CN114686468B - Ion exchange resin selective immobilized enzyme, preparation method and application thereof - Google Patents

Ion exchange resin selective immobilized enzyme, preparation method and application thereof Download PDF

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CN114686468B
CN114686468B CN202210462716.8A CN202210462716A CN114686468B CN 114686468 B CN114686468 B CN 114686468B CN 202210462716 A CN202210462716 A CN 202210462716A CN 114686468 B CN114686468 B CN 114686468B
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ion exchange
exchange resin
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CN114686468A (en
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东为富
王康静
丁重阳
王世波
李婷
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Jiangnan University
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    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01008—Mannose-6-phosphate isomerase (5.3.1.8)

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Abstract

The invention provides an ion exchange resin selective immobilized enzyme, a preparation method and application thereof, belonging to the technical field of immobilized enzyme preparation, wherein the preparation method comprises the following steps: firstly, preprocessing macroporous ion exchange resin; the second step, the ion exchange resin adsorbs transition metal ions; thirdly, adding a buffer solution to react off superfluous active groups; fourth, his-tagged protease is added for immobilization. The ion exchange resin column prepared by the invention has the advantages of simple preparation, low cost, simple and mild reaction conditions and capability of realizing the purification and immobilization processes simultaneously. The immobilized enzyme prepared by the method has firm combination and can perform enzyme catalytic reaction for a plurality of times.

Description

Ion exchange resin selective immobilized enzyme, preparation method and application thereof
Technical Field
The invention belongs to the technical field of preparation of immobilized enzymes, and particularly relates to preparation and application of an ion exchange resin immobilized enzyme.
Background
Compared with free enzyme, the immobilized enzyme can keep the catalytic reaction characteristic of the enzyme, and has the advantages of high storage stability, easy separation and recovery and repeated use. Conventional enzyme immobilization techniques can be divided into four types, adsorption, entrapment, crosslinking, and covalent coupling. Covalent coupling methods provide more stable immobilization by covalently attaching active amino acid residues to the surface of the support than other methods. This method requires a large number of modifiable groups on the support to be chemically coupled to the enzyme molecule, so that the selection of an appropriate support is important for the catalytic efficiency of the immobilized enzyme. The ion exchange resin has high specific surface area, rich surface functional groups, high adsorption capacity, rapid adsorption or desorption speed and certain adsorption selectivity. And the shape and size of the ion exchange resin are such that it can be easily and quantitatively recovered for reuse by simple filtration or elution methods.
Common uses for ion exchange resins include water purification, metal recovery and separation, ion substitution, acid base catalysis, and as sensors or solid electrolytes. The technology of ion exchange resin immobilized enzyme is gradually developed due to the abundant surface functional groups, stable physicochemical properties and moderate particle size and pore structure. CN201410769087.9 uses anion exchange resin to immobilize lipase and apply to esterification reaction; CN201710575802.9 has transaminase immobilized with anion exchange resin and is used in sitagliptin preparation; CN201110035791.8 uses epichlorohydrin to modify the ion exchange resin to obtain an ion exchange resin suitable for enzyme immobilization; however, in the above methods, the ion exchange resin is directly added into the enzyme solution and is covalently connected with the amino acid residue of the enzyme through the modifiable group of the resin, so that the catalytic activity of the immobilized enzyme is affected to a certain extent. Furthermore, these methods have limitations in the selection of enzyme types, and immobilization methods are not specific and selective, so additional purification is required before immobilization.
Disclosure of Invention
In order to solve the problems in the related art, the invention provides a preparation method of ion exchange resin immobilized enzyme and a method for using the ion exchange resin immobilized enzyme in enzyme catalysis, wherein His tag enzyme can be selectively immobilized through simple oscillation adsorption after metal ions are chelated by the ion exchange resin, the enzyme activity is not influenced, and the preparation method is simple in operation, economical and environment-friendly. The method has low requirement on the purity of the enzyme, the variety range of the selectable enzyme is wide, and the prepared immobilized enzyme is firmly combined and can perform multiple enzyme catalytic reactions.
The technical scheme provided by the invention is as follows:
a method for preparing an ion exchange resin selective immobilized enzyme, comprising:
pretreatment of ion exchange resin;
adding the pretreated ion exchange resin into an aqueous solution containing transition metal salt, and oscillating and adsorbing by a shaking table;
adding a buffer solution to react off superfluous active groups on the ion exchange resin;
the ion exchange resin is mixed with the enzyme solution and then immobilized at 4-40 ℃ by stirring or shaking.
