CN108265045A - Magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method and applications - Google Patents
Magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method and applications Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The invention discloses a kind of magnetic flexible carrier CMCC/DHBH Fe3O4Preparation method, include the following steps：Prepare carboxymethyl chitosan schiff bases product；Carboxymethyl chitosan schiff bases product and sodium cyanoborohydride are restored, carboxymethyl chitosan schiff bases reduzate is obtained, then adds in Fe3O4The ultrasonic disperse liquid of nano-particle carries out Magneto separate after stirring 10~14h at room temperature；Magneto separate gains are washed, it is dry, obtain magnetic flexible carrier CMCC/DHBH Fe3O4.The present invention further simultaneously discloses above-mentioned magnetic flexible carrier CMCC/DHBH Fe3O4Purposes：For immobilised enzymes.Magnetic flexible carrier CMCC/DHBH Fe using the present invention3O4Immobilised enzymes has the characteristic for efficiently, easily recycling and reusing.
Description
Technical field
The present invention relates to chemical fields, and in particular to a kind of magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method and its
Using.
Background technology
The biocatalyst that enzyme is had excellent performance as one kind, has the characteristics that efficient, single-minded, reaction condition is mild.Enzyme is urged
Changing organic reaction has solid, region and the chemo-selective of height, is widely used in fine chemistry industry, food, medicine, the energy
And environmental area.Lipase scientific name triglyceride hydrolysis enzyme, in catalytic reaction process, can show excellent chemistry, area
Domain and stereoselectivity.In view of the chiral substrate of lipase has various selective and excellent catalytic, fixation is become
Change and a kind of enzyme the most popular is studied in zymotechnic.
Carboxymethyl chitosan (CMCC) has many advantages, such as water-soluble, safe and non-toxic, degradable.Utilize functions a variety of in CMCC
Group, chelated metal ions；Using carboxymethyl chitosan both sexes and gel characteristic, pH responses material and in recent years can be prepared
Carry out one of research emphasis；Continue the film forming of chitosan, it can be achieved that package to carrier using carboxymethyl chitosan；Utilize carboxylic first
The water solubility of base enclosure glycan, can be in every field (such as：Cosmetics, fresh-keeping, medical, functional material etc.) it is used widely.
3,4- 4-dihydroxy benzaldehyde original pair tea phenol aldehyde belong to catechol derivatives, and wherein catechol is that marine organisms performance is sticked
The main functional group of function realizes the package to magnetic nanometer using catechol.
Invention content
The technical problem to be solved in the present invention is to provide a kind of magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method and
Its purposes；Using magnetic flexible carrier CMCC/DHBH-Fe3O4Immobilised enzymes has the spy for efficiently, easily recycling and reusing
Property.
In order to solve the above technical problem, the present invention provides a kind of magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation side
Method includes the following steps：
1) carboxymethyl chitosan schiff bases product, is prepared：
1.1) carboxymethyl chitosan, is added in into absolute ethyl alcohol, swelling immersion 12h, obtains the carboxylic being swollen at 40~60 DEG C
Methyl chitosan solution；
At room temperature, DMF (reaction dissolvent, n,N-Dimethylformamide) is added in 3,4- 4-dihydroxy benzaldehydes until 3,4-
4-dihydroxy benzaldehyde is completely dissolved, lower later to add in (passing through constant pressure dropping) to the carboxymethyl chitosan solution being swollen；
The mass ratio of the carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehydes is 1~1:4；
1.2) mixed solution obtained by step 1.1), is carried out to sonic oscillation stirring 4~8h (ultrasonic work(at 40~60 DEG C
Rate is 60~100w)；Product after ultrasonic vibration is washed, filtered and dried, obtains the production of carboxymethyl chitosan schiff bases
Object is denoted as CMCC/DHBH；
2), carboxymethyl chitosan schiff bases reduzate is to the package of magnetic nanometer：
2.1), carboxymethyl chitosan schiff bases product and sodium cyanoborohydride are restored, obtain carboxymethyl chitosan schiff bases
Reduzate；The mass ratio of the carboxymethyl chitosan schiff bases product and sodium cyanoborohydride is 1：0.9~1.1 (preferably
1:1)；
2.2), by Fe3O4Nano-particle (grain size is 20~25 nanometers) is added in Tris-HCl solution (10mM, pH=7)
In, 30~90min of ultrasonic disperse；
2.3), the uniform solution of ultrasonic disperse obtained by step 2.2) is added in (being added dropwise to) step 2.1) gains
In (solution of configuration), Magneto separate is carried out after stirring 10~14h at room temperature；
Magneto separate gains are washed, it is dry, obtain magnetic flexible carrier CMCC/DHBH-Fe3O4；
The CMCC/DHBH and Fe in the step 2.2) in step 2.1)3O4With mass ratio 1.8~2.1:1.
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Preparation method improvement, the step 2.1) is：
Carboxymethyl chitosan schiff bases product is soluble in water, and adding in sodium cyanoborohydride stirs evenly it, reacts 4-
8h (preferably 6h)；
The amount ratio of carboxymethyl chitosan schiff bases product and water is 100mg/50 ± 10ml,
The amount ratio of sodium cyanoborohydride and carboxymethyl chitosan schiff bases product is 1:1.
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Preparation method be further improved：
In the step 1.1), the amount ratio of carboxymethyl chitosan and absolute ethyl alcohol is 100mg/20 ± 2ml, 3,4- dihydroxies
The amount ratio of benzaldehyde and DMF are 200mg/20 ± 2mL；
In the step 2.2), Fe3O4The amount ratio of nano-particle and Tris-HCl solution is 50mg/50 ± 5ml.
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Preparation method be further improved：
In the step 1.1), the mass ratio of carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehyde is 1:2；
In the step 1.2), the mixed solution obtained by step 1.1) is subjected to sonic oscillation stirring 8h at 55 DEG C and (is surpassed
Acoustical power is 100w).
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Preparation method be further improved：
In the step 2.3), CMCC/DHBH and Fe3O4With mass ratio 2:1.
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Preparation method be further improved：
In the step 2.2), a length of 60min during ultrasonic disperse.
The present invention also provides the magnetic flexible carrier CMCC/DHBH-Fe being prepared using the above method simultaneously3O4Use
On the way：For immobilised enzymes.
Magnetic flexible carrier CMCC/DHBH-Fe as the present invention3O4Purposes improvement：
Under the system condition (phosphate buffer) of pH=8, by the CMCC/DHBH-Fe of 10mg3O4With 1.8~2.2mg (compared with
Good is 2.1mg) lipase immobilize reaction 6 hours in 38~42 DEG C (preferably 40.5 DEG C).
In terms of existing technologies, the present invention has following technical advantage：
1st, the present invention is combined using flexible covalent method on the basis of forefathers and fixes lipase, is reduced by rigid collision
The problem of enzyme activity is caused to be damaged, at the same also remain to greatest extent resolvase as catalytic activity.
2nd, with reference to the reuse of magnetisable material quick separating, realization enzyme, the problems such as carrier application cost is high is avoided.
3rd, zymoprotein is fixed using covalent coupling method on the carrier of flexibility function base, in addition to having preferable parent
It is aqueous outer, also with good biocompatibility, hypotoxicity, relative moderate price, more reduce space steric effect, simultaneously
Moisture environment around protein is remained again.
4th, operating process is green non-pollution, safe and reliable, easy to operate etc..
In conclusion the present invention, by carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehydes are issued in ultrasonic wave subsidiary conditions
Raw reaction, prepares a kind of carboxymethyl chitosan schiff bases product.Prepared schiff bases product is passed through into sodium cyanoborohydride
Reduction reduces molecular conjugation system, reduces molecular rigidity, flexibility increase.Using catechol stickiness molecule to magnetic nanoparticle
The package of son, prepares a kind of novel magnetic nano material, has the characteristic for efficiently, easily recycling and reusing.That is, present invention profit
The package to magnetic nanometer is realized with the film forming of carboxymethyl chitosan and the adhesion properties of 3,4- 4-dihydroxy benzaldehyde, is formed
Magnetic flexible carrier CMCC/DHBH-Fe3O4, catechol has preferable reactivity with amino under alkaline condition, can be used as one
Kind carrier is to lipase immobilization.The present invention avoids carrier herein in connection with the reuse of magnetisable material quick separating, realization enzyme
The problems such as application cost is high, compared to other flexible carrier preparation methods, with more utility value.
