CN109576237A - L-GLOD solid polypeptide formulation and preparation method thereof - Google Patents
L-GLOD solid polypeptide formulation and preparation method thereof Download PDFInfo
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- CN109576237A CN109576237A CN201910008984.0A CN201910008984A CN109576237A CN 109576237 A CN109576237 A CN 109576237A CN 201910008984 A CN201910008984 A CN 201910008984A CN 109576237 A CN109576237 A CN 109576237A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 90
- 239000007787 solid Substances 0.000 title abstract description 26
- 239000000203 mixture Substances 0.000 title abstract description 23
- 238000009472 formulation Methods 0.000 title abstract description 22
- 229920001184 polypeptide Polymers 0.000 title abstract description 21
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 21
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 144
- 108090000790 Enzymes Proteins 0.000 claims abstract description 144
- 239000003223 protective agent Substances 0.000 claims abstract description 40
- 238000001291 vacuum drying Methods 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 39
- 230000008569 process Effects 0.000 claims description 31
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 16
- 238000007710 freezing Methods 0.000 claims description 16
- 230000008014 freezing Effects 0.000 claims description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 11
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 10
- 229910021529 ammonia Inorganic materials 0.000 claims description 8
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 7
- 229930195725 Mannitol Natural products 0.000 claims description 7
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- 239000000594 mannitol Substances 0.000 claims description 7
- 235000010355 mannitol Nutrition 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 claims description 6
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims description 5
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 claims description 5
- 238000006555 catalytic reaction Methods 0.000 claims description 4
- 235000015110 jellies Nutrition 0.000 claims description 2
- 239000008274 jelly Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 description 129
- 239000000243 solution Substances 0.000 description 60
- 230000000694 effects Effects 0.000 description 27
- 230000002255 enzymatic effect Effects 0.000 description 14
- 239000008363 phosphate buffer Substances 0.000 description 10
- 102000004316 Oxidoreductases Human genes 0.000 description 7
- 108090000854 Oxidoreductases Proteins 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 4
- 108010008292 L-Amino Acid Oxidase Proteins 0.000 description 4
- 102000007070 L-amino-acid oxidase Human genes 0.000 description 4
- 102000018120 Recombinases Human genes 0.000 description 4
- 108010091086 Recombinases Proteins 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- -1 that is Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HIISVQYDQWJITQ-UHFFFAOYSA-N 1h-pyrrole;quinoline Chemical compound C=1C=CNC=1.N1=CC=CC2=CC=CC=C21 HIISVQYDQWJITQ-UHFFFAOYSA-N 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108020004206 Gamma-glutamyltransferase Proteins 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
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000220324 Pyrus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000011868 grain product Nutrition 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001690 micro-dialysis Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000000859 sublimation Methods 0.000 description 1
- 230000008022 sublimation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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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
- C12P13/00—Preparation of nitrogen-containing organic compounds
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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
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03011—L-Glutamate oxidase (1.4.3.11)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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Abstract
The present invention relates to enzyme preparation applied technical fields, more specifically, the invention discloses L-GLOD solid polypeptide formulations and preparation method thereof.First aspect of the present invention provides a kind of preparation method of enzyme preparation, includes the steps that pretreatment, pre-freeze, quick-frozen and vacuum drying；Wherein, the raw material for preparing of the enzyme preparation includes protective agent and L-GLOD liquid.
Description
Technical field
The present invention relates to enzyme preparation applied technical fields, more specifically, the invention discloses L-GLOD solids
Enzyme preparation and preparation method thereof.
Background technique
Enzyme preparation is a kind of protein with biocatalysis performance, what the chemical reaction in catalysis process obtained
Bioactive substance, that is, biological agent is processed into the biological enzyme formulation of different purity and dosage form, is mainly used for catalytic production mistake
Various chemical reactions in journey have mild high catalytic efficiency, high specificity, action condition, reduction energy consumption, reduction chemistry dirty
The features such as dye, and application field is throughout various industries.
Due to the high efficiency of biological products, there is very high application value, but it is vulnerable to environmental factors such as temperature, pH
It influences, so people carry out preservation biological products by the way of low temperature；Cryopreservation involved in vacuum freeze-drying method with
Freeze drying technology is the important technology in field of biology, and is widely used in food in recent years, while being also commercialization
A kind of powerful measure, freeze-drying relatively consume energy it is low, it is very advantageous in economic cost；But there is production in Freeze Drying Technique
The disadvantages of period is long, energy consumption is high, high production cost, so that its extensive use is limited, so influence when in addition to control freeze-drying
Outside factor, the mode for adding some freeze drying protectants reduces active loss, at the same open can be improved biological products storage it is steady
It is qualitative.
L-GLOD (EC1.4.3.1) is a kind of flavo-enzyme using FAD as coenzyme, can not add it is exogenous auxiliary
It helps and aoxidizes Pidolidone deamination under conditions of the factor, generate ammonia, α-ketoglutaric acid and hydrogen peroxide.According to the consumption of oxygen or
The production quantity of hydrogen peroxide can be used for detecting Pidolidone and its related coupling reaction；In biochemistry test, L- paddy ammonia
Acid oxidase can be used for detecting glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease and γ-glutamyl transferase active and blood ammonia, creatinine etc.
Content；In biosensor, L-GLOD is fixed on different carriers, and combination fluid Injection Analysis system,
The technologies such as microdialysis so that the measurement of Pidolidone content easily and fast, high accuracy.Thus high vigor, zymologic property are stablized
L-GLOD be the indispensable key factor of its large-scale application.
L-GLOD is as biocatalyst, although having many advantages, such as that specificity is strong, reaction condition is mild, by
It is at high cost in its, stability is poor, saves difficult, hamper its application significantly；Therefore stable state is made in dglutamic oxidase
Enzyme preparation, it can be improved in the application prospect of the industries such as food processing, the production of α-ketoglutaric acid, biosensor.
Summary of the invention
First aspect of the present invention provides a kind of preparation method of enzyme preparation, including pretreatment, pre-freeze, quick-frozen and vacuum
Dry step；Wherein, the raw material for preparing of the enzyme preparation includes protective agent and L-GLOD enzyme solution.
As a preferred technical solution of the present invention, wherein protective agent is selected from glycerol, mannitol, dithiothreitol (DTT), mountain
Any one or more of combination of pears alcohol, rhamnolipid.
As a preferred technical solution of the present invention, wherein the mass ratio of protective agent and L-GLOD enzyme solution
For (1~20): (80~99).
As a preferred technical solution of the present invention, wherein pre-process as protective agent and L-GLOD enzyme solution
Mixing.
As a preferred technical solution of the present invention, wherein the pre-freezing temperature of pre-freeze process is -5~-25 DEG C, pre-freeze
Time is 0.5~2h.
As a preferred technical solution of the present invention, wherein the quick freezing temperature of quick-frozen process is -30~-65 DEG C, quick-frozen
Time is 0.25~1h.
As a preferred technical solution of the present invention, wherein the vacuum degree of process of vacuum drying is 1~10Pa, vacuum
Dry temperature is -10~-60 DEG C.
The second aspect of the present invention provides a kind of enzyme preparation obtained using the preparation method.
As a preferred technical solution of the present invention, wherein enzyme preparation is powder body material.
Third aspect of the present invention provides the application of the enzyme preparation, and the enzyme preparation is applied to catalysis Pidolidone oxygen
Change enzyme and generates α-ketoglutaric acid, ammonia and hydrogen peroxide；The enzyme preparation is also applied to measurement glutamic acid or measurement glutamic acid is made
Biosensor.
The utility model has the advantages that solid polypeptide formulation designed by the present invention, is made of protective agent and glucose oxidation enzyme solution, by pre-
Processing, pre-freeze (low-temperature treatment) is quick-frozen, and enzyme solution is made powder body material, can greatly reduce the volume of enzyme preparation by vacuum drying,
And enzyme stability can be improved in addition protective agent, facilitates storage and transport；Low energy consumption for the preparation method, simple process, product at
This is low, and the denaturation of the zymoprotein in preparation process and loss of material are few；Bulk density, intensity and the good fluidity of grain products, moisture absorption
Property self raising flour dirt is few.
Detailed description of the invention
Fig. 1: protection agent content influences the performance of enzyme preparation；
Fig. 2: freezing mode influences the performance of enzyme preparation；
Fig. 3: enzyme preparation enzyme activity rate during preservation measures；
Fig. 4: the THERMAL STABILITY of solid polypeptide formulation；
Fig. 5: the pH stability study of solid polypeptide formulation.
Specific embodiment
First aspect of the present invention provides a kind of preparation method of enzyme preparation, including pretreatment, pre-freeze, quick-frozen and vacuum
Dry step；Wherein, the raw material for preparing of the enzyme preparation includes protective agent and L-GLOD enzyme solution.
In one embodiment, the protective agent is selected from glycerol, mannitol, dithiothreitol (DTT), sorbierite, rhamnolipid
Any one or more of combination；Preferably, the protective agent is sorbierite.
Applicants experimentally found that not adding protectant enzyme preparation enzymatic activity cannot obtain in freezing dry process
It is kept to good, and protective agent is added in enzyme solution can effectively reduce the reduction of enzymatic activity, this may be due to protective agent
Physical force is formed with protein surface, so that it is still able to maintain stable structure under water deficit conditions, not loss of activity；This
Outside, it is also possible to which the acute variation of pH in freezing-inhibiting drying process reduces protein inactivation denaturation；It may also be with protein shape
At highly viscous glass state material, its diffusion coefficient is reduced, slows down its metastable conformation interconversion and conformation relaxation, inhibits albumen
The stretching, extension and aggregation of matter, are maintained its space structure.
In addition, applicant also has been surprisingly found that during the experiment, mannitol keeps best to enzyme preparation enzymatic activity, and it is obtained
The product appearance arrived is white sponge open structure, is easily chopped with wall very much from gently rubbing can become powder, have preferable
Quality.
In one embodiment, the mass ratio of the protective agent and L-GLOD enzyme solution is (1~20): (80
~99)；Preferably, the mass ratio of the protective agent and L-GLOD enzyme solution is (2~5): (95~98)；More preferably
The mass ratio of ground, the protective agent and L-GLOD enzyme solution is 2.5:97.5.
In one embodiment, the L-GLOD enzyme solution is the phosphate-buffered of L-GLOD
Liquid；Preferably, the concentration of the phosphate buffer of the L-GLOD is 0.1~2mol/L；It is highly preferred that the L-
The concentration of the phosphate buffer of dglutamic oxidase is 1mol/L.
In one embodiment, the L-GLOD be purchased from sigma company, No. CAS: 39346-34-4.
Applicant by experiment also it can be found that, when the mass ratio of protective agent and L-GLOD enzyme solution is (1~
20): (80~99) are, the enzymatic activity of gained enzyme preparation is preferable, this may due to when protect agent content it is too many when, system viscosity compared with
Greatly, on the one hand extend freezing, drying time, on the other hand gained powder body material is easy with tube wall ining conjunction with, is not easy glass, and raising adds
Work cost also destroys the original structure of powder.
In one embodiment, the pretreatment is that protective agent is mixed with L-GLOD enzyme solution.
In one embodiment, the pre-freezing temperature of the pre-freeze process is -5~-25 DEG C, and the pre-freeze time is 0.5~2h；
Preferably, the pre-freezing temperature of the pre-freeze process is -10~-20 DEG C, and the pre-freeze time is 0.8~1.5h；It is highly preferred that described pre-
The pre-freezing temperature of jelly process is -15 DEG C, and the pre-freeze time is 1h.
In one embodiment, the quick freezing temperature of the quick-frozen process be -30~-65 DEG C, the quick-frozen time be 0.25~
1h；Preferably, the quick freezing temperature of the quick-frozen process is -50 DEG C, and the quick-frozen time is 0.5h.
Pre-freeze is generally first carried out after the pretreatment of the mixture of protective agent and enzyme solution, this is because when enzyme solution moisture is more,
If directly carrying out vacuumize process, the gas being dissolved in water can be made to escape because ambient pressure reduces, bubble is formed, lead to enzyme
There is cavity in powder, influences the quality of the pharmaceutical preparations.
On the other hand, as testing it can be found that the enzymatic activity of the enzyme preparation as obtained by quick-frozen process is preferable, this may be
During quick-frozen, forming minute ice crystals is in branch irregular shape or ball-type, and cell is small, and ice crystal is small, and appearance is good, gap
It is small, and resistance when distillation is big；Rate of sublimation is slower, it is loose readily soluble that product is made, and finished product draws moist also larger, rehydration
It is good, it is advantageous for the preservation of enzyme or viable bacteria；And freezing process slowly, gained lattice is larger, and ice crystal is in hexagonal symmetric form, gained finished product
It is unfavorable to the preservation of enzyme or viable bacteria.
In one embodiment, the vacuum degree of the process of vacuum drying be 1~10Pa, vacuum drying temperature be-
10~-60 DEG C；Preferably, the vacuum degree of the process of vacuum drying is 1.3~8Pa, and vacuum drying temperature is -10~-50
℃；It is highly preferred that the vacuum degree of the process of vacuum drying is 5Pa, vacuum drying temperature is -45 DEG C.
In one embodiment, the vacuum drying time is 5~12h；Preferably, the vacuum drying time is
9h。
The vacuum degree for needing to keep the process of vacuum drying during the preparation process is 1~10Pa, vacuum drying temperature
It is -10~-60 DEG C, otherwise will cause the possibility of article pollution or product preparation failure.
Preferably, in one embodiment, a kind of enzyme preparation the preparation method is as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h.
The second aspect of the present invention provides a kind of enzyme preparation obtained using the preparation method.
In one embodiment, the enzyme preparation is powder body material.
Solid polypeptide formulation product is made in enzyme solution, can greatly reduce the volume of enzyme solution, and adds protective agent that enzyme can be improved is steady
It is qualitative, facilitate storage and transport.
Third aspect of the present invention provides the application of the enzyme preparation, and the enzyme preparation is applied to catalysis Pidolidone oxygen
Change enzyme and generates α-ketoglutaric acid, ammonia and hydrogen peroxide；The enzyme preparation is also applied to measurement glutamic acid or measurement glutamic acid is made
Biosensor.
Embodiment 1
The embodiment of the present invention 1 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is sorbierite；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 2
The embodiment of the present invention 2 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is glycerol；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 3
The embodiment of the present invention 3 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is mannitol；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 4
The embodiment of the present invention 4 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is dithiothreitol (DTT)；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 5
The embodiment of the present invention 5 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is rhamnolipid；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 6
The embodiment of the present invention 6 provides a kind of solid polypeptide formulation, preparation method are as follows:
(1) it pre-processes: L-GLOD liquid is stirred evenly；
(2) pre-freeze: the pre-freeze 1 under conditions of temperature is -15 DEG C；
(3) quick-frozen: pre-freeze 0.5h under the conditions of temperature is -50；
(4) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 7
The embodiment of the present invention 7 provides a kind of enzyme preparation, specific embodiment with embodiment 1, the difference is that, it is described
Protective agent replaces with 1.25:98.75 according to weight ratio with L-GLOD enzyme solution.
Embodiment 8
The embodiment of the present invention 8 provides a kind of enzyme preparation, specific embodiment with embodiment 1, the difference is that, it is described
Protective agent replaces with 5:95 according to weight ratio with L-GLOD enzyme solution.
Embodiment 9
The embodiment of the present invention 9 provides a kind of enzyme preparation, preparation method are as follows:
(1) it pre-processes: protective agent is uniformly mixed with L-GLOD enzyme solution according to weight ratio for 2.5:97.5；
(2) slow to freeze: pre-freeze 12h under the conditions of temperature is -20；
(3) it is dried in vacuo: being 5Pa in vacuum degree, temperature is -45 DEG C of dry 9h；
The protective agent is sorbierite；
The L-GLOD enzyme solution is the phosphate buffer of 1mol/L L-GLOD.
Embodiment 10
The embodiment of the present invention 10 provides a kind of enzyme preparation, specific embodiment with embodiment 9, the difference is that, will
Sorbierite replaces with glycerol.
Embodiment 11
The embodiment of the present invention 11 provides a kind of enzyme preparation, specific embodiment with embodiment 9, the difference is that, will
Sorbierite replaces with mannitol.
Embodiment 12
The embodiment of the present invention 12 provides a kind of enzyme preparation, specific embodiment with embodiment 9, the difference is that, will
Sorbierite replaces with dithiothreitol (DTT).
Embodiment 13
The embodiment of the present invention 13 provides a kind of enzyme preparation, specific embodiment with embodiment 9, the difference is that, will
Sorbierite replaces with rhamnolipid.
Performance Evaluation
The vigor of detection dglutamic oxidase is carried out using aniline development process.By horseradish peroxidase, 4- amino peace is replaced
Pyrrole quinoline, aniline, glutamic acid, phosphate buffer and appropriate enzyme react 10min at 37 DEG C, measure its light absorption value at 550 nm
Variation indicates the enzyme activity size of L-GLOD with the variation of light absorption value；Wherein, an enzyme activity unit definition:
Enzyme amount needed for 1min reaction generates 1 μm of ol hydrogen peroxide at 37 DEG C, and measured with inactivating enzyme solution under the conditions of different temperature
Enzyme activity as control, calculate its enzyme activity (with highest enzyme activity for 100%).
1. different protective agents influence the performance of enzyme preparation: utilizing the Pidolidone oxygen of above method measurement Examples 1 to 6
Change the enzymatic activity of enzyme enzyme solution and gained enzyme preparation, to measure the remaining enzymatic activity probability of Examples 1 to 6, and observes gained enzyme
The surface structure of preparation, the results are shown in Table 1.
2. agent content is protected to influence the performance of enzyme preparation: utilizing the L- of above method measurement embodiment 1, embodiment 6~8
The enzymatic activity of dglutamic oxidase enzyme solution and gained enzyme preparation further judges remaining enzymatic activity probability, to judge protective agent
Influence of the content to enzymatic activity conservation rate, the result is shown in Figure 1.
3. freezing mode influences the performance of enzyme preparation: using said determination method measurement Examples 1 to 5, embodiment 9~
The enzymatic activity of 13 L-GLOD enzyme solution and gained enzyme preparation, thus measure Examples 1 to 5, embodiment 9~13 it is surplus
Remaining enzymatic activity probability, is as a result shown in Fig. 2.
4. enzyme preparation enzyme activity rate during preservation measures: respectively by the L-GLOD enzyme solution of equivalent, embodiment
1,6 gained enzyme preparations save one month in 4 DEG C of refrigerated conditions, and measure its remaining enzymatic activity probability with above-mentioned identical method,
As a result see Fig. 3.
5. the THERMAL STABILITY of solid polypeptide formulation: measuring L- paddy ammonia at a temperature of 27,32,37,42,47,52 DEG C respectively
The relative activity of acid oxidase enzyme solution and embodiment 1, and measurement L-GLOD enzyme solution and embodiment 1 are at 37 DEG C
2,4,6,8, the relative activity under 10h are saved, as a result see Fig. 4, wherein L-GLOD enzyme solution is denoted as free enzyme solution, real
It applies example 1 and is denoted as solid polypeptide formulation.
6. the pH stability study of solid polypeptide formulation: measuring L-GLOD enzyme solution and implementation at different pH respectively
The relative activity of example 1；And measurement L-GLOD enzyme solution and embodiment 1 are placed in the buffer of different pH under 6.0h
Relative activity, as a result see Fig. 5, wherein L-GLOD enzyme solution is denoted as free enzyme solution, and embodiment 1 is denoted as solid enzyme
Preparation.
In addition, by the above 5, test of 6 steps, 1 gained enzyme of available L-GLOD enzyme solution and embodiment
Difference between preparation performance, is shown in Table 2.
Table 1
Table 2
| L-GLOD enzyme solution | Embodiment 1 | |
| Enzyme activity (u/ml) | 32 | 96.5 |
| Optimum temperature (DEG C) | 42 | 42 |
| Temperature tolerance (37 DEG C, 6h) | Enzyme activity keeps 70% | Enzyme activity keeps 82% |
| Optimal pH | 6 | 6 |
| PH tolerance (12h) | Enzyme activity can be down or up rapidly | PH5.0-7.0, enzyme activity are maintained at 80% |
By experiment we can see that: preferred, mannitol, sorbierite, dithiothreitol (DTT), glycerol have enzyme solution activity
Certain protective effect, and it is white that sorbierite, which shows the product appearance better than other protectant protective effects, obtained,
Sponge Porosity structure easily chops with wall from gently rubbing can become powder, have preferable quality very much；And rhamnolipid does not have
Have and play the role of being effectively protected, and oily mater, or even agglomeration condensation occurs in the surface of enzyme preparation product, affects product
Quality；Secondly, protectant content to enzymatic activity and its keeps having a certain impact in enzyme preparation system, add in this experiment
Non-inactivation in the drying process can be guaranteed by adding 2.5% sorbierite that enzyme solution can be effectively protected.
We can also be found through experiments that freezing mode also has large effect to the quality of enzyme preparation, and the present invention provides
Freeze, drying means can effectively keep the activity and its retention property of enzyme preparation；And it is found through experiments that not chilled dry
Dry enzyme solution has grown microbial flora after saving one month, significant to inactivate, and remaining enzyme activity is 48%；Protectant solid is not added
Enzyme powder agent partial inactivation after saving one month, remaining enzyme activity are 28%；And protectant solid enzyme pulvis is added and shows
Good stability, be still able to maintain 82% activity after one month, therefore paddy L- amino acid oxidase enzyme powder agent is prepared as paddy ammonia
The storage of acid oxidase, transport and application are laid a good foundation.
During the experiment as can be seen that knowing that solid polypeptide formulation and L- amino acid oxidase dissociate enzyme solution most by a in 4
Thermophilic degree does not change, remains at 42 DEG C, and enzyme activity variation tendency is similar, has no apparent variation, illustrate lyophilized technique
The optimum temperature of enzymatic reaction is not set to change；By the b in Fig. 4 it is found that after 37 DEG C of heat preservation 10h resolvase and solid enzyme
Preparation is respectively 60% and 72% with respect to enzyme activity, may indicate that thermal stability of the enzyme preparation provided by the invention under the conditions of 37 DEG C
It is better than the stability of L- amino acid oxidase resolvase.
On the other hand, by a in Fig. 5 it is found that the optimal pH of L- amino acid oxidase resolvase enzyme solution and solid polypeptide formulation is equal
Do not change in pH6.0；Resolvase known to b in Fig. 5 is more sensitive compared with solid polypeptide formulation for the variation performance of pH, works as pH
When varying slightly, enzyme activity can be down or up rapidly, and enzyme activity is still kept after solid polypeptide formulation places 6h at pH 5-7
80% or more, the pH stability of solid polypeptide formulation increases compared with resolvase.
Example above-mentioned is merely illustrative, and is used to explain the present invention some features of the method.Appended right is wanted
The range as wide as possible for being intended to require to be contemplated that is sought, and embodiments as presented herein is only according to all possible implementation
The explanation of the embodiment of the combined selection of example.Therefore, the purpose of applicant is that the attached claims are not illustrated this hair
The exemplary selectional restriction of bright feature.Some numberical ranges used also include sub- model in the claims
It encloses, the variation in these ranges should also be construed to be covered by the attached claims in the conceived case.
Claims (10)
1. a kind of preparation method of enzyme preparation, which is characterized in that include the steps that pretreatment, pre-freeze, quick-frozen and vacuum drying；
Wherein, the raw material for preparing of the enzyme preparation includes protective agent and L-GLOD enzyme solution.
2. preparation method according to claim 1, which is characterized in that protective agent be selected from glycerol, mannitol, dithiothreitol (DTT),
Any one or more of combination of sorbierite, rhamnolipid.
3. preparation method according to claim 1, which is characterized in that the mass ratio of protective agent and L-GLOD enzyme solution
For (1~20): (80~99).
4. preparation method according to claim 1, which is characterized in that pre-process as protective agent and L-GLOD enzyme solution
Mixing.
5. preparation method according to claim 1, which is characterized in that the pre-freezing temperature of pre-freeze process is -5~-25 DEG C, pre-freeze
Time is 0.5~2h.
6. preparation method according to claim 1, which is characterized in that the quick freezing temperature of quick-frozen process is -30~-65 DEG C, speed
The jelly time is 0.25~1h.
7. preparation method according to claim 1, which is characterized in that the vacuum degree of process of vacuum drying is 1~10Pa, vacuum
Dry temperature is -10~-60 DEG C.
8. a kind of enzyme preparation obtained using the preparation method as described in any one of claim 1~8.
9. enzyme preparation according to claim 8, which is characterized in that enzyme preparation is powder body material.
10. the application of enzyme preparation as claimed in claim 8, which is characterized in that enzyme preparation is applied to catalysis L-GLOD
Generate α-ketoglutaric acid, ammonia and hydrogen peroxide；The enzyme preparation is also applied to measurement glutamic acid or the life of measurement glutamic acid is made
Object sensor.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4623626A (en) * | 1982-06-29 | 1986-11-18 | Yamasa Shoyu Kabushiki Kaisha | L-glutamic acid oxidase and its production |
| CN1715405A (en) * | 2004-06-28 | 2006-01-04 | 曹卫 | Method for one-step dry powdering molecular biological reagent |
| CN101324568A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Glutamic acid diagnosis/determination reagent kit and method for determining aminoglutaric acid concentration |
| CN104792981A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Enzyme-labeled antibody conjugate stabilizer and application thereof |
-
2019
- 2019-01-04 CN CN201910008984.0A patent/CN109576237A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4623626A (en) * | 1982-06-29 | 1986-11-18 | Yamasa Shoyu Kabushiki Kaisha | L-glutamic acid oxidase and its production |
| CN1715405A (en) * | 2004-06-28 | 2006-01-04 | 曹卫 | Method for one-step dry powdering molecular biological reagent |
| CN101324568A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Glutamic acid diagnosis/determination reagent kit and method for determining aminoglutaric acid concentration |
| CN104792981A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Enzyme-labeled antibody conjugate stabilizer and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 鄢海燕、刘元芬: "《药剂学》", 31 January 2018 * |
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