Further, the ion exchange resin should be a macroporous ion exchange resin, including a strong acid cation resin, a weak acid cation resin, a chelate resin, or the like.
Further, the pretreatment of the ion exchange resin is to remove unpolymerized monomers, porogens, solvents, etc. remaining in the ion exchange resin and to activate the ion exchange resin to a certain extent. The pretreatment time is 4-8 h.
Further, the pretreatment method of the ion exchange resin comprises the following steps: ethanol treatment-acid treatment-alkali treatment-acid treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-acid treatment-alkali treatment-acid treatment; ethanol treatment-alkali treatment-acid treatment-alkali treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-alkali treatment-acid treatment-alkali treatment; the treatment of each step is to soak in the corresponding solution, and the time of each step is the time of equally dividing the total pretreatment time of the ion exchange resin into 4 sections, namely 1-2 hours.
Further, the mass fraction of sodium hydroxide used for the alkali treatment is 2-7%, preferably 3-5%; the mass fraction of hydrochloric acid used for the acid treatment is 2% -7%, preferably 3% -5%.
Further, the transition metal salt is at least one of copper chloride dihydrate, anhydrous copper sulfate, copper sulfate pentahydrate, copper nitrate trihydrate, zinc chloride, zinc sulfate heptahydrate, nickel chloride hexahydrate, nickel sulfate hexahydrate, cobalt chloride hexahydrate or cobalt sulfate heptahydrate.
Further, the concentration of the solution of the transition metal ion is 1000mg/L or less, preferably 300 to 500mg/L.
Further, when the ion exchange resin is added to an aqueous solution containing a transition metal salt, the solid-to-liquid ratio is 0.4 to 10mg/ml, preferably 2 to 4mg/ml.
Further, the shaking table is oscillated for adsorption for 30 min-6 h at a rotation speed of 50 rpm-200 rpm, preferably 100-150 rpm.
Further, the buffer solution is Tris-HCl solution with the concentration of 0.3M-2M, preferably 0.5M-1M; the pH is 6.8 to 10, preferably 6.8 to 8.0.
Further, the enzyme contains a His tag consisting of histidine residues.
Further, the enzyme may be a protease, including single or multiple enzyme systems.
Further, the protease may be an enzyme that itself contains a His tag or an enzyme that itself does not contain a His tag, which requires insertion of a His tag into the N-terminal or C-terminal by gene recombination techniques.
Further, when the ion exchange resin is mixed with the enzyme solution, the solid-liquid ratio is 20-200 mg/ml; the enzyme solution concentration is below 10 mg/ml.
As a second aspect of the present invention, there is provided an ion exchange resin selective immobilized enzyme, which is obtained by the process for producing the ion exchange resin selective immobilized enzyme.
As a third aspect of the present invention, there is provided a method for producing an ion exchange resin column usable for immobilized enzymes, comprising:
pretreating ion exchange resin for 4-8 h;
adding the pretreated ion exchange resin into an aqueous solution containing transition metal salt, and oscillating and adsorbing for 30 min-6 h by a shaking table;
adding a buffer solution to react off redundant active groups on the resin;
loading the above ion exchange resin into chromatography column with sieve plate, washing with deionized water 5-10 times of column volume at flow rate of 5ml/min, storing in 20% ethanol solution, and adding 5-10 times of column volume balancing solution (balancing buffer: 1 XPBS, sodium chloride 0.5 mol.L) -1 Imidazole 20mmol·L -1 pH 7.4) was equilibrated and the flow rate was controlled at 1ml/min.
The invention has the beneficial effects that: the method for modifying and post-treating the ion exchange resin column is simple, the ion exchange resin column obtained by the preparation method can be used for immobilizing the enzyme with the His tag, and the method for immobilizing the enzyme does not influence the enzyme activity; the method has low requirement on the purity of the enzyme, and the variety of the selectable enzyme is wide; the immobilized enzyme prepared by the method has firm combination, can perform enzyme catalytic reaction for a plurality of times, and has obviously improved relative activity compared with free enzyme; the immobilized enzyme ion exchange resin column prepared by the method has simple operation and convenient storage compared with the commercial agarose resin column.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 shows the preparation of an immobilized enzyme ion exchange resin column and a commercially available Co-NTA purification resin column according to example 13.
FIG. 2 shows SDS-PAGE analysis of crude phosphomannose isomerase, immobilized enzyme ion exchange resin column purified enzyme solution prepared in example 13, and Co column purified enzyme solution (Marker is protein molecular weight standard), wherein the enzyme solution obtained in example 13 has clear band at 46.1kD compared with the crude enzyme solution, which proves that the immobilized enzyme material prepared in example can simultaneously immobilize and purify target enzyme.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments are not intended to limit the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The enzyme solution concentration measuring method in the embodiment of the invention comprises the following steps:
after the modified ion exchange resin was incubated with the enzyme for a period of time, 1ml was sampled and the immobilized enzyme concentration was measured after membrane filtration. Adsorption Capacity of enzyme (Q, mg.g) -1 ) Calculated from the following equation:
C 0 : concentration of enzyme solution before adsorption (mg/g)
C: concentration of enzyme solution after adsorption (mg/g)
m: mass of immobilized ion exchange resin (mg)
V: reaction volume (mL)
The enzyme activity determination method in the embodiment of the invention comprises the following steps:
the enzyme activity reaction system consists of 0.5mM MgCl 2 15mM fructose-6-phosphate (substrate), 20mM phosphate solution (pH 7.5), 0.5. Mu.g of enzyme. After 5 minutes of reaction at 30 ℃, the reaction was stopped with a boiling water bath, centrifuged for 10 minutes, and detected by High Performance Liquid Chromatography (HPLC). The unit (U) of enzyme activity is defined as the amount of fructose-6-phosphate converted to mannose-6-phosphate per unit time. The relative enzyme activity of the immobilized enzyme was calculated by measuring the free enzyme activity (U/mg) to 100%.
Example 1
The activated recombinant strain GLpmi-QCDs were cultured overnight, 1% of the inoculum was inoculated into a fresh sterile LB medium containing 30mg/L kanamycin, and when the OD600 reached 0.6-0.8, IPTG was added to give a final concentration of 0.5mM, and the culture was carried out at 25℃for 10 hours at 150 r.min-1. The bacterial solution was centrifuged at 12000 r.min-1 for 5min, resuspended in 1 XPBS (pH=7.4) solution, sonicated in ice-water mixture, and the supernatant was centrifuged at 12000rpm for 20min at 4℃to obtain a crude phosphomannose isomerase solution, which was purified by Co column to obtain purified phosphomannose isomerase.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O is at 500mAnd (3) dissolving in deionized water, taking 100ml of the solution into a 250ml conical flask with a plug, adding 0.2g of pretreated ion exchange resin, and oscillating and adsorbing on a constant temperature shaking table at 25 ℃ at a rotating speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of nickel-loaded D001 ion exchange resin is weighed and dispersed in 10ml of coarse enzyme solution (10 mg/ml) of phosphomannose isomerase, and the immobilized phosphomannose isomerase is obtained by incubation and assembly at 4 ℃, and the loading amount and the enzyme activity of the immobilized phosphomannose isomerase are shown in Table 1.
Example 2
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 3
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.202g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml was taken in a 250ml conical flask with stopper, 0.2g of pretreated ion exchange resin was added, and 150r/mi at 25 ℃The rotation speed of n is oscillated on a constant temperature shaking table for adsorption for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 4
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. 1.012g of NiCl was weighed out 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 5
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 2.025g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin is oscillated in 0.5M Tris-HCl (pH8.0) for 1h at low speedWashing with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 6
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% NaOH solution, 4% HCl solution and 4% NaOH solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 7
Purified phosphomannose isomerase was obtained as in example 1.
20g of D401 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The nickel-loaded D401 ion exchange resin obtained was subjected to low-speed shaking in 0.5M Tris-HCl (pH 8.0) for 1 hour, and then washed with deionized water to neutrality.
0.25g of the above D401 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the loading amount and enzyme activity of the immobilized phosphomannose isomerase were shown in Table 1.
Example 8
Purified phosphomannose isomerase was obtained as in example 1.
20g of D113H ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D113H ion exchange resin was subjected to low-speed shaking in 0.5M Tris-HCl (pH 8.0) for 1 hour, and then washed with deionized water to neutrality.
0.25g of the D113H ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and the immobilized phosphomannose isomerase was obtained by incubation and assembly at 4℃and the loading amount and the enzyme activity of the immobilized phosphomannose isomerase were shown in Table 1.
Example 9
Purified phosphomannose isomerase was obtained as in example 1.
20g of D113H ion exchange resin is soaked in 100ml of deionized water, 4% NaOH solution, 4% HCl solution and 4% NaOH solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D113H ion exchange resin was subjected to low-speed shaking in 0.5M Tris-HCl (pH 8.0) for 1 hour, and then washed with deionized water to neutrality.
0.25g of the D113H ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and the immobilized phosphomannose isomerase was obtained by incubation and assembly at 4℃and the loading amount and the enzyme activity of the immobilized phosphomannose isomerase were shown in Table 1.
Example 10
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.606g CoCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained cobalt-loaded D001 ion exchange resin was subjected to low-speed shaking in 0.5M Tris-HCl (pH 8.0) for 1 hour, and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 11
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 5ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 4℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 12
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
0.25g of the above-mentioned D001 ion exchange resin was weighed and dispersed in 10ml of purified phosphomannose isomerase solution (5 mg/ml), and incubated and assembled at 20℃to obtain immobilized phosphomannose isomerase, and the amount of the immobilized enzyme and the enzyme activity were as shown in Table 1.
Example 13
A crude phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin was oscillated in 0.5M Tris-HCl (pH 8.0) at a low speed for 1 hour and then washed with deionized water to neutrality.
1ml of the above D001 ion exchange resin was put in a 6ml column empty equipped with a sieve plate, washed with 30ml of deionized water at a flow rate of 5ml/min, and equilibrated with an equilibration solution (1 XPBS, sodium chloride 0.5 mol.L) -1 Imidazole 20 mmol.L -1 After equilibration at pH7.4, the crude enzyme solution was added at a flow rate of 1ml/min, followed by elution with an elution solution (1 XPBS, sodium chloride 0.5 mol.L) -1 Imidazole 300 mmol.L -1 pH 7.4) at a flow rate of 1ml/min, the resulting purified enzyme solution bands are shown in FIG. 2。
Comparative example 1
Purified phosphomannose isomerase was obtained as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h, then the solution is washed to be neutral by using deionized water, 0.25g of the D001 ion exchange resin is weighed and dispersed in 10ml of purified mannose phosphate isomerase solution (5 mg/ml), and the immobilized mannose phosphate isomerase is obtained through incubation and assembly at 4 ℃, and the loading amount and the enzyme activity of the immobilized enzyme are shown in the table 1.
Comparative example 2
A crude enzyme solution of phosphomannose isomerase was obtained in the same manner as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.607g NiCl 2 ·6H 2 O was dissolved in 500ml deionized water, 100ml in a 250ml conical flask with stopper was taken, 0.2g of the pretreated ion exchange resin was added, and the adsorption was carried out on a constant temperature shaker at 25℃with shaking at a speed of 150r/min for 1h. The obtained nickel-loaded D001 ion exchange resin is washed by deionized water and then stored in the deionized water.
0.25g of nickel-loaded D001 ion exchange resin is weighed and dispersed in 10ml of coarse enzyme solution (10 mg/ml) of phosphomannose isomerase, and the immobilized phosphomannose isomerase is obtained by incubation and assembly at 4 ℃, and the loading amount and the enzyme activity of the immobilized phosphomannose isomerase are shown in Table 1.
Comparative example 3
A crude enzyme solution of phosphomannose isomerase was obtained in the same manner as in example 1.
20g of D001 ion exchange resin is soaked in 100ml of deionized water, 4% HCl solution, 4% NaOH solution and 4% HCl solution for 1h respectively, then washed to be neutral by deionized water, and dried to constant weight in a vacuum drying oven at 40 ℃. Weigh 0.415g CaCl 2 Dissolving in 500ml deionized water, taking 100ml into 250ml conical flask with plug, adding 0.2g of pretreated ion exchange resin, and stirring at 25deg.C at 150r/minOscillating and adsorbing for 1h on a constant temperature shaking table. The obtained nickel-loaded D001 ion exchange resin is washed by deionized water and then stored in the deionized water.