Description of the drawings
The specific embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 be magnetic nano-particle before modified after grain size distribution, upper figure is Fe3O4The grain size distribution of nano-particle,
Figure below is CMCC/DHBH-Fe3O4Grain size distribution；
Fig. 2 illustrates for single factor test (a- times, b- ultrasonic powers, c- temperature and d- mass ratioes) and derivative degree of substitution relationship
Figure；
Fig. 3 is DHBH (3,4- 4-dihydroxy benzaldehydes), CMCC (carboxymethyl chitosan) and CMCC/DHBH (carboxymethyl chitosans
Sugared schiff bases product) infrared spectrogram；
Fig. 4 is carboxymethyl chitosan (left figure) and modification of polysaccharides --- carboxymethyl chitosan schiff bases product (right figure) scans
Electromicroscopic photograph；
Fig. 5 is magnetic flexible carrier CMCC/DHBH, Fe3O4(Fe3O4Nano-particle) and CMCC/DHBH-Fe3O4Infrared light
Spectrum；
Fig. 6 is the stability schematic diagram of resolvase and immobilised enzymes, and a figures are different time resolvase and immobilization at 40 DEG C
Enzyme stability schematic diagram；B figures are different time resolvase and immobilised enzymes stability schematic diagram at 60 DEG C；
Fig. 7 is CMCC/DHBH-Fe3O4The repeat performance schematic diagram of immobilized lipase；
Fig. 8 is resolvase and immobilised enzymes enzyme activity schematic diagram under different solubility urea liquids.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Magnetic nano-particle Fe used in the present invention3O4Grain size be 20~25 nanometers.
Embodiment 1, a kind of magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method, follow the steps below successively：
1st, carboxymethyl chitosan schiff bases product is prepared：
1.1st, the carboxymethyl chitosan of 100mg is weighed in 100mL three-necked flasks, is added in 20mL absolute ethyl alcohols, is placed on 50
DEG C baking oven swelling impregnate 12h.200mg 3 is weighed later, and 4- 4-dihydroxy benzaldehydes are dissolved in 20mL DMF, are led under room temperature
It crosses constant pressure buret to be added dropwise in the carboxymethyl chitosan that (time for adding is 1-2 minutes) has been swollen, makes carboxymethyl chitosan
The mass ratio of sugar and 3,4- 4-dihydroxy benzaldehydes is 1:2.
1.2nd, it will be placed in supersonic wave cleaning machine equipped with the reaction vessel in step 1.1 mixed solution, carried out at 55 DEG C
Sonic oscillation stirring 8h (ultrasonic power 100w) takes out product and pours into beaker later, absolute ethyl alcohol (50ml) washing, mistake
Filter, dry 12h obtains faint yellow solid powder in 50 DEG C of vacuum drying chambers, that is, obtains carboxymethyl chitosan schiff bases product, production
Object is denoted as CMCC/DHBH.
2nd, carboxymethyl chitosan schiff bases reduzate is to the package of magnetic nanometer：
2.1st, the carboxymethyl chitosan schiff bases product 100mg that step 1.2 prepares gained is taken to be dissolved in 50mL water, is added in
The sodium cyanoborohydride of 100mg, stirs evenly, and reacts 6h under room temperature, and product is also originated in for carboxymethyl chitosan schiff bases
Object；
2.2nd, by 50mg Fe3O4Nano-particle is added in into the Tris-HCl solution (10mM, pH=7) of 50mL, is surpassed
Sound disperses 1h；
2.3rd, the uniform solution of ultrasonic disperse in step 2.2 is added dropwise (time for adding is 1-2 minutes) step 2.1 to match
It in the solution put, is stirred at room temperature after 12h and carries out Magneto separate with magnet, the washing of Magneto separate gains (is first then used into deionized water
It washes, the dosage of ionized water is 50ml/ times, is washed 3 times；Then absolute ethyl alcohol is used, the dosage of absolute ethyl alcohol is 50ml/ times, washes 3 times,
Finally it is washed with distilled water, 50ml/ times, washes 3 times), most after 12h dry in 50 DEG C of vacuum drying chambers, magnetic flexible carrier is obtained,
It is denoted as CMCC/DHBH-Fe3O4。
3rd, characterization and performance test：
3.1st, elemental analysis：
The carboxymethyl chitosan as obtained by accurately weighing in 1.000mg~2.000mg steps 1.2 millionth balance
Schiff bases product is sequentially placed into pallet after being wrapped up with masking foil and waits for sample analysis；Utilize Vario EL type IIIs element point
Analyzer determines the percentage composition of C, N, H in carboxymethyl chitosan schiff bases product, and carries out the calculating of derivative degree of substitution,
Calculation formula is：
DS is degree of substitution；DD is carboxymethyl chitosan deacetylation；MC, MNRespectively carbon, the relative atom matter of nitrogen-atoms
Amount；nCFor carbon atom number in substituent；R is C/N contents in elemental analysis result；
3.2nd, granularmetric analysis
By laser particle analyzer respectively to Fe3O4Nano-particle and CMCC/DHBH-Fe3O4Ultrasonic disperse is carried out, so as to right
Its size distribution is measured, and assay method is：
Sample quality score is 25mg/1000ml, ultrasonic disperse 30min, is filtered later by 0.22 μm of syringe filter,
Sample is 5 times parallel, obtains intensity and particle size values relationship, Fe3O4The grain size intensity distribution of nano-particle is as shown in a figures in Fig. 1.
