CN110129391A - A method of N-acetyl-neuraminate is prepared using fixed double enzymes - Google Patents
A method of N-acetyl-neuraminate is prepared using fixed double enzymes Download PDFInfo
- Publication number
- CN110129391A CN110129391A CN201910334871.XA CN201910334871A CN110129391A CN 110129391 A CN110129391 A CN 110129391A CN 201910334871 A CN201910334871 A CN 201910334871A CN 110129391 A CN110129391 A CN 110129391A
- Authority
- CN
- China
- Prior art keywords
- acetyl
- neuraminate
- immobilization
- glucosamine
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/18—Multi-enzyme systems
-
- 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
- C12N9/88—Lyases (4.)
-
- 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
- C12N9/90—Isomerases (5.)
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/03—Oxo-acid-lyases (4.1.3)
- C12Y401/03003—N-Acetylneuraminate lyase (4.1.3.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
- C12Y501/03008—N-Acylglucosamine 2-epimerase (5.1.3.8)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
It the present invention relates to the use of the method that enzymatic reaction prepares N-acetyl-neuraminate, a method of N-acetyl-neuraminate is prepared using fixed double enzymes, the following steps are included: being 7-9 in PH, temperature is at 22-32 DEG C, by N-Acetyl-D-glucosamine and account for pyruvic acid or acetonate and immobilization N-Acetyl-D-glucosamine -2- epimerase that overall reaction system concentration is 0.2-0.6mol/L and immobilization Neu 5 Ac aldolase is mixed with to obtain the N-acetyl-neuraminate, convert fast and stable, wherein, immobilized bi-enzyme does not use carrier to fix, and preparation is convenient.
Description
Technical field
It the present invention relates to the use of the method that enzymatic reaction prepares N-acetyl-neuraminate, it is a kind of to prepare N- using fixed double enzymes
The method of n acetylneuraminic acid n.
Background technique
Sialic acid is one containing 9 carbon atoms and with the acid amino sugar of pyranose structure.N-acetyl-neuraminate is
The main representative of sialic acid.This kind of compound can be coupled to the terminal position of glycoprotein and glycolipid, rise in biometric identification process
Very important effect.Such as among the process of influenza infection host cell, the sialidase of influenza virus can be known
Other N-acetyl-neuraminate and its derivative, and pass through the infection cell in conjunction with sialic acid and release the virus replicated.Such as
Fruit inhibits its sialidase, then disease caused by virus can be treated.According to the structure of sialic acid, its class is designed
Like object, the sialidase of virus is combined using this analog, is allowed to lose the chance in conjunction with host cell surface sialic acid,
So virus can be effectively suppressed.New antiviral drug zanamavir as Ge Lansu company releases is exactly N- acetyl mind
Analog through propylhomoserin, it has good antiviral activity.Synthesize N-acetyl-neuraminate analog and the N- containing oligosaccharides
The inhibition for the leukocyte that n acetylneuraminic acid n mediates neuraminidase, erythrocyte agglutination element and selectin in research
In it is quite important.
N-acetyl-neuraminate can be obtained from natural resources such as milk, yolk, but these source contents are low, be not suitable for
A large amount of preparations.Chemical method or enzymatic clarification N-acetyl-neuraminate can be used in industrial production.However, since chemical method needs
Duplicate radical protection and deprotection step, so preparing the relatively inexpensive convenience of N-acetyl-neuraminate using enzyme process.But at present
Enzyme process prepare the enzymatic conversion that N-acetyl-neuraminate generally uses free state, the enzyme in this method does not have immobilised enzymes to stablize,
It cannot recycle, and the reaction time used is longer, inefficiency.In addition, being unfavorable for separating using the enzymatic conversion of free state
Product.Application No. is the patents of CN200510025745.4 to propose " a method of prepare N- acetyl-D- neuraminic acid ", uses
There is N-terminal the affine carrier immobilized enzyme of metal-chelating of the N- acetyl-D- neuraminic acid aldolase of histidine residues to be urged
Change, but after carrier is added in immobilised enzymes, carrier may react with reaction system, easily cause pollution, and conversion ratio is low, enzyme
It must be firmly combined with carrier, enzymic catalytic reaction stability is poor.
Summary of the invention
In view of the above problems, the present invention proposes a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes, it is fixed
Carrier is not needed when double enzymes, enzymic catalytic reaction is stablized quick.
In order to achieve the above object, the present invention is using technical solution once: a kind of using fixed double enzyme preparation N- acetyl nerves
The method of propylhomoserin, comprising the following steps: in pH be 7-9, temperature is that N-Acetyl-D-glucosamine and will account for overall reaction at 22-32 DEG C
Pyruvic acid or acetonate and immobilization N-Acetyl-D-glucosamine -2- epimerase that system concentration is 0.2-0.6mol/L and
Immobilization Neu 5 Ac aldolase is mixed with to obtain the N-acetyl-neuraminate；The immobilization N- acetyl grape
The immobilization process of osamine -2- epimerase and immobilization Neu 5 Ac aldolase are as follows: by N-Acetyl-D-glucosamine -
2- epimerase enzyme solution is 1:(1-4 by total enzyme activity ratio with Neu 5 Ac aldolase enzyme solution) it mixes, ice-water bath is cooling
Afterwards, while stirring be added mass fraction be 50-75% ammonium sulfate formed precipitating, precipitating is collected by centrifugation, then addition crosslinking agent into
Row crosslinking is fixed, and immobilization N-Acetyl-D-glucosamine -2- epimerase and immobilization Neu 5 Ac aldolase are formed；
N-Acetyl-D-glucosamine -2- epimerase the enzyme solution and Neu 5 Ac aldolase can also individually be solidified.
The present invention is using ammonium sulfate cooperation crosslinking agent to N-Acetyl-D-glucosamine -2- epimerase and N- acetyl nerve ammonia
Sour aldolase immobilizes, and two kinds of enzymes can mix crosslinking immobilization, can also individually using ammonium sulfate cooperate crosslinking agent into
Row crosslinking immobilization, obtained activity of the immobilized enzyme is strong, itself immobilization is stablized, and combines and stablizes between molecule.
The number of the N-Acetyl-D-glucosamine -2- epimerase is E.C.5.1.3.8, the N- acetyl nerve ammonia
The number of sour aldolase is E.C.4.1.3.3, combines when immobilization more stable between molecule, and enzymatic activity is higher.
Preferably, when crosslinking is fixed, temperature is 15-20 DEG C, crosslinking time 3h, and temperature is unsuitable excessively high, avoids being crosslinked
Enzyme loses activity in journey, simultaneously because the combination between enzyme interior molecules is needed when immobilization, because this time is unsuitable too short, it is ensured that
The stability of immobilised enzymes itself.
Preferably, the crosslinking agent is the glutaraldehyde solution that mass fraction is 25%, and glutaraldehyde contains there are two carbonyl, is easy
It reacts, is crosslinked with the polar group in N-Acetyl-D-glucosamine -2- epimerase and Neu 5 Ac aldolase
Closer cross-linked structure can be obtained afterwards, improve the strength and stability of immobilised enzymes original appearance.
It is further preferred that when crosslinking is fixed, the mass fraction of glutaraldehyde solution maintains 0.05-0.5% in enzyme solution, penta
Dialdehyde solution concentration is unsuitable excessively high, avoids the formation of mixed and disorderly polymer.
Preferably, the acetonate is Sodium Pyruvate, and concentration in the reaction system is 0.3-0.4mol/L, to reaction
The influence of system is smaller, and the conversion ratio of final N-acetyl-neuraminate is higher.
Preferably, in immobilization process, the N-Acetyl-D-glucosamine -2- epimerase enzyme solution and N- acetyl nerve ammonia
The total enzyme activity ratio of sour aldolase enzyme solution is 1-2.
Preferably, when preparing N-acetyl-neuraminate, pH 7, temperature is 30 DEG C.
Preferably, when preparing N-acetyl-neuraminate, the enzyme activity of immobilization N-Acetyl-D-glucosamine -2- epimerase is
70-120U/g, the enzyme activity of immobilization Neu 5 Ac aldolase are 30-60U/g.
Preferably, when preparing N-acetyl-neuraminate, pyruvic acid or acetonate are added when reaction proceeds to plateau,
The concentration of pyruvic acid or acetonate is set to maintain 0.3-0.4mol/L.
The beneficial effects of the present invention are: the present invention prepares N- acetyl mind using the fixed double enzymes of carrier-free immobilization technology
Through propylhomoserin, by carrying out being crosslinked fixation to N-Acetyl-D-glucosamine -2- epimerase and Neu 5 Ac aldolase, no
Carrier is needed, catalysis will not be reacted and generate pollution, the double enzymes of obtained fixation have higher activity, and self structure is stablized, energy
Multiple Reusability, the conversion ratio of N-acetyl-neuraminate greatly improves and stablizes in catalytic process.
Specific embodiment
This specific implementation method is only explanation of the invention, is not limitation of the present invention.Those skilled in the art
Member's any change made after having read specification of the invention, as long as within the scope of the claims, it all will be by
To the protection of Patent Law.
Embodiment 1:
A method of N-acetyl-neuraminate, immobilization N-Acetyl-D-glucosamine -2- epimerism are prepared using fixed double enzymes
The immobilization process of enzyme and immobilization Neu 5 Ac aldolase are as follows: the N- acetyl grape for E.C.5.1.3.8 will be numbered
The Neu 5 Ac aldolase enzyme solution that osamine -2- epimerase enzyme solution and number are E.C.4.1.3.3 is by total enzyme activity ratio
For 1:2 mixing, after ice-water bath is cooling, the ammonium sulfate that mass fraction is 75% is added while stirring and forms precipitating, it is heavy to be collected after centrifugation
It forms sediment, the glutaraldehyde solution that mass fraction is 25% is then added while stirring, 3h is reacted at 15 DEG C and carries out crosslinking fixation, is formed
Immobilization N-Acetyl-D-glucosamine -2- epimerase and immobilization Neu 5 Ac aldolase.
The process of N-acetyl-neuraminate is prepared the following steps are included: being 7 in pH, temperature is at 30 DEG C, by N- acetyl Portugal
Grapes glucosamine and account for Sodium Pyruvate that overall reaction system concentration is 0.3mol/L and the immobilization N- acetyl glucosamine that enzyme activity is 70U/g
The immobilization Neu 5 Ac aldolase that amine -2- epimerase and enzyme activity are 30U/g is mixed with to obtain the N- second
Acyl neuraminic acid adds Sodium Pyruvate when reaction proceeds to plateau, its concentration is made to maintain 0.3mol/L.
Embodiment 2:
A method of N-acetyl-neuraminate, immobilization N-Acetyl-D-glucosamine -2- epimerism are prepared using fixed double enzymes
The immobilization process of enzyme and immobilization Neu 5 Ac aldolase are as follows: by N-Acetyl-D-glucosamine -2- epimerase enzyme
Liquid is that 1:3 is mixed by total enzyme activity ratio with Neu 5 Ac aldolase enzyme solution, and after ice-water bath is cooling, quality is added while stirring
The ammonium sulfate that score is 50% forms precipitating, and precipitating is collected by centrifugation, and the glutaraldehyde solution that sole mass score is 25% is then added,
3h is reacted at 18 DEG C and carries out crosslinking fixation, and when crosslinking is fixed, the mass fraction of glutaraldehyde solution maintains 0.2% in enzyme solution, shape
At immobilization N-Acetyl-D-glucosamine -2- epimerase and immobilization Neu 5 Ac aldolase.
The process of N-acetyl-neuraminate is prepared the following steps are included: being 9 in pH, temperature is at 22 DEG C, by N- acetyl Portugal
Grapes glucosamine and account for pyruvic acid that overall reaction system concentration is 0.6mol/L and the immobilization N- acetyl glucosamine that enzyme activity is 120U/g
The immobilization Neu 5 Ac aldolase that amine -2- epimerase and enzyme activity are 60U/g is mixed with to obtain the N- second
Acyl neuraminic acid.
Embodiment 3:
A method of N-acetyl-neuraminate, immobilization N-Acetyl-D-glucosamine -2- epimerism are prepared using fixed double enzymes
The immobilization process of enzyme and immobilization Neu 5 Ac aldolase are as follows: by N-Acetyl-D-glucosamine -2- epimerase enzyme
Liquid is that 1:4 is mixed by total enzyme activity ratio with Neu 5 Ac aldolase enzyme solution, and after ice-water bath is cooling, quality is added while stirring
The ammonium sulfate that score is 60% forms precipitating, and precipitating is collected by centrifugation, and the glutaraldehyde solution that sole mass score is 25% is then added,
3h is reacted at 18 DEG C and carries out crosslinking fixation, and when crosslinking is fixed, the mass fraction of glutaraldehyde solution maintains 0.5% in enzyme solution, shape
At immobilization N-Acetyl-D-glucosamine -2- epimerase and immobilization Neu 5 Ac aldolase.
The process of N-acetyl-neuraminate is prepared the following steps are included: being 8 in pH, temperature is at 25 DEG C, by N- acetyl Portugal
Grapes glucosamine and account for pyruvic acid that overall reaction system concentration is 0.6mol/L and the immobilization N- acetyl glucosamine that enzyme activity is 100U/g
The immobilization Neu 5 Ac aldolase that amine -2- epimerase and enzyme activity are 45U/g is mixed with to obtain the N- second
Acyl neuraminic acid.
Embodiment 4:
A method of N-acetyl-neuraminate, immobilization N-Acetyl-D-glucosamine -2- epimerism are prepared using fixed double enzymes
Enzyme immobilizatio process are as follows: by N-Acetyl-D-glucosamine -2- epimerase enzyme solution after ice-water bath is cooling, be added while stirring
The ammonium sulfate that mass fraction is 50% forms precipitating, and precipitating is collected by centrifugation, and the glutaraldehyde that sole mass score is 25% is then added
Solution reacts 3h at 18 DEG C and carries out crosslinking fixation, and when crosslinking is fixed, the mass fraction of glutaraldehyde solution is maintained in enzyme solution
0.05%, form immobilization N-Acetyl-D-glucosamine -2- epimerase.
The immobilization process of immobilization Neu 5 Ac aldolase are as follows: Neu 5 Ac aldolase enzyme solution exists
After ice-water bath is cooling, the ammonium sulfate that mass fraction is 50% is added while stirring and forms precipitating, precipitating is collected by centrifugation, is then added
The glutaraldehyde solution that sole mass score is 25% reacts 3h at 18 DEG C and carries out crosslinking fixation, when crosslinking is fixed, penta in enzyme solution
The mass fraction of dialdehyde solution maintains 0.05%, forms immobilization Neu 5 Ac aldolase.
The process of N-acetyl-neuraminate is prepared the following steps are included: being 9 in pH, temperature is at 22 DEG C, by N- acetyl Portugal
Grapes glucosamine and account for pyruvic acid that overall reaction system concentration is 0.6mol/L and the immobilization N- acetyl glucosamine that enzyme activity is 120U/g
The immobilization Neu 5 Ac aldolase that amine -2- epimerase and enzyme activity are 60U/g is mixed with to obtain the N- second
Acyl neuraminic acid.
Embodiment 5:
A method of N-acetyl-neuraminate, immobilization N-Acetyl-D-glucosamine -2- epimerism are prepared using fixed double enzymes
Enzyme immobilizatio process are as follows: by N-Acetyl-D-glucosamine -2- epimerase enzyme solution after ice-water bath is cooling, be added while stirring
The ammonium sulfate that mass fraction is 60% forms precipitating, and precipitating is collected by centrifugation, and the glutaraldehyde that sole mass score is 25% is then added
Solution reacts 3h at 20 DEG C and carries out crosslinking fixation, and when crosslinking is fixed, the mass fraction of glutaraldehyde solution is maintained in enzyme solution
0.1%, form immobilization N-Acetyl-D-glucosamine -2- epimerase.
The immobilization process of immobilization Neu 5 Ac aldolase are as follows: Neu 5 Ac aldolase enzyme solution exists
After ice-water bath is cooling, the ammonium sulfate that mass fraction is 60% is added while stirring and forms precipitating, precipitating is collected by centrifugation, is then added
The glutaraldehyde solution that sole mass score is 25% reacts 3h at 20 DEG C and carries out crosslinking fixation, when crosslinking is fixed, penta in enzyme solution
The mass fraction of dialdehyde solution maintains 0.1%, forms immobilization Neu 5 Ac aldolase.
The process of N-acetyl-neuraminate is prepared the following steps are included: being 8 in pH, temperature is at 25 DEG C, by N- acetyl Portugal
Grapes glucosamine and account for pyruvic acid that overall reaction system concentration is 0.6mol/L and the immobilization N- acetyl glucosamine that enzyme activity is 100U/g
The immobilization Neu 5 Ac aldolase that amine -2- epimerase and enzyme activity are 45U/g is mixed with to obtain the N- second
Acyl neuraminic acid.
Comparative example 1:
A method of preparing N-acetyl-neuraminate using fixed double enzymes, prepare N-acetyl-neuraminate process include with
Lower step: being 7 in pH, and temperature is by N-Acetyl-D-glucosamine and to account for overall reaction system concentration at 30 DEG C as the third of 0.3mol/L
The free state that the immobilization N-Acetyl-D-glucosamine -2- epimerase and enzyme activity that ketone acid sodium and enzyme activity are 70U/g are 30U/g
Neu 5 Ac aldolase is mixed with to obtain the N-acetyl-neuraminate, when reaction proceeds to plateau, adds
Sodium Pyruvate makes its concentration maintain 0.3mol/L.
Comparative example 2:
A method of N-acetyl-neuraminate being prepared using fixed double enzymes, it includes following for preparing the process of N-acetyl-neuraminate
Step: being 9 in pH, and temperature is by N-Acetyl-D-glucosamine and to account for the acetone that overall reaction system concentration is 0.6mol/L at 22 DEG C
The immobilization N- that the N-Acetyl-D-glucosamine -2- epimerase and enzyme activity for the free state that acid is 120U/g with enzyme activity are 60U/g
Acetylneuraminate aldolase is mixed with to obtain the N-acetyl-neuraminate.
Comparative example 3:
A method of preparing N-acetyl-neuraminate using fixed double enzymes, prepare N-acetyl-neuraminate process include with
Lower step: being 8 in pH, and temperature is by N-Acetyl-D-glucosamine and to account for overall reaction system concentration at 25 DEG C as the third of 0.6mol/L
The free state that the N-Acetyl-D-glucosamine -2- epimerase and enzyme activity for the free state that ketone acid and enzyme activity are 100U/g are 45U/g
Neu 5 Ac aldolase be mixed with to obtain the N-acetyl-neuraminate.
Comparative example 4:
A method of N-acetyl-neuraminate being prepared using fixed double enzymes, it includes following for preparing the process of N-acetyl-neuraminate
Step: being 9 in pH, and temperature is by N-Acetyl-D-glucosamine and to account for the acetone that overall reaction system concentration is 0.6mol/L at 22 DEG C
The N- for the free state that the immobilization N-Acetyl-D-glucosamine -2- epimerase and enzyme activity that acid is 120U/g with enzyme activity are 60U/g
Acetylneuraminate aldolase is mixed with to obtain the N-acetyl-neuraminate.
Prepare immobilization N-Acetyl-D-glucosamine -2- epimerase process are as follows: by N-Acetyl-D-glucosamine -2- difference to
The ammonium sulfate that mass fraction is 50% is added while stirring and forms precipitating, it is heavy to be collected by centrifugation after ice-water bath is cooling for isomerase enzyme solution
It forms sediment, the glutaraldehyde solution that sole mass score is 25% is then added, 3h is reacted at 18 DEG C and carries out crosslinking fixation, crosslinking is fixed
When, the mass fraction of glutaraldehyde solution maintains 0.05% in enzyme solution, forms immobilization N-Acetyl-D-glucosamine -2- epimerism
Enzyme.
Comparative example 5:
A method of N-acetyl-neuraminate being prepared using fixed double enzymes, it includes following for preparing the process of N-acetyl-neuraminate
Step: being 8 in pH, and temperature is by N-Acetyl-D-glucosamine and to account for the acetone that overall reaction system concentration is 0.6mol/L at 25 DEG C
The immobilization N- that the N-Acetyl-D-glucosamine -2- epimerase and enzyme activity for the free state that acid is 100U/g with enzyme activity are 45U/g
Acetylneuraminate aldolase is mixed with to obtain the N-acetyl-neuraminate.
The immobilization process of immobilization Neu 5 Ac aldolase are as follows: Neu 5 Ac aldolase enzyme solution exists
After ice-water bath is cooling, the ammonium sulfate that mass fraction is 60% is added while stirring and forms precipitating, precipitating is collected by centrifugation, is then added
The glutaraldehyde solution that sole mass score is 25% reacts 3h at 20 DEG C and carries out crosslinking fixation, when crosslinking is fixed, penta in enzyme solution
The mass fraction of dialdehyde solution maintains 0.1%, forms immobilization Neu 5 Ac aldolase.
The conversion process of embodiment 1-5 and comparative example 1-5 are recorded, with reactant N-Acetyl-D-glucosamine 80%
Conversion ratio is the time required for criterion calculation conversion reaction, while recording the 21st time after same group of enzyme solution is used continuously 20 times
The time required to 80% conversion ratio is completed in reaction, data as shown in the table are obtained:
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes, which comprises the following steps: be in pH
7-9, temperature are by N-Acetyl-D-glucosamine and to account for the pyruvic acid that overall reaction system concentration is 0.2-0.6mol/L at 22-32 DEG C
Or acetonate is mixed with immobilization N-Acetyl-D-glucosamine -2- epimerase and immobilization Neu 5 Ac aldolase
The N-acetyl-neuraminate is prepared；Immobilization N-Acetyl-D-glucosamine -2- the epimerase and immobilization N- second
The immobilization process of acyl neuraminic acid aldolase are as follows: by N-Acetyl-D-glucosamine -2- epimerase enzyme solution and N- acetyl nerve
Propylhomoserin aldolase enzyme solution is 1:(1-4 by total enzyme activity ratio) mixing, after ice-water bath is cooling, it is 50- that mass fraction is added while stirring
75% ammonium sulfate forms precipitating, and precipitating is collected by centrifugation, and crosslinking agent is then added and carries out crosslinking fixation, forms immobilization N- acetyl
Gucosamine -2- epimerase and immobilization Neu 5 Ac aldolase.
2. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
When crosslinking is fixed, temperature is 15-20 DEG C, crosslinking time 3h.
3. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
The crosslinking agent is the glutaraldehyde solution that mass fraction is 25%.
4. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 3, it is characterised in that:
When crosslinking is fixed, the mass fraction of glutaraldehyde solution maintains 0.05-0.5% in enzyme solution.
5. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
The acetonate is Sodium Pyruvate, and concentration in the reaction system is 0.3-0.4mol/L.
6. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
In immobilization process, the N-Acetyl-D-glucosamine -2- epimerase enzyme solution and Neu 5 Ac aldolase enzyme solution
Total enzyme activity ratio is 1-2.
7. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
When preparing N-acetyl-neuraminate, pH 7, temperature is 30 DEG C.
8. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
When preparing N-acetyl-neuraminate, the enzyme activity of immobilization N-Acetyl-D-glucosamine -2- epimerase is 70-120U/g, fixed
The enzyme activity for changing Neu 5 Ac aldolase is 30-60U/g.
9. a kind of method for preparing N-acetyl-neuraminate using fixed double enzymes according to claim 1, it is characterised in that:
When preparing N-acetyl-neuraminate, pyruvic acid or acetonate are added when reaction proceeds to plateau, make pyruvic acid or acetone
The concentration of hydrochlorate maintains 0.3-0.4mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910334871.XA CN110129391A (en) | 2019-04-24 | 2019-04-24 | A method of N-acetyl-neuraminate is prepared using fixed double enzymes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910334871.XA CN110129391A (en) | 2019-04-24 | 2019-04-24 | A method of N-acetyl-neuraminate is prepared using fixed double enzymes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110129391A true CN110129391A (en) | 2019-08-16 |
Family
ID=67570985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910334871.XA Pending CN110129391A (en) | 2019-04-24 | 2019-04-24 | A method of N-acetyl-neuraminate is prepared using fixed double enzymes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110129391A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507658A (en) * | 2022-04-02 | 2022-05-17 | 深圳瑞德林生物技术有限公司 | Enzyme coexpression system and application thereof in sialic acid synthesis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5071750A (en) * | 1989-11-15 | 1991-12-10 | Forschungszentrum Juelich Gmbh | Enzymatic process for preparing n-acetylneuraminic acid |
| CN101165190A (en) * | 2006-10-17 | 2008-04-23 | 中国科学院上海生命科学研究院 | Preparation for N-acetylneuraminic acid by immobilization double-enzyme method |
| CN106011184A (en) * | 2016-06-21 | 2016-10-12 | 华南农业大学 | Noncellular synthetic biology based preparation method of 2-phenylethanol and application |
-
2019
- 2019-04-24 CN CN201910334871.XA patent/CN110129391A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5071750A (en) * | 1989-11-15 | 1991-12-10 | Forschungszentrum Juelich Gmbh | Enzymatic process for preparing n-acetylneuraminic acid |
| CN101165190A (en) * | 2006-10-17 | 2008-04-23 | 中国科学院上海生命科学研究院 | Preparation for N-acetylneuraminic acid by immobilization double-enzyme method |
| CN106011184A (en) * | 2016-06-21 | 2016-10-12 | 华南农业大学 | Noncellular synthetic biology based preparation method of 2-phenylethanol and application |
Non-Patent Citations (2)
| Title |
|---|
| MARÍA INMACULADA GARCÍA-GARCÍA ET AL.: "New stabilized FastPrep-CLEAs for sialic acid synthesis", 《BIORESOURCE TECHNOLOGY》 * |
| 饶娆: "N-乙酰-D-神经氨酸的酶法制备", 《中国优秀博硕士学位论文全文数据库 (硕士) 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507658A (en) * | 2022-04-02 | 2022-05-17 | 深圳瑞德林生物技术有限公司 | Enzyme coexpression system and application thereof in sialic acid synthesis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE69232138T2 (en) | METHOD FOR INSULATING GLYCOSYL TRANSFERASES | |
| Wang et al. | Enzymatic production of lactulose and 1-lactulose: current state and perspectives | |
| Mandels et al. | The use of adsorbed cellulase in the continuous conversion of cellulose to glucose | |
| FI85285C (en) | Crosslinked, water-insoluble glucose isomerase and process for its preparation | |
| US5071750A (en) | Enzymatic process for preparing n-acetylneuraminic acid | |
| CN110129391A (en) | A method of N-acetyl-neuraminate is prepared using fixed double enzymes | |
| JPS6219835B2 (en) | ||
| EP0049801B1 (en) | Sucrose-mutase, immobilized sucrose-mutase and use of this immobilized sucrose-mutase for the production of isomaltulose(6-o-alpha-d-glucopyranosido-d fructose) | |
| US20240262858A1 (en) | Scalable production of polyribonucleotides of controlled size | |
| US5296360A (en) | Process for producing ganglioside GM1 | |
| US5275939A (en) | Process for producing asialo GM1 | |
| CN1224703C (en) | Process for preparing magnetic microsphere immobilized cellulase | |
| EP0259598A2 (en) | Process for the enzymatic synthesis of a trisaccharide containing N-acetylneuraminic acid | |
| CN111607584A (en) | Method for immobilizing marine cyclodextrin glucosyltransferase by resin | |
| SU1041567A1 (en) | Method for preparing water-soluble immobilized proteolytic complex | |
| CN112626060A (en) | Immobilized multienzyme system for producing inositol and method for producing inositol | |
| JPS623795A (en) | Production of branched cyclodextrin | |
| Hollo et al. | Enzyme engineering in starch industry | |
| CN108642036A (en) | A kind of preparation method for the ordered mesoporous aluminium oxide and immobilized fructosyltransferase that nickel adulterates | |
| KR20150041627A (en) | Enzymes immobilised on styrene-divinyl benzene polymer matrices and the use thereof in industrial productions | |
| CN110818753B (en) | Method for recycling crystallized trehalose mother liquor | |
| SU1571047A1 (en) | Method of obtaining water-soluble oligosaccharides | |
| US5190869A (en) | Process for obtaining sorbitol and gluconic acid or gluconate usingzymomonas mobilis | |
| KR0130938B1 (en) | Preparation process of high concentrated galacto-oligo-saccharide | |
| KR940010307B1 (en) | Preparation method of high concentrated fructooligosaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200413 Address after: Room 1212-12, floor 12, building 3, No. 1366, Hongfeng Road, Kangshan street, Huzhou City, Zhejiang Province Applicant after: ZHEJIANG ZHENGSHUO BIO-TECH Co.,Ltd. Address before: 313000 Room A102, Building 1236 Longwangshan Road, Huzhou Economic and Technological Development Zone, Huzhou City, Zhejiang Province Applicant before: Huzhou Shuobang Biotechnology Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190816 |
|
| RJ01 | Rejection of invention patent application after publication |