer flask, and adding NaH2PO4-Na2HPO4100mL of a buffer (50mM, pH8.0), 2mL of a 25% glutaraldehyde solution were added, and the mixture was shaken at 150rpm on a shaker at 25 ℃ for 3 hours. The carrier is filtered off, washed 5 times with 30mL of the same buffer solution, and filtered to dryness for later use based on the washing of residual glutaraldehyde.
Immobilization of chitosanase
Adding NaH into 1g of the activated carrier2PO4-Na2HPO45mL of buffer solution (50mM, pH8.0), 10mL of chitosanase enzyme solution (the enzyme activity of the enzyme solution is 32.6U/mL) containing 326U in total is added, after uniform mixing, the bottle mouth is sealed, and the bottle is placed on a shaking table at 25 ℃ and under 150rpm to oscillate for 20 hours;
filtering out the resin carrier, washing with pure water until the ninhydrin detection effluent is negative, washing with 1mol/L NaCl solution until ninhydrin detection is negative, and collecting the carrier.
Adding 100mM sodium borohydride solution with the final volume percentage of 3% into the resin carrier of the cross-linked chitosanase, continuously stirring, standing for 3 hours at the temperature of 2-8 ℃, and further reducing covalent bonds; collecting the carrier, and detecting the enzyme activity, wherein the enzyme activity of the immobilized enzyme is 99.78U, and the activity recovery rate of the immobilized enzyme is 30.61%;
when the obtained immobilized chitosanase is reused, the enzyme activity is reduced to 40.21U after the first use and the loss is 60 percent, the enzyme activity of the immobilized enzyme is 7.93U after the second use and the enzyme activity loss is more than 92 percent.
Claims (10)
1. A chitosanase immobilization method is characterized in that: the method comprises the following specific steps:
(1) activation of the support
Weighing a certain amount of resin ESR-3 carrier, adding the carrier into a buffer solution, simultaneously adding a cross-linking agent with a certain concentration, and placing the carrier on a shaking table for oscillation to react for 2-5 h; taking out the carrier, washing the resin by buffer solution, and filtering the resin to dryness for later use by taking the eluent as the standard without the cross-linking agent;
(2) immobilization of chitosanase
Weighing a proper amount of activated resin carrier, adding the buffer solution, wherein the buffer solution can be used for immersing all the carriers; adding a proper amount of chitosanase enzyme liquid with activity, uniformly mixing, and then placing on a shaking table to oscillate until the chitosanase is immobilized; filtering out the carrier immobilized with the chitosanase, respectively washing with pure water and sodium chloride solution, filtering out the carrier after washing, adding sodium borohydride/potassium solution, stirring and filtering, collecting the carrier, and detecting the enzyme activity to obtain the immobilized chitosanase.
2. The method for immobilizing chitosanase according to claim 1, wherein:
the buffer solution in the step (1) is an alkaline buffer solution with the pH value of more than 7.0, and the buffer solution can be used for immersing all carriers;
the cross-linking agent is one of glutaraldehyde, hexamethylene diamine, maleic anhydride, double nitrogen benzidine-2, 2' -disulfuric acid, hexamethylene isocyanate, 1, 5-difluoro-2, 4-nitro-benzenesulfone and dimethyl adipimidate, and the dosage of the cross-linking agent is 0.2-5% of the volume dosage of the buffer solution.
3. The method for immobilizing chitosanase according to claim 1 or 2, wherein:
the alkaline buffer solution in the step (1) is one of a phosphate buffer solution, a Tris buffer solution and a sodium borate-sodium hydroxide buffer solution; the cross-linking agent is glutaraldehyde.
4. The method for immobilizing chitosanase according to claim 1, wherein:
adding a proper amount of chitosanase enzyme liquid with activity in the step (2), uniformly mixing, and placing on a shaking table to oscillate for more than 20 hours; wherein the mass-volume ratio of the resin carrier to the chitosanase enzyme solution is 1g:5mL-1g:30mL, and the activity of the chitosanase enzyme solution is 10-300U/mL.
5. The method for immobilizing chitosanase according to claim 1, wherein:
the cleaning process in the step (2) is as follows:
filtering out the carrier immobilized with the chitosanase, and washing with pure water until the effluent liquid of ninhydrin detection is negative; finally, washing with 1mol/L sodium chloride solution until ninhydrin test shows negative, and filtering out the carrier.
6. The method for immobilizing chitosanase according to claim 1, wherein: the dosage of the buffer solution in the step (1) is that the mass-to-volume ratio of the resin carrier to the buffer solution is 1g:0.8mL-1g:10 mL;
the dosage of the buffer solution in the step (2) is that the mass-to-volume ratio of the resin carrier to the buffer solution is 1g:0.8mL-1g:10 mL.
7. The method for immobilizing chitosanase according to claim 1, wherein:
the concentration of the sodium borohydride/potassium borohydride solution adopted in the step (2) is 0.2mM-0.5mM, and the dosage is 1g:2mL-1g:10mL of the mass-volume ratio of the resin carrier mass to the sodium borohydride/potassium borohydride solution.
8. The method for immobilizing chitosanase according to claim 1, wherein:
the relevant steps for hydrolyzing chitosan by using immobilized chitosanase are as follows:
preparing chitosan substrate with concentration of 1-6 wt%, adding 1-3 vt% acetic acid solution, swelling, adjusting pH value to 4.0-6.0, performing enzymolysis reaction at 50-60 deg.C for 20-130min according to certain mass of immobilized enzyme, sampling and detecting: the reaction is stopped when the reaction solution is dropped into 2 percent NaOH solution and no white flocculent precipitate is generated.
9. The method for immobilizing chitosanase according to claim 8, wherein: the dosage of the immobilized enzyme is as follows: the total enzyme activity of the immobilized chitosanase required to be added for 1g of chitosan is 8-50U.
10. An immobilized chitosanase obtained by the method of immobilizing chitosanase according to claim 1.
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| CN101508985A (en) * | 2009-03-27 | 2009-08-19 | 江西师范大学 | Process for producing chitosanase carrier and fixing method for the chitosanase |
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| CN101508985A (en) * | 2009-03-27 | 2009-08-19 | 江西师范大学 | Process for producing chitosanase carrier and fixing method for the chitosanase |
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