By the CMCC/DHBH-Fe of gained in step 2.33O4Granularmetric analysis, grain size intensity point are carried out according to the method described above
Butut is as shown in b figures in Fig. 1.
Magnetic nano-particle rear grain size distribution variation before modified as seen from Figure 1, for Fe3O4Nano-particle (changes
Magnetic nano-particle before property), particle diameter distribution is wider, there is small peak appearance, and intensity is uneven, and reflected intensity proportion is smaller,
It is not very uniform to illustrate nanometer particle size distribution, the main reason is that magnetic nano-particle surface has higher Gibbs free energys,
Reuniting effect is huge, causes the uneven (Fe of particle diameter distribution3O4Grain size average out to：562nm), for modified final product CMCC/
DHBH-Fe3O4(i.e. modified magnetic nano-particle) particle diameter distribution is relatively narrow, and particle size is uniformly distributed, and grain size intensity is more bright
Aobvious and proportion is larger, particle size values average out to 207.7nm.
3.3rd, infrared spectrum analysis：Including the infrared spectrum to the carboxymethyl chitosan schiff bases product of gained in step 1
Magnetic flexible carrier (the CMCC/DHBH-Fe of gained in analysis and step 23O4) infrared spectrum analysis.
3.3.1, the infrared spectrum of carboxymethyl chitosan schiff bases product is as shown in figure 3, DHBH represents 3,4- dihydroxies in Fig. 3
Benzaldehyde, CMCC represent carboxymethyl chitosan, and CMCC/DHBH represents the carboxymethyl chitosan schiff bases production of gained in step 1
Object.
From the figure 3, it may be seen that carboxymethyl chitosan contains following characteristics peak：3436cm-1- the NH at place2Stretching vibration with-OH is inhaled
Receive peak, 2919cm-1The C-H absorption of vibrations at place, 1604cm-1With 1405cm-1The antisymmetric vibration of-the COO- at place and pair of-COO-
Claim absorption of vibrations, after schiff base reaction occurs by 3,4- 4-dihydroxy benzaldehydes, in 1635cm-1There is apparent characteristic peak,
It is the 1382cm caused by the stretching vibration of C=N double bonds-1- the CH at place2Flexural vibrations peak is strengthened.Pass through rear substance peak shape before modified
The comparison comparison of CMCC/DHBH substance peak shapes (i.e. CMCC with) can illustrate to be modified to work(, catechol structure has succeeded
It is linked on the side chain of carboxymethyl chitosan.
Fig. 4 left figures are CMCC (that is, carboxymethyl chitosan before modified) electron microscope, and right figure is CMCC/DHBH (that is, modified
Carboxymethyl chitosan) electron microscope, it compares and understands that there is surface smooth structure, warp without the polysaccharide under the conditions of modified 1000 times
Cross it is modified after, rough surface, case shell structure distribution is uneven, has that surface porosity is porous, and in cellular, form is not advised
Whole, this is because sticking ligand 3, the introducing of 4- 4-dihydroxy benzaldehydes changes original existing space structure in its molecule, breaks
It is broken the crystal structure of polysaccharide, configuration of surface shows as coarse, is unevenly distributed.With reference to infrared spectrum amide bonding analysis, from another
Outer one side can prove to be modified successfully.
3.3.2, magnetic flexible carrier (CMCC/DHBH-Fe3O4) infrared spectrum as shown in figure 5, CMCC/DHBH tables in Fig. 5
Show the carboxymethyl chitosan schiff bases product of gained in step 1, Fe3O4Represent ferroferric oxide magnetic nano-particles, CMCC/
DHBH-Fe3O4Represent the magnetic flexible carrier of gained in step 2.
As shown in Figure 5, in CMCC/DHBH-Fe3O4It can be seen that the stretching vibration of apparent Fe-O keys is inhaled on infrared line
Receive peak (579cm-1), illustrate there is Fe in sample3O4Presence.In 3440cm-1And 1640cm-1]The absorption peak at place is due to-OH
Caused by-FeOO- stretching vibrations, Fe is further illustrated3O4There are some active groups-OH for nanoparticle surface.Pass through comparison
Magnetic Nano microsphere (CMCC/DHBH-Fe3O4)、Fe3O4With the infared spectrum of CMCC/DHBH, it is possible to find Fe3O4In infrared spectrum
Fe-O is in 580cm-1Have absorption peak, after being modified by reduzate, Fe3O4Characteristic peak red have been moved to 590cm-1.Comparison
Infrared spectrogram (i.e. Fe after before modified3O4With the infrared spectrogram of CMCC/DHBH), it is possible to find：Peak intensity size and peak position
It all changes, shows package success of the carboxymethyl chitosan modified product to magnetic nanometer, and its active force is hydrogen bond.
Experiment one, reaction time, ultrasonic power, reaction temperature and mass ratio are to carboxymethyl chitosan schiff bases Product formation
Influence：
Under the premise of unitary variant is changed, taken according to the analysis of carboxymethyl chitosan schiff bases product elemental and derivative
The calculating of Dai Du, differential responses time (2-12h) and relational graph such as Fig. 2 (a) of derivative degree of substitution are shown, different ultrasonic power
Relational graph such as Fig. 2 (b) of (20-100w) and derivative degree of substitution are shown, and differential responses temperature (30-55 DEG C) replaces with derivative
Shown in the relational graph of degree such as Fig. 2 (c), different quality ratio (1：1-6) and shown in the relational graph of derivative degree of substitution such as Fig. 2 (d).On
State mass ratio of the mass ratio for carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehydes.
As shown in Figure 2, ultrasonic power influences the formation of carboxymethyl chitosan schiff bases product smaller, and what is be affected is anti-
It between seasonable, is primarily due to over time, the reaction interval of ligand (3,4- 4-dihydroxy benzaldehyde) and carboxymethyl chitosan
Degree aggravation, overlong time, amount of ligand are reduced, and carboxymethyl chitosan may occur fraction autohemagglutination, degree of substitution is caused to reduce.From figure
It can be seen that in 2：Ultrasonic power is 100w, reaction time 8h, 55 DEG C of reaction temperature, carboxymethyl chitosan and 3,4- dihydroxy
The mass ratio of benzaldehyde is 2:When 1, degree of substitution is up to：53.06%.It is comprehensive with reference to the analysis to infrared light collection of illustrative plates and electron microscope
Conjunction Fig. 2, which can be analyzed, show that degree of substitution is relatively low, the main reason is that carboxymethyl chitosan is macromolecular substances, deacetylation (carboxylic first
The content of free amino group in base chitosan molecule chain) it is relatively low, cause the active amino that can participate in reaction less, therefore carboxymethyl shell
Glycan schiff bases product is relatively low.
Embodiment 2, immobilised enzymes
Weigh the CMCC/DHBH-Fe of 1 gained of embodiment3O410mg adds in 10mL 0.05M phosphate buffers (pH=
8.0), ultrasound, one evening of immersion add in the lipase solution 0.21ml of a concentration of 10mg/ml, then in 40.5 DEG C of temperature shaking tables
Reaction 6 hours.After immobilized reactant, Magneto separate immobilised enzymes, and repeatedly washed with phosphate buffer and be not associated with removing
Lipase up, until it can't detect free-fat enzyme in cleaning solution.By Copoloid-spectrophotometry to embodiment institute
The immobilised enzymes of preparation measures enzyme activity, enzyme activity 413.276U/g.
Experiment two, enzyme activity experiment：
Study the thermal stability of immobilised enzymes and resolvase：A certain amount of immobilised enzymes (2 gained of embodiment) is weighed, other
In the case that condition is identical, resolvase (0.1mL) and immobilized lipase (are individually placed to 40 DEG C and 60 DEG C obtained by embodiment 2
(0.05M, pH=8) keeps the temperature 120min in phosphate buffer solution, is sampled every 30min in 40 DEG C of catalyzing hydrolysis substrate solution (3mL
Phosphate buffer solution (0.05M, pH=8) and 1mL olive oil) 10min, measure immobilised enzymes according to Copoloid-spectrophotometry
Enzyme activity, and corresponding relative activity is calculated, the thermal stability of resolvase and immobilised enzymes is studied.Concrete outcome such as Fig. 6
It is shown：
Immobilized lipase still has higher stability in the case where temperature is 40 DEG C and 60 DEG C processing.When temperature is 40 DEG C
When, enzyme activity is on a declining curve with the extension of soaking time, and when temperature is 60 DEG C, in 180min, immobilised enzymes residual is lived
Power is 37% (CMCC/DHBH-Fe3O4) and the vigor of resolvase only remains 28% at this time, this may be because lipase and modified support
With reference to rear, certain changes, which have occurred, in the microenvironment of the space conformation of enzyme active center position and enzyme molecule causes thermal denaturation resistant that can increase
By force.It can be seen that the lipase specific ionization lipase after fixed has higher thermal stability, this has enzyme in practical applications
There is important meaning.
Experiment three, easily recycling and reuse：
Study CMCC/DHBH-Fe3O4The reusability of immobilised enzymes in the case where other conditions are identical, weighs one
Determine immobilised enzymes catalyzing hydrolysis substrate solution 10min (amount ratio of immobilised enzymes is with above-mentioned experiment two) at 45 DEG C of quality,
After one wheel of reaction, Magneto separate is simultaneously washed with phosphate buffer solution (0.05M, pH=8), and it is anti-to continue on for next round hydrolysis
It should.It is reused many times and calculates its relative activity, as shown in Figure 7.
CMCC/DHBH-Fe3O4After immobilized lipase, there is preferable reusability.After reusing every time
CMCC/DHBH-Fe3O4Enzyme activity beginning higher, the CMCC/DHBH-Fe of reservation3O4After recycling 10 times, retain enzyme activity
Still have 53.88%, for resolvase cannot be reused, immobilised enzymes can be repeatedly used, hence it is evident that be carried
The high service efficiency of enzyme, reduces cost.Good repeat performance, can not only be effectively reduced cost, also make continuous
Catalysis reaction process is designed to possibility, has actual application value.
Experiment four, influence of the denaturant to enzyme activity：
Urea is a kind of denaturant, by CMCC/DHBH-Fe in embodiment 23O4The immobilized lipase of acquisition and free fat
Fat enzyme, is added separately in the urea liquid of various concentration, and processing 1h is placed in the case where other conditions are identical, under room temperature,
Residual enzyme activity is measured respectively, so as to study influence of the different urea concentrations (0-7M) to immobilized lipase, as a result such as Fig. 8 institutes
Show；
Denaturant using urea as representative can lead to the folding of zymoprotein, serious to limit so as to which enzyme deactivation be made even to inactivate
The application of enzyme in the industrial production is made.Resolvase is when urea concentration is 5M, resolvase enzyme activity residue 24.55%, and consolidates
Surely it is 43.37% to change enzyme enzyme activity, and immobilised enzymes enzyme activity is all higher than resolvase under different urea solubility, and this demonstrate fixations
Change enzyme to urea-denatured dose with better tolerance.Lipase significantly increases the resistivity of denaturant after immobilization
By force, covalent immobilization enzyme can provide higher activation energy for enzyme molecule and prevent the change of enzyme tertiary structure.
The influence of experiment five, metal ion and organic reagent to enzyme activity：
By immobilized lipase (CMCC/DHBH-Fe in embodiment 23O4The immobilized lipase of acquisition) and free-fat enzyme
It is placed in the salting liquid (concentration 2mg/ml) containing different metal ions and the organic reagent of different quality containing, in other conditions
Processing 1h is placed in the case of identical, under room temperature, measures residual enzyme activity respectively.Not handle experimental group as control.It is as follows
Shown in table 1；
The influence of table 1, organic reagent and metal ion to immobilised enzymes and resolvase
Resolvase and immobilized lipase application study are found by heavy metal ion and organic reagent：Immobilised enzymes exists
Preferable stability is shown in metal ion solution, while activator has apparent activation to immobilised enzymes, and pressed down
Preparation has smaller effect to immobilised enzymes.Influence for free-fat enzyme and immobilized lipase organic solvent solubility compared with
Greatly, the study found that the organic solvent reagent of 90% solubility has free-fat enzyme and immobilized lipase inhibiting effect, but solid
Immobilized lipase can keep preferable enzyme activity.
Comparative example 1, by carboxymethyl chitosan schiff bases product obtained by 1 step 1) of embodiment without 1 step 2) of embodiment
In 2.1 reduction, remaining is with embodiment 1, and products therefrom is known as carboxymethyl chitosan magnetic nano-particle, according to above-described embodiment
2 the methods immobilize lipase experiment.
The sample of embodiment 1 is changed to chitosan by comparative example 2, and carrier is changed to ferroso-ferric oxide, prepares products therefrom as magnetic
Property chitosan, according to 2 the method for above-described embodiment immobilize lipase experiment.
Ligand in 1 step 1) of embodiment is changed to chitosan by comparative example 3, remaining is the same as embodiment 1；The product of gained claims
For Chitosan Schiff-base product magnetic Nano microsphere.Lipase experiment is immobilized according to 2 the method for above-described embodiment.
Solvent DMF in 1 step 1) of embodiment is changed to ethyl alcohol by comparative example 4；Remaining is the same as embodiment 1；The product of gained claims
For CMCC-DHBH-Fe3O4.Lipase experiment is immobilized according to 2 the method for above-described embodiment.
Ligand 3,4- 4-dihydroxy benzaldehydes in 1 step 1) of embodiment are changed to vanillic aldehyde by comparative example 5；Remaining is the same as implementation
Example 1；The product of gained is known as carboxymethyl chitosan sugar vanillin magnetic nano-particle.It is carried out according to 2 the method for above-described embodiment
Immobilized lipase is tested.
Above-mentioned comparative example and the correction data of the oxidised maize starch modified magnetic polycaprolactam polyamine composite nano-microsphere of the present invention
Such as the following table 2；
Table 2
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deform.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (8)
1. magnetic flexible carrier CMCC/DHBH-Fe3O4Preparation method, it is characterized in that including the following steps：
1) carboxymethyl chitosan schiff bases product, is prepared：
1.1) carboxymethyl chitosan, is added in into absolute ethyl alcohol, swelling immersion 12h, obtains the carboxymethyl being swollen at 40~60 DEG C
Chitosan solution；
At room temperature, DMF is added in 3,4- 4-dihydroxy benzaldehydes until 3,4- 4-dihydroxy benzaldehydes are completely dissolved, it is lower later to add in
Into the carboxymethyl chitosan solution being swollen；
The mass ratio of the carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehydes is 1~1:4；
1.2) mixed solution obtained by step 1.1), at 40~60 DEG C is carried out to sonic oscillation and stirs 4~8h；By ultrasonic vibration
Product afterwards is washed, filtered and is dried, and is obtained carboxymethyl chitosan schiff bases product, is denoted as CMCC/DHBH；
2), carboxymethyl chitosan schiff bases reduzate is to the package of magnetic nanometer：
2.1), carboxymethyl chitosan schiff bases product and sodium cyanoborohydride are restored, obtain the reduction of carboxymethyl chitosan schiff bases
Product；The mass ratio of the carboxymethyl chitosan schiff bases product and sodium cyanoborohydride is 1：0.9~1.1；
2.2), by Fe3O4Nano-particle is added in into Tris-HCl solution, 30~90min of ultrasonic disperse；
2.3), the uniform solution of ultrasonic disperse obtained by step 2.2) is added in into step 2.1) gains, is stirred at room temperature
Magneto separate is carried out after 10~14h；
Magneto separate gains are washed, it is dry, obtain magnetic flexible carrier CMCC/DHBH-Fe3O4；
The CMCC/DHBH and Fe in the step 2.2) in step 2.1)3O4With mass ratio 1.8~2.1:1.
2. magnetic flexible carrier CMCC/DHBH-Fe according to claim 13O4Preparation method, it is characterized in that, the step
2.1) it is：
Carboxymethyl chitosan schiff bases product is soluble in water, and adding in sodium cyanoborohydride stirs evenly it, reacts 4-8h；
The amount ratio of carboxymethyl chitosan schiff bases product and water is 100mg/50 ± 10ml,
The amount ratio of sodium cyanoborohydride and carboxymethyl chitosan schiff bases product is 1:1.
3. magnetic flexible carrier CMCC/DHBH-Fe according to claim 1 or 23O4Preparation method, it is characterized in that,
In the step 1.1), the amount ratio of carboxymethyl chitosan and absolute ethyl alcohol is 100mg/20 ± 2ml, 3,4- dihydroxy benzenes
The amount ratio of formaldehyde and DMF are 200mg/20 ± 2mL；
In the step 2.2), Fe3O4The amount ratio of nano-particle and Tris-HCl solution is 50mg/50 ± 5ml.
4. magnetic flexible carrier CMCC/DHBH-Fe according to claim 33O4Preparation method, it is characterized in that：
In the step 1.1), the mass ratio of carboxymethyl chitosan and 3,4- 4-dihydroxy benzaldehyde is 1:2；
In the step 1.2), the mixed solution obtained by step 1.1) is carried out to sonic oscillation stirring 8h at 55 DEG C.
5. magnetic flexible carrier CMCC/DHBH-Fe according to claim 43O4Preparation method, it is characterized in that：
In the step 2.3), CMCC/DHBH and Fe3O4With mass ratio 2:1.
6. magnetic flexible carrier CMCC/DHBH-Fe according to claim 53O4Preparation method, it is characterized in that：
In the step 2.2), a length of 60min during ultrasonic disperse.
7. the magnetic flexible carrier CMCC/DHBH-Fe being prepared such as claim 1~6 either method3O4Purposes, feature
It is：For immobilised enzymes.
8. magnetic flexible carrier CMCC/DHBH-Fe according to claim 73O4Purposes, it is characterized in that：
Under the system condition of pH=8, by the CMCC/DHBH-Fe of 10mg3O4Lipase with 1.8~2.2mg is in 38~42 DEG C
Immobilize reaction 6 hours.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695442A (en) * | 2015-12-02 | 2016-06-22 | 浙江工商大学 | Modified magnetic chitosan microspheres for enzyme immobilization and preparation method and application thereof |
| CN107254460A (en) * | 2017-06-23 | 2017-10-17 | 广东海洋大学 | A kind of carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and its application |
| CN108159482A (en) * | 2018-01-02 | 2018-06-15 | 上海其胜生物制剂有限公司 | A kind of injectable natural hydrogel system with temperature-sensing property and high bioadhesion and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105695442A (en) * | 2015-12-02 | 2016-06-22 | 浙江工商大学 | Modified magnetic chitosan microspheres for enzyme immobilization and preparation method and application thereof |
| CN107254460A (en) * | 2017-06-23 | 2017-10-17 | 广东海洋大学 | A kind of carboxymethyl chitosan/sodium alginate composite sponge immobilized papain and its application |
| CN108159482A (en) * | 2018-01-02 | 2018-06-15 | 上海其胜生物制剂有限公司 | A kind of injectable natural hydrogel system with temperature-sensing property and high bioadhesion and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| JUN SUN等: "Stability and activity of immobilized trypsin on carboxymethyl chitosanfunctionalized magnetic nanoparticles cross-linked with carbodiimide and glutaraldehyde", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| WANG JIANGTAO等: "Preparation of soluble p-aminobenzoyl chitosan ester by Schiff’s base and antibacterial activity of the derivatives", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 蔡晓敏: "新型磁柔性载体的制备及固定化脂肪酶的应用研究", 《中国优秀硕士学位全文库基础科学辑》 * |
| 谭玉龙: "基于疏水化修饰羧甲基壳聚糖自组装纳米粒的制备及应用", 《中国优秀硕士学位论文全文库医药卫生科技辑》 * |
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