CN101285060B - Process of chitosan-arginine resin anion immobilizing chymotrypsin - Google Patents

Abstract

A method for immobilizing chymotrypsin with chitosan-arginine anion resin relates to a method for immobilizing enzyme with chitosan derivatives. The invention uses commercially available chitosan as a raw material, and firstly prepares a cross-linked chitosan resin. Then prepare chitosan-arginine anion resin, and finally carry out Schiff reaction to immobilize chymotrypsin to obtain the finished product. The invention has the advantages of simple method, convenient operation, mild reaction conditions, low production cost, high stability of immobilized enzyme, high enzyme activity recovery rate of 89.95%, and long half-life, that is, the half-life at 75°C is as long as 18 hours, and the half-life at 4°C Up to 56 days, the optimum pH value is only 5.92. The product prepared by the invention can be widely used in various aqueous phase and non-aqueous phase enzymatic reactions in industries such as medicine, food, and light industry.

Description

Method for immobilizing chymotrypsin with chitosan-arginine anion resin

technical field

The invention belongs to the technical field of enzyme immobilization, in particular to a method for immobilizing enzymes with chitosan derivatives.

technical background

Studies have shown that: chymotrypsin has the functions of digesting pus and necrotic tissue, eliminating wounds, local anti-inflammation, promoting hematoma absorption, and wound healing in medicine; it has the functions of decomposing protein and depilating leather in food and light industries. At present, the free enzymes used mainly have the disadvantages of high cost, low utilization rate, and the discharged waste enzymes pollute the environment. Therefore, the development and use of a new type of immobilized chymotrypsin with a longer half-life has important economic and social value.

Chitosan is a natural polymer material that is abundant in nature and cheap and easy to obtain. Because of its good film-forming, moisturizing, adsorption, and antibacterial properties, it is widely used in medicine, food, light industry and other industries. And because chitosan is easy to biodegrade, safe and non-toxic, has good biocompatibility, and is easy to carry out chemical modification, especially chitosan derivatives have the characteristics of good hydrophilicity, high rigidity, and good mechanical properties, which are excellent. Carriers for immobilized enzymes.

Existing methods for immobilizing enzymes include embedding, adsorption, covalent binding and cross-linking. The embedding method refers to embedding enzymes in polymer gel fine grids or semi-permeable membranes. The preparation process is simple and the reaction conditions are relatively mild, and high enzyme activity recovery can be obtained, because only small molecules can pass through polymers. Gel grid, and diffusion resistance will lead to reduced enzyme activity, so the embedding method is only suitable for enzymes that act on small molecule substrates and products. The adsorption method refers to the immobilization method in which the enzyme is adsorbed on an insoluble carrier. This method is easy to operate, with mild conditions, little loss of enzyme activity, and the carrier is cheap and easy to obtain, and can be used repeatedly. However, the interaction between the enzyme and the carrier is weak. , easy to fall off. The covalent binding method refers to the immobilization method in which the enzyme and the carrier are covalently bonded. The immobilized enzyme is not easy to fall off and can be used continuously for a long time, but the activation of the carrier is more complicated, and the binding method is more intense. The reaction caused a large loss of enzyme activity. The cross-linking method refers to the immobilization method of cross-linking between the enzyme and the carrier with a bifunctional or multifunctional reagent. The cross-linking method has good stability of the immobilized enzyme, such as "Chitosan" published in the first issue of the Journal of Ningbo University in March 1999. Preparation of immobilized trypsin and research on its physical and chemical properties” discloses the method of immobilizing trypsin on the chitosan obtained from chitosan crude product under the action of cross-linking agent glutaraldehyde The disadvantage of this method is that it is difficult to obtain pure chitosan from the crude product of chitosan, and chitosan is directly used as a carrier. When the macromolecule of trypsin is close to it, the steric hindrance is large, and the efficiency of direct crosslinking and immobilization of the enzyme is low. It is easy to cause self-crosslinking of trypsin molecules. Another example is the "Preparation Method of Nanomagnetic Chitosan Immobilized Enzyme" patent with publication number CN1904043A, which discloses the use of nanomagnetic chitosan containing acid chloride groups on the surface to react with enzyme protein molecules under the action of a magnetic field to prepare immobilized enzymes. method; the disadvantage of this method is that it is difficult to prepare a nano-magnetic chitosan carrier with a certain particle size, and at the same time, the nano-magnetic chitosan is easily oxidized and precipitated during use, resulting in a decrease in enzyme activity.

Contents of the invention

The purpose of the present invention is to provide a method for immobilizing chymotrypsin with chitosan-arginine anion resin aiming at the shortcomings of the existing chitosan resin immobilization enzyme method. The method has the characteristics of simple operation, high recovery rate of enzyme activity, mild reaction conditions, and low preparation cost. At the same time, compared with free chymotrypsin, the thermal stability of immobilized chymotrypsin is increased, the applicable pH value is lowered, and the half-life is extended. It can be used repeatedly, reducing pollution and making full use of resources.

Mechanism of the present invention: because chitosan molecular chain contains a large amount of free amino groups, these amino groups have a lone pair of electrons, have very strong nucleophilicity, and glutaraldehyde is a kind of bifunctional reagent commonly used, can combine with chitosan The amino group of the sugar undergoes a cross-linking reaction to form a Schiff base structure, and the cross-linking mainly occurs between molecules; dicyclohexylcarbodiimide (DCC) is a commonly used peptide condensation agent, which can activate the carboxyl group, that is, strengthen the The electrophilicity of the carbon atom makes it easy for the nucleophilic non-ionized amino group to attack it, resulting in a condensation reaction to form a peptide bond. Using glutaraldehyde as a cross-linking agent again, the free amino groups on the surface of chymotrypsin and the residual amino groups of arginine undergo a shiff reaction to obtain chitosan-arginine anion resin immobilized chymotrypsin.

The object of the present invention is achieved like this: a kind of method for chitosan-arginine anion resin immobilization chymotrypsin, take commercially available chitosan as raw material, first prepare cross-linked chitosan microsphere, then prepare The chitosan-arginine anion resin is finally subjected to Schiff reaction to immobilize chymotrypsin to obtain the finished product. The specific method steps are as follows:

(1) Preparation of cross-linked chitosan resin

Using commercially available chitosan as a raw material, first add chitosan to dilute hydrochloric acid with a mass percentage of 1-5% hydrochloric acid, stir and dissolve, and prepare a chitosan acid sol with a mass percentage of chitosan of 1-4% . Finally, according to the ratio of chitosan acidic sol: liquid paraffin volume ratio of 1: 1 ~ 4, slowly add chitosan acidic sol into liquid paraffin, stir for 20 ~ 30min, and heat up to 35 ~ 45 ° C to obtain the shell After the polycan paraffin solution, add Tween 80 which accounts for 0.05-0.25% of the volume fraction of the chitosan paraffin solution, emulsifies for 20-30 minutes, and then adds 0.05-0.3% glutaraldehyde which accounts for the volume fraction of the chitosan paraffin solution. The cross-linking agent makes some of the amino groups of chitosan molecules participate in the cross-linking reaction, so that the linear chitosan molecules are transformed into chitosan microspheres. Then add 0.01-0.1% calcium carbonate porogen in the mass fraction of the chitosan paraffin wax solution, and carry out pore-forming reaction for 50-70 minutes to prepare a suspension of chitosan microspheres. Finally, adjust the pH value of the chitosan microsphere suspension to 9-10 with sodium hydroxide solution, heat to 55-75° C., stir and solidify for 2-4 hours, then filter, discard the filtrate, and collect the filter residue. The filter residue is washed with ethanol for 3 to 6 times, and then the calcium carbonate in the filter residue is dissolved with dilute hydrochloric acid with a mass fraction of 1 to 5%, and then washed with distilled water until neutral to prepare a cross-linked chitosan resin.

(2) Preparation of chitosan-arginine anion resin

After the first step (1) is completed, the cross-linked chitosan resin prepared by the first step is added to the arginine solution with a mass fraction of 0.5 to 2.0% arginine and shaken for 20 to 30 minutes to make the chitosan The resin is suspended in the arginine solution, and the chitosan-arginine solution with the chitosan mass fraction of 0.5-2.5% is prepared. Then by the arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is the ratio of 1: 0.5～2.5, in the chitosan-arginine solution, add DCC is connected with a peptide condensing agent, and the condensation reaction is carried out for 20 to 40 minutes, so that another part of the amino group in the chitosan resin and the carboxyl group of arginine undergo a condensation reaction, and the chitosan-arginine resin is prepared. Finally, after filtration, discard the filtrate and collect the filter residue. Wash the filter residue with ethanol for 3 to 6 times, then dialyze with distilled water for 8 to 12 hours, then filter, discard the filtrate, and collect the filter residue to prepare the chitosan-arginine anion resin.

(3) Immobilization of chymotrypsin

After step (2) is completed, first add chymotrypsin into the phosphate buffer solution with a pH value of 4.5 to 6.0, and stir evenly to prepare chymotrypsin with a molar concentration of 0.05 to 0.08mol/L. Lactase Phosphate Solution. Chitosan-arginine anion resin prepared by the step (2): the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is the ratio of 1: 15～25, the chitosan-arginine Add arginine anion resin to the chymotrypsin phosphate solution, shake for 20 to 40 minutes in a constant temperature water bath at 30-60°C, and suspend the chitosan-arginine anion resin in the chymotrypsin phosphate solution . Then by glutaraldehyde: the mass ratio of chitosan-arginine anion resin is the ratio of 1: 6～14, glutaraldehyde is added to the chymotrypsin phosphate of suspension chitosan-arginine anion resin solution, and placed in an oscillator to vibrate, carry out Schiff reaction for 50 to 70 minutes, carry out suction filtration, discard the filtrate, collect the resin filter residue, wash the resin filter residue with phosphate buffer saline for 4 to 7 times, and remove free pancreatin The protease is then suction filtered, and the filtrate is discarded, and the chitosan-arginine anion immobilized chymotrypsin filter residue is collected to obtain the finished product.

After the present invention adopts the above-mentioned technical scheme, it mainly has the following characteristics:

(1) The immobilization effect is good. Since there is a long aliphatic chain structure-(CH ₂ ) ₄ between the guanidinium group and the carboxyl group in the arginine molecule, when the chitosan resin reacts with arginine, it is equivalent to connecting many "cantilevers" to the chitosan resin , using it as a carrier for immobilized enzymes avoids the disadvantages of high steric hindrance when the enzyme protein macromolecule is close to the carrier, low efficiency of direct cross-linking immobilized enzymes, and easy to cause self-crosslinking of enzyme molecules, so the effect of immobilized enzymes is better ; Carrying out the enzymatic reaction with the immobilized enzyme is more conducive to the contact between the immobilized enzyme and the reaction medium, and improves the reaction efficiency.

(2) The stability of the immobilized enzyme is high. The method of the present invention adopts the glutaraldehyde cross-linking method to fix chymotrypsin, increases the binding force between the enzyme and the carrier, improves the mechanical strength of the immobilized enzyme, and avoids the defect that the enzyme molecule falls off from the carrier during use, so that the enzyme The stability has been further enhanced.

(3) The applicable pH value is lowered. The optimum pH value of free chymotrypsin is 8.0, while that of immobilized chymotrypsin is only 5.92, which further expands the scope of use of chymotrypsin.

(4) Low production cost. The method of the invention adopts the chitosan-arginine anion resin immobilized chymotrypsin to be more easily separated from the organic solvent, is more conducive to the recovery and utilization of the enzyme, improves the use efficiency of the enzyme, and reduces the production cost.

(5) The recovery rate of enzyme activity is high and the half-life is long. The thermal stability of the immobilized chymotrypsin is increased, and the recovery rate of the enzyme activity is as high as 89.95%. The half-life is extended, that is, the half-life at 75°C is as long as 18 hours, and the half-life at 4°C is as long as 56 days.

(6) The method of the present invention is simple, easy to operate, and the reaction conditions are mild in the production process, which is convenient for popularization and application.

The chitosan-arginine anion resin immobilized chymotrypsin prepared by the method of the invention can be widely used in various water phase and non-aqueous phase enzymatic reactions in medicine, food, light industry and other industries.

Detailed ways

The present invention will be further described below in combination with specific embodiments.

Embodiment one

A kind of concrete steps of the method for chitosan-arginine anion resin immobilization chymotrypsin are as follows:

(1) Preparation of cross-linked chitosan resin

Using commercially available chitosan as a raw material, the chitosan is firstly added into dilute hydrochloric acid with a mass percentage of 2% hydrochloric acid, stirred and dissolved, and a chitosan acid sol with a mass percentage of chitosan of 2% is prepared. Afterwards, the volume ratio of chitosan acid sol: liquid paraffin is 1: 1, chitosan acid sol is slowly added in liquid paraffin, stirred for 25min, and heated to 40°C to obtain the chitosan paraffin solution, Adding and accounting for chitosan paraffin solution volume fraction is 0.1% Tween 80 again, carry out emulsification 25min, then add and account for chitosan paraffin solution volume fraction and be 0.15% glutaraldehyde cross-linking agent, make the part amino group of chitosan molecule Participate in the cross-linking reaction, thereby converting linear chitosan molecules into chitosan microspheres. Then add 0.05% calcium carbonate pore-forming agent accounting for the mass fraction of the chitosan paraffin solution, and carry out pore-forming reaction for 60 minutes to prepare a suspension of chitosan microspheres. Finally, adjust the pH value of the chitosan microsphere suspension to 9.5 with sodium hydroxide solution, heat to 60° C., stir and solidify for 3 hours, then filter, discard the filtrate, and collect the filter residue. The filter residue was first washed with ethanol for 5 times, and then the calcium carbonate in the filter residue was dissolved with 3% dilute hydrochloric acid, and then washed with distilled water until neutral to prepare a cross-linked chitosan resin.

(2) Preparation of chitosan-arginine anion resin

After the first (1) step was completed, the cross-linked chitosan resin prepared by the (1) step was added to the arginine mass fraction of 1% arginine solution, and vibrated for 25 minutes to suspend the chitosan resin in the arginine solution. A chitosan-arginine solution with a chitosan mass fraction of 1.5% was prepared. Then by the arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is the ratio of 1: 1, in the chitosan-arginine solution, add DCC to connect Peptide condensing agent, carry out condensation reaction 30min, make another part of amino group in chitosan resin and the carboxyl group of arginine produce condensation reaction, just prepare chitosan-arginine resin. Finally, after filtration, discard the filtrate and collect the filter residue. The filter residue was first washed with ethanol for 4 times, then dialyzed with distilled water for 10 hours, filtered again, the filtrate was discarded, and the filter residue was collected to prepare the chitosan-arginine anion resin.

(3) Immobilization of chymotrypsin

After step (2) is completed, first add chymotrypsin to the phosphate buffer with a pH value of 5.9, stir evenly, and then prepare chymotrypsin phosphate with a molar concentration of chymotrypsin of 0.06mol/L solution. Chitosan-arginine anion resin prepared by the step (2): the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is the ratio of 1: 20, the chitosan-arginine Add the anion resin to the chymotrypsin phosphate solution, shake for 30 minutes under the condition of a constant temperature water bath at 45° C., and suspend the chitosan-arginine anion resin in the chymotrypsin phosphate solution. Then by glutaraldehyde: the mass ratio of chitosan-arginine anion resin is the ratio of 1: 8, glutaraldehyde is added in the chymotrypsin phosphate solution of suspended chitosan-arginine anion resin , and placed in an oscillator to oscillate, carry out Schiff reaction for 60min, carry out suction filtration, discard the filtrate, collect the resin filter residue, wash the resin filter residue 6 times with phosphate buffer saline, remove free chymotrypsin, and then suction filter, Then discard the filtrate, and then collect chitosan-arginine anion-immobilized chymotrypsin filter residue to obtain the finished product.

Embodiment two

A kind of concrete steps of the method for chitosan-arginine anion resin immobilization chymotrypsin are as follows:

(1) Preparation of cross-linked chitosan resin

Same as Example 1, the feature is that chitosan is added into dilute hydrochloric acid with a mass percentage of 1% hydrochloric acid to prepare a chitosan acidic sol with a mass percentage of chitosan. Chitosan acidic sol: the volume ratio of liquid paraffin is 1: 2, stir for 20min, and heat up to 35°C, then add Tween 80 accounting for 0.05% of the chitosan paraffin solution volume fraction, emulsify for 20min, and then add The volume fraction of the chitosan paraffin solution is 0.05% glutaraldehyde crosslinking agent, and then the chitosan paraffin solution mass fraction is 0.01% calcium carbonate porogen, and the pore-forming reaction is carried out for 50 minutes. Use sodium hydroxide solution to adjust the pH value of the chitosan microsphere suspension to 9, heat to 55 ° C, stir and solidify for 2 hours, wash the filter residue with ethanol for 3 times, and then dissolve the filter residue with 1% dilute hydrochloric acid. of calcium carbonate.

(2) Preparation of chitosan-arginine anion resin

Same as Example 1, the feature is: the mass fraction of arginine in the arginine solution is 0.5%, shake for 20 minutes, and prepare a chitosan-arginine solution with a mass fraction of chitosan of 0.5%. The molar ratio of arginine: dicyclohexylcarbodiimide (DCC) in the chitosan-arginine solution is 1:0.5, carry out the condensation reaction for 20 minutes, wash the filter residue with ethanol for 3 times, and then dialyze with distilled water for 8 hours .

(3) Immobilization of chymotrypsin

Same as Example 1, the feature is: firstly add chymotrypsin into the phosphate buffer with a pH value of 4.5 to prepare a chymotrypsin phosphate solution with a molar concentration of chymotrypsin of 0.05 mol/L. Chitosan-arginine anion resin prepared in (2): the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is 1: 15, under the constant temperature water bath condition of 30 ℃, shake 20min, The mass ratio of glutaraldehyde: chitosan-arginine anion resin was 1:6, Schiff reaction was carried out for 50 min, and the resin filter residue was washed 4 times with phosphate buffer saline.

Embodiment Three

A kind of concrete steps of the method for chitosan-arginine anion resin immobilization chymotrypsin are as follows:

(1) Preparation of cross-linked chitosan resin

Same as Example 1, the feature is that chitosan is added into dilute hydrochloric acid with a mass percentage of 5% hydrochloric acid to prepare a chitosan acidic sol with a mass percentage of chitosan of 4%. Chitosan acidic sol: the volume ratio of liquid paraffin is 1: 4, stir for 30min, and heat up to 45°C, then add Tween 80 accounting for 0.25% of the chitosan paraffin solution volume fraction, emulsify for 30min, and then add The volume fraction of the chitosan paraffin solution is 0.3% glutaraldehyde crosslinking agent, and the mass fraction of the chitosan paraffin solution is 0.1% calcium carbonate porogen, and the pore-forming reaction is carried out for 70 minutes. Use sodium hydroxide solution to adjust the pH value of the chitosan microsphere suspension to 10, heat to 75°C, stir and solidify for 4 hours, wash the filter residue with ethanol for 6 times, and then dissolve the filter residue with 5% dilute hydrochloric acid. of calcium carbonate.

(2) Preparation of chitosan-arginine anion resin

Same as Example 1, the feature is: the mass fraction of arginine in the arginine solution is 2.0%, shake for 30 minutes, and prepare a chitosan-arginine solution with a mass fraction of chitosan of 2.5%. The molar ratio of arginine: dicyclohexylcarbodiimide (DCC) in the chitosan-arginine solution is 1: 2.5, carry out the condensation reaction for 40 minutes, wash the filter residue with ethanol for 6 times, and then dialyze with distilled water for 12 hours .

(3) Immobilization of chymotrypsin

Same as Example 1, the feature is: firstly add chymotrypsin into the phosphate buffer with a pH value of 6.0 to prepare a chymotrypsin phosphate solution with a molar concentration of chymotrypsin of 0.08 mol/L. The chitosan-arginine anion resin prepared in the 2nd step: the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is 1: 25, under the constant temperature water bath condition of 60 ℃, shake 40min, The mass ratio of glutaraldehyde: chitosan-arginine anion resin was 1:14. After Schiff reaction was carried out for 70 min, the resin filter residue was washed 7 times with phosphate buffer saline.

Experimental results

The products prepared in Examples 1-3 were tested for optimum catalytic temperature, optimum catalytic pH value, enzyme activity recovery rate and half-life of chitosan-arginine anion resin immobilized chymotrypsin. The test results are as follows:

It can be seen from the above table that the thermal stability of chitosan-arginine anion resin immobilized chymotrypsin is higher than that of free chymotrypsin, and the optimum catalytic temperature of immobilized chymotrypsin is 70°C, which is higher than that of free chymotrypsin. Lactase increased by 10°C; the optimal catalytic pH value of immobilized chymotrypsin was 5.92, which was 2.08 lower than that of free chymotrypsin, which expanded the application range of immobilized chymotrypsin; The live recovery rate is as high as 89.95%, the half-life at 75°C is as long as 18 hours, and the half-life at 4°C is as long as 56 days, which improves the utilization efficiency of immobilized chymotrypsin, reduces pollution, and makes full use of resources.

Claims (4)

1. a method for chitosan-arginine anion resin immobilization chymotrypsin, is characterized in that concrete method step is as follows: (1) Preparation of cross-linked chitosan resin Using commercially available chitosan as raw material, first add chitosan to dilute hydrochloric acid with a mass percentage of 1-5% hydrochloric acid to prepare a chitosan acid sol with a mass percentage of chitosan of 1-4%, and then press the shell The volume ratio of polysaccharide acid sol: liquid paraffin is 1:1-4, slowly add chitosan acid sol into liquid paraffin, stir for 20-30min, and heat up to 35-45°C to obtain chitosan paraffin After the solution, add Tween 80 accounting for 0.05-0.25% of the volume fraction of the chitosan paraffin solution, emulsify for 20-30 minutes, and then add 0.05-0.3% of the volume fraction of the chitosan paraffin solution glutaraldehyde crosslinking agent , then add 0.01-0.1% calcium carbonate pore-forming agent accounting for the mass fraction of the chitosan paraffin wax solution, carry out the pore-forming reaction for 50-70 minutes to obtain a suspension of chitosan microspheres, and finally adjust the chitosan with sodium hydroxide solution The pH value of the microsphere suspension is 9-10, and heated to 55-75°C, stirred and solidified for 2-4 hours, then filtered, discarded the filtrate, collected the filter residue, washed the filter residue with ethanol for 3-6 times, and then used the mass fraction Dissolve the calcium carbonate in the filter residue with 1-5% dilute hydrochloric acid, then wash with distilled water until neutral; (2) Preparation of chitosan-arginine anion resin After step (1) is completed, add the cross-linked chitosan resin prepared in step (1) into arginine solution with a mass fraction of 0.5-2.0% arginine, shake for 20-30 minutes, and prepare chitosan The sugar mass fraction is the chitosan-arginine solution of 0.5～2.5%, then according to the mol ratio of arginine in the chitosan-arginine solution: dicyclohexylcarbodiimide is 1: 0.5～2.5 Ratio, in the chitosan-arginine solution, add dicyclohexyl carbodiimide peptide condensing agent, carry out condensation reaction for 20-40min, finally after filtering, discard the filtrate, collect the filter residue, wash the filter residue with ethanol for 3 ~6 times, then dialyze with distilled water for 8~12h, then filter, then discard the filtrate, and then collect the filter residue; (3) Immobilization of chymotrypsin After step (2) is completed, first add chymotrypsin into the phosphate buffer solution with a pH value of 4.5 to 6.0, and stir evenly to prepare chymotrypsin with a molar concentration of 0.05 to 0.08mol/L. Lactase phosphate solution, the chitosan-arginine anion resin prepared by (2) step: the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is the ratio of 1: 15～25, Add the chitosan-arginine anion resin into the chymotrypsin phosphate solution, shake it for 20-40 minutes under the condition of constant temperature water bath at 30-60°C, and then press glutaraldehyde: chitosan-arginine anion The mass ratio of the resin is a ratio of 1:6 to 14, glutaraldehyde is added to the chymotrypsin phosphate solution of the suspended chitosan-arginine anion resin, and placed in a shaker to vibrate, and the Schiff After reacting for 50 to 70 minutes, perform suction filtration, discard the filtrate, collect the resin filter residue, wash the resin filter residue with phosphate buffer for 4 to 7 times, then suction filter, then discard the filtrate, and then collect chitosan-arginine anion fixation Chymotrypsin filter residue. 2. according to the method for chitosan-arginine anion resin immobilization chymotrypsin according to claim 1, it is characterized in that concrete method step is as follows: In the (1) step, adding chitosan to hydrochloric acid mass percentage is in 2% dilute hydrochloric acid, prepares the chitosan acid sol that chitosan mass percentage is 2%, chitosan acid sol: the volume ratio of liquid paraffin 1:1, stirred for 25min, and heated up to 40°C, then added Tween 80 accounting for 0.1% of the chitosan paraffin solution volume fraction, emulsified for 25min, and then added 0.15% chitosan paraffin solution volume fraction Glutaraldehyde cross-linking agent, then add 0.05% calcium carbonate pore forming agent accounting for the mass fraction of chitosan paraffin wax solution, carry out pore forming reaction for 60min, adjust the pH value of chitosan microsphere suspension with sodium hydroxide solution to be 9.5 , and heated to 60°C, stirred and solidified for 3 hours, washed the filter residue 5 times with ethanol, and then dissolved the calcium carbonate in the filter residue with 3% dilute hydrochloric acid; In the (2) step, the mass fraction of arginine in the arginine solution is 1%, shakes 25min, prepares the chitosan-arginine solution that the mass fraction of chitosan is 1.5%, the chitosan-arginine The molar ratio of arginine in the acid solution: dicyclohexylcarbodiimide is 1: 1, carry out the condensation reaction for 30 minutes, wash the filter residue with ethanol for 4 times, and then dialyze with distilled water for 10 hours; In the (3) step, first adding chymotrypsin to pH value is 5.9 phosphate buffer saline, the molar concentration of preparing chymotrypsin is the chymotrypsin phosphate solution of 0.06mol/L, the first (2 ) step prepared chitosan-arginine anion resin: the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is 1: 20, under the constant temperature water bath condition of 45 ℃, shake 30min, glutaraldehyde : The mass ratio of chitosan-arginine anion resin is 1: 8, after carrying out Schiff reaction for 60min, the resin filter residue is washed 6 times with phosphate buffer saline. 3. according to the method for chitosan-arginine anion resin immobilization chymotrypsin according to claim 1, it is characterized in that concrete method step is as follows: In the (1) step, adding chitosan to hydrochloric acid mass percentage is in 1% dilute hydrochloric acid, prepares the chitosan acid sol that chitosan mass percentage is 1%, chitosan acid sol: the volume ratio of liquid paraffin 1:2, stirred for 20min, and heated up to 35°C, then added Tween 80 accounting for 0.05% of the chitosan paraffin solution volume fraction, emulsified for 20min, and then added 0.05% Tween 80 accounting for the chitosan paraffin solution volume fraction glutaraldehyde cross-linking agent, then add 0.01% calcium carbonate pore-forming agent accounting for the mass fraction of chitosan paraffin wax solution, carry out pore-forming reaction 50min, adjust the pH value of chitosan microsphere suspension with sodium hydroxide solution to be 9 , and heated to 55°C, stirred and solidified for 2 hours, washed the filter residue three times with ethanol, and then dissolved the calcium carbonate in the filter residue with a mass fraction of 1% dilute hydrochloric acid; In the (2) step, the arginine mass fraction is 0.5% in the arginine solution, shakes 20min, prepares the chitosan-arginine solution that the chitosan mass fraction is 0.5%, chitosan-arginine The molar ratio of arginine in the acid solution: dicyclohexylcarbodiimide is 1:0.5, carry out the condensation reaction for 20 minutes, wash the filter residue with ethanol for 3 times, and then dialyze with distilled water for 8 hours; In the (3) step, first adding chymotrypsin to pH value is 4.5 phosphate buffer saline, the molar concentration of preparing chymotrypsin is the chymotrypsin phosphate solution of 0.05mol/L, the first (2 ) step prepared chitosan-arginine anion resin: the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is 1: 15, under the constant temperature water bath condition of 30 ℃, shake 20min, glutaraldehyde : The mass ratio of chitosan-arginine anion resin is 1: 6, after Schiff reaction is carried out for 50min, the resin filter residue is washed 4 times with phosphate buffer saline. 4. according to the method for chitosan-arginine anion resin immobilization chymotrypsin according to claim 1, it is characterized in that concrete method step is as follows: In the (1) step, adding chitosan to hydrochloric acid mass percentage is in 5% dilute hydrochloric acid, prepares the chitosan acid sol that chitosan mass percentage is 4%, chitosan acid sol: the volume ratio of liquid paraffin 1:4, stirred for 30min, and heated up to 45°C, then added Tween 80 that accounted for 0.25% of the chitosan paraffin solution volume fraction, emulsified for 30min, and then added 0.3% Tween 80 accounted for chitosan paraffin solution volume fraction glutaraldehyde cross-linking agent, then add 0.1% calcium carbonate pore-forming agent accounting for the mass fraction of chitosan paraffin wax solution, carry out pore-forming reaction 70min, adjust the pH value of chitosan microsphere suspension with sodium hydroxide solution to be 10 , and heated to 75°C, stirred and solidified for 4 hours, washed the filter residue 6 times with ethanol, and then dissolved the calcium carbonate in the filter residue with a mass fraction of 5% dilute hydrochloric acid; In the (2) step, the arginine mass fraction is 2.0% in the arginine solution, shakes 30min, prepares the chitosan-arginine solution that the chitosan mass fraction is 2.5%, chitosan-arginine The molar ratio of arginine in the acid solution: dicyclohexylcarbodiimide is 1: 2.5, carry out the condensation reaction for 40 minutes, wash the filter residue with ethanol for 6 times, and then dialyze with distilled water for 12 hours; In the (3) step, first adding chymotrypsin to pH value is 6.0 phosphate buffer saline, the molar concentration of preparing chymotrypsin is the chymotrypsin phosphate solution of 0.08mol/L; ) step prepared chitosan-arginine anion resin: the mass ratio of chymotrypsin in the chymotrypsin phosphate solution is 1: 25, under the constant temperature water bath condition of 60 ℃, shake 40min; Glutaraldehyde : The mass ratio of chitosan-arginine anion resin is 1: 14, after carrying out Schiff reaction for 70min, the resin filter residue is washed 7 times with phosphate buffer saline.