CN110129308A - The mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase reactor and its application - Google Patents

The mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase reactor and its application Download PDF

Info

Publication number
CN110129308A
CN110129308A CN201910393910.3A CN201910393910A CN110129308A CN 110129308 A CN110129308 A CN 110129308A CN 201910393910 A CN201910393910 A CN 201910393910A CN 110129308 A CN110129308 A CN 110129308A
Authority
CN
China
Prior art keywords
dendroid
cpo
dsp
chloroperoxidase
reactor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910393910.3A
Other languages
Chinese (zh)
Other versions
CN110129308B (en
Inventor
蒋育澄
王兰兰
宋艺超
胡满成
李淑妮
翟全国
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shaanxi Normal University
Original Assignee
Shaanxi Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shaanxi Normal University filed Critical Shaanxi Normal University
Priority to CN201910393910.3A priority Critical patent/CN110129308B/en
Publication of CN110129308A publication Critical patent/CN110129308A/en
Application granted granted Critical
Publication of CN110129308B publication Critical patent/CN110129308B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • 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
    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/0101—Chloride peroxidase (1.11.1.10)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/36—Organic compounds containing halogen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Hydrology & Water Resources (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a kind of mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase reactor and its application, the enzyme reactor are the mesoporous SiO of dendroid for modifying surface amino groups2Nanoparticle and the chitosan crosslinked of hyaluronic acid or protonated amino are used as carrier afterwards, are obtained by electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.Enzyme reactor used carrier of the present invention duct loads negative electrical charge or positive charge abundant, while rich in functional groups such as hydroxyls, having greatly reinforced the binding force between carrier and enzyme molecule, having improved the load capacity of enzyme.The enzyme reactor has good catalytic activity, thermal stability and reusability, and tolerance is good in the organic solvents such as DMF, methanol, acetonitrile, and for the drugs such as lavo-ofloxacin and rifaximin in degrading waste water, degradation speed is fast, degradation rate is high.

Description

The mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase Reactor and its application
Technical field
The invention belongs to fixation techniques for enzyme fields, and in particular to load charge abundant in a kind of duct, rich simultaneously The mesoporous SiO of the dendroid of the functional groups such as hydroxyl2Immobilization chloroperoxidase reactor and application.
Background technique
Biological enzyme is the organic matter with catalytic action generated by living cells, and most of is protein, it is a kind of nothing Malicious, environment amenable biocatalyst.Biological enzyme efficiency is very high, has high degree of specificity, and a kind of enzyme can only be catalyzed A kind of or a kind of chemical reaction, reaction item are mild.But resolvase is easy in environment such as high temperature, strong acid and strong base, organic solvents Inactivation, can effectively improve enzyme to the tolerance of temperature, acid-base property, organic solvent by the way of immobilised enzymes.And it is fixed Change the features such as enzyme is green, recyclable due to it, is with a wide range of applications in terms of sewage treatment.
Chloroperoxidase (CPO) be separated from marine fungi Caldariomyces fumago one kind it is blood red Plain peroxidase (42kDa), has both a variety of enzymes such as heme peroxidases, catalase and cytochrome P-450 Catalytic characteristics, be presently believed to be most widely used enzyme in peroxiredoxins.CPO can be catalyzed a variety of organic anti- It answers, such as: halogenation, peroxidization, hydroxylating, epoxidation reaction, sulphonation oxidation reaction, so CPO has very greatly Application potential.
Mesoporous silicon oxide refers to earth silicon material of the pore size between 2~50nm, with high specific surface area, greatly Pore volume and orderly pore network, have stronger chemical stability, thermal stability and operational stability.In three-dimensional The dendroid mesoporous silicon oxide aperture of heart radiation is adjustable, and is vertical pore structure, has in terms of enzyme immobilizatio extensive Application prospect.But since silica itself is electrically weaker, the electrostatic interaction between CPO is relatively weak, repeats Enzyme molecule is easy leakage in use process.
Summary of the invention
It is high that the object of the present invention is to provide a kind of catalytic activity, thermal stability, reusability and organic solvent tolerance It is good, and the regulatable mesoporous SiO of functionalization dendroid of surface charge2Immobilization chloroperoxidase reactor, and it is anti-for the enzyme Device is answered to provide new application.
For above-mentioned purpose, enzyme reactor of the present invention is the mesoporous SiO of dendroid for modifying surface amino groups2It receives Rice corpuscles and the chitosan crosslinked of hyaluronic acid or protonated amino are used as carrier afterwards, are fixed by electrostatic interaction and hydrogen bond action Chloroperoxidase obtains.
In above-mentioned enzyme reactor, the compound that surface amino groups modification uses is 3-aminopropyltriethoxysilane, four Asias Any one in five amine of ethyl, N- aminoethyl -3- aminopropyltriethoxy dimethoxysilane.
In above-mentioned enzyme reactor, the hyaluronic acid is to pass through n-hydroxysuccinimide under conditions of pH is 8~10 Sulfonate sodium and the effect of 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride, the dendroid with surface amino groups modification Mesoporous SiO2Nanoparticle crosslinking；The mesoporous SiO of dendroid of the surface amino groups modification2After nanoparticle cross-linked-hyaluronic acid, Under conditions of pH is 3~4, pass through electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.
In above-mentioned enzyme reactor, the chitosan of the protonated amino is the branch by glutaraldehyde and surface amino groups modification The mesoporous SiO of shape2Nanoparticle crosslinking；The mesoporous SiO of dendroid of the surface amino groups modification2After nanoparticle cross-linked chitosan, Under conditions of pH is 5~6, pass through electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.
The mesoporous SiO of functionalization dendroid of surface charge regulation of the present invention2Immobilization chloroperoxidase reactor is available In degradation lavo-ofloxacin and degradation rifaximin.
Compared with prior art, the invention has the following advantages:
1, of the invention by the mesoporous SiO of dendroid2Nanoparticle by after amido modified, then with hyaluronic acid or amide proton The chitosan of change is further crosslinked, so that the mesoporous SiO of dendroid2Duct loads negative electrical charge or positive charge abundant, is rich in simultaneously The functional groups such as hydroxyl make chloroperoxidase pass through electrostatic interaction and hydrogen bond knot then using it as carrier by adjusting pH value of solution The mode of conjunction is fixed on the mesoporous SiO of dendroid2In duct, the binding force between carrier and enzyme molecule is greatly reinforced, has been improved The supported quantity of enzyme.
2, enzyme reactor of the present invention had not only been able to maintain the catalytic activity of chloroperoxidase but also some of resolvase can be overcome to lack Point improves the stability and operational stability of enzyme molecular structure, overcomes the drawbacks of enzyme molecule leaks during reusing, Reuse number is improved, and enzyme reactor tolerance in the organic solvents such as DMF, methanol, acetonitrile is good.
3, enzyme reactor of the present invention is i.e. reachable within a short period of time for lavo-ofloxacin and rifaximin in degrading waste water To preferable degradation effect.
Detailed description of the invention
Fig. 1 is the Flied emission transmission electron microscope picture of DSP-HA prepared by embodiment 1.
Fig. 2 is the laser co-focusing micrograph of CPO@DSP-HA prepared by embodiment 1.
Fig. 3 is the Flied emission transmission electron microscope picture of DSP-CHIT prepared by embodiment 2.
Fig. 4 is CPO@DSP-CHIT laser co-focusing micrograph prepared by embodiment 2.
Fig. 5 is influence diagram of the temperature to CPO@DSP-HA and CPO@DSP-CHIT catalytic activity.
Fig. 6 is reusability figure of the CPO@DSP-HA in buffer solution.
Fig. 7 is reusability figure of the CPO@DSP-CHIT in buffer solution.
Fig. 8 is influence diagram of the methanol to CPO@DSP-HA and CPO@DSP-CHIT catalytic activity.
Fig. 9 is influence diagram of the acetonitrile to CPO@DSP-HA and CPO@DSP-CHIT catalytic activity.
Figure 10 is influence diagram of the DMF to CPO@DSP-HA and CPO@DSP-CHIT catalytic activity.
Figure 11 is the effect picture that various concentration lavo-ofloxacin is degraded by CPO@DSP-HA.
Figure 12 is the effect picture that various concentration lavo-ofloxacin is degraded by CPO@DSP-CHIT.
Figure 13 is the effect picture that various concentration rifaximin is degraded by CPO@DSP-HA.
Figure 14 is the effect picture that various concentration rifaximin is degraded by CPO@DSP-CHIT.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to These embodiments.
The mesoporous SiO of following dendroid2Nanoparticle (DSP) is according to document " Shen D K, Yang J P, Li X M, et al.Biphase Stratification Approach to Three-Dimensional Dendritic Biodegradable Mesoporous Silica Nanospheres[J].Nano Letters,2014,14(2):923.” Disclosed in method be prepared.
Embodiment 1
1g DSP nanoparticle is distributed in 30mL dry toluene, 0.5mL volumetric concentration is added dropwise thereto is 97% 3- aminopropyl triethoxysilane aqueous solution, flow back 15h at 110 DEG C, the centrifuge separation of gained mixture, clear with acetone It washes three times, is dried in vacuo 12h at room temperature, obtain the mesoporous SiO of amido modified dendroid2(DSP-NH2)。
By 100mg DSP-NH2It is dispersed in 100mL deionized water, forms solution a；Add in 20mL deionized water Enter 0.37g n-hydroxysuccinimide sulfonate sodium and 0.20g 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride Salt dissolves solid, and the aqueous solution of 60mL hyaluronic acid containing 0.113g (HA) is then added into the solution, is sufficiently mixed rear shape At solution b；Solution a and solution b are uniformly mixed, adjusting mixed solution to pH with triethylamine is 9,12h is stirred at 38 DEG C, from Heart separation, product is cleaned three times with deionized water, 12h is dried in vacuo at room temperature, obtains the dendroid of cross-linked-hyaluronic acid Mesoporous SiO2(DSP-HA), Fig. 1 is the transmission electron microscope picture of gained DSP-HA.
PBS buffer solution and 100 μ L CPO solution (2.0 × 10 that 1400 μ L pH are 3 are added into 10mg DSP-HA- 5mmol·L-1, pH=4.0), 5min is ultrasonically treated to be uniformly dispersed, and then vibrates for 24 hours at 20 DEG C, CPO is enable sufficiently to consolidate It is fixed.Product is centrifugated after reaction, with pH be 3 PBS buffer solution eccentric cleaning three times, vacuum is dry at room temperature It is dry, obtain the mesoporous SiO of functionalization dendroid of surface-crosslinked hyaluronic acid2Immobilization chloroperoxidase reactor (CPO@ DSP-HA), Fig. 2 is the laser co-focusing micrograph of CPO@DSP-HA.After tested, chloroperoxidase in gained enzyme reactor Supported quantity is 30.68mgg-1。
Embodiment 2
DSP-NH is prepared according to the method for embodiment 12.By 100mg DSP-NH2It is dispersed in 2.5mL0.1molL- 1In the glutaraldehyde water solution that the PBS buffer solution and 100 μ L volume fractions that pH is 7.5 are 50%, it is stored at room temperature 1h, then will Mixed solution centrifuge separation, uses 0.1molL-1The PBS buffer solution eccentric cleaning that pH is 7.5 three times, removes supernatant, obtains It is crosslinked the DSP-NH of glutaraldehyde2Dispersion liquid.50mg chitosan (CHIT) is added to 2.5mL 0.1molL-1HCL aqueous solution In, it is completely dissolved chitosan solid；Later by the DSP-NH of above-mentioned crosslinking glutaraldehyde2Dispersion liquid is mixed with chitosan solution, Stir 12h at room temperature, be centrifugated, by product with pH be 8 PBS buffer solution eccentric cleaning three times, vacuum at room temperature It is dry, obtain the mesoporous SiO of dendroid of cross-linked chitosan2(DSP-CHIT), Fig. 3 is the transmission electron microscope picture of gained DSP-CHIT.
Be added into 10mg DSP-CHIT 1400 μ L pH be 5 PBS buffer solution and 100 μ L CPO solution (2.0 × 10-5mmol·L-1, pH=4.0), 5min is ultrasonically treated to be uniformly dispersed, is then vibrated at 20 DEG C for 24 hours, passes through CPO quiet Electro ultrafiltration and hydrogen bond action are sufficiently fixed.Product is centrifugated after reaction, is centrifuged with the PBS buffer solution that pH is 5 clear It washes three times, is dried in vacuo at room temperature, obtain the surface mesoporous SiO of chitosan functionalization dendroid2Fixed chloroperoxidase is anti- It answers device (CPO@DSP-CHIT), Fig. 4 is the laser co-focusing micrograph of CPO@DSP-CHIT.After tested, in gained enzyme reactor The supported quantity of chloroperoxidase is 31.12mgg-1。
Inventor has carried out performance to the CPO@DSP-CHIT of the CPO@DSP-HA prepared of embodiment 1 and the preparation of embodiment 2 Test, specific test are as follows:
1, catalytic activity is tested
Urging for enzyme reactor is investigated to be catalyzed the chloro- 5,5- dimethyl of 2--hydroresorcinol (MCD) as model reaction Change activity, specific steps are as follows: 5 μ L is taken to dissociate CPO solution (2.0 × 10-5mmol·L-1, pH=4.0), 5mg CPO@DSP-HA, 5mg CPO@DSP-CHIT is added separately to 1420 μ L 0.1molL-1The PBS buffer solution of pH=2.75 and 50 μ L 2.5mmol·L-1In the mixed liquor of MCD aqueous solution, 30 μ L0.1molL are then added-1H2O2Aqueous solution is placed in shaking table and shakes 15min is swung, is taken out, supernatant absorbance value at 278nm is measured with ultraviolet specrophotometer after centrifugation, is denoted as At.It will separately wait PBS buffer solution, MCD aqueous solution and the H of amount2O2Aqueous solution is sufficiently mixed, and is measured its absorbance value at 278nm, is denoted as A0.The conversion ratio of MCD is calculated by following formula:
A in formulat: absorbance value of the supernatant in t moment；A0: the absorbance value of MCD when equal concentration reaction 0min.As a result it shows To show, the catalytic activity by the CPO that dissociates is in terms of 100%, and CPO@DSP-HA and CPO@DSP-CHIT remains with higher catalytic activity, About 95.34% and 96.72%.
2, thermal stability is tested
Respectively by CPO@DSP-HA, CPO@DSP-CHIT with etc. enzyme amount free CPO (2 μ L, 2.0 × 10-5mmol·L-1) It is taken out after incubating 1h under different temperatures (50~90 DEG C), is cooled to room temperature, measure it using the model reaction of catalysis MCD Catalytic activity will be considered as 100% to the maximum conversion of MCD, with the catalytic activity and its relative activity pair at a temperature of other Time mapping, to indicate the thermal stability of CPO@DSP-HA and CPO@DSP-CHIT, is as a result shown in Fig. 5.
As seen from Figure 5, the residual activity of two kinds of enzyme reactor catalysis MCD chlorination reactions declines as temperature increases, At a temperature of each, the equal specific ionization CPO high of the retentive activity of enzyme reactor.Wherein, CPO@DSP-HA and CPO@DSP-CHIT exists It can keep 73.40% and 58.28% catalytic activity at 70 DEG C after placement 1h respectively；After 80 DEG C of placement 1h, CPO@DSP-HA 47.65% and 35.25% catalytic activity can still be kept respectively with CPO@DSP-CHIT, compared with free CPO, at high temperature all With good thermal stability.
3, reusability is tested
5mg CPO@DSP-HA and CPO@DSP-CHIT is taken to be added to 1420 μ L 0.1molL respectively-1PH is 2.75 PBS buffer solution and 50 μ L 2.5molL-1In the mixed liquor of MCD aqueous solution, 30 μ L0.1mmolL are then added-1H2O2Water Solution is placed in shaking table and vibrates 15min, takes out, and measures supernatant absorbance at 278nm with ultraviolet specrophotometer after centrifugation Value.After each reaction, reaction solution is centrifugated, after drawing out supernatant, continuously add equivalent PBS buffer solution, MCD aqueous solution and H2O2Aqueous solution is to start reaction next time.The catalytic activity used for the first time is considered as 100%, by after It catalytic activity each time and compares, is indicated with residual activity for the first time.As a result see Fig. 6 and Fig. 7.
As seen from the figure, after CPO@DSP-HA is reused 12 times, 56.11% catalytic activity can still be retained；CPO@DSP- After CHIT is reused 16 times, it can still retain 62.48% catalytic activity, illustrate that its reusability is good.
4, organic solvent tolerance is tested
(1) to methanol tolerance
By CPO@DSP-HA, CPO@DSP-CHIT with etc. enzyme amount dissociate CPO solution (2 μ L, 2.0 × 10-5mmol·L-1) point Not Jia Ru 1.5mL different volumes concentration methanol aqueous solution (volume fraction 0%, 5%, 10%, 15%, 20%, 25%, 30%) in, 1h is placed at room temperature for for being catalyzed MCD chlorination reaction.CPO@DSP-HA, CPO@DSP- when will be added without methanol aqueous solution CHIT and free CPO solution are respectively seen as 100% to the conversion ratio of MCD, respectively with CPO@DSP- under different methanol volumetric concentrations The relative activity of HA, CPO@DSP-CHIT and free CPO solution to methanol volumetric concentration map, come indicate CPO@DSP-HA, The methanol tolerance of CPO@DSP-CHIT and free CPO, are as a result shown in Fig. 8.
As seen from Figure 8, after the methanol aqueous solution processing that volumetric concentration is 30%, the residual activity for the CPO that dissociates is insufficient 20%, and the residual activity of CPO@DSP-HA and CPO@DSP-CHIT can reach 88.56% and 85.32% or so.
(2) to acetonitrile tolerance
By CPO@DSP-HA, CPO@DSP-CHIT with etc. enzyme amount dissociate CPO solution (2 μ L, 2.0 × 10-5mmol·L-1) point It Jia Ru not 1.5mL different volumes concentration acetonitrile solution (volume fraction 0%, 5%, 10%, 15%, 20%, 25%, 30%) In, 1h is placed at room temperature for for being catalyzed MCD chlorination reaction.CPO@DSP-HA, CPO@DSP-CHIT when will be added without acetonitrile solution 100% is respectively seen as to the conversion ratio of MCD with free CPO solution, respectively with CPO@DSP-HA under different acetonitrile volumetric concentrations, The relative activity of CPO@DSP-CHIT and free CPO solution maps to acetonitrile volumetric concentration, to indicate CPO@DSP-HA, CPO@ The acetonitrile tolerance of DSP-CHIT and free CPO, is as a result shown in Fig. 9.
As seen from Figure 9, after the acetonitrile solution processing that volumetric concentration is 20%, free CPO is almost inactivated, and CPO@DSP-HA and CPO@DSP-CHIT can still retain 88.46% and 83.93% or so activity.
(3) to N,N-dimethylformamide (DMF) tolerance
By CPO@DSP-HA, CPO@DSP-CHIT with etc. enzyme amount dissociate CPO solution (2 μ L, 2.0 × 10-5mmol·L-1) point Not Jia Ru 1.5mL different volumes concentration DMF aqueous solution (volume fraction 0%, 5%, 10%, 15%, 20%, 25%, 30%) in, 1h is placed at room temperature for for being catalyzed MCD chlorination reaction.CPO@DSP-HA, CPO@DSP- when will be added without DMF aqueous solution CHIT and free CPO solution are respectively seen as 100% to the conversion ratio of MCD, respectively with CPO@DSP- under different DMF volumetric concentrations The relative activity of HA, CPO@DSP-CHIT and free CPO solution maps to DMF volumetric concentration, to indicate CPO@DSP-HA, CPO@ The DMF tolerance of DSP-CHIT and free CPO, the result is shown in Figure 10.
As seen from Figure 10, after the DMF aqueous solution processing that volumetric concentration is 15%, the residual activity of free CPO is almost connect It is bordering on zero, and the residual activity of CPO@DSP-HA and CPO@DSP-CHIT can still respectively reach 82.15%, 73.13%.
5, the electrostatic drive power combined between CPO and carrier
By DSP, DSP-NH2, DSP-HA and DSP-CHIT the PBS buffering of distinguishing ultrasonic disperse to pH=3 and pH=5 it is molten In liquid, its Zeta potential at different pH is measured with laser particle analyzer, such as table 1.
Table 1
As shown in Table 1, in the PBS buffer solution that pH is 3 and pH is 5, DSP is negatively charged, after amido modified, DSP-NH2Electrical property in two kinds of buffer solutions becomes just.Due to the presence of free carboxy in hyaluronic acid, DSP-HA is in negative Electrically, when PBS buffer solution pH is 3, the electrical property of CPO is positive, and mainly passes through electrostatic interaction between CPO and carrier DSP-HA In conjunction with；Become-NH since protonation occurs in acid condition for the amino in chitosan chain3 +, DSP-CHIT is in strong electropositive, when When PBS buffer solution pH is 5, the electrical property of CPO is negative, and is mainly combined by electrostatic interaction between CPO and carrier DSP-HA.
Embodiment 2
CPO@DSP-HA and CPO@DSP-CHIT degradation drug
1, degradation lavo-ofloxacin
5mg CPO@DSP-HA or CPO@DSP-CHIT, 2480 μ L0.1molL are added in 10mL centrifuge tube-1PH is 2.75 PBS buffer solution, 500 μ L various concentration lavo-ofloxacins standard solution (make levofloxacin in end reaction system The initial concentration of star is 10 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL), it is eventually adding 20 μ L 0.1molL-1H2O2Water Solution starting reaction.25min is reacted at room temperature under magnetic stirring, is extracted with ethyl acetate after reaction 3 times.Finally with rotation Extract liquor whole evaporative removal is used trifluoroacetic acid aqueous solution sample dissolution later, obtains study by evaporimeter.Study has through 0.22 μm For high performance liquid chromatography (HPLC-15C) analysis measurement, high-efficient liquid phase chromatogram determining condition after the filtering of machine phase filter membrane are as follows: etc. Acetonitrile-KH is used under gradient mode2PO4Aqueous solution (V/V=20:70) is mobile phase, flow velocity 1.0mL/min, Detection wavelength 294nm, 40 DEG C of column temperature, 20 μ L of sample volume.
The calculating of degradation rate (η) is according to formula are as follows: η=(C0-Ct)/C0× 100%, C in formulatIndicate enzyme rear left oxygen fluorine Concentration of the Sha Xing in t moment, C0The levofloxacin concentration of reaction system when indicating not enzyme.
Experimental result is shown, when lavo-ofloxacin initial concentration is 10 μ g/mL, CPO@DSP-HA and CPO@DSP-CHIT Degradation rate to lavo-ofloxacin is respectively 97.52% and 96.93%；When levofloxacin concentration is 20 μ g/mL, CPO@DSP- HA and CPO@DSP-CHIT is respectively 96.21% and 96.52% to the degradation rate of lavo-ofloxacin；When lavo-ofloxacin is initially dense When degree is 50 μ g/mL, CPO@DSP-HA and CPO@DSP-CHIT is respectively 95.49% He to the degradation rate of lavo-ofloxacin 95.34%；When levofloxacin concentration is 100 μ g/mL, degradation of CPO@DSP-HA and CPO the@DSP-CHIT to lavo-ofloxacin Rate is respectively 87.17% and 87.04%.
Three kinds of school periphery sewage source (artificial lake water, Hei Heshui, sanitary sewage) is chosen to be applied to as experiment water sample The reaction system of enzyme reactor catalytic degradation lavo-ofloxacin.It is separately added into 5mg CPO@DSP-HA or CPO@DSP-CHIT The lavo-ofloxacin solution (being prepared with water sample) of three kinds of water samples and various concentration is eventually adding 10 μ L 0.1molL-1H2O2 Aqueous solution, holding final volume are 1500 μ L, react 25min under magnetic stirring and are allowed to sufficiently react.Second is used after reaction Acetoacetic ester extracts 3 times.Finally with Rotary Evaporators by extract liquor whole evaporative removal, then dissolved with isometric trifluoroacetic acid aqueous solution Sample obtains study.Study is analyzed after 0.22 μm of organic phase filter membrane filtering for high performance liquid chromatography (HPLC-15C) Measurement, the result is shown in Figure 11 and 12.
As seen from the figure, when lavo-ofloxacin initial concentration is 20 μ g/mL in water sample, CPO@DSP-HA and CPO@DSP- CHIT can reach 80% or more to the degradation rate of lavo-ofloxacin in three kinds of water sources in the degradation time of 25min；When left oxygen When Flucloxacillin initial concentration reaches 100 μ g/mL, degradation of the CPO DSP-HA to the lavo-ofloxacin in Heihe water and artificial lake water Rate can achieve 80.17% and 85.45% respectively, can reach 60.32% to the degradation rate of lavo-ofloxacin in sanitary sewage, CPO DSP-CHIT can achieve 78.62% He to the degradation rate of the lavo-ofloxacin in Heihe water and artificial lake water respectively 82.18%, 71.36% can reach to the degradation rate of lavo-ofloxacin in sanitary sewage.It can be seen that CPO@DSP-HA and CPO@ DSP-CHIT can reach preferable degradation effect to the lavo-ofloxacin in different quality.
2, degradation rifaximin
It is with methanol and acetonitrile, 0.075mol/L potassium dihydrogen phosphate aqueous solution, 1.0mol/L aqueous solution of citric acid volume ratio The mixed solution of 30:30:36:4 is solvent, and the rifaximin titer for preparing various concentration (makes sharp good fortune in end reaction system Former times bright initial concentration is 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL)；Take five 10mL from Heart pipe be separately added into 500 μ L rifaximin titers, the PBS buffer solution of 2400 μ L pH=2.75,5mg CPO@DSP-HA or CPO@DSP-CHIT、100μL 0.1mol/L H2O2Aqueous solution reacts at room temperature 25min.3 are extracted with ethyl acetate after reaction It is secondary, with Rotary Evaporators by extract liquor whole evaporative removal, trifluoroacetic acid aqueous solution sample dissolution is then used, study is obtained.Study warp For high performance liquid chromatography (HPLC-15C) analysis measurement after 0.22 μm of organic phase filter membrane filtering.High performance liquid chromatography measurement Condition are as follows: using methanol and acetonitrile, 0.075mol/L potassium dihydrogen phosphate aqueous solution, 1.0mol/L citron sour water under constant gradient mode Liquor capacity is mobile phase than the mixed solution for 30:30:36:4；Testing conditions are as follows: flow velocity 1.0mLmin-1, Detection wavelength 254nm, column temperature room temperature, 20 μ L of sample volume.
Experimental result is shown, when rifaximin initial concentration is 10 μ g/mL, CPO@DSP-HA and CPO@DSP-CHIT couple The degradation rate of rifaximin is respectively 95.60% and 95.52%；When rifaximin concentration be 20 μ g/mL when, CPO@DSP-HA and CPO@DSP-CHIT is respectively 93.25% and 94.21% to the degradation rate of rifaximin；When rifaximin concentration is 50 μ g/mL When, CPO@DSP-HA and CPO@DSP-CHIT is respectively 90.33% and 91.49% to the degradation rate of rifaximin；When sharp good fortune former times When bright initial concentration is 100 μ g/mL, CPO@DSP-HA and CPO@DSP-CHIT is respectively to the degradation rate of rifaximin 86.12% and 88.10%.
Three kinds of school periphery sewage source (artificial lake water, Hei Heshui, sanitary sewage) is chosen to be applied to as experiment water sample The reaction system of enzyme reactor catalytic degradation rifaximin.Three are separately added into 5mg CPO@DSP-HA or CPO@DSP-CHIT The rifaximin solution of kind water sample and various concentration, is eventually adding 10 μ L0.1molL-1H2O2, keep final volume be 1500 μ L react 25min under magnetic stirring and are allowed to sufficiently react.It is extracted with ethyl acetate 3 times, is steamed with rotation after reaction Instrument is sent out by extract liquor whole evaporative removal, trifluoroacetic acid aqueous solution sample dissolution is then used, obtains study.Study is organic through 0.22 μm For high performance liquid chromatography (HPLC-15C) analysis measurement, the result is shown in Figure 13 and 14 after the filtering of phase filter membrane.
As seen from the figure, when rifaximin initial concentration is 20 μ g/mL in water sample, in the 25min time CPO@DSP-HA and CPO@DSP-CHIT is all up to 90% or more the degradation rate of rifaximin, and degradation effect is good；When rifaximin in water sample When initial concentration reaches 100 μ g/mL, CPO DSP-HA respectively can be with to the degradation rate of rifaximin in Heihe water and artificial lake water Reach 82.32% and 88.55%, 76.32% can reach to the degradation rate of rifaximin in sanitary sewage；CPO@DSP-CHIT couple The degradation rate of Heihe water and the rifaximin in artificial lake water can achieve 83.36% and 88.62% respectively, in sanitary sewage The degradation rate of rifaximin can reach 78.25%.

Claims (8)

1. a kind of mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase reactor, feature exist In: the enzyme reactor is the mesoporous SiO of dendroid for modifying surface amino groups2Nanoparticle and hyaluronic acid or protonated amino It is used as carrier after chitosan crosslinked, is obtained by electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.
2. the mesoporous SiO of functionalization dendroid of surface charge regulation according to claim 12Immobilization chloroperoxidase Reactor, it is characterised in that: the compound that the surface amino groups modification uses is 3-aminopropyltriethoxysilane, four sub- second Any one in five amine of base, N- aminoethyl -3- aminopropyltriethoxy dimethoxysilane.
3. the mesoporous SiO of functionalization dendroid of surface charge regulation according to claim 12Immobilization chloroperoxidase Reactor, it is characterised in that: the hyaluronic acid passes through n-hydroxysuccinimide sulfonic acid under conditions of pH is 8~10 Sodium salt and the effect of 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride, it is mesoporous with the dendroid of surface amino groups modification SiO2Nanoparticle crosslinking.
4. the mesoporous SiO of functionalization dendroid of surface charge regulation according to claim 32Immobilization chloroperoxidase Reactor, it is characterised in that: the mesoporous SiO of dendroid of the surface amino groups modification2After nanoparticle and cross-linking hyaluronic acid, Under conditions of pH is 3~4, pass through electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.
5. the mesoporous SiO of functionalization dendroid of surface charge regulation according to claim 12Immobilization chloroperoxidase Reactor, it is characterised in that: the chitosan of the protonated amino is situated between by the dendroid of glutaraldehyde and surface amino groups modification Hole SiO2Nanoparticle crosslinking.
6. the mesoporous SiO of functionalization dendroid of surface charge regulation according to claim 52Immobilization chloroperoxidase Reactor, it is characterised in that: the mesoporous SiO of dendroid of the surface amino groups modification2The shell of nanoparticle and protonated amino is poly- After sugar crosslinking, under conditions of pH is 5~6, pass through electrostatic interaction and the fixed chloroperoxidase of hydrogen bond action.
7. the mesoporous SiO of functionalization dendroid of surface charge regulation described in claim 12The reaction of immobilization chloroperoxidase Application of the device in degradation lavo-ofloxacin.
8. the mesoporous SiO of functionalization dendroid of surface charge regulation described in claim 12The reaction of immobilization chloroperoxidase Application of the device in degradation rifaximin.
CN201910393910.3A 2019-05-13 2019-05-13 Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof Active CN110129308B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910393910.3A CN110129308B (en) 2019-05-13 2019-05-13 Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910393910.3A CN110129308B (en) 2019-05-13 2019-05-13 Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof

Publications (2)

Publication Number Publication Date
CN110129308A true CN110129308A (en) 2019-08-16
CN110129308B CN110129308B (en) 2023-04-07

Family

ID=67573333

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910393910.3A Active CN110129308B (en) 2019-05-13 2019-05-13 Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof

Country Status (1)

Country Link
CN (1) CN110129308B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110692630A (en) * 2019-09-23 2020-01-17 南京高正农用化工有限公司 Floating carrier for floating large granules and preparation method thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274085A (en) * 2014-07-11 2016-01-27 宁波大学 Method for preparing block macroporous composite immobilized laccase constructed by CS/SiO2 ultrathin membrane
CN105907743A (en) * 2016-06-23 2016-08-31 陕西师范大学 Preparation and application of hollow spherical-segment-shaped mesoporous silica/chloroperoxidase nanoreactor
CN107865972A (en) * 2017-09-20 2018-04-03 中南民族大学 A kind of preparation method and application for the multi-functional film controlling type targeted nano carrier for having tracer and targeted drug conveying effect concurrently
CN109280660A (en) * 2018-10-29 2019-01-29 陕西师范大学 Fixed chloroperoxidase reactor and the application simultaneously of surface modified magnetic halloysite nanotubes inside and outside wall

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105274085A (en) * 2014-07-11 2016-01-27 宁波大学 Method for preparing block macroporous composite immobilized laccase constructed by CS/SiO2 ultrathin membrane
CN105907743A (en) * 2016-06-23 2016-08-31 陕西师范大学 Preparation and application of hollow spherical-segment-shaped mesoporous silica/chloroperoxidase nanoreactor
CN107865972A (en) * 2017-09-20 2018-04-03 中南民族大学 A kind of preparation method and application for the multi-functional film controlling type targeted nano carrier for having tracer and targeted drug conveying effect concurrently
CN109280660A (en) * 2018-10-29 2019-01-29 陕西师范大学 Fixed chloroperoxidase reactor and the application simultaneously of surface modified magnetic halloysite nanotubes inside and outside wall

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DENGKE SHEN ET AL.: "Biphase Stratification Approach to Three-Dimensional Dendritic Biodegradable Mesoporous Silica Nanospheres", 《NANO LETT.》 *
LUCA MEDDA ET AL.: "Adsorption of Lysozyme on Hyaluronic Acid Functionalized SBA-15 Mesoporous Silica: A Possible Bioadhesive Depot System", 《LANGMUIR》 *
YICHAO SONG ET AL.: "Construction of nano-composites by enzyme entrapped in mesoporous dendritic silica particles for efficient biocatalytic degradation of antibiotics in wastewater", 《CHEMICAL ENGINEERING JOURNAL》 *
刘晓贞等: "二氧化硅/壳聚糖大孔复合材料固定化漆酶及其对2,4-二氯苯酚的降解", 《高校化学工程学报》 *
孙文雅: "新型介孔硅固定化脂肪酶及其催化性能研究", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 *
张博雅等: "氨基二氧化硅固定化纤维素酶及其特性", 《北京大学学报（自然科学版）》 *
李晓红: "氯过氧化物酶催化降解抗生素的研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110692630A (en) * 2019-09-23 2020-01-17 南京高正农用化工有限公司 Floating carrier for floating large granules and preparation method thereof

Also Published As

Publication number Publication date
CN110129308B (en) 2023-04-07

Similar Documents

Publication Publication Date Title
Wu et al. Electrospun blend nanofiber membrane consisting of polyurethane, amidoxime polyarcylonitrile, and β-cyclodextrin as high-performance carrier/support for efficient and reusable immobilization of laccase
Fopase et al. Strategies, challenges and opportunities of enzyme immobilization on porous silicon for biosensing applications
Bagheri et al. Ionic liquid-based preparation of cellulose− dendrimer films as solid supports for enzyme immobilization
CN102260662B (en) Carrier for immobilized enzyme and application thereof and the carrier for being fixed with enzyme
Tarasi et al. Laccase immobilization onto magnetic β-cyclodextrin-modified chitosan: improved enzyme stability and efficient performance for phenolic compounds elimination
US7311926B2 (en) Biocomposite materials and methods for making the same
Chao et al. Template-free in situ encapsulation of enzymes in hollow covalent organic framework capsules for the electrochemical analysis of biomarkers
Schoenfeld et al. Investigations on diffusion limitations of biocatalyzed reactions in amphiphilic polymer conetworks in organic solvents
Bilal et al. Graphene-based nanoarchitectures as ideal supporting materials to develop multifunctional nanobiocatalytic systems for strengthening the biotechnology industry
WO2008058963A1 (en) Immobilization of membrane proteins onto supports via an amphiphile
Wan et al. Immobilization of Candida antarctic lipase B on MWNTs modified by ionic liquids with different functional groups
CN106994333B (en) The preparation method of the cystamine modification biological charcoal of adsorbing heavy metal cadmium ion
Zou et al. Enhancing bio-catalytic activity and stability of lipase nanogel by functional ionic liquids modification
CN109266639B (en) Dual immobilized enzyme and preparation method and application thereof
Zhao et al. Multimeric immobilization of alcohol oxidase on electrospun fibers for valid tests of alcoholic saliva
Rodriguez-Abetxuko et al. Carrierless immobilization route for highly robust metal–organic hybrid enzymes
CN110129308A (en) The mesoporous SiO of functionalization dendroid of surface charge regulation2Immobilization chloroperoxidase reactor and its application
Musyanovych et al. Grafting of amino functional monomer onto initiator-modified polystyrene particles
US7723084B2 (en) Fibrous protein-immobilization systems
Akgöl et al. New generation polymeric nanospheres for catalase immobilization
KR20130064909A (en) Branched polymer microspheres with silica shell
CN109280660A (en) Fixed chloroperoxidase reactor and the application simultaneously of surface modified magnetic halloysite nanotubes inside and outside wall
CN111701581B (en) Low-density lipoprotein, cholesterol and triglyceride adsorbent and preparation method and application thereof
CN100371372C (en) Phospholipid modified acrylon nano fiber composite film and its preparation and immobilized enzyme thereof
CN106977746A (en) Ultrabranching polyamide or derivatives thereof mesoporous molecular sieve composite material, its preparation method and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant