CN100398646C - Preparation method of a novel affinity carrier - Google Patents

Abstract

The invention discloses a metal chelating affinity carrier and a preparation method thereof. The metal chelating affinity carrier uses agar powder as a matrix, cross-links a chelating agent, and chelates metal ions. Its preparation includes extracting agar powder with acid buffer solution and alkaline buffer solution respectively, activating the extracted agar powder with activator, cross-linking with chelating agent, and chelating with metal ions. The metal chelating affinity carrier of the present invention is low in cost, easy to manufacture, and can be used not only as a purification carrier, but also as an immobilization carrier, and its performance as an affinity carrier is comparable to or even much better than that of commercially available carriers .

Description

Preparation method of a novel affinity carrier

technical field

The invention relates to a method for preparing an affinity carrier using agar powder as a substrate, and a method for preparing an industrial enzyme.

Background technique

Immobilized Metal-Chelated Affinity Chromatography, also known as Immobilized Metal-Chelated Affinity Chromatography (IMAC), is a common method widely used in protein purification in recent years. Its principle is to use Cu ²⁺ , Co ²⁺ , Ni ²⁺ , Zn ²⁺ and other transition metal ions coordinate with histidine, tryptophan or cysteine on the protein surface. Due to the different types, positions and spatial conformations of these amino acids on the protein surface, they have different affinity with metal chelates, so they can be selectively separated and purified.

The development of genetic engineering has greatly promoted the application of IMAC technology. Usually, genetic engineering technology is used to connect a polypeptide segment at the N-terminal or C-terminal of the target recombinant protein, mostly polyhistidine, which will hardly change the enzyme activity and stability, and can improve the binding of the target protein to the carrier force. IMAC technology makes protein purification more convenient. Due to its simplicity, speed, specificity and high efficiency, it has been gradually applied in various fields of life sciences, especially in the downstream processing of current biotechnology. Moreover, one-step purification and immobilization of various proteins and industrial enzymes can be realized.

With people's increasing emphasis on environmental protection and in order to meet the requirements of reducing costs, chemical synthesis is gradually replaced by enzymatic synthesis. For the purification of enzymes, many companies have introduced many immobilized metal chelation affinity chromatography carriers, such as Clontech, GE healthcare, Qiagen and other companies. The binding capacity of these carriers to proteins tagged with histidine is shown in Table 1.

Table 1: Vehicles marketed by various companies

company Matrix Chelating group Clontech TALON Resin IDA Clontech TALON superflow Resin IDA GE healthcare Hitrap chelating hp IDA

Qiagen Ni-NTA agarose NTA Qiagen Ni-NTA superflow column NTA

These carriers are all prepared with agarose as the matrix. The carriers have high mechanical strength and can quickly and simply purify the target protein. They can be used not only for laboratory research, but also for large-scale industrial production. However, these carriers are expensive, and the cost is high when applied in large quantities, and it is difficult to promote them in cost-sensitive industrial production processes (such as industrial enzyme purification and immobilization).

Therefore, it would still be of great interest to the art to have a carrier that is less expensive and still retains even better performance.

Contents of the invention

One aspect of the present invention relates to an affinity carrier, which uses agar powder as a matrix, and the agar powder is cross-linked with a chelating agent, wherein the mass ratio of the agar powder matrix to the chelating agent is 800-1200:10-45.

Another aspect of the present invention relates to a metal chelating affinity carrier, which is based on agar powder, and the agar powder is cross-linked with a chelating agent, and the chelating agent is chelated with metal ions, wherein the agar powder matrix, The mass ratio of the chelating agent to the metal ion is 800-1200:10-45:1-15.

Another aspect of the present invention relates to the preparation method of the metal chelating affinity carrier taking agar powder as matrix, the method comprising:

(1) extract agar powder with the acid buffer solution of pH3.0-6.8, then extract agar powder with the alkaline buffer solution of pH8.0-10.0, drain, obtain wet agar powder;

(2) the wet agar powder obtained in step (1) with the activator treatment step (1) that can make the solid carrier with the OH group have a reactive functional group;

(3) the agar powder obtained in step (2) is cross-linked with the chelating agent to obtain the agar powder cross-linked with the chelating agent; and

(4) chelating the agar powder obtained in step (3) with metal ions to obtain the metal chelating affinity carrier.

The invention also relates to the use of the agar powder in the preparation of the metal chelating affinity carrier.

Detailed ways

In principle, any polymer with free hydroxyl groups, such as agarose, agar powder, chitosan, cellulose, etc., can be used as a matrix for IMAC preparation. Although cellulose is cheap and easy to obtain, its non-specific adsorption is serious, which affects the protein purification effect. Agarose is composed of neutral chain polysaccharides combined with D-galactose and 3,6-anhydro-L-galactose. It has no charge on the surface, low non-specific adsorption, and has a large number of rich chemical groups (such as OH functional group), easy to modify, so it is commonly used in the prior art to prepare IMAC carriers, but the price is relatively expensive, which limits its industrial-scale application.

Agar powder is inexpensive and consists of agarose and pectin. The pectin in it is reported to have a strong ability to adsorb proteins non-specifically. If it is used for affinity chromatography, it will affect the separation effect. So far, there is no report on the preparation of carriers with agar powder as the matrix. We prepared a metal chelating affinity carrier with agar powder as the matrix, and tested it with a variety of industrial enzymes, and found that the purification and immobilization results were good, and there was no serious non-specific adsorption phenomenon.

Therefore, the present invention relates to a metal chelating affinity carrier, which is prepared from agar powder as a base. The agar powder is firstly extracted with a buffer, then activated with an activator, and then interacted with a chelating agent and a salt solution of metal ions successively, so as to prepare the metal chelating affinity carrier used in the present invention. In the metal chelating affinity carrier of the present invention, the mass ratio of agar powder, chelating agent and metal ion is usually 800-1200: 10-45: 1-15, more usually 900-1000: 15-35: 3-10, More typically 1000:18-33:5-9. In a preferred embodiment, the agar powder matrix cross-linked with the chelating agent may not chelate metal ions first, and then chelate the required metal ions when used.

The present invention has no special limitation on the buffer solution used for extracting agar powder. Generally, acidic buffer solution with pH 3.0-6.8, preferably 4.0-6.0 is used for extraction, and then alkaline buffer solution with pH 7.5-11.0, preferably pH 8.0-10.0 is used for extraction. The present invention also has no special limitation on the concentration of the buffer solution, which is usually 0.02-0.08 mol/L, preferably 0.03-0.06 mol/L, more preferably 0.04-0.05 mol/L. Commonly used acid buffers include acetate buffer and phosphate buffer, and alkaline buffers include sodium carbonate-sodium bicarbonate buffer.

In a preferred embodiment, acetic acid buffer is added in the ratio of 1 gram of agar powder 30-70 milliliters of buffer, the pH range of this acetate buffer is 3.0-6.0, preferably 4.0-5.0, more preferably 4.0, which The concentration is 0.03-0.06 mol/L, preferably 0.04 mol/L. Usually two extractions are carried out with acetic acid buffer solution. Of course, the number of extractions depends on the actual situation, for example, it can be 3 or 4 times, or it can be 1 time. When extracting multiple times, the interval for changing the buffer is generally 2-5 hours, preferably 2-3 hours. Besides acetate buffer, other buffers can also be used, such as phosphate buffer, the pH of which is usually 6.8 and the concentration is usually 0.03 mol/L.

After extraction with acetic acid buffer, it is usually washed with water for several times, such as 2-3 times, and then left overnight. Then extract with sodium carbonate-sodium bicarbonate buffer. The pH of the sodium carbonate-sodium bicarbonate buffer used in the present invention is usually 8.0-10.0, preferably 9.0, and its concentration is usually 0.03-0.07mol/L, usually 0.05mol/L. Typically, stir for 5-6 hours after addition of buffer and leave overnight. The precipitate can then be filtered and washed with water to drain. It can be extracted again with sodium carbonate-sodium bicarbonate buffer, that is, the extraction process of sodium carbonate-sodium bicarbonate buffer is repeated once. In a preferred embodiment, the extraction is carried out at 40° C. with pH 9.0, 0.05 mol/L sodium carbonate-sodium bicarbonate buffer solution.

The extracted agar powder can then be used to prepare the affinity carrier of the present invention. First, the wet agar powder obtained by the above-mentioned extraction and draining can be treated with an activator. