CN114350647B - Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase - Google Patents
Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase Download PDFInfo
- Publication number
- CN114350647B CN114350647B CN202111593054.XA CN202111593054A CN114350647B CN 114350647 B CN114350647 B CN 114350647B CN 202111593054 A CN202111593054 A CN 202111593054A CN 114350647 B CN114350647 B CN 114350647B
- Authority
- CN
- China
- Prior art keywords
- glucose isomerase
- soluble polymer
- type nonionic
- aam
- nonionic water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108700040099 Xylose isomerases Proteins 0.000 title claims abstract description 51
- 229920003169 water-soluble polymer Polymers 0.000 title claims abstract description 16
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 12
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 claims abstract description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 229920000642 polymer Polymers 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
- 239000000178 monomer Substances 0.000 claims description 8
- 238000006116 polymerization reaction Methods 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229920006389 polyphenyl polymer Polymers 0.000 claims description 2
- 230000007704 transition Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 9
- 238000012712 reversible addition−fragmentation chain-transfer polymerization Methods 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 230000000694 effects Effects 0.000 description 16
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000008103 glucose Substances 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 238000006317 isomerization reaction Methods 0.000 description 8
- 229930091371 Fructose Natural products 0.000 description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 7
- 239000005715 Fructose Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- XIXNSLABECPEMI-VURMDHGXSA-N (z)-2-[2-[(2-methylpropan-2-yl)oxycarbonylamino]-1,3-thiazol-4-yl]pent-2-enoic acid Chemical compound CC\C=C(/C(O)=O)C1=CSC(NC(=O)OC(C)(C)C)=N1 XIXNSLABECPEMI-VURMDHGXSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 235000019534 high fructose corn syrup Nutrition 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- QWMJEUJXWVZSAG-UHFFFAOYSA-N (4-ethenylphenyl)boronic acid Chemical compound OB(O)C1=CC=C(C=C)C=C1 QWMJEUJXWVZSAG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 125000005619 boric acid group Chemical group 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
- 238000000502 dialysis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- PJVXUVWGSCCGHT-ZPYZYFCMSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(3s,4r,5r)-1,3,4,5,6-pentahydroxyhexan-2-one Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO PJVXUVWGSCCGHT-ZPYZYFCMSA-N 0.000 description 1
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 1
- XTHRTLHUMOKINM-UHFFFAOYSA-N benzylsulfanylmethanedithioic acid Chemical compound SC(=S)SCC1=CC=CC=C1 XTHRTLHUMOKINM-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000011557 critical solution Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021433 fructose syrup Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides an application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase, belonging to the technical field of immobilized enzyme; in the invention, UCST type nonionic water-soluble polymer which is polymerized with VBA block after the reversible addition-fragmentation chain transfer polymerization of acrylamide AAM and acrylonitrile AN is adopted to fix glucose isomerase; the UCST type nonionic water-soluble polymer can be used under the condition of existence of salt and has low hysteresis, and has good application on glucose isomerase.
Description
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to application of UCST type nonionic water-soluble polymers in immobilized glucose isomerase.
Background
Glucose Isomerase (GI), also known as xylose isomerase, can isomerise glucose into fructose, and is one of the key enzymes for preparing fructose-glucose syrup from starch in the food industry. Large scale conversion and utilization of glucose is closely related to the healthy life of contemporary people. With the development of economy and the maturation of the market, the consumption of High Fructose Corn Syrup (HFCS) increases year by year, and the market competition is also becoming vigorous. Currently, HFCS may be produced using GI isomerization. However, the method is difficult to mass-produce due to factors such as poor separation technology, low enzyme yield of the strain, and the like, and has the problems of high production cost and the like. The liquid enzyme preparation has the defects of incapability of recycling and low use efficiency.
Immobilization refers to a technique of limiting enzyme molecules to a certain spatial range by using a physical or chemical method to perform catalytic reaction. On the basis of keeping the activity of the enzyme molecules, the tolerance of the enzyme molecules to external factors is enhanced through immobilization, so that the enzyme can be recycled, and the production cost is reduced. The immobilized enzyme has rapid development and various kinds, and can be divided into adsorption, embedding, chemical crosslinking and covalent bonding methods, and the different methods have different lengths, so that the immobilized enzyme can be screened by combining the characteristics and the use environment of the enzyme. However, the existing immobilization technology has the problems of poor biocompatibility, fewer surface functional groups, difficult degradation in a natural state and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase. In the invention, UCST type nonionic water-soluble polymer which is polymerized with VBA block after the reversible addition-fragmentation chain transfer polymerization of acrylamide AAM and acrylonitrile AN is adopted to fix glucose isomerase; the UCST type nonionic water-soluble polymer can be used under the condition of existence of salt and has low hysteresis, and has good application on glucose isomerase.
The invention provides an application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase.
Further, the chemical formula of the UCST type nonionic water-soluble polymer is mVBA x-b-P(AAMk-co-ANn, wherein m and b represent VBA and two monomers are combined in a block polymerization mode, wherein x represents the polymerization degree of polyphenyl boric acid, k and n respectively represent the polymerization degree of acrylamide and acrylonitrile in the polymer, the range of x, k and n is between 1 and 100, and P represents polymerization; the structural formula of the polymer is as follows:
Further, the high critical dissolution temperature of the UCST type nonionic water-soluble polymer can be adjusted within 0-100 ℃, and the phase transition temperature hysteresis is within 2 ℃.
Further, the application is as follows:
mVBA x-b-P(AAMk-co-ANn) is dissolved in water, glutaraldehyde is added and stirred, the precipitate is removed by low-temperature centrifugation after stirring, the precipitate and glucose isomerase are dissolved in phosphate buffer solution, immobilization reaction is carried out, and immobilized glucose isomerase is obtained by low-temperature centrifugation and washing after the reaction is finished.
Further, the mVBA x-b-P(AAMk-co-ANn) water and glutaraldehyde in a ratio of 30mg to 1ml:30mg.
Further, the ratio of the amount of the precipitate, glucose isomerase and phosphate buffer was 20mg:1mg:1mL.
Further, the stirring condition is 50 ℃ stirring reaction for 4 hours.
Further, the immobilization reaction conditions were stirring at 50℃for 2 hours.
Further, both low temperature centrifuges were at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that:
In the invention, amino and boric acid groups existing in UCST type nonionic water-based polymer structure are used for realizing double-step immobilization, and the immobilization capacity and the mechanical strength are improved. The finished product (Glase) fixed into the granule has the characteristics of high activity, continuous production, automation and controllability, and the product is easy to separate and refine, thereby improving the quality of the product, reducing the production cost and powerfully promoting the development of the production of the high fructose syrup.
In the invention, UCST polymer is combined with glucose isomerase to construct immobilized enzyme, so that the immobilized enzyme can be separated, recovered and recycled under the regulation of external stimulus factors, and the stability of the enzyme is improved. The amino and boric acid groups in UCST-type nonionic water-soluble polymer can be activated by glutaraldehyde to realize double immobilization, so that the immobilization capacity and the mechanical strength are improved. The immobilized glucose isomerase prepared by the invention has better temperature stability than free enzyme, and the immobilized glucose isomerase still maintains more than seventy percent of activity after being circularly catalyzed for seven times.
Drawings
FIG. 1 is a schematic diagram of the synthesis of polymer mVBA x-b-P(AAMk-co-ANn); in the figure, step a is BTPA of the synthesis process, b is the synthesis process of the random monomer, and c is the synthesis process of the temperature-sensitive polymer mVBA x-b-P(AAMm-co-ANn).
FIG. 2 is a graph showing the immobilization of immobilized glucose isomerase prepared under different glutaraldehyde concentration (a), mVBA x-b-P(AAMk-co-ANn concentration (b), pH (c) and immobilization time (d).
FIG. 3 is a graph showing the effect of different conditions on immobilized glucose isomerase-catalyzed glucose isomerization; wherein a is a metal ion, b is a pH value, c is a temperature, and d is a time.
FIG. 4 is a graph showing the storage stability of immobilized glucose isomerase.
FIG. 5 is a graph showing the cycling stability of immobilized glucose isomerase.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
Example 1: UCST-type polymer mVBA 6-b-P(AAM69-co-AN24)
Nonionic polymers mVBA 6-b-P(AAM69-co-AN24 with high critical solution temperatures are synthesized using a reversible addition-fragmentation chain transfer polymerization mechanism). Firstly, synthesizing a random monomer P (AAM 69-co-AN24), then, inserting a 4-vinyl phenylboronic acid block into the random monomer, wherein a detailed synthesis flow chart is shown in fig. 1, a is a BTPA synthesis process, b is a random monomer P (AAM 69-co-AN24), and c is a preparation process of a target polymer mVBA 6-b-P(AAM69-co-AN24). The method comprises the following specific steps:
(1) Preparation of Benzyl Trithiocarbonate (BTPA):
3.25g of potassium hydroxide is weighed and dissolved in 31.25ml of water and stirred, then 2.5ml of 3-mercaptopropionic acid and 4.5ml of carbon disulfide are dripped into the mixture, the mixture is reacted for 5 hours at room temperature in an oil bath, then 6g of benzyl bromide is dripped into the mixture within 1 hour, the temperature of the oil bath is raised to 85.0 ℃ and the mixture is refluxed for 12 hours. Naturally cooling the solution to room temperature after the reaction is finished, adding 70ml of chloroform and 6ml of concentrated hydrochloric acid into the reaction solution, and stirring while adding to obtain a mixed solution; the mixed solution was then rinsed with deionized water until the upper phase was clear, the upper phase was removed, the lower phase was concentrated to an orange oily liquid, 4ml of methylene chloride was added, the mixture was frozen in a refrigerator to reduce the temperature, the product was crystallized, and then filtered through a buchner funnel and the solids were washed with clear water to remove the solvent, finally obtaining a yellow powder sample.
(2) Preparation of initiator AIBN (azobisisobutyronitrile):
adding azobisisobutyronitrile into 50ml, heating to 50deg.C, stirring to dissolve AIBN (about 5 min), immediately filtering to remove insoluble substances, placing into refrigerator, crystallizing to obtain needle-like product, filtering, vacuum drying, drying at room temperature, and sealing and storing in refrigerator.
(3) Preparation of random monomer P (AAM 69-co-AN24):
BTPA 0.11g, AIBN 0.02g, acrylamide 2g and acrylonitrile 0.6g are weighed, put into a single-neck flask, added with 15ml DMF for ultrasonic treatment, discharged from air and reacted for 24 hours at 70 ℃ in an oil bath by a nitrogen protection device. After the reaction is finished, the mixture is placed into an ice water bath, cooled and poured into a beaker with the existing methanol, the dosage of the methanol is 10 times of that of DMF, the mixture is stirred and filtered to obtain solid, the solid is placed into a vacuum drying oven for drying, the solid is taken out, dissolved in a dialysis bag by adding water, dialyzed for 24 hours, and the yellow powdery solid, namely P (AAM 69-co-AN24) is obtained through freeze drying.
(4) MVBA 6-b-P(AAM69-co-AN24) preparation:
Weighing 0.38g of 4-vinylphenylboronic acid, putting the random monomer P (AAM 69-co-AN24) 0.75g,AIBN 0.1163g synthesized in the step (3) into a single-neck flask, adding 20ml of DMF (dimethyl formamide) into the flask, performing ultrasonic treatment, exhausting air, adding a nitrogen protection device, and reacting for 24 hours at 70 ℃. After the reaction is finished, the mixture is placed into an ice-water bath, cooled and poured into a beaker with the existing methanol, the dosage of the methanol is 10 times that of DMF, the mixture is centrifuged to obtain white solid, the white solid is placed for 8 hours, water is added for direct dialysis, and the white powdery solid is obtained through freeze drying, namely mVBA 6-b-P(AAM69-co-AN24.
Example 2: preparation of immobilized glucose isomerase
First, 0.3g mVBA 6-b-P(AAM69-co-AN24) was dissolved in 1mL of water, glutaraldehyde was added, and stirred at 45℃for 4 hours. Then, the precipitate was taken and washed with 1mL of deionized water at a low temperature of 4 ℃ for ten minutes, the operation was repeated twice to remove remaining glutaraldehyde, next, the precipitate and glucose isomerase were dissolved in 1mL of phosphate buffer solution at ph=8, the immobilization reaction was carried out at 50 ℃ for 2 hours, after that, the precipitate was taken and washed with 1mL of phosphate buffer solution at ph=8 at a low temperature of 4 ℃ for ten minutes, the operation was repeated twice to obtain immobilized glucose isomerase, and the cloud point after immobilization was 46.7 ℃.
Example 3: optimized condition exploration of immobilized glucose isomerase
In this example, different immobilized glucose isomerase was prepared by changing glutaraldehyde concentration, polymer concentration, pH and immobilization time, and optimizing conditions of immobilized glucose isomerase were explored by examining the properties.
(1) Optimization of glutaraldehyde concentration:
The preparation of immobilized glucose isomerase was as shown in example 2, with the following differences: the concentration of glutaraldehyde was modified to 10, 20, 30, 40, 50, 60mg, respectively, and then the immobilization amount of immobilized glucose isomerase prepared under the condition of adding glutaraldehyde of different concentrations was examined.
As shown in fig. 2a, continued increase of glutaraldehyde as glutaraldehyde concentration increases to saturation at glutaraldehyde concentration 30mg results in reduced affinity for the carrier substrate, and it can be seen that the immobilization is optimal at glutaraldehyde concentration 30 mg/mL.
(2) MVBA x-b-P(AAMk-co-ANn) optimization of concentration:
The preparation of immobilized glucose isomerase was as shown in example 2, with the following differences: mVBA x-b-P(AAMk-co-ANn) were modified to 10, 20, 30, 40, 50, 60mg, respectively, and then the immobilized glucose isomerase prepared under the condition of adding mVBA x-b-P(AAMk-co-ANn) at different concentrations was examined.
As shown in fig. 2b, with mVBA x-b-P(AAMk-co-ANn) concentration increase, the solids loading was first rising and then falling, with the maximum solids loading at 30mg/mL polymer. This is because mVBA x-b-P(AAMk-co-ANn) the limited enzyme-accessible site at 30mg/mL has reached saturation and cannot access more enzyme.
(3) Optimization of pH value:
The preparation of immobilized glucose isomerase was as shown in example 2, with the following differences: the pH values were modified to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, respectively, and then the immobilization amounts of immobilized glucose isomerase prepared under the conditions of different pH values were examined.
As shown in fig. 2c, the pH change has little effect on the immobilized enzyme, but the immobilization is significantly higher than other pH values at ph=8, so we choose ph=8 as the optimal pH value.
(4) Optimization of immobilization time:
The preparation of immobilized glucose isomerase was as shown in example 2, with the following differences: the immobilization time was modified to 0.5, 1,2, 3, 4h, respectively, and then the immobilization amounts of immobilized glucose isomerase prepared under the conditions of different immobilization times were examined.
As shown in FIG. 2d, the immobilization time is 2h, and the immobilization amount is reduced after saturation, so that the immobilization time is preferably 2 h.
In summary, the optimal conditions for immobilized glucose isomerase are: glutaraldehyde concentration 30mg/mL, polymer concentration 30mg/mL, pH=8, time 2h.
Example 4: immobilized glucose isomerase catalyzed glucose isomerization
In this example, glucose (1.44 g) was used as an isomerization target, and the catalytic activity of immobilized glucose isomerase was evaluated. 200mg of the sample was dispersed in 10mL of glucose solution. Samples of 500. Mu.L were taken at set time intervals (10 min). The sample was subjected to high-phase liquid phase measurement for fructose content.
Fig. 3 is a graph showing the effect of different conditions on immobilized glucose isomerase to catalyze glucose isomerization, and fig. 3a shows that different metal ions have different catalytic effects on immobilized glucose isomerase, but Co 2+ ions can effectively promote glucose isomerization effect. Fig. 3b is the effect of PH on glucose isomerization, and it can be seen that fructose yields can be maximized at ph=7. FIG. 3c is the effect of temperature on isomerised fructose, it being seen that the fructose yield is highest at a temperature of 85 ℃. FIG. 3d shows the effect of time on fructose yield, and it was found that with increasing time, fructose yield increased, preferably 3h time was chosen in view of efficiency.
Fig. 4 shows the search of optimized conditions of the catalytic glucose isomerization experiment, and the research on metal ions, PH, temperature and catalytic time is used to obtain the optimized immobilization conditions, namely, the metal ions are Co 2+, ph=7 and the catalytic time is 3 hours at 85 ℃.
Example 5: storage stability and recycling stability of immobilized glucose isomerase
To investigate the storage stability of the synthesized UCST-GI and the free GI, the samples were stored at constant temperature in a refrigerator at 4℃and the enzyme activities were taken out and measured at fixed intervals, and a relative enzyme activity data map was obtained based on the optimal enzyme activities.
As shown in fig. 4, the immobilized GI was relatively more active than the free GI. These results clearly demonstrate that UCST-GI has good storage stability and great potential for industrial application.
Recycling stability is an important reference standard for measuring the success of enzyme immobilization. As shown in FIG. 5, the immobilized enzyme still has 76.53% relative enzyme activity after catalyzing glucose to repeat 10 times. In some cases, the enzyme activity is inevitably lost in the separation process, and the hydrogen bonding effect becomes weak with the increase of the cycle times, so that the immobilized enzyme is difficult to completely separate. Therefore, the immobilized glucose isomerase which is recycled for a plurality of times in the experiment can reduce the cost of industrial application.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (6)
1. The application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase;
The chemical formula of the UCST type nonionic water-soluble polymer is mVBA x-b-P(AAMk-co-ANn), wherein m and b represent VBA and two monomers are combined in a block polymerization mode, wherein x represents the polymerization degree of polyphenyl boric acid, k and n respectively represent the polymerization degree of acrylamide and acrylonitrile in the polymer, the ranges of x, k and n are all 1-100, and P represents polymerization; the structural formula of the polymer is as follows:
；
The high critical dissolution temperature of the UCST type nonionic water-soluble polymer can be adjusted within 0-100 ℃, and the phase transition temperature hysteresis is within 2 ℃;
The application is as follows:
mVBA x-b-P(AAMk-co-ANn) is dissolved in water, glutaraldehyde is added and stirred, the precipitate is obtained by low-temperature centrifugation after stirring, the precipitate and glucose isomerase are dissolved in phosphate buffer solution and are subjected to immobilization reaction, and immobilized glucose isomerase is obtained by low-temperature centrifugation and washing after the reaction.
2. The use according to claim 1, wherein the dosage ratio of mVBA x-b-P(AAMk-co-ANn), water and glutaraldehyde is 30mg:1ml:30mg.
3. The use according to claim 1, characterized in that the ratio of the amount of precipitate, glucose isomerase and phosphate buffer is 20mg:1mg:1mL.
4. The use according to claim 1, wherein the stirring conditions are 50 ℃ stirring for 4 hours.
5. The use according to claim 1, wherein the immobilization reaction conditions are 50 ℃ stirring reaction for 2 hours.
6. Use according to claim 1, characterized in that both low-temperature centrifuges are centrifuges at 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111593054.XA CN114350647B (en) | 2021-12-23 | 2021-12-23 | Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111593054.XA CN114350647B (en) | 2021-12-23 | 2021-12-23 | Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114350647A CN114350647A (en) | 2022-04-15 |
| CN114350647B true CN114350647B (en) | 2024-05-10 |
Family
ID=81101473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111593054.XA Active CN114350647B (en) | 2021-12-23 | 2021-12-23 | Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114350647B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4275156A (en) * | 1979-01-23 | 1981-06-23 | Toray Industries, Inc. | Glucose isomerase immobilized product and process for preparing same |
| US4371612A (en) * | 1978-02-17 | 1983-02-01 | Toyo Jozo Company, Ltd. | Immobilization of biological material with an acrylonitrile polymer |
| CN1281038A (en) * | 1999-07-16 | 2001-01-24 | 中国科学院化工冶金研究所 | Microbead-shaped porous carrier with skeleton of multi-element copolymer for immobilized enzyme and its preparing process |
| CN108642040A (en) * | 2018-04-12 | 2018-10-12 | 江苏大学 | Solvable-insoluble UCST type PMAAc carriers of one kind, its immobilised enzymes and application |
| CN111440785A (en) * | 2020-02-28 | 2020-07-24 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
| CN112480340A (en) * | 2020-11-13 | 2021-03-12 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
| CN113214440A (en) * | 2021-04-30 | 2021-08-06 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
| CN113512153A (en) * | 2021-04-15 | 2021-10-19 | 陕西科技大学 | Method for preparing novel UCST type temperature-sensitive polymer immobilized enzyme |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8822550B2 (en) * | 2010-03-23 | 2014-09-02 | Kyushu University, National University Corporation | Temperature-, pH- or salt concentration-sensitive separation material and use thereof |
-
2021
- 2021-12-23 CN CN202111593054.XA patent/CN114350647B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4371612A (en) * | 1978-02-17 | 1983-02-01 | Toyo Jozo Company, Ltd. | Immobilization of biological material with an acrylonitrile polymer |
| US4275156A (en) * | 1979-01-23 | 1981-06-23 | Toray Industries, Inc. | Glucose isomerase immobilized product and process for preparing same |
| CN1281038A (en) * | 1999-07-16 | 2001-01-24 | 中国科学院化工冶金研究所 | Microbead-shaped porous carrier with skeleton of multi-element copolymer for immobilized enzyme and its preparing process |
| CN108642040A (en) * | 2018-04-12 | 2018-10-12 | 江苏大学 | Solvable-insoluble UCST type PMAAc carriers of one kind, its immobilised enzymes and application |
| CN111440785A (en) * | 2020-02-28 | 2020-07-24 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
| CN112480340A (en) * | 2020-11-13 | 2021-03-12 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
| CN113512153A (en) * | 2021-04-15 | 2021-10-19 | 陕西科技大学 | Method for preparing novel UCST type temperature-sensitive polymer immobilized enzyme |
| CN113214440A (en) * | 2021-04-30 | 2021-08-06 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports;Rodrigo Torres，等;Journal of Biotechnology;20051231;第120卷;第396–401页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114350647A (en) | 2022-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Preparation of graphene nano-sheet bonded PDA/MOF microcapsules with immobilized glucose oxidase as a mimetic multi-enzyme system for electrochemical sensing of glucose | |
| CN108130321B (en) | Hydrogel containing protease-inorganic hybrid nanoflower as well as preparation method and application of hydrogel | |
| EP1316599B1 (en) | Magnetic particles and process for producing the same | |
| CN106525796B (en) | Recyclable fluorescence sensor for detecting microcystin and application method thereof | |
| Yang et al. | Fluorescent core-shell SiO2@ vertical covalent organic frameworks nanosheets for sensing application | |
| CN114350647B (en) | Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase | |
| CN102516456B (en) | Yeast surface atom transfer imprinted adsorbent, and preparation method and application thereof | |
| CN107254014B (en) | A kind of complex solidifying enzyme carrier material and its preparation method and application | |
| JP2020536508A (en) | Transaminase mutants and their use | |
| Mirzaei et al. | Immobilization of papain on nitrogen-doped graphene quantum dots improves the enzymatic properties and makes it a biosensor for cystatin C | |
| CN110129030B (en) | Preparation and application of block polymer nano-material fluorescent probe with stimulus responsiveness | |
| Pastinen et al. | Cross-linked glucose isomerase crystals as a liquid chromatographic separation material | |
| CN113214440B (en) | UCST nonionic water-soluble polymer, and preparation method and application thereof | |
| Gruesbeck et al. | Insolubilized glucoamylase enzyme system for continuous production of dextrose | |
| CN108949854B (en) | Process for producing extracellular polysaccharide by fermentation of immobilized lactobacillus plantarum | |
| CN116376890A (en) | Preparation method of graphene oxide hydrogel immobilized enzyme | |
| CN106636294B (en) | Process for producing unnatural amino acid product by immobilized double-enzyme coupling reaction | |
| Pollak et al. | Immobilization of synthetically useful enzymes by condensation polymerization | |
| CN109182480A (en) | Application of the metal-organic framework material in polymerase chain reaction | |
| CN112986361B (en) | Application of electrochemical biosensor based on gold-graphene quantum dots in detection of ctDNA in cells | |
| Freeman | [18] Gel entrapment of whole cells in cross-linked prepolymerized polyacrylamide-hydrazide gels | |
| EP0542292A2 (en) | Method for producing 3'-phosphoadenosine 5'-phosphosulfate | |
| CN108841816A (en) | Organic-inorganic composite immobilized cell and its preparation method and application | |
| CN101525603A (en) | Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof | |
| CN1931913A (en) | Surface functionalizing process of magnetic polystyrene microsphere |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Bin Inventor after: Han Juan Inventor after: Wu Jiacong Inventor after: Song Yongqing Inventor after: Mao Yanli Inventor after: Yang Liuqing Inventor before: Song Yongqing Inventor before: Wang Bin Inventor before: Han Juan Inventor before: Wu Jiacong Inventor before: Mao Yanli Inventor before: Yang Liuqing |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |