CN109750025A - A kind of hydrogel cladding dendroid silica immobilization CPO enzyme reactor and its preparation method and application - Google Patents
A kind of hydrogel cladding dendroid silica immobilization CPO enzyme reactor and its preparation method and application Download PDFInfo
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- CN109750025A CN109750025A CN201811630107.9A CN201811630107A CN109750025A CN 109750025 A CN109750025 A CN 109750025A CN 201811630107 A CN201811630107 A CN 201811630107A CN 109750025 A CN109750025 A CN 109750025A
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Abstract
The invention discloses a kind of hydrogel cladding dendroid silica immobilization CPO enzyme reactors and its preparation method and application, synthesis has the radial dendroid silica spherical shape nanoparticle in open center first, CPO is embedded in the duct of carrier as solid phase carrier, one layer of hydrogel thin film is finally coated outside carrier, do not influence immobilized enzyme catalysis it is active under the premise of, improve enzyme in physical absorption fixed form be easy leakage the problem of.Fixing means of the present invention is simple, reduce the cost of enzyme, with objective economic benefit, with covalent stationary phase ratio, the loss of activity of enzyme is less, the catalytic activity of immobilised enzymes is the 91.45% of resolvase, and 90% catalytic activity is able to maintain after reusing 10 times, and 50% activity is still able to maintain after reusing 20 times；With this enzyme reactor degradation lavo-ofloxacin, when lavo-ofloxacin content reaches 100 μ g/mL, degradation rate reaches 83.05%；With this enzyme reactor degradation rifaximin, when rifaximin content reaches 50 μ g/mL, degradation rate reaches 85.70%.
Description
Technical field
The invention belongs to fixation techniques for enzyme fields, and in particular to a kind of dendroid silica of hydrogel cladding is received
The preparation method and application of rice corpuscles immobilization chloroperoxidase reactor and the enzyme reactor.
Background technique
Enzyme is a kind of protein with catalysis generated by biological cell, has single-minded height for catalysis reaction
Effect, speed is fast, mild condition, it is environmental-friendly the features such as, therefore be widely used in synthesizing chemistry, food, pharmacy, wastewater treatment etc.
Industry.And there is also drawbacks in practical applications for resolvase, for example make in high temperature, organic solvent, strong acid and strong base, mechanical external force
It is all easy to cause enzyme denaturation to inactivate under equal environment, causes catalytic efficiency to reduce, it is difficult to recycle and reuse.Enzyme immobilization technology
It is the effective ways for inhibiting drawback.Compared with resolvase, immobilised enzymes has the operational stability for improving enzyme, in the reaction system
Substrate easy to accomplish is separated with product, it can be achieved that repeatedly using, to reduce a variety of advantages such as use cost of enzyme.
However, designing and preparing the problem of aspect remains some generalities at present in immobilised enzymes: with it is free
Enzyme is compared, and the stability of immobilised enzymes is improved often premised on sacrificing partially catalyzed activity and cost, because are as follows:
(1) interaction during enzyme immobilization between enzyme and carrier inevitably changes enzyme molecule surface to a certain extent
The conformation of amino acid residue and the microenvironment of active site, both factors result in immobilised enzymes and are catalyzed compared with resolvase
Activity is declined；(2) in order to overcome the obscission of immobilised enzymes enzyme molecule in use, need to reinforce enzyme molecule with
Interaction between carrier, but it is stronger often to interact, and the change of the structure and conformation of enzyme molecule is bigger, thus enzyme activity
Property loss it is bigger.
Therefore, the immobilized enzyme reactor one for having both high catalytic activity, high stability and high reusability how is prepared
It is directly a challenge.
Chloroperoxidase (CPO) is a kind of ferroheme glucoproteinase separated from marine fungi.It has peroxide
The catalytic activity of compound enzyme, catalase and cytochrome P-450, can widely effectively catalytic halogenation, oxidation, peroxide
Change, epoxidation, hydroxylation reaction and reaction of high enantioselectivity etc..
Metaporous silicon dioxide material is easy to synthesize on a large scale, has adjustable aperture size and structure, specific surface area is high, hole
Gap rate is high, and thermal stability is high, and mechanical stability is high, easily carries out selectively functionalized in channel surfaces or particle surface.Wherein, it sets
Dendritic porous silica spheres shape nanoparticle due to having the three-dimensional radial dendroid superstructure in open center, thus
With higher hole permeability, bigger pore volume, accessible property of better particle inner surface etc..
Hydrogel (Hydrogel) is the gel using water as decentralized medium.Water soluble polymer with cross-linked network
It is middle to introduce a part of hydrophobic grouping and hydrophilic residue, hydrophilic residue and water molecules, hydrone is connected to netted inside, and
The cross-linked polymer of hydrophobic residue water-swellable is a kind of good coating.
Sodium alginate is a kind of sodium salt of polyanionic polysaccharide (alginic acid) extracted from natural brown alga, by β-D-
Two kinds of structural unit chains of mannuronic acid (M sugar) and α-L- guluronic acid (G sugar) link.Contain a large amount of hydroxyl in molecule
Base and carboxyl have very strong hydrophily, and be a kind of ideal hydrogel material, advantage is as follows: (1) gel network, which ensure that, contains
The microenvironment of water is conducive to the survival of large biological molecule；(2) large biological molecule mild condition is embedded, organic solvent is not needed, has
Help the existence of large biological molecule；(3) there is relatively high gel aperture, facilitate the diffusion of large biological molecule；(4) can lead to
Cross the aperture of different coating process control gels；(5) under general physiological condition, alginic acid can decompose and biology drop
Solution.
Summary of the invention
It is an object of the invention to overcome problems of the prior art, one kind is provided and has both high catalytic activity, Gao Wen
The dendroid silica immobilization chloroperoxidase reactor of the hydrogel cladding of qualitative and high reusability, and should
The preparation method of enzyme reactor, and new application is provided for the enzyme reactor.
The fixed chloroperoxidase reactor of silica used by above-mentioned technical problem is solved to be wrapped up by hydrogel,
Middle silica is dendroid mesoporous silicon oxide.
Above-mentioned hydrogel is the hydrophilic high mol compound of natural polysaccharide or synthesis, wherein the natural polysaccharide includes
Any one in lentinan, chitosan, hyaluronic acid or its salt, alginic acid or its salt, preferably sodium alginate.
The preparation method of above-mentioned silica immobilization chloroperoxidase reactor is made of following step:
1, dendroid mesoporous silicon oxide is added in the PBS buffer solution that pH is 2~3, chlorine peroxide is then added
Enzyme solutions, isothermal vibration, centrifuge separation, and solid is cleaned with PBS buffer solution, to remove loose chlorine peroxide
Enzyme obtains dendroid silica immobilization chloroperoxidase.
2, dendroid silica immobilization chloroperoxidase is added in the PBS buffer solution that pH is 6~6.5, ultrasound
It is uniformly dispersed, hydrogel solution is then added and carries out cross-linking reaction, centrifuge separation after having reacted obtains the branch of hydrogel cladding
Shape silica immobilization chloroperoxidase reactor.
In above-mentioned steps 1, preferably dendroid mesoporous silicon oxide and quality-volume ratio of chloroperoxidase solution is
The μ of 1mg:2~5 L, the pH of the chloroperoxidase solution are 5~6, wherein the concentration of chloroperoxidase be 0.20~
0.30nmol/L。
In above-mentioned steps 1, the temperature preferably shaken is 10~20 DEG C, and the concussion time is 40~70min.
In above-mentioned rapid 2, the preferred quality of the chloroperoxidase Yu hydrogel, crosslinking agent of the immobilization of dendroid silica
Than for 1:0.4~2:0.5~2, wherein preferred hydrogel sodium alginate, crosslinking agent CaCl2。
The dendroid silica immobilization chloroperoxidase reactor of subject hydrogel cladding is in left oxygen fluorine of degrading
The application of Sha Xingzhong.
The dendroid silica immobilization chloroperoxidase reactor of subject hydrogel cladding is in sharp good fortune former times of degrading
Application in bright.
Compared with prior art, the invention has the following advantages:
CPO has been immobilized on titanium dioxide by way of physical absorption using dendroid silica as carrier by the present invention
In the duct of silicon nano spherical particle, prepared CPO immobilized enzyme reactor has both high catalytic activity, high stability and Gao Chong
Multiple usability.Reason is: (1) compared with covalent bond, immobilized enzyme can reduce enzyme immobilization mistake by the way of physical absorption
Interaction in journey between enzyme and carrier, thus reduce enzyme molecule surface amino groups acid residue conformation and active site it is micro-
The change of environment thus can make more enzymatic activitys be retained；(2) the dendroid silica prepared in the present invention
In aperture be in macropore range, match more important is the size with CPO, it can be achieved that single array of enzyme molecule is fixed, from
And avoid and assemble since a large amount of enzyme molecules are present in duct under the pressure of the confinement pressure in duct, and this aggregation can
Substrate channels in enzyme molecule are caused to be blocked and influence its catalytic activity；(3) dendroid silica has in three-dimensional opening
The radial dendroid superstructure of the heart, thus have higher hole permeability, bigger pore volume, better particle inner surface can
Contact etc., thus compared with traditional silicon dioxide carrier, the supported quantity of enzyme increases, while being also beneficial to substrate and enzyme point
The contact of son, that is, be conducive to catalytic activity；Meanwhile in the table of dendroid silica immobilization chloroperoxidase in the present invention
Bread covers one layer of hydrogel thin film, solve in physisorphtion due to the interaction force between enzyme and carrier it is weaker caused by
Immobilised enzymes is easy the problem of leakage, further improves the reusability of immobilised enzymes.Although the presence of this tunic is no different
The resistance to mass tranfer of substrate be will increase so as to cause the active loss of partially catalyzed.But by lentinan, acrylic acid, polypropylene
The hydrogel thin film of the formation such as amide and sodium alginate is screened, it is found that the effect of Sodium Alginate Hydrogel Films is best.This
The preparation method of the Sodium Alginate Hydrogel Films provided in invention can reach the mesoporous hole of hydrogel network Yu dendroid silica
Diameter and substrate size match, to reduce resistance to mass tranfer of the substrate when jumping aquagel membrane to the full extent, thus most
Retain the catalytic activity of immobilised enzymes in big degree.In fact, Sodium Alginate Hydrogel Films cladding after immobilised enzymes catalytic activity
It is substantially consistent with before cladding.
The present invention is using dendroid porous silica spheres shape nanoparticle as the fixed CPO of carrier, prepared CPO enzyme
Reactor has both high catalytic activity, high stability and high reusability.Immobilised enzymes can retain free enzymatic activity
91.45%, after reusing 10 times, remaining catalytic activity is 90%, the use of catalytic activity remaining after 20 times is 50%.With this enzyme
Reactor degradation lavo-ofloxacin significant effect, when lavo-ofloxacin content reaches 100 μ g/mL, degradation rate reaches
83.05%.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of SA@CPO@DMSNs prepared by embodiment 1.
Fig. 2 is the catalytic activity comparison diagram of free CPO and SA@CPO@DMSNs conversion MCD.
Fig. 3 is the reusability figure of SA@CPO@DMSNs.
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.
Following dendroid mesoporous silicon oxide is according to document " Weston D E.Horizontal Refraction in
a Three-dimensional Medium of Variable Stratification[J].Proceedings of the
Physical Society, 2002,78 (1): method disclosed in 46. " is prepared, specific the preparation method comprises the following steps: holding in 100mL
6g hexadecyltrimethylammonium chloride, 0.16mL triethanolamine and 36mL deionized water are added in measuring bottle, in magnetic stirring apparatus
In 60 DEG C, under 150rpm revolving speed stirring 1h until dissolution, is then added dropwise 1mL ethyl orthosilicate, 19mL hexamethylene, obtains
Mixed liquor water-bath 12h under 60 DEG C, 150rpm revolving speed, is cooled to room temperature after having reacted, and removes upper oil phase with liquid-transfering gun,
Lower layer's water phase is centrifugated product, and is cleaned 3 times with ethyl alcohol.Product is transferred in crucible later, 550 DEG C of items in Muffle furnace
5h is calcined under part to remove template, obtains dendroid mesoporous silicon oxide (DMSNs).
Embodiment 1
1. weighing 5mg DMSNs to be added in the PBS buffer solution of 1450 μ L pH=2.5, and it is molten that the free CPO of 10 μ L is added
Liquid (0.28nmol/L, pH=4.0) is placed in 20 DEG C of concussion 1h in isothermal vibration shaking table, is then centrifuged with the revolving speed of 6000rmp
Supernatant and solid are separated, and are carried out cleaning 2~3 times with the PBS buffer solution of pH=2.5 to solid by 3min, to remove
The loose CPO in the surface DMSNs obtains the chloroperoxidase (CPO@DMSNs) of dendroid silica immobilization.
2. take 5mg CPO@DMSNs to be added in the PBS buffer solution of 600 μ L 20mM pH=6.5, ultrasonic disperse 20min,
Then sodium alginate (SA) aqueous solution of 400 μ L 20mg/mL and the CaCl of 500 μ L 0.1mol/L is added2Aqueous solution, in room temperature
Lower reaction 12h is centrifugated 5min after having reacted with the revolving speed of 10000rmp, obtains the dendroid titanium dioxide of sodium alginate cladding
Silicon immobilization chloroperoxidase reactor (see Fig. 1) is denoted as SA@CPO@DMSNs.
The SA@CPO@DMSNs of preparation is tested for the property, specific test is as follows:
1. catalytic activity is tested
The catalytic activity of SA@CPO@DMSNs generates 2 by the catalysis chloro- 5,5- dimethyl of 2--hydroresorcinol (MCD),
Chloro- dimethyl -1 5,5- 2- bis-, the chlorination reaction of hydroresorcinol (DCD) measure, the specific steps are as follows: at 25 DEG C, take two
A quartz colorimetric utensil is separately added into 5 μ L free CPO solution (0.28nmol/L) and 5mg SA@CPO@DMSNs, 1420 μ L is added
0.1mol·L-1The PBS buffer solution of pH=2.75,50 μ L0.1molL-1Then 30 μ L 0.1molL are added in MCD aqueous solution-1H2O2Aqueous solution initiation reaction, reaction total system volume are 1.5mL.With absorbance value at ultraviolet specrophotometer measurement 278nm
Variation to determine the catalytic activity of free CPO and SA@CPO@DMSNs.
The conversion ratio of MCD is calculated by following formula:
A in formulat: absorbance value of the supernatant in t moment after enzyme；A0: the absorbance value of reaction system when not enzyme.Knot
Fruit sees Fig. 2.
From Figure 2 it can be seen that being in terms of 100% by the catalytic activity for the CPO that dissociates, SA@CPO@DMSNs remains with higher catalysis and lives
Property, about 91.45%.
2. reusability is tested
Catalysis is characterized by MCD model reaction, the specific steps are as follows: at 25 DEG C, 5mg is added in quartz colorimetric utensil
SA@CPO@DMSNs、1420μL 0.1mol·L-1The PBS buffer solution of pH=2.75,50 μ L0.1molL-1MCD aqueous solution, so
After 30 μ L 0.1molL are added-1H2O2Aqueous solution initiation reaction, reaction total system volume are 1.5mL.Use uv-spectrophotometric
Absorbance value at meter measurement 278nm.
After each reaction, reaction solution is centrifuged to 1min under 6000rpm revolving speed to separate supernatant, draws out supernatant
After liquid, PBS buffer solution, MCD aqueous solution and the H of equivalent are continuously added2O2Aqueous solution is to start reaction next time.It will be for the first time
Activity using SA@CPO@DMSNs is considered as 100%, by the catalytic activity of SA@CPO@DMSNs and first time are right each time later
Than being indicated with residual activity.As a result see Fig. 3.
As seen from Figure 3, after reusing 10 times, the residual activity of SA@CPO@DMSNs is still up to 90%, reuses 20
After secondary, residual activity reaches 50%.
Embodiment 2
The SA@CPO@DMSNs degradation lavo-ofloxacin prepared using embodiment 1
5mg SA@CPO@DMSNs, 2480 μ L PBS buffer solution liquid (0.1molL are added in 10mL centrifuge tube-1, pH=
2.75), the standard solution (10 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL) of 500 μ L various concentration lavo-ofloxacins, most
After 20 μ L0.1molL are added-1H2O2Aqueous solution starting reaction.25min is reacted at room temperature under magnetic stirring, is used after reaction
Ethyl acetate extracts 3 times.Finally with Rotary Evaporators then extract liquor all evaporations, removal are dissolved into sample with trifluoroacetic acid aqueous solution
Product obtain study.Study is surveyed after 0.22 μm of organic phase filter membrane filtering for high performance liquid chromatography (HPLC-15C) analysis
It is fixed, high-efficient liquid phase chromatogram determining condition are as follows: acetonitrile-KH is used under constant gradient mode2PO4Solution (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
η=(A0-At)/A0× 100%
A in formulat: absorbance value of the supernatant in t moment after enzyme；A0: the absorbance value of reaction system when not enzyme.
Experimental result shows that when levofloxacin concentration is 10 μ g/mL, SA@CPO@DMSNs is to lavo-ofloxacin hydrochloride
Degradation rate is 95.00%；When levofloxacin concentration is 20 μ g/mL, degradation of the SA@CPO@DMSNs to lavo-ofloxacin hydrochloride
Rate is 93.50%；When levofloxacin concentration is 50 μ g/mL, SA@CPO@DMSNs is to the degradation rate of lavo-ofloxacin hydrochloride
92.50%；When levofloxacin concentration is 100 μ g/mL, SA@CPO@DMSNs is to the degradation rate of lavo-ofloxacin hydrochloride
83.05%.
Embodiment 3
The SA@CPO@DMSNs degradation rifaximin prepared using embodiment 1
(1) mobile phase is prepared
Weigh KH2PO43.4g, dissolves and the constant volume in 250mL volumetric flask, weighing NaAc 2.05g dissolve and in 250mL
Then constant volume in volumetric flask mixes the two in equal volume.Use 0.1molL-1It is 2.6 that aqueous citric acid solution, which adjusts pH value of solution, this
Solution is buffer.Using acetonitrile, methanol, buffer volume ratio=44:16:40 as mobile phase.
(2) degradation rifaximin
Using distilled water as solvent, prepare various concentration rifaximin titer (10 μ g/mL, 20 μ g/mL, 30 μ g/mL,
30 μ g/mL, 40 μ g/mL, 50 μ g/mL) sample；Take five 10mL centrifuge tubes be separately added into 500 μ L rifaximin titers,
2400 μ LPBS buffer solutions (pH=2.75), 5mg SA@CPO@DMSNs, the H that 100 μ L mass fractions are 30%2O2Solution, room
Temperature reaction 30min.Solid material is removed with magnet after reaction, product (3mL × 3 time) are extracted with ethyl acetate, is steamed using rotation
Hair extracts product, then uses mobile phase sample dissolution, obtains study.Study is after 0.22 μm of organic phase filter membrane filters
It analyzes and measures for high performance liquid chromatography (HPLC-15C).
High-efficient liquid phase chromatogram determining condition are as follows: acetonitrile-methanol-buffer volume ratio=44:16 is used under constant gradient mode:
40 be mobile phase, flow velocity 1.0mLmin-1, Detection wavelength 254nm, column temperature is room temperature, 20 μ L of sample volume.
Experimental result shows that when rifaximin concentration is 10 μ g/mL, SA@CPO@DMSNs is to the degradation rate of rifaximin
87.00%；When rifaximin concentration is 20 μ g/mL, SA@CPO@DMSNs is 88.50% to the degradation rate of salt rifaximin；Benefit
When good fortune former times bright concentration is 50 μ g/mL, SA@CPO@DMSNs is 85.70% to the degradation rate of rifaximin.
Embodiment 4
The method for preparing SA@CPO@DMSNs using embodiment 1 investigates different SA contents to CPO enzyme reactor catalytic effect
Influence (using the catalytic conversion of MCD as activity rating index), such as table 1.
Table 1
| SA:CPO@DMSNs (mass ratio) | 2:5 | 4:5 | 6:5 | 8:5 | 10:5 |
| The catalytic activity of CPO enzyme reactor | 90.01% | 90.73% | 91.05% | 92.01% | 89.77% |
When the mass ratio of SA and CPO@DMSNs reaches 8:5, the film thickness of formation is suitable for that can reach optimal catalysis to live
Property.In the range of SA/CPO@DMSNs (mass ratio) 2:5 to 10:5, enzyme reactor can keep greater activity.
Embodiment 5
The method for preparing SA@CPO@DMSNs using embodiment 1 investigates different hydrogels to CPO enzyme reaction catalytic effect
It influences, such as table 2.
Table 2
| Type of hydrogel | Sodium alginate | Chitosan | Hyaluronic acid | Lentinan |
| The catalytic activity of CPO enzyme reactor | 92.01% | 91.77% | 91.40% | 90.33% |
Embodiment 6
The method for preparing SA@CPO@DMSNs using embodiment 1, investigates the species of metal ion of different alginates to CPO
The influence of enzyme reactor catalytic effect, such as table 3.
Table 3
| Metal ion | Na+ | K+ | Ca2+ | Zn2+ | Mg2+ | Al3+ |
| The catalytic activity of CPO enzyme reactor | 92.01% | 90.84% | 90.88% | 85.30% | 72.84% | 70.25% |
It is to be understood that foregoing invention content and specific embodiment are intended to prove technical solution provided by the present invention
Practical application should not be construed as limiting the scope of the present invention.Those skilled in the art are in spirit and principles of the present invention
It is interior, when can various modifications may be made, equivalent replacement or improve.
Claims (12)
1. a kind of fixed chloroperoxidase reactor of silica, it is characterised in that: the chlorine mistake of the silica immobilization
Oxide enzyme is wrapped up by hydrogel, and wherein silica is dendroid mesoporous silicon oxide.
2. silica immobilization chloroperoxidase reactor according to claim 1, it is characterised in that: the hydrogel
For natural polysaccharide or the hydrophilic high mol compound of synthesis.
3. silica immobilization chloroperoxidase reactor according to claim 2, it is characterised in that: the hydrogel
For any one in natural polysaccharide, including lentinan, chitosan, hyaluronic acid or its salt, alginic acid or its salt.
4. silica immobilization chloroperoxidase reactor according to claim 3, it is characterised in that: the hydrogel
For sodium alginate.
5. a kind of system of silica immobilization chloroperoxidase reactor described in any one according to claim 1~4
Preparation Method, it is characterised in that it is prepared by the following method to obtain:
(1) dendroid mesoporous silicon oxide is added in the PBS buffer solution that pH is 2~3, it is molten that chloroperoxidase is then added
Liquid, isothermal vibration, centrifuge separation, and solid is cleaned with PBS buffer solution, to remove loose chloroperoxidase,
Obtain dendroid silica immobilization chloroperoxidase；
(2) dendroid silica immobilization chloroperoxidase is added in the PBS buffer solution that pH is 6~6.5, ultrasonic disperse
Uniformly, hydrogel is then added to react with cross-linking agents, centrifuge separation after having reacted obtains the dendroid two of hydrogel cladding
Silica immobilization chloroperoxidase reactor.
6. preparation method according to claim 5, it is characterised in that: in step (1), the dendroid mesoporous silicon oxide
Quality-volume ratio with chloroperoxidase solution is the μ of 1mg:2~5 L.
7. preparation method according to claim 6, it is characterised in that: the pH of the chloroperoxidase solution is 5~6,
Wherein the concentration of chloroperoxidase is 0.20~0.30nmol/L.
8. preparation method according to claim 5, it is characterised in that: in step (1), the temperature of concussion is 10~20 DEG C,
The concussion time is 40~70min.
9. preparation method according to claim 5, it is characterised in that: in step (2), the dendroid silica is fixed
The mass ratio for changing chloroperoxidase and hydrogel, crosslinking agent is 1:0.4~2:0.5~2.
10. preparation method according to claim 5, it is characterised in that: the hydrogel is sodium alginate, and crosslinking agent is
CaCl2。
11. any silica immobilization chloroperoxidase reactor according to claims 1 to 4 is in left oxygen fluorine of degrading
The application of Sha Xingzhong.
12. any silica immobilization chloroperoxidase reactor according to claims 1 to 4 is in sharp good fortune former times of degrading
Application in bright.
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| CN115011575A (en) * | 2022-07-11 | 2022-09-06 | 陕西师范大学 | Chloroperoxidase nano-reactor constructed by sodium alginate-coated iron oxyhydroxide hollow microcapsules and application |
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