CN109402104B - Immobilization method of cephalosporin acylase and immobilized enzyme - Google Patents
Immobilization method of cephalosporin acylase and immobilized enzyme Download PDFInfo
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- C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)
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Abstract
The invention provides an immobilization method of cephalosporin acylase, belonging to the technical field of enzyme immobilization, and the method comprises the following steps: 1) dropwise adding the bridging body solution into the carrier solution, carrying out a first condensation reaction for 2-5 h, carrying out first solid-liquid separation, and collecting solid phase components to obtain a carrier with a bridging body; 2) mixing and stirring the carrier with the bridging body obtained in the step 1) and a cephalosporin acylase aqueous solution to obtain a mixed material liquid, dropwise adding triethylamine into the mixed material liquid to perform a second condensation reaction for 20-40 h, performing second solid-liquid separation, and collecting a solid phase component which is immobilized cephalosporin acylase; the bridge is halogen acyl halide with a main chain containing 2-8 carbon atoms; the carrier is amino resin. The cephalosporin acylase prepared by the method has high activity and good stability.
Description
Technical Field
The invention belongs to the technical field of enzyme immobilization, and particularly relates to an immobilization method of cephalosporin acylase and immobilized enzyme.
Background
The immobilized enzyme is a new technology developed in the 60's of the 20 th century, and the immobilized enzyme is an enzyme which plays a catalytic role in a certain spatial range and can be repeatedly and continuously used.
The existing methods for immobilizing enzymes mainly comprise an adsorption method, an embedding method, a crosslinking method and a covalent bonding method. The adsorption method is the earliest appearing immobilization method, and can be divided into two types, namely ion exchange adsorption and physical adsorption. The adsorption method has mild conditions, and can not change the conformation of the enzyme to a great extent, so that the catalytic performance of the enzyme can not be greatly influenced; however, the binding force between the enzyme and the carrier is weak, and the enzyme is easily separated from the carrier and pollutes the catalytic reaction product under special conditions, such as high salt concentration, high temperature and the like. The entrapping method is an immobilization method in which an enzyme is entrapped in the pores of a polymer. The embedding method can be classified into a mesh type and a microcapsule type according to the type of the embedding form. The method of embedding enzyme by using fine meshes of carriers such as polyacrylamide, polyvinyl alcohol, starch, gelatin, alginic acid and the like is called as mesh type embedding. The microcapsule type is a microcapsule in which an enzyme is embedded in a polymeric semipermeable membrane. The entrapment method is prone to problems such as enzyme leakage and diffusion limitation. The cross-linking method is to produce immobilized enzyme by using multifunctional cross-linking reagents, such as glutaraldehyde, etc., to form covalent bonds between enzyme molecules or between enzyme molecules and carrier molecules under different cross-linking conditions. However, covalent imine bonds formed by using a cross-linking agent, such as glutaraldehyde, are easily broken by water, and the enzyme is easily separated from the carrier and lost. The covalent bonding method is an enzyme immobilization method in which a chemical covalent bond is formed between an active functional group on the surface of a carrier and an unnecessary group on an enzyme molecule, thereby realizing irreversible bonding. In the prior art, epoxy resin, 1, 3-dihalogenated alkane, 1, 4-dihalogenated alkane and 1, 5-dihalogenated alkane are adopted to realize covalent binding of a carrier and an enzyme; there are disadvantages in that: 1. the epoxy resin is adopted, because the electrophilic activity of the epoxy group is not high, the reaction condition is violent, and the enzyme is easy to inactivate; 2. when the disubstituted halogenated alkane is used as the bridging body ligase and the carrier, because the electrophilic activities of the carbons at the two ends of the halogenated alkane used as the bridging body are the same, the carbons at the two ends of the halogenated alkane are easily combined with the carrier or the enzyme, and the connection between the carrier and the enzyme cannot be realized.
Disclosure of Invention
In view of the above, the invention provides an immobilization method of cephalosporin acylase and an immobilized enzyme, wherein the immobilization method can effectively realize the connection between a carrier and an enzyme, the connection condition is mild, the enzyme is not easy to inactivate, and the obtained immobilized enzyme has good stability.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for immobilizing cephalosporin acylase, which comprises the following steps:
1) dropwise adding the bridging body solution into the carrier solution, carrying out a first condensation reaction for 2-5 h, carrying out first solid-liquid separation, and collecting solid phase components to obtain a carrier with a bridging body;
2) mixing and stirring the carrier with the bridging body obtained in the step 1) and a cephalosporin acylase aqueous solution to obtain a mixed material liquid, dropwise adding triethylamine into the mixed material liquid to perform a second condensation reaction for 20-40 h, performing second solid-liquid separation, and collecting a solid phase component which is immobilized cephalosporin acylase;
the bridge is halogen acyl halide with a main chain containing 2-8 carbon atoms;
the carrier is amino resin.
Preferably, the bridge is haloacetyl halide, halopropionyl halide, halobutyl halide or haloamyl halide.
Preferably, the dropping time in the step 1) is 20-60 min, and the temperature of the dropping and the first condensation reaction is independently-10-40 ℃.
Preferably, the mass ratio of the bridging body to the carrier is (0.05-0.2): 1.
preferably, the mass ratio of the carrier with the bridge to the cephalosporin acylase aqueous solution in the step 2) is 1 (1-5), and the enzyme activity of the cephalosporin acylase aqueous solution is 80-100U/m L.
Preferably, the mass ratio of the carrier with the bridging body to triethylamine in the step 2) is 1: (0.1-0.5).
Preferably, the first solid-liquid separation method in the step 1) is suction filtration, and the filter cake after suction filtration is washed with an organic solvent and water in sequence to obtain the carrier with the bridging body.
Preferably, the carrier solution comprises an amino resin, an acid-binding agent, a water-miscible aprotic polar solvent and water; the mass ratio of the amino resin, the acid-binding agent, the water-miscible aprotic polar solvent and water is 1: (0.1-0.5): (5-20): (2-5).
Preferably, the acid-binding agent is triethylamine, sodium bicarbonate or sodium carbonate.
Preferably, the bridging body solution comprises a bridging body and a solvent, and the mass ratio of the bridging body to the solvent is (0.05-0.2): (0.1-1), wherein the solvent is an aprotic polar solvent which is mutually soluble with water.
The invention also provides the immobilized cephalosporin acylase prepared by the immobilization method of the cephalosporin acylase.
The invention has the beneficial effects that: according to the immobilization method of cephalosporin acylase provided by the invention, halogen acyl halide with a main chain containing 2-8 carbon atoms is selected as a bridging body, the difference of electrophilic activities of carbon at two ends of the bridging body is large, the cephalosporin acylase is immobilized according to the defined feeding sequence and reaction conditions in the invention, two ends of the bridging agent can be prevented from being simultaneously connected with a carrier or simultaneously connected with an enzyme, the effective connection between the carrier and the enzyme can be realized, and meanwhile, the connection conditions are mild, so that the enzyme is not easily inactivated; the method adopts covalent bond combination, and the immobilized enzyme has good stability. According to the records of the embodiment, the immobilized cephalosporin acylase provided by the invention has high enzyme activity (125U/g), the enzyme activity is kept about 120U/g after 20 batches of cephalosporin C sodium salt are cracked, no obvious reduction occurs, and the stability is good.
Drawings
FIG. 1 is a flow chart showing a method for immobilizing cephalosporin acylase according to the present invention.
Detailed Description
The invention provides an immobilization method of cephalosporin acylase, which comprises the following steps: 1) dropwise adding the bridging body solution into the carrier solution, carrying out a first condensation reaction for 2-5 h, carrying out first solid-liquid separation, and collecting solid phase components to obtain a carrier with a bridging body; 2) mixing and stirring the carrier with the bridging body obtained in the step 1) and a cephalosporin acylase aqueous solution to obtain a mixed material liquid, dropwise adding triethylamine into the mixed material liquid to perform a second condensation reaction for 20-40 h, performing second solid-liquid separation, and collecting a solid phase component which is immobilized cephalosporin acylase; the bridge is halogen acyl halide with a main chain containing 2-8 carbon atoms; the carrier is amino resin.
The carrier solution comprises amino resin, an acid binding agent, a water-miscible aprotic polar solvent and water, wherein the mass ratio of the amino resin to the acid binding agent to the water-miscible aprotic polar solvent to the water is 1 (0.1-0.5): (5-20): 2-5), preferably 1 (0.2-0.4): 8-15): 3-4.
In the invention, the carrier solution is prepared by the following method: mixing the amino resin, the aprotic polar solvent mutually soluble with water and water under the condition of stirring, and then adding an acid binding agent and stirring to obtain the carrier solution. In the present invention, the rotation speed of the stirring is preferably 40 to 100rpm, more preferably 50 to 90rpm, and the stirring time is not particularly limited, and the stirring is preferably performed uniformly. The temperature of the carrier solution is reduced to-10-40 ℃ in the stirring process. In the invention, the temperature reduction is preferably performed by using ice brine.
In the invention, the bridging body solution comprises a bridging body and a solvent, wherein the bridging body is halogen acyl halide with a main chain containing 2-8 carbon atoms, preferably halogen acetyl halide, halogen propyl acyl halide, halogen butyl acyl halide or halogen amyl acyl halide; the solvent is an aprotic polar solvent which is mutually soluble with water, and the aprotic polar solvent which is mutually soluble with water is acetone, acetonitrile or tetrahydrofuran. In the invention, the mass ratio of the bridging body to the solvent is (0.05-0.2): (0.1-1), preferably (0.1-0.18): (0.2-0.8). The bridge solution in the present invention is obtained by mixing the bridge with a solvent.
The method comprises the steps of dropwise adding a bridging body solution into a carrier solution, carrying out a first condensation reaction for 2-5 hours, carrying out first solid-liquid separation, and collecting solid-phase components to obtain the carrier with the bridging body. In the present invention, the mass ratio of the bridge to the carrier is preferably (0.05 to 0.2): 1, more preferably (0.1 to 0.15): 1; in the invention, the dripping time is preferably 20-60 min, and more preferably 30-50 min; the preferable temperature of the dropwise addition is-10-40 ℃, and the more preferable temperature is-9-0 ℃. In the invention, the time of the first condensation reaction is preferably 3-4 h, and the temperature of the first condensation reaction is preferably-10-40 ℃, and more preferably-9-0 ℃. In the first condensation reaction process of the present invention, electrophilic attack of an amino group by an acid halide forms an amide bond. After the first condensation reaction is finished, carrying out first solid-liquid separation, and collecting solid phase components to obtain the carrier with the bridging body. In the invention, the first solid-liquid separation method is preferably suction filtration, and the filter cake after suction filtration is washed by an organic solvent and water in sequence to obtain the carrier with the bridging body. In the present invention, the organic solvent is preferably acetone, the number of times of washing with the organic solvent is preferably 1 to 3 times, more preferably 2 times, and the number of times of washing with water is preferably 1 to 3 times, more preferably 2 times. The mass of the organic solvent and the water used for washing is not particularly limited, and the washing can be realized, and in the specific implementation process of the invention, the mass of the organic solvent and the water is independently 2-5 times of that of the filter cake. The filter cake after washing is the carrier with the bridging body.
The method comprises the steps of obtaining a carrier with a bridge, mixing and stirring the carrier with the bridge and a cephalosporin acylase aqueous solution to obtain a mixed material liquid, wherein the enzyme activity of the cephalosporin acylase aqueous solution is preferably 80-100U/m L, more preferably 85-95U/m L, the cephalosporin acylase aqueous solution is prepared by mixing and concentrating cephalosporin acylase and water, the source of the cephalosporin acylase is not specially required, the cephalosporin acylase for cephalosporin C cracking is adopted, the mass ratio of the carrier with the bridge to the cephalosporin acylase aqueous solution is preferably 1 (1-5), more preferably 1 (2-4), the rotation speed of mixing and stirring is preferably 40-100 rpm, more preferably 50-90 rpm, the time of mixing and stirring is not specially limited, the mixing and stirring is preferably carried out for realizing uniform mixing, the temperature of mixing and stirring is preferably-10-40 ℃, more preferably 0-10 ℃, the mixing and stirring is preferably carried out by dropwise adding triethylamine to the mixed material liquid for a second time of 0.5-10-hour, the triethylamine and the mixed material liquid is preferably carried out a solid-liquid-solid-liquid-phase reaction, the triethylamine and the triethylamine is preferably carried out a solid-liquid-solid-liquid-solid-liquid-phase reaction, the triethylamine-liquid-solid-liquid-solid-liquid-solid-liquid-solid-liquid-solid-liquid-solid-liquid-solid-liquid-solid-liquid-is preferably obtained, the triethylamine is preferably obtained by a triethylamine, the triethylamine.
The invention provides the immobilized cephalosporin acylase prepared by the preparation method, the wet enzyme activity of the cephalosporin acylase is 125-130U/g, the enzyme activity is not obviously reduced after 20 batches of cephalosporin C sodium are acylated and cracked by the immobilized cephalosporin, and the stability is good.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Step 1) connection of bridge and carrier:
a. acetone 600m L, purified water 60m L, 30g of amino resin and triethylamine 3m L are added under the stirring of 60rpm, and the temperature is reduced to-10 to-5 ℃.
b.2.2g chloroacetyl chloride was dissolved in 10m L acetone.
c. The temperature is controlled between minus 10 ℃ and minus 5 ℃, and the acetone solution of the chloracetyl chloride is dripped into the amino resin solution. The dripping time is 20-60 min, the temperature is controlled to be-10 to-5 ℃, and the first condensation reaction is carried out for 2-5 h.
d. Suction filtration, the filter cake was washed twice with 100m L acetone and twice with 100m L purified water, the carrier already carrying the bridge.
Step 2) connecting the carrier with the bridge and cephalosporin acylase.
a, 50m L (enzyme activity 100U/m L) cephalosporin acylase aqueous solution, stirring and adding 31g of carrier with a bridging body, stirring at the speed of 30-50rpm, and cooling to 0-10 ℃.
b.2.7m L triethylamine was dissolved in 10m L water.
c. And controlling the reaction temperature to be 0-10 ℃, dropwise adding the triethylamine aqueous solution into the cephalosporin acylase aqueous solution for 20-60 min, controlling the temperature to be 0-10 ℃, and carrying out a second condensation reaction for 8-10 h.
d. And (5) carrying out suction filtration, and washing with purified water of 200m L for three times to obtain the immobilized cephalosporin acylase.
An immobilized enzyme cracking experiment, namely adding 30g of immobilized cephalosporin acylase into a 1000m L cephalosporin C cracking experiment device, dissolving 10g of cephalosporin C sodium salt into 400m L of water, adjusting the pH to 8.5 by ammonia water, controlling the temperature to be 20-25 ℃, controlling the pH to be 8.5-9.0 by using an automatic control ammonia adding device, terminating the reaction when 0.00025% of 10g of cephalosporin C sodium remains in liquid phase tracking reaction, retaining the immobilized enzyme in the experiment device through a filter screen below the experiment device, continuing the next batch production, and collecting and crystallizing filtrate to prepare 7-aminocephalosporanic acid (7-ACA).
The results of the performance test of the immobilized cephalosporin acylase are shown in Table 1.
TABLE 1 results of examining the performance of the immobilized cephalosporin acylase prepared in example 1
As can be seen from the data in Table 1, the immobilized cephalosporin acylase provided by the embodiment has high enzyme activity, the enzyme activity is not obviously reduced after 20 batches of cephalosporin C sodium are cracked, and the stability is good.
Example 2
Step 1) connection of bridge and carrier:
a. acetonitrile 600m L, purified water 60m L, 30g of amino resin and 3m L of triethylamine are added under the stirring of 50rpm, the temperature is reduced to minus 10 to minus 5℃ under the stirring
b.4.5g of bromobutyryl bromide were dissolved in 10m L acetonitrile.
c. And (3) controlling the temperature to be-8 to-2 ℃, and dropwise adding the acetonitrile solution of the bromobutyryl bromide into the amino resin solution. The dripping time is 20-40 min, and the temperature is controlled to be-8 to-2 ℃ to react for 2-3 h.
d. Suction filtration, the filter cake was washed twice with 100m L acetonitrile and twice with 100m L purified water, the carrier already carrying the bridge.
Step 2) connecting the carrier with the bridge and cephalosporin acylase.
a, 50m L (enzyme activity 100U/m L) cephalosporin acylase aqueous solution, stirring and adding 31g of carrier with a bridging body, stirring at the speed of 30-60 rpm, and cooling to 0-15 ℃.
b.2.7m L triethylamine was dissolved in 10m L water.
c. And controlling the reaction temperature to be 0-10 ℃, dropwise adding the triethylamine aqueous solution into the cephalosporin acylase aqueous solution for 20-40 min. Controlling the temperature to be 0-15 ℃, and reacting for 8-20 h.
d. And (5) carrying out suction filtration, and washing with purified water of 200m L for three times to obtain the immobilized cephalosporin acylase.
An immobilized enzyme cracking experiment, namely adding 30g of immobilized cephalosporin acylase into a 1000m L cephalosporin C cracking experiment device, dissolving 10g of cephalosporin C sodium salt into 400m L of water, adjusting the pH to 8.5 with ammonia water, controlling the temperature to be 20-25 ℃, controlling the pH to be 8.5-9.0 by using an automatic control ammonia adding device, terminating the reaction when 0.00025% of 10g of cephalosporin C sodium remains in liquid phase tracking reaction, retaining the immobilized enzyme in the experiment device through a filter screen below the experiment device, continuing the next batch of production, and collecting and crystallizing filtrate to prepare 7-aminocephalosporanic acid (7-ACA).
The results of the performance test of the immobilized cephalosporin acylase are shown in Table 2.
TABLE 2 results of examining the performance of the immobilized cephalosporin acylase prepared in example 2
As can be seen from the data in Table 2, the immobilized cephalosporin acylase provided by the invention has high enzyme activity, the enzyme activity is not obviously reduced after 20 batches of cephalosporin C sodium salts are cracked, and the stability is good.
Example 3
Step 1) connection of bridge and carrier:
a. 600m of tetrahydrofuran L, 60m of purified water L and 30g of amino resin are added under stirring at 70rpm, 3m of triethylamine L is added, and the mixture is stirred and cooled to-5-0 ℃.
b.3.3g of chlorohexanoyl chloride were dissolved in 10m L of tetrahydrofuran.
c. And (3) controlling the temperature to be-5-0 ℃, and dropwise adding a tetrahydrofuran solution of chlorohexanoyl chloride into the amino resin solution. The dropping time is 20-50 min, and the temperature is controlled to be-5-0 ℃ to react for 2-6 h.
d. Suction filtration was carried out, and the filter cake was washed twice with 100m L tetrahydrofuran and twice with 100m L purified water, the carrier having the bridge.
Step 2) connecting the carrier with the bridge and cephalosporin acylase.
a, 50m L (enzyme activity 100U/m L) cephalosporin acylase aqueous solution, stirring and adding 31g of carrier with a bridging body, stirring at 40-70 rpm, and cooling to 10-20 ℃.
b.2.7m L triethylamine was dissolved in 10m L water.
c. And controlling the reaction temperature to be 10-20 ℃, dropwise adding the triethylamine aqueous solution into the cephalosporin acylase aqueous solution for 20-50 min, and controlling the temperature to be 10-20 ℃ to react for 10-20 h.
d. And (5) carrying out suction filtration, and washing with purified water of 200m L for three times to obtain the immobilized cephalosporin acylase.
An immobilized enzyme cracking experiment, namely adding 30g of immobilized cephalosporin acylase into a 1000m L cephalosporin C cracking experiment device, dissolving 10g of cephalosporin C sodium salt into 400m L of water, adjusting the pH to 8.5 by ammonia water, controlling the temperature to be 20-25 ℃, controlling the pH to be 8.5-9.0 by using an automatic control ammonia adding device, terminating the reaction when 0.00025% of 10g of cephalosporin C sodium remains in liquid phase tracking reaction, retaining the immobilized enzyme in the experiment device through a filter screen below the experiment device, continuing the next batch production, and collecting and crystallizing filtrate to prepare 7-aminocephalosporanic acid (7-ACA).
The results of the performance test of the immobilized cephalosporin acylase are shown in Table 3.
TABLE 3 results of examining the performance of the immobilized cephalosporin acylase prepared in example 3
As can be seen from the data in Table 3, the immobilized cephalosporin acylase provided by the invention has high enzyme activity, no obvious reduction of the enzyme activity after cracking 20 batches of cephalosporin C sodium salts, and good stability.
As can be seen from the above examples, the method for immobilizing cephalosporin acylase provided by the invention is simple to operate, and the obtained immobilized cephalosporin acylase has high enzymatic activity and good stability.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A method for immobilizing cephalosporin acylase, which comprises the following steps:
1) dropwise adding the bridging body solution into the carrier solution, carrying out a first condensation reaction for 2-5 h, carrying out first solid-liquid separation, and collecting solid phase components to obtain a carrier with a bridging body;
2) mixing and stirring the carrier with the bridging body obtained in the step 1) and a cephalosporin acylase aqueous solution to obtain a mixed material liquid, dropwise adding triethylamine into the mixed material liquid to perform a second condensation reaction for 8-20 h, performing second solid-liquid separation, and collecting a solid phase component which is immobilized cephalosporin acylase;
the bridging body is chloracetyl chloride or bromobutyryl bromide;
the carrier is amino resin;
the dripping time in the step 1) is 20-60 min, and the temperature of the dripping and the first condensation reaction is independently-10-0 ℃;
the mass ratio of the bridging body to the carrier is (0.05-0.2): 1;
the mass ratio of the carrier with the bridge to the cephalosporin acylase aqueous solution in the step 2) is 1 (1-5), and the enzyme activity of the cephalosporin acylase aqueous solution is 80-100U/m L;
the mass ratio of the carrier with the bridging body to triethylamine in the step 2) is 1: (0.1 to 0.5);
the carrier solution comprises amino resin, an acid-binding agent, an aprotic polar solvent mutually soluble with water and water; the mass ratio of the amino resin, the acid-binding agent, the water-miscible aprotic polar solvent and water is 1: (0.1-0.5): (5-20): (2-5), wherein the acid-binding agent is triethylamine, sodium bicarbonate or sodium carbonate;
the bridging body solution comprises a bridging body and a solvent, and the mass ratio of the bridging body to the solvent is (0.05-0.2): (0.1-1), wherein the solvent is an aprotic polar solvent which is mutually soluble with water.
2. The method for immobilizing cephalosporin acylase according to claim 1, wherein the first solid-liquid separation in step 1) is suction filtration, and the filter cake after suction filtration is washed with an organic solvent and water in sequence to obtain the carrier with the bridge.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4057685A (en) * | 1972-02-02 | 1977-11-08 | Abbott Laboratories | Chemically modified endotoxin immunizing agent |
| CN101798372A (en) * | 2010-04-13 | 2010-08-11 | 苏州大学 | Polymer microsphere and preparation method thereof |
| CN101848988A (en) * | 2007-11-19 | 2010-09-29 | 波兰纽克大学有机化学学院 | Lovastatin esterase enzyme immobilized on solid support, process for enzyme immobilization, use of immobilized enzyme, biocatalytic flow reactor and process for preparation and/or purification of simvastatin |
| CN106148313A (en) * | 2015-04-09 | 2016-11-23 | 中国医药工业研究总院 | A kind of immobilization cephalosporin C acrylase and preparation method thereof |
-
2018
- 2018-11-08 CN CN201811325606.7A patent/CN109402104B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4057685A (en) * | 1972-02-02 | 1977-11-08 | Abbott Laboratories | Chemically modified endotoxin immunizing agent |
| CN101848988A (en) * | 2007-11-19 | 2010-09-29 | 波兰纽克大学有机化学学院 | Lovastatin esterase enzyme immobilized on solid support, process for enzyme immobilization, use of immobilized enzyme, biocatalytic flow reactor and process for preparation and/or purification of simvastatin |
| CN101798372A (en) * | 2010-04-13 | 2010-08-11 | 苏州大学 | Polymer microsphere and preparation method thereof |
| CN106148313A (en) * | 2015-04-09 | 2016-11-23 | 中国医药工业研究总院 | A kind of immobilization cephalosporin C acrylase and preparation method thereof |
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