CN117535278A - Immobilized enzyme and method for preparing D-allose by using immobilized enzyme - Google Patents
Immobilized enzyme and method for preparing D-allose by using immobilized enzyme Download PDFInfo
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 59
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 48
- 102000004190 Enzymes Human genes 0.000 claims abstract description 48
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 73
- 239000008055 phosphate buffer solution Substances 0.000 claims description 24
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 238000005406 washing Methods 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 15
- 238000004132 cross linking Methods 0.000 claims description 14
- 239000011259 mixed solution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 12
- 239000001110 calcium chloride Substances 0.000 claims description 12
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 12
- 108010001469 L-rhamnose isomerase Proteins 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 10
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 9
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 9
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 9
- 235000010413 sodium alginate Nutrition 0.000 claims description 9
- 239000000661 sodium alginate Substances 0.000 claims description 9
- 229940005550 sodium alginate Drugs 0.000 claims description 9
- 239000004327 boric acid Substances 0.000 claims description 8
- 230000005484 gravity Effects 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 239000012510 hollow fiber Substances 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000006555 catalytic reaction Methods 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 241000672609 Escherichia coli BL21 Species 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000012047 saturated solution Substances 0.000 claims description 2
- 230000003248 secreting effect Effects 0.000 claims description 2
- 230000003100 immobilizing effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000004064 recycling Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 9
- 239000012798 spherical particle Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000001952 enzyme assay Methods 0.000 description 2
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- 230000002194 synthesizing effect Effects 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 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 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
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- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
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- 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
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/084—Polymers containing vinyl alcohol units
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01014—L-Rhamnose isomerase (5.3.1.14)
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Abstract
The invention discloses immobilized enzyme and a method for preparing D-allose by using the immobilized enzyme, which relate to the technical field of biology. The immobilized enzyme has mild immobilization conditions, high enzyme immobilization rate, simple process and recycling after simple treatment, greatly reduces the dosage of enzyme needed by multiple allose preparation and reduces the production cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an immobilized enzyme and a method for preparing D-allose by using the immobilized enzyme.
Background
D-allose is a hexose, a rare monosaccharide, a white odorless crystalline solid, a non-caloric sugar, and no toxicity. Chemical formula CH 2 OH(CHOH) 4 CHO, isolated from the leaves of an african shrub, but has very low extraction yields, the artificial synthesis of D-allose is becoming more urgent. D-allose is soluble in water but hardly soluble in methanol, and D-allose is the C-3 epimer of glucose. D-allose exists mainly in a cyclic form and consists of beta-D-allo-1.5-pyranose (77.5%), alpha-D-allo-1.5-pyranose (14%), beta-D-allo-1.4-furanose (5%) and alpha-D-allo-1.4-furanose (3.5%), wherein beta-D-allo-1.5-pyranose is the main component.
In recent years, D-allose has attracted attention because of its many pharmaceutical activities, and inhibition of canceration by D-allose, especially under oxidative stress conditions, can inhibit proliferation of various cancer cell lines including cervical cancer, hepatocellular carcinoma, ovarian, head-neck, skin and prostate cancer. And the combination of D-allose and radiation in cancer treatment improves the cure rate of tumors. D-allose can also be used as an antioxidant and has a certain potential as a therapeutic agent in pharmaceutical formulations and as a functional ingredient in formulas. D-allose as an anti-inflammatory agent inhibits ischemia-reperfusion injury, inhibits segmental neutrophil production, and reduces platelet count. The use of D-allose in combination with low doses of FK506 significantly increases allograft survival while reducing tissue damage. In addition, dextran has a cryoprotecting effect on cells. Therefore, the D-allose has a certain application prospect in surgical operation and transplantation.
However, the source of D-allose mainly originates from chemical synthesis and biological synthesis, and the chemical synthesis has many defects such as complex synthesis path, more byproducts, large environmental pollution and high separation and purification difficulty, so that the biological synthesis is more competitive when the allose is synthesized, and the allose is produced by microbial fermentation, so that the cost is low and the environment is protected.
The current research on D-allose mainly carries out researches on engineering bacteria for producing D-allose, a construction method and application thereof, and the like. However, the new synthetic route not only requires complicated extraction and purification of enzyme molecules to be used for catalytic reaction, but also results in high production cost of the enzyme; the biological enzyme is a water-soluble molecule, and the water-soluble enzyme molecule is difficult to recycle after the catalytic reaction is finished, so that the enzyme is wasted. These factors result in high production costs of the new path for synthesizing D-allose, and development of an enzyme immobilization method is needed to immobilize the enzyme in the new path for synthesizing D-allose, so that the enzyme can be reused, and the production cost is reduced.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows: aiming at the defects existing in the prior art, the preparation method of the immobilized enzyme is provided, the obtained immobilized enzyme can be reused, and the production cost is low.
In order to solve the first technical problem, the technical scheme of the invention is as follows:
a method for preparing immobilized enzyme, comprising the following steps:
a: inoculating the escherichia coli engineering bacteria expressing the L-rhamnose isomerase into a seed culture medium according to the inoculum size of 2-5% v/v, and culturing for 8-10h at the temperature of 30-40 ℃ to obtain a culture solution;
inoculating the culture solution into a fermentation culture medium with an inoculum size of 2-5% v/v for culturing for 30-40h to obtain a fermentation broth;
b: crushing the fermentation liquor by a homogenizer to obtain a crushing liquor, adding disodium hydrogen phosphate accounting for 3-5 per mill v/v and calcium chloride accounting for 1-5 per mill v/v of the volume of the crushing liquor, adjusting the pH value to 6.0-7.0 by using sodium hydroxide, adding diatomite accounting for 1-5% g/L of the volume of the crushing liquor, and filtering by using a plate frame to obtain a crude enzyme solution;
c: feeding the crude enzyme solution into a hollow fiber membrane, and concentrating for 5-10 times to obtain concentrated crude enzyme solution;
d: adding 0.01-0.05% v/v glutaraldehyde solution into the concentrated crude enzyme solution, crosslinking for 0.5-1.5h at 20-50 ℃, and then respectively washing for 3 times with phosphate buffer solution with pH of 7.0 and purified water to obtain crosslinked enzyme solution;
e: 7-9% of polyvinyl alcohol and 2-4% of sodium alginate by volume ratio 1:1 to 1.5, and obtaining a mixed solution, wherein the cross-linking enzyme solution and the mixed solution are mixed according to the volume ratio of 1: mixing evenly 2-4, dripping into boric acid saturated solution of 1.8-5.8%wt calcium chloride, standing for 10-15h at 2-10 ℃, filtering, washing 3 times with phosphate buffer with pH of 7.0, and washing 3 times with purified water to obtain immobilized enzyme.
Preferably, the host bacterium of the escherichia coli engineering bacterium for expressing the L-rhamnose isomerase is escherichia coli BL21, the secretory expression vector plasmid is pET-28a, the gene sequence of the target gene is shown as SEQ ID No. 1, and the method specifically comprises the following steps: GTGAATAAACACAAAAAGATCCCATTACGCAAATGTGTAGTGACTGGTGAAATGAAGCCTA AAAAGGAACTGATCCGAGTTGTACGGTCGAAAGAAGGCGAAATCTCAGTAGACCCGACCGGAAAAAAGAATGGGCGGGGTGCTTATTTAACGCTGGATAAAGAGTGCATCTTAGCAGCAAAAAAGAAAAACACTTTGCAAAATCAATTTCAATCACAAATCGATGACCAGATTTTCGATGAATTGCTGGAACTGGCGGAAAAGGTGAAAAAATAA.
Preferably, the fermentation conditions in step a are: the temperature is 34-40 ℃ and the wind speed is 1.0-2.0m 3 And/h, the rotating speed is 200-300rpm, the pressure is 0.1-0.3Mpa, the pH value is regulated to 7.0+/-0.1 by ammonia water, and the dissolved oxygen is 20-30%.
Preferably, the parameters of the homogenizer in the step B are that the pressure is 40-45Mpa, the cycle times are 2-5, and the temperature is controlled below 30 ℃.
The pore diameter of the hollow fiber membrane in the step C ranges from 50000 Da to 100000Da.
And C, adding the concentrated crude enzyme solution in the step C into a gravity purification column, eluting with 0.1-0.5mol/L imidazole-containing phosphate buffer salt, collecting eluent, and adding the eluent into the glutaraldehyde solution in the step D for crosslinking reaction.
And E, adjusting the pH value of the saturated boric acid solution containing calcium chloride to 6.0-7.0 by using a sodium carbonate solution before adding the cross-linking enzyme solution and the mixed solution in a dropwise manner.
The second technical problem to be solved by the invention is as follows: aiming at the defects existing in the prior art, the method for preparing the D-allose by the immobilized enzyme is provided, the immobilized enzyme can be recycled, and the production cost is low.
In order to solve the second technical problem, the technical scheme of the invention is as follows:
a method for preparing D-allose by immobilized enzyme, comprising the following steps:
f: preparing a D-psicose buffer solution with the concentration of 50-100g/L by using a phosphate buffer solution with the pH of 7, then adding the immobilized enzyme in claim 1, and stirring;
g: continuously adding 0.1-0.5mol/L Tris-HCl buffer solution to maintain the pH value of the reaction system to 6.0-9.0, and carrying out enzyme catalytic reaction at 60-80 ℃ to obtain D-allose.
The addition amount of the immobilized enzyme in the step F accounts for 3-10% g/mL of the volume of the D-psicose solution.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the invention has mild immobilization condition and high enzyme immobilization rate.
2. The immobilized enzyme has excellent catalytic activity and good stability, is used for allose production, has simple and convenient process, can be recycled through simple treatment, greatly reduces the dosage of enzyme required by repeated allose preparation, and reduces the production cost.
3. The immobilized enzyme is granular, has the advantages of difficult leakage and good stability, has small repression effect on the transfer of substrates and products, and is beneficial to the catalytic reaction of the immobilized enzyme on the substrates.
4. The immobilized enzyme has good thermal stability, wider pH stability and 75.6 percent activity after being continuously used for 51 batches.
Drawings
FIG. 1 is a graph showing the thermostability of free enzyme (L-rhamnose isomerase) and immobilized enzyme;
FIG. 2 is a graph showing pH stability of free enzyme (L-rhamnose isomerase) and immobilized enzyme;
FIG. 3 is a graph showing the activity of the immobilized enzyme after repeated use;
FIG. 4 is a liquid phase detection chromatogram of D-allose in example 2.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
(1) Inoculating the escherichia coli engineering bacteria expressing the L-rhamnose isomerase into a seed culture medium according to the inoculum size of 2% v/v, and culturing for 8 hours at the temperature of 30 ℃ to obtain a culture solution;
inoculating the culture solution into a fermentation culture medium for culturing for 30 hours according to the inoculum size of 2% v/v to obtain a fermentation solution;
taking 60L of fermentation liquor, crushing the fermentation liquor in a homogenizer under the pressure of 40MPa for 2 times, keeping the temperature of the circulating liquor below 30 ℃, collecting 70L of crushing liquor, adding 210ml of disodium hydrogen phosphate and 70ml of calcium chloride into the crushing liquor, adjusting the pH value of the crushing liquor to 7 by using sodium hydroxide solution, adding 3.5g of diatomite, feeding the solution into a plate frame, and collecting 80L of filtrate.
(2) Concentrating the filtrate collected in the step (1) by using a hollow fiber membrane, collecting 8.0L of concentrated solution, purifying by using a gravity purification column, washing the gravity purification column by using 8.0L of phosphate buffer solution, eluting by using 0.1mol/L of phosphate buffer solution containing imidazole, and collecting 12.0L of eluent.
(3) And (3) adding 1.2ml of glutaraldehyde solution into the eluent collected in the step (2), crosslinking for 1.5h at 20 ℃, washing three times with phosphate buffer solution with pH=7.0, and washing three times with pure water to obtain a crosslinked enzyme solution.
(4) 7% wt of polyvinyl alcohol and 2% wt of sodium alginate are prepared, and the prepared polyvinyl alcohol and sodium alginate are mixed according to the weight ratio of 1:1 to obtain a mixed solution, and mixing the obtained mixed solution and the cross-linking enzyme solution obtained in the step (3) according to the volume ratio of 1:2, then dripping into 4%wt boric acid solution containing calcium chloride (the pH value of the boric acid solution is regulated to 6.0 by sodium carbonate solution), standing for 10 hours at the temperature of 2 ℃, then filtering to form immobilized spherical particles, washing the immobilized spherical particles with phosphate buffer solution for three times, and washing the immobilized spherical particles with pure water for three times again to obtain the immobilized enzyme.
(5) Preparing a D-psicose buffer solution with the concentration of 80g/L by using a phosphate buffer solution with the pH of 7, then adding the immobilized enzyme in the step (4), wherein the adding amount of the immobilized enzyme accounts for 5% g/mL of the volume of the D-psicose buffer solution, converting at the pH of 6 and the temperature of 80 ℃, collecting a conversion solution, and detecting the content of the D-psicose, wherein the conversion rate is 27.3%.
Example 2
(1) Inoculating the escherichia coli engineering bacteria expressing the L-rhamnose isomerase into a seed culture medium according to the inoculum size of 3% v/v, and culturing for 10 hours at 35 ℃ to obtain a culture solution;
inoculating the culture solution into a fermentation culture medium for culturing for 35 hours according to the inoculum size of 3% v/v to obtain a fermentation solution;
taking 60L of fermentation liquor, crushing the fermentation liquor in a homogenizer under the pressure of 42MPa for 3 times, keeping the temperature of the circulating liquor below 30 ℃, collecting 70L of crushed liquor, adding 280ml of disodium hydrogen phosphate and 210ml of calcium chloride into the crushed liquor, adjusting the pH value of the crushed liquor to 7 by using sodium hydroxide solution, adding 2.1g of diatomite, feeding the crushed liquor into a plate frame, and collecting 82L of filtrate.
(2) Concentrating the filtrate collected in the step (1) by using a hollow fiber membrane, collecting 8.2L of concentrated solution, purifying by using a gravity purification column, washing the gravity purification column by using 8.2L of phosphate buffer solution, eluting by using 0.3mol/L of phosphate buffer solution containing imidazole, and collecting 12.5L of eluent.
(3) Adding 1.2ml of glutaraldehyde solution into the eluent collected in the step (2), crosslinking for 1.0h at 30 ℃, washing three times with phosphate buffer solution with pH=7.0, and washing three times with pure water to obtain a crosslinked enzyme solution.
(4) 8% of polyvinyl alcohol and 3% of sodium alginate by weight are prepared, and the prepared polyvinyl alcohol and sodium alginate solution are mixed according to the weight ratio of 1:1.2, obtaining a mixed solution, and mixing the obtained mixed solution and the cross-linking enzyme solution obtained in the step (3) according to the volume ratio of 1:4, then dripping the mixture into 1.8%wt boric acid solution containing calcium chloride (the pH value of the boric acid solution is regulated to 6.0 by sodium carbonate solution), standing the mixture for 12 hours at the temperature of 4 ℃, then filtering the mixture to form immobilized spherical particles, washing the immobilized spherical particles with phosphate buffer solution for three times, and washing the immobilized spherical particles with pure water for three times again to obtain the immobilized enzyme.
(5) Preparing a D-psicose buffer solution with the concentration of 50g/L by using a phosphate buffer solution with the pH of 7, then adding the immobilized enzyme in the step (4), wherein the addition amount of the immobilized enzyme accounts for 10% g/mL of the volume of the D-psicose buffer solution, converting at the pH of 7 and the temperature of 70 ℃, collecting a conversion solution, detecting the content of psicose, and the conversion rate is 30.6%.
Example 3
(1) Inoculating the escherichia coli engineering bacteria expressing the L-rhamnose isomerase into a seed culture medium according to an inoculum size of 5% v/v, and culturing for 9 hours at 40 ℃ to obtain a culture solution;
inoculating the culture solution into a fermentation culture medium for culturing for 40 hours according to the inoculum size of 5% v/v to obtain a fermentation solution;
taking 60L of fermentation liquor, crushing the fermentation liquor in a homogenizer under the pressure of 45MPa for 5 times, keeping the temperature of the circulating liquor below 30 ℃, collecting 70L of crushing liquor, adding 350ml of disodium hydrogen phosphate and 350ml of calcium chloride into the crushing liquor, adjusting the pH value of the crushing liquor to 7 by using sodium hydroxide solution, adding 2.1g of diatomite, entering a plate frame, and collecting 84L of filtrate.
(2) Concentrating the filtrate collected in the step (1) by using a hollow fiber membrane, collecting 8.4L of concentrated solution, purifying by using a gravity purification column, washing the gravity purification column by using 8.4L of phosphate buffer solution, eluting by using 0.5mol/L of phosphate buffer solution containing imidazole, and collecting 13.0L of eluent.
(3) And (3) adding 6.5ml of glutaraldehyde solution into the eluent collected in the step (2), crosslinking for 1.0h at 50 ℃, washing three times with phosphate buffer solution with pH=7.0, and washing three times with pure water to obtain a crosslinked enzyme solution.
(4) 9% by weight of polyvinyl alcohol and 4% by weight of sodium alginate are prepared, and the prepared polyvinyl alcohol and sodium alginate solution are mixed according to the weight ratio of 1:1.5, obtaining a mixed solution, and mixing the obtained mixed solution and the cross-linking enzyme solution obtained in the step (3) according to the volume ratio of 1:3, then dripping the mixture into boric acid solution containing calcium chloride (the pH value of the boric acid solution is regulated to 6.0 by sodium carbonate solution), standing for 15 hours at the temperature of 10 ℃, filtering the mixture to form immobilized spherical particles, washing the immobilized spherical particles with phosphate buffer solution for three times, and washing the immobilized spherical particles with pure water for three times again to obtain the immobilized enzyme.
(5) Preparing a D-psicose buffer solution with the concentration of 100g/L by using a phosphate buffer solution with the pH of 7, then adding immobilized enzyme in the step (4), wherein the addition amount of the immobilized enzyme accounts for 3%g/mL of the volume of the D-psicose buffer solution, converting at the pH of 9 and the temperature of 60 ℃, collecting a conversion solution, and detecting the content of the D-psicose, wherein the conversion rate is 26.3%.
Example 4
Free enzyme activity assay: 200 mu L of properly diluted pure enzyme solution (L-rhamnose isomerase) is added into 800 mu L of phosphate buffer solution containing D-psicose, the pH value of the phosphate buffer solution is 7, the final concentration of the D-psicose is 50g/L, after shaking and mixing, the mixture is added into a 70 ℃ water bath kettle, the mixture is reacted for 10min, inactivation treatment is carried out, and the sample is taken to measure the content of the D-psicose.
Immobilized enzyme activity assay: weighing 0.5g of immobilized enzyme in example 1, adding into 10ml of phosphate buffer solution containing psicose, leading the pH value of the phosphate buffer solution to be 7, leading the final concentration of D-psicose to be 50g/L, adding into a 70 ℃ water bath kettle after shaking and mixing uniformly, reacting for 10min, inactivating, sampling and measuring the content of D-psicose.
The relative enzyme activity was defined as 100% of the relative enzyme activity as the group with the highest D-allose content.
The free enzyme and immobilized enzyme were placed in buffers, each in a 50-80℃water bath, then cooled to room temperature, and then tested according to the two assay methods described above, respectively, as shown in FIG. 1.
Example 5
The immobilized enzyme and the free enzyme (L-rhamnose isomerase) were placed in phosphate buffers of different pH and placed in a refrigerator at 4℃for 24 hours, after which the test was carried out according to the method in example 4, see FIG. 2.
Example 6
The same preparation method of immobilized enzyme as in example 2, preparing 50g/L of D-psicose solution by using phosphate buffer solution with pH of 7, adding immobilized enzyme, converting at 70 ℃ under the condition of pH of 7, recovering the converted immobilized enzyme, continuously converting under the same condition, continuously converting for 51 times in sequence, measuring the D-psicose content in the feed liquid after each conversion, and taking the D-psicose content in the feed liquid after the first conversion as 100% relative enzyme activity, wherein the D-psicose content in the feed liquid after the first conversion is shown in figure 3.
Comparative example 1
Except for the removal of step (3) under the same conditions as in example 2, the allose content in the converted solution was determined by sampling and the conversion was 20.1%
Comparative example 2
The procedure was as in example 2, except that the mixed solution in step (4) was changed to one of polyvinyl alcohol, and the other conditions were the same, and the allose content in the converted solution was measured by sampling, and the conversion was 19.2%.
Comparative example 3
The difference from example 2 is that the mixed solution in the step (4) is changed into one of sodium alginate, the rest conditions are the same, and the allose content in the conversion solution is measured by sampling, and the conversion rate is 18.6%.
By way of examples and comparative examples, the following results were obtained:
1. the immobilized enzyme prepared by the invention has stable enzyme activity, and the conversion rate is maintained above 25%.
2. The immobilized enzyme prepared by the invention has good stability at the temperature lower than 70 ℃, and the free enzyme has good stability at the temperature lower than 65 ℃, which indicates that the immobilized enzyme prepared by the invention has good thermal stability.
3. The pH stability range of the immobilized enzyme prepared by the invention is 6.5-8.5, and the pH stability range of the free enzyme is 7-8, which shows that the immobilized enzyme prepared by the invention has good pH tolerance.
4. The immobilized enzyme prepared by the invention has the catalytic activity of more than 75.6% after being continuously converted for 51 batches at the pH of 7 and the temperature of 70 ℃.
In summary, the immobilized enzyme has the advantages of high enzyme activity, good thermal stability, good pH tolerance and high recycling rate.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (9)
1. The preparation method of the immobilized enzyme is characterized by comprising the following steps:
a: inoculating the escherichia coli engineering bacteria expressing the L-rhamnose isomerase into a seed culture medium according to the inoculum size of 2-5% v/v, and culturing for 8-10h at the temperature of 30-40 ℃ to obtain a culture solution;
inoculating the culture solution into a fermentation culture medium with an inoculum size of 2-5% v/v for culturing for 30-40h to obtain a fermentation broth;
b: crushing the fermentation liquor by a homogenizer to obtain a crushing liquor, adding disodium hydrogen phosphate accounting for 3-5 per mill v/v and calcium chloride accounting for 1-5 per mill v/v of the volume of the crushing liquor, adjusting the pH value to 6.0-7.0 by using sodium hydroxide, adding diatomite accounting for 1-5% g/L of the volume of the crushing liquor, and filtering by using a plate frame to obtain a crude enzyme solution;
c: feeding the crude enzyme solution into a hollow fiber membrane, and concentrating for 5-10 times to obtain concentrated crude enzyme solution;
d: adding 0.01-0.05% v/v glutaraldehyde solution into the concentrated crude enzyme solution, crosslinking for 0.5-1.5h at 20-50 ℃, and then respectively washing for 3 times with phosphate buffer solution with pH of 7.0 and purified water to obtain crosslinked enzyme solution;
e: 7-9% of polyvinyl alcohol and 2-4% of sodium alginate by volume ratio 1:1 to 1.5, and obtaining a mixed solution, wherein the cross-linking enzyme solution and the mixed solution are mixed according to the volume ratio of 1: mixing evenly 2-4, dripping into 1.8-5.8%wt boric acid saturated solution of calcium chloride, stirring and immobilizing for 10-15h at 2-10 ℃, filtering, washing 3 times with phosphate buffer with pH of 7.0, and washing 3 times with purified water to obtain immobilized enzyme.
2. The method for preparing an immobilized enzyme according to claim 1, wherein: the host bacterium of the escherichia coli engineering bacterium for expressing the L-rhamnose isomerase is escherichia coli BL21, the secretory expression vector plasmid is pET-28a, and the gene sequence of the target gene is shown as SEQ ID No. 1.
3. The method of claim 1, wherein the fermentation conditions in step a are: the temperature is 34-40 ℃ and the wind speed is 1.0-2.0m 3 And/h, the rotating speed is 200-300rpm, the pressure is 0.1-0.3Mpa, the pH value is regulated to 7.0+/-0.1 by ammonia water, and the dissolved oxygen is 20-30%.
4. The method for preparing an immobilized enzyme according to claim 1, wherein: the parameters of the homogenizer in the step B are that the pressure is 40-45Mpa, the cycle times are 2-5, and the temperature is controlled below 30 ℃.
5. The method for preparing an immobilized enzyme according to claim 1, wherein: the pore diameter of the hollow fiber membrane in the step C ranges from 50000 Da to 100000Da.
6. The method for preparing an immobilized enzyme according to claim 1, wherein: and C, adding the concentrated crude enzyme solution in the step C into a gravity purification column, eluting with 0.1-0.5mol/L imidazole-containing phosphate buffer salt, collecting eluent, and adding the eluent into the glutaraldehyde solution in the step D for crosslinking reaction.
7. The method for preparing an immobilized enzyme according to claim 1, wherein: and E, adjusting the pH value of the saturated boric acid solution containing calcium chloride to 6.0-7.0 by using a sodium carbonate solution before adding the cross-linking enzyme solution and the mixed solution in a dropwise manner.
8. A method for preparing D-allose by immobilized enzyme, which is characterized by comprising the following steps:
f: preparing a D-psicose buffer solution with the concentration of 50-100g/L by using a phosphate buffer solution with the pH of 7, then adding the immobilized enzyme in claim 1, and stirring;
g: continuously adding 0.1-0.5mol/L Tris-HCl buffer solution to maintain the pH value of the reaction system to 6.0-9.0, and carrying out enzyme catalytic reaction at 60-80 ℃ to obtain D-allose.
9. The method for preparing D-allose by using an immobilized enzyme according to claim 8, wherein: the addition amount of the immobilized enzyme in the step F is 3-10% g/mL of the volume of the D-psicose buffer solution.
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