CN107177583A - A kind of immobilization is acylated enzyme stabilizers and the method for improving immobilization acylase storage stability - Google Patents
A kind of immobilization is acylated enzyme stabilizers and the method for improving immobilization acylase storage stability Download PDFInfo
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- CN107177583A CN107177583A CN201710624003.6A CN201710624003A CN107177583A CN 107177583 A CN107177583 A CN 107177583A CN 201710624003 A CN201710624003 A CN 201710624003A CN 107177583 A CN107177583 A CN 107177583A
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- immobilization
- acylase
- methyl
- storage stability
- enzyme
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 52
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 49
- 239000003381 stabilizer Substances 0.000 title claims abstract description 16
- 238000003860 storage Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 15
- 239000003899 bactericide agent Substances 0.000 claims abstract description 14
- 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 abstract description 11
- 150000001298 alcohols Chemical class 0.000 claims abstract description 6
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 6
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 6
- 150000003839 salts Chemical class 0.000 claims abstract description 6
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 claims description 25
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical group COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 claims description 25
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical group SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 19
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 9
- 229930091371 Fructose Natural products 0.000 claims description 6
- 239000005715 Fructose Substances 0.000 claims description 6
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 2
- VHJLVAABSRFDPM-IMJSIDKUSA-N L-1,4-dithiothreitol Chemical compound SC[C@H](O)[C@@H](O)CS VHJLVAABSRFDPM-IMJSIDKUSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 27
- 230000001681 protective effect Effects 0.000 abstract description 11
- 239000007788 liquid Substances 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 3
- 239000000243 solution Substances 0.000 abstract 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 23
- 108010073038 Penicillin Amidase Proteins 0.000 description 16
- 108700023418 Amidases Proteins 0.000 description 14
- 102000005922 amidase Human genes 0.000 description 14
- 230000014759 maintenance of location Effects 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 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 4
- HSHGZXNAXBPPDL-HZGVNTEJSA-N 7beta-aminocephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H]([NH3+])[C@@H]12 HSHGZXNAXBPPDL-HZGVNTEJSA-N 0.000 description 3
- 229930186147 Cephalosporin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical compound COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 239000002132 β-lactam antibiotic Substances 0.000 description 2
- 229940124586 β-lactam antibiotics Drugs 0.000 description 2
- 241000194107 Bacillus megaterium Species 0.000 description 1
- HOKIDJSKDBPKTQ-GLXFQSAKSA-N Cephalosporin C Natural products S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 description 1
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-threitol Chemical compound OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- HOKIDJSKDBPKTQ-GLXFQSAKSA-M cephalosporin C(1-) Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H]([NH3+])C([O-])=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-M 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000005204 hydroxybenzenes Chemical class 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002633 protecting effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 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
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
- C12N9/84—Penicillin amidase (3.5.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention belongs to field of medicaments, and in particular to a kind of immobilization is acylated enzyme stabilizers and the method for improving immobilization acylase storage stability, and it includes following component：PH7.0 9.0, solution based on 0.01 0.03mol/L Tris Hcl solution, the stabilizer contains the one or more in anticorrosion and bactericidal agent 0.1wt%~0.5wt%, and alcohols 1wt%~5wt%, carbohydrate 1wt%~5wt%, 0.5~2mmol/L of reducing agent, 0.01~0.05mol/L of salt.The protection liquid that the present invention is provided is best to enzyme activity protective effect, and its addition very little influences little to storage cost, helps to develop immobilised enzymes protection liquid cheap and easy to get.There is certain directive significance for the storage protection of immobilization acylase in pharmaceuticals industry, its storage stability can be improved, so as to extend its service life reduction production cost.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of immobilization is acylated enzyme stabilizers and improves the storage of immobilization acylase
The method for depositing stability.
Background technology
PA ase, also known as penicillin amidase or penicillin amidohydrolase.Mainly from EHEC born of the same parents
Interior enzyme and bacillus megaterium ectoenzyme are obtained, the enzyme large-scale application in the pass of industrial production β-lactam antibiotics
Key intermediate and semi-synthetic β-lactam antibiotics.To the efficient immobilization of PA ase to improve it to temperature, pH
Value, the applicability in terms of solvent polarity and the stability of reuse repeatedly, widen PA ase and apply in the industry
Inevitable choice and key.
7-amino-cephalosporanic acid（7-aminocephalosporanic acid, 7-ACA）It is to synthesize cephalosporin analog antibiotic most
Important intermediate, typically has cephalosporin to slough side chain molecule by chemical method or biological enzyme and is made.But due to chemistry
Method has the shortcomings that complex process, severe reaction conditions, high pollution, by biological enzyme institute easy to operate, energy-saving and environmental protection
Substitution.Biological enzyme can be divided into two step enzyme methods and a step enzyme method again at present, and heretofore described immobilization cynnematin is acylated
Enzyme is used enzyme in a step enzyme method.
It is well known that liquid enzymes are existed easily affected by environment and inactivated in industrial production and application process, after use
It is difficult to the drawbacks of recycling etc. is very big, thus it is general all by being fixed of enzyme in current practice, to facilitate it to transport
It is defeated, use and store.
However, during the storage of immobilised enzymes or find often through enzyme after a while activity just have it is certain
Loss.And by immobilised enzymes be immersed in containing be in protectant solution it is a kind of improve its storage stability effective ways, and
This method has the advantages that simple to operate and with low cost.Conventional protective agent has preservative, carbohydrate, alcohols, reducing agent and salt
Class etc..
The content of the invention
Immobilization acylase storage stability can be improved it is an object of the invention to provide one kind and improves immobilization acylase
The method of storage stability, makes the immobilised enzymes being immersed in protection liquid remain to keep preferable after longer period of time is stored
Enzyme activity.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of immobilization is acylated enzyme stabilizers, and the stabilizer contains anticorrosion and bactericidal agent 0.1wt%~0.5wt%, and alcohols 1wt%
One or more in~5wt%, carbohydrate 1wt%~5wt%, 0.5~2mmol/L of reducing agent, 0.01~0.05mol/L of salt.
Preferably, stabilizer solution based on 0.01-0.03Mol/L Tris-Hcl, the anticorrosion and bactericidal agent
For methyl p-hydroxybenzoate, carbohydrate is fructose, and alcohols is glycerine or inositol, and reducing agent is dithiothreitol (DTT), and salt is MgSO4
Or phosphate.
Preferably, the pH of the base soln is 7.0-9.0.
Preferably, including following component：0.01-0.03mol/L Tris-Hcl base soln（pH8.0）And 0.2wt%
Methyl p-hydroxybenzoate.
Preferably, including following component：PH8.0,0.02mol/L Tris-Hcl base soln, 2mmol/L two sulphur
Threitol and 0.2wt% methyl p-hydroxybenzoates.
A kind of method for improving immobilization acylase storage stability, it is characterised in that based on immobilization acylase,
In the solution for being immersed in aforementioned stable agent, in 4-25 DEG C of preservation.Preferably, storage temperature is 4 DEG C.
The advantages of the present invention：
The protection liquid that the present invention is provided is best to enzyme activity protective effect, and its addition very little influences little to storage cost,
Help to develop immobilised enzymes protection liquid cheap and easy to get.
Added with dithiothreitol (DTT)（DTT）The acylated enzyme protection liquid of immobilization head, can effectively suppress the growth of microorganism
And breeding, it is preferable to the protecting effect of immobilization acylase, stored 12 months at being stored 9 weeks and 4 DEG C at 25 DEG C, enzyme activity can be protected
Rate is stayed to improve a lot.DTT used is the reducing agent of disulfide bond, can keep the reduction-state of-SH in enzyme, available for prevention egg
In the protein molecule that is formed between cysteine in white matter or intermolecular disulfide bond, additionally also have to enzyme activity significantly
Activation, therefore can have preferable protective effect to immobilization acylase enzyme activity.
Brief description of the drawings
Fig. 1 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate（Embodiment 1）Protection to immobilization Cephalosporin Acylases
Effect.
Fig. 2 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and inositol（Embodiment 2）To immobilization cephalo bacterium at 25 DEG C
The protective effect of plain acylase.
Fig. 3 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and MgSO4（Embodiment 3）To immobilization cephalo bacterium at 25 DEG C
The protective effect of plain acylase.
Fig. 4 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and fructose（Embodiment 4）To immobilization cephalo bacterium at 25 DEG C
The protective effect of plain acylase.
Fig. 5 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and glycerine（Embodiment 5）To immobilization cephalo bacterium at 25 DEG C
The protective effect of plain acylase.
Fig. 6 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and DTT（Embodiment 6）To immobilization cynnematin at 25 DEG C
The protective effect of acylase.
Fig. 7 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and phosphate（Embodiment 7）To immobilization cephalo at 25 DEG C
The protective effect of rhzomorph acylase.
Fig. 8 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and DTT（Embodiment 8）Respectively to fixation at 4 DEG C and 25 DEG C
Change the protective effect of Cephalosporin Acylases.
Fig. 9 is anticorrosion and bactericidal agent methyl p-hydroxybenzoate and DTT（Embodiment 9）Respectively to fixation at 4 DEG C and 25 DEG C
Change the protective effect of PA ase.
In Fig. 1-7,1 is control, during 2 are embodiment, Fig. 8-9, and 1 is 25 DEG C of preservations, and 2 be 4 DEG C of preservations.
Embodiment
1 experiment reagent：
Resin, Xi'an Lan Xiao scientific ＆ technical corporation；Methyl p-hydroxybenzoate, fructose, glycerine, inositol, dithiothreitol (DTT)（DTT）, on
Hai Shenggong；MgSO4 and phosphate, Tianjin Reagent Company.
2 experimental facilities
High pressure homogenizer, ATS；PH meter, METTLER；Shaking table, Shanghai new talent；Vacuum pump using circulatory water, Henan Gongyi；Electronic balance,
Shanghai balance equipment factory.
3 test methods
Solution based on 0.01-0.03mol/L Tris-Hcl solution is respectively configured, 2 ‰ para hydroxybenzenes are separately added into thereto
Methyl formate, 2% inositol, 0.5mM MgSO4, 2% fructose, 5% glycerine, 1mM DTT, one kind in 0.02mol/L PBS or several
Kind.By immobilization Cephalosporin Acylases（PA ase）It is immersed in the various protection liquid of configuration, to be not added with any guarantor
The immobilised enzymes of liquid is protected as control, is investigated under certain temperature, after certain time, immobilised enzymes enzyme activity retention rate.
4 enzyme activity determinations
（1）Cephalosporin Acylases enzyme activity determination
Enzyme activity is defined：One enzyme activity international unit refers under rated condition, per minute to hydrolyze 1 micromolar cephalosporonic acid institute
Need the amount of cephalosporin C acrylase.
2.0gCPC sodium salts are weighed, is dissolved in 100mL 0.02mol/L, pH8.0 kaliumphosphate buffer, uses 0.1mol/L
NaOH solution regulation pH to 8.0, temperature control is at 25 DEG C.A certain amount of immobilised enzymes is taken to add in the solution, with 0.1mol/L's
NaOH solution is titrated, and it is 8.0 to control reaction pH, reacts 10min, and the NaOH solution of record consumption obtains volume VNaOH。
。
（2）Penicillin Acylase Activity is determined
It is accurate to weigh sample 500mg, the benzylpenicillin potassium solution for the 100ml 4% for being preheating to 28 DEG C is added, stirring, control is opened
28 DEG C of reaction temperature, adjusts pH to 8.0, control reaction pH8.0 with 0.1 mol/L NaOH solutions in reaction, records about 6 minutes left sides
Right NaOH consumption volume（ml）.
Hydrolysis vigor（28℃）Calculate
。
V：The NaOH volumes consumed in titration（ml）
C：The molar concentration of NaOH solution（mol/L）
W：The weight of sample（g）
T：Titration time（min）.
Comparative example 1
By ferment and purified acquisition being fixed of enzyme liquid, being fixed Cephalosporin Acylases, its enzyme activity for 100~
200U/g.Be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 80%, 44%, 35%, 21%.
Embodiment 1
Immobilization Cephalosporin Acylases are immersed in the 0.01mol/L Tris-Hcl containing 2 ‰ methyl p-hydroxybenzoates
（pH9.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 99%, 55%, 51%, 48%.
Embodiment 2
Immobilization Cephalosporin Acylases are immersed in the 0.02mol/L Tris- containing 2% inositol, 2 ‰ methyl p-hydroxybenzoates
Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 72%, 57%, 52%,
50%。
Embodiment 3
Immobilization Cephalosporin Acylases are immersed in MgSO containing 0.5mM4, 2 ‰ methyl p-hydroxybenzoates 0.03mol/L
Tris-Hcl（pH7.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 74%, 55%,
47%、43%。
Embodiment 4
Immobilization Cephalosporin Acylases are immersed in the 0.02mol/L Tris- containing 2% fructose, 2 ‰ methyl p-hydroxybenzoates
Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 79%, 70%, 67%,
46%。
Embodiment 5
Immobilization Cephalosporin Acylases are immersed in the 0.02mol/L Tris- containing 5% glycerine, 2 ‰ methyl p-hydroxybenzoates
Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 91%, 70%, 69%,
63%。
Embodiment 6
Immobilization Cephalosporin Acylases are immersed in the 0.02mol/L of DTT containing 1mM, 2 ‰ methyl p-hydroxybenzoates
Tris-Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 95%, 91%,
88%、87%。
Embodiment 7
Immobilization Cephalosporin Acylases are immersed in the solution of PBS containing 0.02mol/L, 2 ‰ methyl p-hydroxybenzoates
（pH8.0）In, be placed at 25 DEG C and preserve 1,3,6,9 weeks, its enzyme activity retention rate is respectively 63%, 48%, 47%, 43%.
Embodiment 8
Immobilization Cephalosporin Acylases are immersed in the 0.02mol/L of DTT containing 1mM, 2 ‰ methyl p-hydroxybenzoates
Tris-Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9,12 months, its enzyme activity retention rate is respectively 90%,
81%、72%、60%、48%.Be placed at 4 DEG C and preserve 1,3,6,9,12 months, its enzyme activity retention rate is respectively 99%, 97%,
94%、89%、85%。
Embodiment 9
Immobilized penicillin acylated enzyme is immersed in the 0.02mol/L Tris- of DTT containing 1mM, 2 ‰ methyl p-hydroxybenzoates
Hcl（pH8.0）In solution, be placed at 25 DEG C and preserve 1,3,6,9,12 months, its enzyme activity retention rate is respectively 98%, 89%,
82%、75%、66%.Be placed at 4 DEG C and preserve 1,3,6,9,12 months, its enzyme activity retention rate is respectively 99%, 98%, 95%,
93%、91%。
Claims (7)
1. a kind of immobilization is acylated enzyme stabilizers, it is characterised in that the stabilizer contains anticorrosion and bactericidal agent 0.1wt%~0.5wt%,
And in alcohols 1wt%~5wt%, carbohydrate 1wt%~5wt%, 0.5~2mmol/L of reducing agent, 0.01~0.05mol/L of salt
It is one or more of.
2. stabilizer according to claim 1, it is characterised in that the stabilizer is with 0.01-0.03Mol/L Tris-
Solution based on Hcl, the anticorrosion and bactericidal agent is methyl p-hydroxybenzoate, and carbohydrate is fructose, and alcohols is glycerine or inositol, is gone back
Former agent is dithiothreitol (DTT), and salt is MgSO4Or phosphate.
3. stabilizer according to claim 2, it is characterised in that the pH of the base soln is 7.0-9.0, preferably pH is
8.0。
4. stabilizer according to claim 2, it is characterised in that including following component：PH8.0,0.01-0.03mol/
L Tris-Hcl base soln and 0.2wt% methyl p-hydroxybenzoates.
5. stabilizer according to claim 2, it is characterised in that including following component：PH8.0,0.02mol/L Tris-
Hcl base soln, 2mmol/L dithiothreitol (DTT) and 0.2wt% methyl p-hydroxybenzoates.
6. a kind of method for improving immobilization acylase storage stability, it is characterised in that based on immobilization acylase, will
It is immersed in the solution of any one of the claim 1-5 stabilizers, in 0-25 DEG C of preservation.
7. method according to claim 6, it is characterised in that storage temperature is 4 DEG C.
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| CN111876402A (en) * | 2020-06-30 | 2020-11-03 | 伊犁川宁生物技术有限公司 | Preparation method of immobilized cephalosporin C acylase |
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