CN106244574A - A kind of aminoacylates enzyme immobilization carrier and preparation method thereof - Google Patents

A kind of aminoacylates enzyme immobilization carrier and preparation method thereof Download PDF

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Publication number
CN106244574A
CN106244574A CN201610694759.3A CN201610694759A CN106244574A CN 106244574 A CN106244574 A CN 106244574A CN 201610694759 A CN201610694759 A CN 201610694759A CN 106244574 A CN106244574 A CN 106244574A
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China
Prior art keywords
aqueous phase
enzyme immobilization
parts
carrier
reaction system
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CN201610694759.3A
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Chinese (zh)
Inventor
王有
杨景然
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Yingjing Lihong Technology Promotion Services Co Ltd
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Yingjing Lihong Technology Promotion Services Co Ltd
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Priority to CN201610694759.3A priority Critical patent/CN106244574A/en
Publication of CN106244574A publication Critical patent/CN106244574A/en
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
    • C08F212/34—Monomers containing two or more unsaturated aliphatic radicals
    • C08F212/36—Divinylbenzene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L25/00—Compositions of, homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Compositions of derivatives of such polymers
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • 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)
    • 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)
    • C12Y305/01014—Aminoacylase (3.5.1.14)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2205/00—Polymer mixtures characterised by other features
    • C08L2205/03—Polymer mixtures characterised by other features containing three or more polymers in a blend

Abstract

The present invention relates to a kind of aminoacylates enzyme immobilization carrier and preparation method thereof, belong to technical field of enzyme immobilization.The carrier of the present invention includes glytidyl methacrylate, divinylbenzene, acrylonitrile, toluene, 2 ethyl hexanols, dibenzoyl peroxide, polyvinyl alcohol, gelatin, NaCl, CaCO3, deionized water.This matrix resin reacts with diethylamine again, prepares aminoacylates enzyme immobilization carrier.The activity of aminoacylates enzyme immobilizatio gained immobilized enzyme is carried out between 1000 1200u/g with this carrier.

Description

A kind of aminoacylates enzyme immobilization carrier and preparation method thereof
Technical field
The present invention relates to a kind of enzyme immobilization carrier and preparation method thereof, it is more particularly related to a kind of amino Acylase fixation support and preparation method thereof, belongs to technical field of enzyme immobilization.
Background technology
There is plurality of advantages: the stability of enzyme can be increased thus extend the service life of enzyme after liquid enzyme immobilization；At enzyme Catalytic reaction can be facilitated after terminating and separated by filters with product and reactant liquor by enzyme, has both reduced and has operated into This, turn avoid the enzyme pollution to catalysate；Enzymatic column operation can be realized, improve enzyme catalysis efficiency etc..
At present, producing amino acid whose main method is microbe fermentation method and chemical synthesis.Along with medical industry, food Industry and Peptide systhesis the reach of science, demand amino acid whose to optical voidness sharply increases.And the ammonia that chemical synthesis produces Base acid is usually racemic mixture, needs to carry out optical resolution.Solution enzyme splits DL aminoacid, and separation and purification of products is stranded Difficulty, is unfavorable for industrialized production.
Summary of the invention
Present invention seek to address that separation and purification of products difficulty, the problem being unfavorable for industrialized production in prior art, it is provided that A kind of aminoacylates enzyme immobilization carrier, this carrier can be applicable to produce amino acid whose amino-acylase, increases the stability of enzyme Thus extend the service life of enzyme, improve enzymatic activity, and beneficially separation and purification of products.
In order to realize foregoing invention purpose, its concrete technical scheme is as follows:
A kind of aminoacylates enzyme immobilization carrier, it is characterised in that: include carrier matrix resin and diethylamine, described carrier base Body resin includes following raw materials by weight:
Oil phase:
Glytidyl methacrylate 30-40 part
Divinylbenzene 40-50 part
Acrylonitrile 5-10 part
Toluene 50-60 part
2-Ethylhexyl Alcohol 50-60 part
Dibenzoyl peroxide 1-3 part；
Aqueous phase:
Polyvinyl alcohol 5-10 part
Gelatin 5-10 part
NaCl 100-200 part
CaCO315 parts
Deionized water 1000 parts.
Currently preferred, described polyvinyl alcohol is PVA1788.
A kind of preparation method of aminoacylates enzyme immobilization carrier, it is characterised in that: comprise the following steps that:
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensation is added with deionized water In pipe and charge door there-necked flask；System temperature is risen to 40-50 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 100-200 rev/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 72- 76 DEG C, until bead sizing；
D, polymerization reaction system temperature is raised to 80 DEG C, insulation 2-3 hour；Polymerization reaction system is warming up to 85 DEG C, insulation 2-3 hour；Polymerization reaction system is warming up to 90 DEG C, insulation 2-3 hour；Reaction system is cooled to 30-50 DEG C, leaches Resin bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.
The Advantageous Effects that the present invention brings:
The present invention uses environmentally-friendly technique process for suspension polymerization, with glytidyl methacrylate as primary raw material, with diethyl Alkenyl benzene is cross-linking agent, by adding the Third monomer acrylonitrile mechanical strength with increase carrier, prepares in the presence of porogen Macropore zymophore matrix resin.This matrix resin reacts with diethylamine again, prepares aminoacylates enzyme immobilization carrier.With this Carrier carries out the activity of aminoacylates enzyme immobilizatio gained immobilized enzyme between 1000-1200u/g.
Detailed description of the invention
Embodiment 1
A kind of aminoacylates enzyme immobilization carrier, including carrier matrix resin and diethylamine, described carrier matrix resin includes Following raw materials by weight:
Oil phase:
Glytidyl methacrylate 30 parts
Divinylbenzene 40 parts
Acrylonitrile 5 parts
Toluene 50 parts
2-Ethylhexyl Alcohol 50 parts
Dibenzoyl peroxide 1 part；
Aqueous phase:
Polyvinyl alcohol 5 parts
5 parts of gelatin
NaCl 100 parts
CaCO315 parts
Deionized water 1000 parts.
Embodiment 2
A kind of aminoacylates enzyme immobilization carrier, including carrier matrix resin and diethylamine, described carrier matrix resin includes Following raw materials by weight:
Oil phase:
Glytidyl methacrylate 40 parts
Divinylbenzene 50 parts
Acrylonitrile 10 parts
Toluene 60 parts
2-Ethylhexyl Alcohol 60 parts
Dibenzoyl peroxide 3 parts；
Aqueous phase:
Polyvinyl alcohol 10 parts
10 parts of gelatin
NaCl 200 parts
CaCO315 parts
Deionized water 1000 parts.
Embodiment 3
A kind of aminoacylates enzyme immobilization carrier, including carrier matrix resin and diethylamine, described carrier matrix resin includes Following raw materials by weight:
Oil phase:
Glytidyl methacrylate 35 parts
Divinylbenzene 45 parts
Acrylonitrile 7.5 parts
Toluene 55 parts
2-Ethylhexyl Alcohol 55 parts
Dibenzoyl peroxide 1-3 part；
Aqueous phase:
Polyvinyl alcohol 7.5 parts
7.5 parts of gelatin
NaCl 150 parts
CaCO315 parts
Deionized water 1000 parts.
Embodiment 4
A kind of aminoacylates enzyme immobilization carrier, including carrier matrix resin and diethylamine, described carrier matrix resin includes Following raw materials by weight:
Oil phase:
Glytidyl methacrylate 32 parts
Divinylbenzene 46 parts
Acrylonitrile 9 parts
Toluene 59 parts
2-Ethylhexyl Alcohol 53 parts
Dibenzoyl peroxide 1.5 parts；
Aqueous phase:
Polyvinyl alcohol 6 parts
8 parts of gelatin
NaCl 105 parts
CaCO315 parts
Deionized water 1000 parts.
Embodiment 5
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensation is added with deionized water In pipe and charge door there-necked flask；System temperature is risen to 40 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 100 revs/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 72 DEG C, directly Shape to bead；
D, polymerization reaction system temperature is raised to 80 DEG C, is incubated 2 hours；Polymerization reaction system is warming up to 85 DEG C, is incubated 2 Hour；Polymerization reaction system is warming up to 90 DEG C, is incubated 2 hours；Reaction system is cooled to 30 DEG C, leaches resin bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.
Embodiment 6
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensation is added with deionized water In pipe and charge door there-necked flask；System temperature is risen to 50 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 200 revs/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 76 DEG C, directly Shape to bead；
D, polymerization reaction system temperature is raised to 80 DEG C, is incubated 3 hours；Polymerization reaction system is warming up to 85 DEG C, is incubated 3 Hour；Polymerization reaction system is warming up to 90 DEG C, is incubated 3 hours；Reaction system is cooled to 50 DEG C, leaches resin bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.
Embodiment 7
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensation is added with deionized water In pipe and charge door there-necked flask；System temperature is risen to 45 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 150 revs/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 74 DEG C, directly Shape to bead；
D, polymerization reaction system temperature is raised to 80 DEG C, is incubated 2.5 hours；Polymerization reaction system is warming up to 85 DEG C, insulation 2.5 hours；Polymerization reaction system is warming up to 90 DEG C, is incubated 2.5 hours；Reaction system is cooled to 40 DEG C, leaches tree Fat bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.
Embodiment 8
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensation is added with deionized water In pipe and charge door there-necked flask；System temperature is risen to 41 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 120 revs/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 73 DEG C, directly Shape to bead；
D, polymerization reaction system temperature is raised to 80 DEG C, is incubated 2.2 hours；Polymerization reaction system is warming up to 85 DEG C, insulation 2.8 hours；Polymerization reaction system is warming up to 90 DEG C, is incubated 2.7 hours；Reaction system is cooled to 36 DEG C, leaches tree Fat bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.

Claims (3)

1. an aminoacylates enzyme immobilization carrier, it is characterised in that: include carrier matrix resin and diethylamine, described carrier Matrix resin includes following raw materials by weight:
Oil phase:
Glytidyl methacrylate 30-40 part
Divinylbenzene 40-50 part
Acrylonitrile 5-10 part
Toluene 50-60 part
2-Ethylhexyl Alcohol 50-60 part
Dibenzoyl peroxide 1-3 part；
Aqueous phase:
Polyvinyl alcohol 5-10 part
Gelatin 5-10 part
NaCl 100-200 part
CaCO315 parts
Deionized water 1000 parts.
A kind of aminoacylates enzyme immobilization carrier the most according to claim 1, it is characterised in that: described polyvinyl alcohol is PVA1788。
The preparation method of a kind of aminoacylates enzyme immobilization carrier the most according to claim 1, it is characterised in that: include with Lower processing step:
A, accurately weigh the glytidyl methacrylate in oil phase raw material, divinylbenzene, acrylonitrile, first according to recipe requirements Benzene, 2-Ethylhexyl Alcohol and dibenzoyl peroxide are sufficiently mixed uniformly in container, standby；
B, according to aqueous phase recipe requirements, by polyvinyl alcohol, gelatin, NaCl, CaCO3A tool stirring, condensing tube is added with deionized water And in charge door there-necked flask；System temperature is risen to 40-50 DEG C, starts stirring until each component of aqueous phase is sufficiently mixed dissolving；
C, oil mixture step A prepared are poured in the aqueous phase there-necked flask that step B prepares；Keep reactor stirs Speed be 100-200 rev/min, make oil phase form uniform bead and be dispersed in the aqueous phase prepared in advance；It is warming up to 72- 76 DEG C, until bead sizing；
D, polymerization reaction system temperature is raised to 80 DEG C, insulation 2-3 hour；Polymerization reaction system is warming up to 85 DEG C, insulation 2-3 hour；Polymerization reaction system is warming up to 90 DEG C, insulation 2-3 hour；Reaction system is cooled to 30-50 DEG C, leaches Resin bead；
E, employing ethanol carry out drip washing to resin, and the impurity of removing resin air-dries and i.e. obtains aminoacylates enzyme immobilization carrier.
CN201610694759.3A 2016-08-22 2016-08-22 A kind of aminoacylates enzyme immobilization carrier and preparation method thereof Pending CN106244574A (en)

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Applications Claiming Priority (1)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106810635A (en) * 2017-01-19 2017-06-09 中蓝晨光化工研究设计院有限公司 A kind of aminoacylates enzyme immobilization carrier and preparation method thereof
CN107641623A (en) * 2017-05-08 2018-01-30 天津南开和成科技有限公司 Primary amine base immobilized enzyme vector and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1900127A (en) * 2006-07-27 2007-01-24 汕头市铭虹化工有限公司 Acrylic resin and its preparing method
CN105131168A (en) * 2015-10-23 2015-12-09 蓝星（成都）新材料有限公司 Macropore hydroxyethyl methylacrylate resin and preparing method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1900127A (en) * 2006-07-27 2007-01-24 汕头市铭虹化工有限公司 Acrylic resin and its preparing method
CN105131168A (en) * 2015-10-23 2015-12-09 蓝星（成都）新材料有限公司 Macropore hydroxyethyl methylacrylate resin and preparing method thereof

Non-Patent Citations (4)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106810635A (en) * 2017-01-19 2017-06-09 中蓝晨光化工研究设计院有限公司 A kind of aminoacylates enzyme immobilization carrier and preparation method thereof
CN107641623A (en) * 2017-05-08 2018-01-30 天津南开和成科技有限公司 Primary amine base immobilized enzyme vector and preparation method thereof
CN107641623B (en) * 2017-05-08 2020-07-10 天津南开和成科技有限公司 Primary amino immobilized enzyme carrier and preparation method thereof

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