CN109456959A - A kind of method of modified magnetic graphene oxide immobilized lipase - Google Patents
A kind of method of modified magnetic graphene oxide immobilized lipase Download PDFInfo
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- CN109456959A CN109456959A CN201811580131.6A CN201811580131A CN109456959A CN 109456959 A CN109456959 A CN 109456959A CN 201811580131 A CN201811580131 A CN 201811580131A CN 109456959 A CN109456959 A CN 109456959A
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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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The present invention provides a kind of method in modified magnetic graphene oxide immobilized lipase.Magnetic oxygenated graphene is carried out using polyethylene glycol diamines amido modified and in conjunction with the carboxyl of lipase, the load factor of modified magnetic graphite oxide improves 9.61%, enzyme activity is 3519.3U/g, saturation magnetization is 23.8emu/g, therefore the activity of load capacity and enzyme can be improved in modified magnetic graphite oxide, since magnetic carrier is reusable, it is effectively reduced production cost, overcomes the defect of resolvase in process of production.
Description
Technical field
The present invention relates to a kind of methods of modified magnetic graphene oxide immobilized lipase.
Background technique
The enzyme of natural oil can be catalytically decomposed in lipase, according to its source can be divided into animal tallow enzyme, plant fat enzyme and
Microbial lipase, the type of microbial lipase is more, source is wide, is easy to cultivate, therefore is the main source of commercial fatty enzyme,
It is widely used in the fields such as food industry, pharmaceuticals industry and biodiesel.Lipase can be shown under suitable reaction condition
High catalytic performance out, but the activity of enzyme is greatly lowered, stability is poor in the industrial production, not reproducible use, separates and is stranded
Difficulty substantially reduces the application of resolvase in actual production.In order to overcome the defect of above-mentioned resolvase in actual production, can incite somebody to action
Enzyme is fettered on the support by physically or chemically means and keeps certain activity by its immobilization.Immobilised enzymes is certain
Improve its zymologic property, reusable and be easily isolated in degree, can effectively reduce industrial production cost.
Graphene oxide is a kind of derivative of graphene, and a large amount of oxygen-containing group is contained on biggish specific surface area, its surface
Group such as epoxy group, carboxyl, hydroxyl, highly-water-soluble, good biocompatibility, excellent stability and high absorption capacity, make oxygen
Graphite alkene has preferable application prospect in terms of the fixed carrier of enzyme.For the ease of the separation of immobilised enzymes and reaction system,
Magnetic Fe can be introduced in graphene oxide3O4.If enzyme and graphene oxide are directly connected to will limit the movement of limit enzyme molecule, meeting
Slow down reaction speed.Result of study shows the increased activity of immobilised enzymes in the presence of spacer.Therefore the present invention is first with poly- second
Two hydro diamines (PEGA) are used as spacer modified magnetic graphene oxide, and then modified carrier is used further to immobilized lipase.
This hair utilizes polyethylene glycol diamines modified magnetic graphene oxide, is then applied to fatty enzyme immobilizatio, improves
The load capacity of enzyme, activity and operational stability, are easy to isolating and purifying for enzyme, reduce its cost in industrialized production.
Summary of the invention
The present invention is achieved by the following scheme: a certain amount of polyethylene glycol diamines being first added to magnetic oxygenated graphene
In solution, Magnetic Isolation after a certain period of time is reacted, a certain amount of lipase is then added into carrier, reaction certain time completes rouge
Fat enzyme immobilizatio.
Specific embodiment
Specific embodiment 1: a kind of method of modified magnetic graphene oxide immobilized lipase, passes through following steps
It realizes:
Step 1: weighing 1g graphene oxide, 250mL, 0.1mol/LNaOH solution are added to ultrasound 45min in beaker,
It is centrifugally separating to obtain powder and with water repeated flushing, adjusting its pH value is 6, then, the distilled water of 100mL is added to separation
In powder, 30min is dispersed in ultrasound, under nitrogen protection, 5.40g FeCl will be contained3·6H2O and 2.65g FeCl2·
4H2The 40ml distilled water of O is added dropwise in suspension, the NH of fast drop 10mL 25%3·H2O, adjusting suspension pH value is 12,
At 85 DEG C, 45min is stirred, is cooled to room temperature, magnet separation is simultaneously washed repeatedly under ultrasound with methanol repeatedly, and obtained solid exists
It is dry under vacuum condition；
Step 2: the magnetic oxygenated graphene of 30mg is measured in 10mL, 0.01mol/L, the phosphate buffered saline of pH7.0
Middle immersion 30min adds the NaOH solution of 6mL, 3mol/L, is ultrasonically treated 3h, HCl solution is added dropwise to neutrality, magnet separates simultaneously
Repeatedly washing, vacuum drying, measure the magnetic oxygenated graphene aqueous solution of 10mL, 1mg/mL, then be added 30,35,40,45,
50mg polyethylene glycol diamines are ultrasonically treated 5min, and 9.6mg 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride is added
Salt (EDC) is ultrasonically treated 30min, then adds 38.5mg EDC and is stirred to react 10,11,12,13,14h, sulfydryl second is added
Alcohol terminates reaction, and magnet separation is washed repeatedly and dried under vacuum conditions；
Step 3: 10mg modified magnetic graphene oxide is added to 3mL, the phosphate-buffered of 0.02mol/L, pH6.5
In solution, the Novozyme435 of 1,2,3,4,5mg is added, is stirred to react 4,6,8,10,12h, magnet separates immobilised enzymes, phosphorus
Phthalate buffer is repeatedly rinsed and is dried in vacuo, and measures the load factor of carrier, the enzyme activity of immobilised enzymes and saturation magnetization.
Specific embodiment 2: present embodiment and one difference of specific embodiment are to adjust poly- second two in step 2
Hydro diamine content is 35,40,45mg, and other compositions and step are identical as specific implementation method one.
Specific embodiment 3: present embodiment and one difference of specific embodiment are to adjust stirring in step 2 instead
It is 11,12,13h between seasonable, other compositions and step are identical as specific implementation method one.
Specific embodiment 4: present embodiment is to adjust in step 3 with one difference of specific embodiment
The additive amount of Novozyme435 is 3,4,5mg, and other compositions and step are identical as specific implementation method one.
Specific embodiment 5: present embodiment and one difference of specific embodiment are to adjust stirring in step 3 instead
4,6,8h between seasonable, other compositions and step are identical as specific implementation method one.
Claims (5)
1. a kind of method of modified magnetic graphene oxide immobilized lipase, it is characterised in that polyethylene glycol diamines modified magnetic
Graphene oxide increases the load capacity of carrier and the activity of enzyme, and steps are as follows for specific method:
Step 1: 1g graphene oxide is weighed, 250mL, 0.1mol/LNaOH solution are added to ultrasound 45min in beaker, centrifugation
Isolated powder and with water repeated flushing, adjusting its pH value is 6, then, the distilled water of 100mL is added to the powder of separation
In, 30min is dispersed in ultrasound, under nitrogen protection, 5.40g FeCl will be contained3·6H2O and 2.65g FeCl2·4H2O's
40ml distilled water is added dropwise in suspension, the NH of fast drop 10mL 25%3·H2O, adjusting suspension pH value is 12,85
DEG C, 45min is stirred, is cooled to room temperature, magnet separation is simultaneously washed repeatedly under ultrasound with methanol repeatedly, and obtained solid is in vacuum
Under the conditions of it is dry；
Step 2: it measures the magnetic oxygenated graphene of 30mg and is soaked in 10mL, 0.01mol/L, the phosphate buffered saline of pH7.0
30min is steeped, the NaOH solution of 6mL, 3mol/L are added, is ultrasonically treated 3h, HCl solution is added dropwise to neutrality, magnet separation is simultaneously multiple
Washing, vacuum drying measure the magnetic oxygenated graphene aqueous solution of 10mL, 1mg/mL, be then added 30,35,40,45,50mg it is poly-
Glycol diamine is ultrasonically treated 5min, and 9.6mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride is added
(EDC), it is ultrasonically treated 30min, 38.5mg EDC is then added and is stirred to react 10,11,12,13,14h, mercaptoethanol is added
Reaction is terminated, magnet separation is washed repeatedly and dried under vacuum conditions；
Step 3: 10mg modified magnetic graphene oxide is added to 3mL, the phosphate buffer solution of 0.02mol/L, pH6.5
In, the Novozyme435 of 1,2,3,4,5mg is added, is stirred to react 4,6,8,10,12h, magnet separates immobilised enzymes, phosphate
Buffer is repeatedly rinsed and is dried in vacuo, and measures the load capacity of carrier, the enzyme activity of immobilised enzymes and saturation magnetization.
2. a kind of method of modified magnetic graphene oxide immobilized lipase according to claim 1, it is characterised in that
It is 35,40,45mg that polyethylene glycol diamines content is adjusted in step 2.
3. a kind of method of modified magnetic graphene oxide immobilized lipase according to claim 1, it is characterised in that
It is adjusted in step 2 and is stirred to react the time as 11,12,13h.
4. a kind of method of modified magnetic graphene oxide immobilized lipase according to claim 1, it is characterised in that
The additive amount that Novozyme435 is adjusted in step 3 is 3,4,5mg.
5. a kind of method of modified magnetic graphene oxide immobilized lipase according to claim 1, it is characterised in that
Time 4,6,8h are stirred to react in step 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172148A (en) * | 2019-12-20 | 2020-05-19 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Immobilized biological enzyme and preparation method thereof |
| CN113061595A (en) * | 2021-03-17 | 2021-07-02 | 东北农业大学 | Method for immobilizing phospholipase C by using epoxy group modified magnetic chitosan nanoparticles as carrier |
Citations (3)
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| CN105441419A (en) * | 2015-12-22 | 2016-03-30 | 陕西师范大学 | Magnetic-graphene-oxide-immobilized CPO (chloroperoxidase), and preparation method and application thereof |
| CN105602931A (en) * | 2016-03-11 | 2016-05-25 | 东北农业大学 | Method for synthesizing carbon chain triacylglycerol under supercritical CO2 system based on magnetic immobilized enzyme method |
| CN108424905A (en) * | 2018-02-08 | 2018-08-21 | 江苏大学 | A kind of magnetic oxygenated graphene complex microsphere of multi-arm and its preparation method and application |
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2018
- 2018-12-24 CN CN201811580131.6A patent/CN109456959A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441419A (en) * | 2015-12-22 | 2016-03-30 | 陕西师范大学 | Magnetic-graphene-oxide-immobilized CPO (chloroperoxidase), and preparation method and application thereof |
| CN105602931A (en) * | 2016-03-11 | 2016-05-25 | 东北农业大学 | Method for synthesizing carbon chain triacylglycerol under supercritical CO2 system based on magnetic immobilized enzyme method |
| CN108424905A (en) * | 2018-02-08 | 2018-08-21 | 江苏大学 | A kind of magnetic oxygenated graphene complex microsphere of multi-arm and its preparation method and application |
Non-Patent Citations (2)
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| BAI XUE等: "Binary adsorption of 17β-estradiol and bisphenol A on superparamagnetic aminofunctionalized graphene oxide nanocomposites", 《MATERIALS CHEMISTRY AND PHYSICS》 * |
| XIE WENLEI等: "Immobilization of Candida rugosa lipase onto graphene oxide Fe3O4", 《ENERGY CONVERSION AND MANAGEMENT》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172148A (en) * | 2019-12-20 | 2020-05-19 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Immobilized biological enzyme and preparation method thereof |
| CN113061595A (en) * | 2021-03-17 | 2021-07-02 | 东北农业大学 | Method for immobilizing phospholipase C by using epoxy group modified magnetic chitosan nanoparticles as carrier |
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Application publication date: 20190312 |