CN106520747A - Novel immobilized enzyme preparation, and preparation method and application thereof - Google Patents
Novel immobilized enzyme preparation, and preparation method and application thereof Download PDFInfo
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- CN106520747A CN106520747A CN201610864278.2A CN201610864278A CN106520747A CN 106520747 A CN106520747 A CN 106520747A CN 201610864278 A CN201610864278 A CN 201610864278A CN 106520747 A CN106520747 A CN 106520747A
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- 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
Abstract
The invention belongs to the field of biological catalysis, and particularly relates to a novel immobilized enzyme preparation, and a preparation method and application thereof. The preparation method of the novel immobilized enzyme preparation comprises the following steps: combining nano magnetic chitosan microspheres and a metal-organic framework material having so many properties such as multiple pores, large specific area, multiple metal sites and the like through graft copolymerization to obtain a nano magnetic chitosan-metal organic framework material having a core-shell structure with a porous network structure; and mixing the nano magnetic chitosan-metal organic framework material used as a carrier and an enzyme solution, and immobilizing at 0-25 DEG C for 1-12 hours to obtain the novel immobilized enzyme preparation. The method can immobilize enzymes such as lipase, proteinase, peroxidase and the like, and has the advantages of low cost, high immobilization efficiency, favorable enzyme stability, high enzyme loading amount and high enzyme recovery rate; no bifunctional reagent is used in the immobilization process; and the method is simple to operate, low in cost and mild in reaction.
Description
Technical field
The invention belongs to biocatalysis field, and in particular to a kind of new immobilised enzymes preparation and preparation method thereof with should
With.
Background technology
Enzyme is a kind of biocatalyst for being produced, being catalyzed particular organisms chemical reaction by living cells.Enzyme preparation is enzyme Jing
Cross purification, processing after the biological products with catalysis, the various chemical reactions being mainly used in during catalytic production, tool
The features such as having gentle high catalytic efficiency, high specificity, action condition, reducing energy consumption, reduce chemical contamination, its application time
Cloth food (bread baking industry, flour deep processing, fruit processing industry etc.), weaving, feed, detergent, papermaking, leather, medicine with
And the aspect such as energy development, environmental protection.
Immobilised enzymes (immobilized enzyme) is a kind of new technology that the sixties in 20th century grows up.It is so-called solid
Surely change enzyme, refer to and play catalytic action in certain spatial dimension, and the enzyme that can be used repeatedly and continuously.Usual enzymic catalytic reaction
All carry out in aqueous, and immobilised enzymes is, with either physically or chemically processing, to make insoluble by water-soluble enzyme
In water, but still the state with enzymatic activity.The research of the enzyme immobilization technology of system starts from the 1950's, German
Grubhofer and Schleith take the lead in poly- aminostyryl resin diazotising, then combine with amylase, pepsin etc.,
Make earliest immobilised enzymes.But it is until first international enzyme engineering (Enzyme Engineering) meeting in 1971, this
Technology is just officially named enzyme immobilization technology.Research of the people to immobilised enzymes afterwards achieves considerable progress, including
Immobilization technology, carrier material and its application in biochemical process and enzyme preparation etc..
Immobilised enzymes is had the advantage that compared with water-soluble enzyme：1. immobilised enzymes is reusable, and make enzyme uses effect
Rate is improved, use cost is reduced；2. immobilised enzymes is easily separated with reaction system, simplifies purifying technique, and product yield
High, quality is good；3. as a rule, enzyme Jing immobilizations rear stability is improved；4. the catalytic reaction process of immobilised enzymes
It is more easy to control；5. immobilised enzymes has certain mechanical strength, can be to act on substrate solution with stirring or in the way of filling post, just
In the serialization and automation mechanized operation of enzymic catalytic reaction；6. immobilised enzymes is more suitable for the use of multi-enzyme system compared with resolvase,
Not only greatly improve enzymic catalytic reaction speed using the cooperative effect in multi-enzyme system, but also can control to react by one
It is fixed sequentially to carry out.
According to physicochemical property and the purposes of resolvase, enzyme immobilizatio method can be divided into physical method and chemical method.Thing
Reason method mainly includes investment, absorption method；Chemical method mainly has chemical crosslink technique and covalent bond method.Currently, due to fixation
The method for changing enzyme is excessively complicated, and efficiency is low, high cost, or it is necessary not meet food processing institute using poisonous chemical reagent
The standard of the economy and safety of satisfaction, all these applications for all limiting immobilized lipase zymotechnic in the food industry.
The content of the invention
In order to overcome in prior art enzyme immobilization method excessively complexity, low efficiency, high cost, be difficult to recycle, bear
Loading capability is little to wait not enough and shortcoming, and the primary and foremost purpose of the present invention is to provide a kind of preparation method of new immobilised enzymes preparation.
Another object of the present invention is to provide the new immobilised enzymes preparation that above-mentioned preparation method is prepared, the enzyme system
The good stability of agent enzyme, load enzyme amount are high, safety non-toxic.
It is still another object of the present invention to provide the application of above-mentioned new immobilised enzymes preparation.
The purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of new immobilised enzymes preparation, comprises the steps of：
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution；Then add in shitosan acetic acid solution
Enter ferroferric oxide magnetic fluid, 40～50 DEG C of 40～60min of ultrasonic reaction obtain product 1；By product 1 add atoleine/
Be well mixed in Span-80 mixed liquors, then crosslinking agent penta 2 is added under conditions of 60～70 DEG C of waters bath with thermostatic control, quick stirring
Aldehyde, and reaction system pH is adjusted for 9～11, water bath with thermostatic control continues 1～2h of reaction, and removal of impurities is dried, and obtains nano-magnetic shell and gathers
Sugar；
(2) will be gamma-cyclodextrin and Potassium Benzoate soluble in water, it is subsequently adding methyl alcohol mixing, 60～80 DEG C of 12～24h of reaction
Leading to nitrogen bubble afterwards makes first alcohol and water volatilize naturally, separates out crystallization, filters, and washing is dried, obtains metal-organic framework materials；
(3) nano-magnetic chitin obtained in step (1) is added into 1～2h of basification in NaOH solution, is subsequently adding
40～60 DEG C of 8～10h of reaction of metal-organic framework materials obtained in step (2), obtain the organic bone of nano-magnetic chitin-metal
Frame material；
(4) above-mentioned nano-magnetic chitin-metal-organic framework materials are mixed as carrier with enzyme liquid, at 0～25 DEG C
At a temperature of 1～24h of immobilization, obtain new immobilised enzymes preparation；
In shitosan acetic acid solution described in step (1), the mass fraction of shitosan is preferably 1～5%, the matter of acetic acid
Amount fraction is preferably 1～10%；
In shitosan acetic acid solution described in step (1), the mass fraction of shitosan is more preferably 3%, acetic acid
Mass fraction is more preferably 5%；
Ferroferric oxide magnetic fluid described in step (1) is preferably (1 with the mass ratio of shitosan acetic acid solution:2)～
(1:5)；
Atoleine described in step (1) is preferably (20 with the volume ratio of Span-80:1)～(30:1)；
Product 1 described in step (1) is preferably (1 with the volume ratio of atoleine/Span-80 mixed liquors:3)～(1:
5)；
The rotating speed of the quick stirring described in step (1) is preferably 500～800r/min；
The consumption of the glutaraldehyde described in step (1) is preferably the 5%～10% of overall reaction system volume；
The time that water bath with thermostatic control described in step (1) continues to react is preferably 1.5h；
Removal of impurities described in step (1) is preferably first filtration under diminished pressure and then successively with petroleum ether, absolute ethyl alcohol extracting 3～5
It is secondary；
The condition of the drying described in step (1) is preferably：It is vacuum dried under the conditions of 30～60 DEG C；
The mol ratio of gamma-cyclodextrin and Potassium Benzoate described in step (2) is preferably (1:2)～(1:8)；
Methyl alcohol described in step (2) is preferably (3 with the volume ratio of water:1)～(3:2)；
The condition of the reaction described in step (2) is preferably 70 DEG C of reaction 20h；
The mass fraction of the NaOH solution described in step (3) is preferably 8～10%；
The shitosan in nano-magnetic chitin described in step (3) is pasted with the γ-ring in metal-organic framework materials
The mol ratio of essence is preferably (1:5)～(1:20)；
The condition of the reaction described in step (3) is preferably 50 DEG C of reaction 9h；
Enzyme described in step (4) is preferably lipase, protease, peroxidase etc.；
The condition of the solidification described in step (4) is preferably 15～25 DEG C of 4～20h of immobilization；
A kind of new immobilised enzymes preparation, is prepared by above-mentioned preparation method；
Application of the described new immobilised enzymes preparation in biocatalysis field；
The principle of the present invention：
Using magnetic Nano microsphere and metal-organic framework materials (MOF) loose structure can very effectively immobilized enzyme,
But the former cannot further improve its load capacity, be because increase carrier specific surface area while inevitably reduce it is micro-
The diameter in hole, so that reduce its load capacity；The latter is difficult to recycling, can only be separated using the means of centrifugation, and
The method is unfavorable for the recycling of immobilised enzymes and can cause certain loss to enzyme activity；The present invention will have magnetic receiving
Rice microsphere nano chitosan magnetic and the metal with many performances such as porous, bigger serface and many metallic sites-organic bone
Frame material is combined by way of graft copolymerization, and is further acted on by hydrogen bond and electrostatic force etc., will be with the organic bone of metal
The structurized gamma-cyclodextrin adsorbed close of frame is constructed a kind of with porous network structure on the surface of rice chitosan magnetic
The enzyme preparation carrier of core shell structure, the carrier have very big specific surface area and good load capacity, and with active official
The combination beneficial to zymoprotein can be rolled into a ball, enzyme can be fixed on carrier by simple embedded mode or other combinations, so as to
To immobilized enzyme preparation.
The present invention is had the following advantages relative to prior art and effect：
(1) nano-magnetic chitin-metal-organic framework materials obtained in the present invention can act as the carrier of immobilised enzymes,
Can a series of enzymes such as immobilized lipase, protease, peroxidase well, cost is relatively low, immobilization efficiency is high, enzyme
Good stability, load enzyme amount are high, and the enzyme activity rate of recovery is high.
(2) present invention does not use the bifunctional reagents such as glutaraldehyde, formaldehyde, simple to operate, cost during immobilised enzymes
Low, reaction is gentle.
(3) enzyme preparation safety non-toxic obtained in the present invention, can be applicable to food and the medical field (treatment not comprising disease
With diagnosis).
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this.
Embodiment 1
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution, wherein, the mass fraction of shitosan
For 3%, the mass fraction of acetic acid is 5%；Then ferroferric oxide magnetic fluid is added in shitosan acetic acid solution, 45 DEG C of ultrasounds are anti-
50min is answered, product 1 is obtained, wherein, ferroferric oxide magnetic fluid is 1 with the mass ratio of shitosan acetic acid solution:3；By product 1
(atoleine is 25 with the volume ratio of Span-80 to add atoleine/Span-80 mixed liquors:1) be well mixed in, product 1 with
The volume ratio of atoleine/Span-80 mixed liquors is 1:4；Then the condition for quickly stirring in 65 DEG C of waters bath with thermostatic control, 600r/min
It is lower to add crosslinking agent glutaraldehyde the 8% of overall reaction system volume (consumption of glutaraldehyde for), and reaction system pH is adjusted for 10, it is permanent
Tepidarium continues reaction 1.5h；Then filtration under diminished pressure, is extracted 4 times with petroleum ether, absolute ethyl alcohol, the vacuum under the conditions of 50 DEG C successively
It is dried, obtains nano-magnetic chitin；
(2) by gamma-cyclodextrin and Potassium Benzoate according to mol ratio 1:5 is soluble in water, is subsequently adding methyl alcohol (methyl alcohol and water
Volume ratio is 3:1) mix, leading to nitrogen bubble after 70 DEG C of reaction 20h makes first alcohol and water volatilize naturally, separates out crystallization, filters, wash
Wash, be dried, obtain metal-organic framework materials；
(3) nano-magnetic chitin obtained in step (1) is added into basification in the NaOH solution that mass fraction is 9%
1.5h, is subsequently adding 50 DEG C of reaction 9h of metal-organic framework materials obtained in step (2), wherein, in nano-magnetic chitin
The mol ratio of the gamma-cyclodextrin in shitosan and metal-organic framework materials is 1:15；Obtaining nano-magnetic chitin-metal has
Machine framework material；
(4) using above-mentioned nano-magnetic chitin-metal-organic framework materials as carrier and lipase from Aspergillus Niger liquid
(20mg/mL) mix, immobilization 8h at a temperature of 20 DEG C, then with the enzyme in distillation water washing unlockedization to carrier, obtain
To new immobilised enzymes preparation.
The present embodiment immobilised enzymes efficiency is 92.6%, and the enzyme activity rate of recovery is 98%, obtained new immobilised enzymes preparation
The load capacity of middle lipase from Aspergillus Niger is 200.5mg enzymes/g carriers.
Embodiment 2
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution, wherein, the mass fraction of shitosan
For 1%, the mass fraction of acetic acid is 1%；Then ferroferric oxide magnetic fluid is added in shitosan acetic acid solution, 40 DEG C of ultrasounds are anti-
60min is answered, product 1 is obtained, wherein, ferroferric oxide magnetic fluid is 1 with the mass ratio of shitosan acetic acid solution:2；By product 1
(atoleine is 30 with the volume ratio of Span-80 to add atoleine/Span-80 mixed liquors:1) be well mixed in, product 1 with
The volume ratio of atoleine/Span-80 mixed liquors is 1:5；Then the condition for quickly stirring in 70 DEG C of waters bath with thermostatic control, 500r/min
It is lower to add crosslinking agent glutaraldehyde the 5% of overall reaction system volume (consumption of glutaraldehyde for), and reaction system pH is adjusted for 11, it is permanent
Tepidarium continues reaction 2h；Then filtration under diminished pressure, is extracted 3 times with petroleum ether, absolute ethyl alcohol successively, and under the conditions of 30 DEG C, vacuum is done
It is dry, obtain nano-magnetic chitin；
(2) by gamma-cyclodextrin and Potassium Benzoate according to mol ratio 1:2 is soluble in water, is subsequently adding methyl alcohol (methyl alcohol and water
Volume ratio is 3:2) mix, leading to nitrogen bubble after 60 DEG C of reaction 24h makes first alcohol and water volatilize naturally, separates out crystallization, filters, wash
Wash, be dried, obtain metal-organic framework materials；
(3) nano-magnetic chitin obtained in step (1) is added into basification in the NaOH solution that mass fraction is 8%
2h, is subsequently adding 60 DEG C of reaction 8h of metal-organic framework materials obtained in step (2), wherein, the shell in nano-magnetic chitin
The mol ratio of the gamma-cyclodextrin in glycan and metal-organic framework materials is 1:20；Obtain nano-magnetic chitin-metal organic
Framework material；
(4) using above-mentioned nano-magnetic chitin-metal-organic framework materials as carrier and lipase from Aspergillus Niger liquid
(20mg/mL) mix, immobilization 20h at a temperature of 15 DEG C obtains new immobilised enzymes preparation.
The present embodiment immobilised enzymes efficiency is 89.2%, and the enzyme activity rate of recovery is 94%, obtained new immobilised enzymes preparation
The load capacity of middle lipase from Aspergillus Niger is 186.3mg enzymes/g carriers.
Embodiment 3
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution, wherein, the mass fraction of shitosan
For 5%, the mass fraction of acetic acid is 10%；Then ferroferric oxide magnetic fluid is added in shitosan acetic acid solution, 50 DEG C ultrasonic
Reaction 40min, obtains product 1, and wherein, ferroferric oxide magnetic fluid is 1 with the mass ratio of shitosan acetic acid solution:5；By product
1 adds atoleine/Span-80 mixed liquors, and (atoleine is 20 with the volume ratio of Span-80:1) it is well mixed in, product 1
Volume ratio with atoleine/Span-80 mixed liquors is 1:3；Then the bar for quickly stirring in 60 DEG C of waters bath with thermostatic control, 800r/min
Add crosslinking agent glutaraldehyde the 10% of overall reaction system volume (consumption of glutaraldehyde for) under part, and reaction system pH is adjusted for 9,
Water bath with thermostatic control continues reaction 1h；Then filtration under diminished pressure, is extracted 5 times with petroleum ether, absolute ethyl alcohol, the vacuum under the conditions of 60 DEG C successively
It is dried, obtains nano-magnetic chitin；
(2) by gamma-cyclodextrin and Potassium Benzoate according to mol ratio 1:8 is soluble in water, is subsequently adding methyl alcohol (methyl alcohol and water
Volume ratio is 3:1) mix, leading to nitrogen bubble after 80 DEG C of reaction 12h makes first alcohol and water volatilize naturally, separates out crystallization, filters, wash
Wash, be dried, obtain metal-organic framework materials；
(3) nano-magnetic chitin obtained in step (1) is added in the NaOH solution that mass fraction is 10% at alkalization
Reason 1h, is subsequently adding 40 DEG C of reaction 10h of metal-organic framework materials obtained in step (2), wherein, in nano-magnetic chitin
The mol ratio of the gamma-cyclodextrin in shitosan and metal-organic framework materials is 1:5；Obtaining nano-magnetic chitin-metal has
Machine framework material；
(4) using above-mentioned nano-magnetic chitin-metal-organic framework materials as carrier and lipase from Aspergillus Niger liquid
(20mg/mL) mix, immobilization 4h at a temperature of 25 DEG C obtains new immobilised enzymes preparation.
The present embodiment immobilised enzymes efficiency is 88.9%, and the enzyme activity rate of recovery is 90.2%, obtained new immobilised enzymes system
In agent, the load capacity of lipase from Aspergillus Niger is 169.4mg enzymes/g carriers.
Comparative example
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution, wherein, the mass fraction of shitosan
For 3%, the mass fraction of acetic acid is 5%；Then ferroferric oxide magnetic fluid is added in shitosan acetic acid solution, 45 DEG C of ultrasounds are anti-
50min is answered, product 1 is obtained, wherein, ferroferric oxide magnetic fluid is 1 with the mass ratio of shitosan acetic acid solution:3；By product 1
(atoleine is 25 with the volume ratio of Span-80 to add atoleine/Span-80 mixed liquors:1) be well mixed in, product 1 with
The volume ratio of atoleine/Span-80 mixed liquors is 1:4；Then add under conditions of 65 DEG C of waters bath with thermostatic control, quick stirring and hand over
Connection agent glutaraldehyde the 8% of overall reaction system volume (consumption of glutaraldehyde for), and adjust reaction system pH for 10, water bath with thermostatic control after
Continuous reaction 1.5h；Then filtration under diminished pressure, is extracted 4 times with petroleum ether, absolute ethyl alcohol successively, is vacuum dried, obtains under the conditions of 50 DEG C
To nano-magnetic chitin；
(2) above-mentioned nano-magnetic chitin is mixed as carrier with lipase from Aspergillus Niger liquid (20mg/mL), at 20 DEG C
At a temperature of immobilization 8h, being fixed enzyme preparation.
The present embodiment immobilised enzymes efficiency is 70.8%, and the enzyme activity rate of recovery is 73.5%, in obtained immobilised enzymes preparation
The load capacity of lipase from Aspergillus Niger is 80.6mg enzymes/g carriers.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of new immobilised enzymes preparation, it is characterised in that comprise the steps of：
(1) shitosan is dissolved in acetum, obtains shitosan acetic acid solution；Then four are added in shitosan acetic acid solution
Fe 3 O magnetic fluid, 40～50 DEG C of 40～60min of ultrasonic reaction, obtains product 1；Product 1 is added into atoleine/Span-
Be well mixed in 80 mixed liquors, then crosslinking agent glutaraldehyde is added under conditions of 60～70 DEG C of waters bath with thermostatic control, quick stirring, and
It is 9～11 to adjust reaction system pH, and water bath with thermostatic control continues 1～2h of reaction, and removal of impurities is dried, obtains nano-magnetic chitin；
(2) will be gamma-cyclodextrin and Potassium Benzoate soluble in water, methyl alcohol mixing is subsequently adding, is led to after 60～80 DEG C of 12～24h of reaction
Nitrogen bubble makes first alcohol and water volatilize naturally, separates out crystallization, filters, and washing is dried, obtains metal-organic framework materials；
(3) nano-magnetic chitin obtained in step (1) is added into 1～2h of basification in NaOH solution, is subsequently adding step
(2) 40～60 DEG C of 8～10h of reaction of metal-organic framework materials obtained in, obtain nano-magnetic chitin-metallic organic framework material
Material；
(4) above-mentioned nano-magnetic chitin-metal-organic framework materials are mixed as carrier with enzyme liquid, in 0～25 DEG C of temperature
Lower 1～the 24h of immobilization of degree, obtains new immobilised enzymes preparation.
2. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
In shitosan acetic acid solution described in step (1), the mass fraction of shitosan is 1～5%, and the mass fraction of acetic acid is 1
～10%.
3. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
Ferroferric oxide magnetic fluid described in step (1) is (1 with the mass ratio of shitosan acetic acid solution:2)～(1:5).
4. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
Atoleine described in step (1) is (20 with the volume ratio of Span-80:1)～(30:1).
5. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
The consumption of the glutaraldehyde described in step (1) for overall reaction system volume 5%～10%.
6. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
The mol ratio of gamma-cyclodextrin and Potassium Benzoate described in step (2) is (1:2)～(1:8).
7. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
The gamma-cyclodextrin in the shitosan in nano-magnetic chitin and metal-organic framework materials described in step (3)
Mol ratio is (1:5)～(1:20).
8. the preparation method of new immobilised enzymes preparation according to claim 1, it is characterised in that：
Enzyme described in step (4) is at least one in lipase, protease and peroxidase.
9. a kind of new immobilised enzymes preparation, it is characterised in that prepared by the preparation method described in any one of claim 1～8
Obtain.
10. application of the new immobilised enzymes preparation described in claim 9 in biocatalysis field.
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