0.25g of nickel-loaded D001 ion exchange resin is weighed and dispersed in 10ml of coarse enzyme solution of phosphomannose isomerase, and the immobilized phosphomannose isomerase is obtained by incubation and assembly at 4 ℃, and the loading amount and the enzyme activity of the immobilized phosphomannose isomerase are shown in Table 1.
Comparative example 4
A crude enzyme solution of phosphomannose isomerase was obtained in the same manner as in example 1.
The Co-NTA purification resin column was equilibrated with an equilibration solution (1 XPBS, sodium chloride 0.5 mol.L) -1 Imidazole 20 mmol.L -1 After equilibration at pH7.4, the crude enzyme solution was added at a flow rate of 1ml/min, followed by elution with an elution solution (1 XPBS, sodium chloride 0.5 mol.L) -1 Imidazole 300 mmol.L -1 pH 7.4) was eluted at a flow rate of 1ml/min, and the resulting purified enzyme solution bands were shown in FIG. 2.
TABLE 1 immobilized enzyme Loading and relative enzyme Activity
Ion load Q (mg/g) Recovery of enzyme activity (%)
Example 1 122 55
Example 2 122 89
Example 3 50 71
Example 4 141 88
Example 5 169 84
Example 6 120 80
Example 7 120 85
Example 8 98 80
Example 9 125 85
Example 10 121 67
Example 11 122 87
Example 12 122 74
Example 13 -- --
Comparative example 1 -- 48
Comparative example 2 120 45
Comparative example 3 119 0
Comparative example 4 -- --
The recovery rate of the immobilized enzyme prepared by the invention is 55-89%, the relative enzyme activity can reach 89% of the free enzyme, and compared with the unblocked ion exchange resin (comparative example 2) and the ion exchange resin which does not chelate transition metal (comparative example 1 and comparative example 3), the recovery rate of the enzyme is greatly improved. Since the ion exchange resin and the enzyme complete the immobilization process through His tag, the activity of the immobilized enzyme is maximally maintained. Compared with comparative example 4, it was confirmed that the prepared ion exchange resin column was selective for enzyme immobilization and that both immobilization and purification processes could be performed simultaneously.
In conclusion, compared with the comparative example method, the immobilized enzyme material has obvious advantages in the aspect of enzyme activity recovery rate; the prepared chelate transition metal ion exchange resin can be loaded with His tag enzyme to obtain immobilized enzyme, and the obtained immobilized enzyme can keep good enzyme activity; the prepared ion exchange resin column has selectivity for enzyme immobilization, and can simultaneously carry out immobilization and purification processes.
The foregoing description of the preferred embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify for specific embodiments and applications without departing from the true spirit and scope of the present invention, and therefore, all such modifications, equivalents, and improvements that fall within the true spirit and scope of the present invention should be considered to be within the scope of the following claims.

Claims (8)

1. The preparation method of the ion exchange resin selective immobilized enzyme is characterized by comprising the following steps:
pretreatment of ion exchange resin;
adding the pretreated ion exchange resin into an aqueous solution containing transition metal salt, and oscillating and adsorbing by a shaking table;
adding a buffer solution to react off superfluous active groups on the ion exchange resin;
mixing ion exchange resin with enzyme solution, stirring or oscillating, and immobilizing at 4-40deg.C;
the ion exchange resin is macroporous ion exchange resin and comprises strong acid cation resin, weak acid cation resin or chelate resin; the pretreatment of the ion exchange resin is to remove unpolymerized monomers, pore-forming agents and solvents remained in the ion exchange resin and activate the ion exchange resin; the total pretreatment time is 4-8 hours;
the pretreatment method of the ion exchange resin is one of the following modes: ethanol treatment-acid treatment-alkali treatment-acid treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-acid treatment-alkali treatment-acid treatment; ethanol treatment-alkali treatment-acid treatment-alkali treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-alkali treatment-acid treatment-alkali treatment; each step of treatment is to soak in the corresponding solution, and the time of each step of treatment is the time of dividing the total pretreatment time of the ion exchange resin into 4 sections, namely 1-2 hours; the mass fraction of sodium hydroxide used for the alkali treatment is 2% -7%; the mass fraction of hydrochloric acid used for acid treatment is 2% -7%;
the transition metal salt is at least one of nickel chloride hexahydrate, nickel sulfate hexahydrate, cobalt chloride hexahydrate or cobalt sulfate heptahydrate.
2. The method for producing an ion exchange resin selective immobilized enzyme according to claim 1, wherein the concentration of the solution of the transition metal ion is 1000mg/L or less; when the ion exchange resin is added into the aqueous solution dissolved with the transition metal salt, the solid-liquid ratio is 0.4-10 mg/ml; the shaking table oscillates and adsorbs for 30 min-6 h, and the rotation speed is 50-200 rpm.
3. The method for preparing an ion exchange resin selective immobilized enzyme according to claim 1, wherein the buffer solution is Tris-HCl solution with a concentration of 0.3M to 2M and a ph of 8.
4. The method for preparing an ion exchange resin selective immobilized enzyme according to claim 1, wherein in the pretreatment of the ion exchange resin, the mass fraction of sodium hydroxide used for alkali treatment is 3-5%, and the mass fraction of hydrochloric acid used for acid treatment is 3-5%; the concentration of the solution of the transition metal ions is 300-500 mg/L; when the ion exchange resin is added into the water solution dissolved with the transition metal salt, the solid-liquid ratio is 2-4 mg/ml; the shaking table shaking speed is 100-150 rpm; the concentration of the buffer solution is 0.5M-1M; the buffer pH was 8.
5. The method for producing an ion exchange resin selective immobilization enzyme according to claim 1, wherein the enzyme contains a His tag composed of histidine residues.
6. The method for preparing an ion exchange resin selective immobilized enzyme according to claim 5, wherein the enzyme is a protease comprising a single enzyme or a multiple enzyme system; the protease is an enzyme containing a His tag or an enzyme not containing a His tag, and the enzyme not containing the His tag needs to insert the His tag into an N end or a C end through a gene recombination technology; when the ion exchange resin is mixed with the enzyme solution, the solid-liquid ratio is 20-200 mg/ml; the enzyme solution concentration is below 10 mg/ml.
7. An ion exchange resin selective immobilization enzyme, characterized in that the ion exchange resin selective immobilization enzyme is obtained by the method for producing an ion exchange resin selective immobilization enzyme according to any one of claims 1 to 6.
8. A method for preparing an ion exchange resin column for immobilized enzyme, comprising the steps of:
pretreating ion exchange resin for 4-8 h;
adding the pretreated ion exchange resin into an aqueous solution containing transition metal salt, and oscillating and adsorbing for 30 min-6 h by a shaking table;
adding a buffer solution to react off redundant active groups on the resin;
loading the ion exchange resin into a chromatographic column with a sieve plate, flushing with deionized water with a volume which is 5-10 times of the column volume at a flow rate of 5ml/min, then storing in 20% ethanol solution, balancing with balancing solution with a volume which is 5-10 times of the column volume before use, and controlling the flow rate to be 1ml/min; equilibration buffer: 1 XPBS, sodium chloride 0.5 mol.L -1 Imidazole 20 mmol.L -1 ，pH7.4；
The ion exchange resin is macroporous ion exchange resin and comprises strong acid cation resin, weak acid cation resin or chelate resin; the pretreatment of the ion exchange resin is to remove unpolymerized monomers, pore-forming agents and solvents remained in the ion exchange resin and activate the ion exchange resin; the total pretreatment time is 4-8 hours;
the pretreatment method of the ion exchange resin is one of the following modes: ethanol treatment-acid treatment-alkali treatment-acid treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-acid treatment-alkali treatment-acid treatment; ethanol treatment-alkali treatment-acid treatment-alkali treatment; saturated saline water treatment, acid treatment, alkali treatment and acid treatment; deionized water treatment-alkali treatment-acid treatment-alkali treatment; each step of treatment is to soak in the corresponding solution, and the time of each step of treatment is the time of dividing the total pretreatment time of the ion exchange resin into 4 sections, namely 1-2 hours; the mass fraction of sodium hydroxide used for the alkali treatment is 2% -7%; the mass fraction of hydrochloric acid used for acid treatment is 2% -7%;
the transition metal salt is at least one of nickel chloride hexahydrate, nickel sulfate hexahydrate, cobalt chloride hexahydrate or cobalt sulfate heptahydrate.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533711A (en) * 2012-02-22 2012-07-04 江南大学 Method for immobilizing D-tagatose 3-epimerase (DTE)

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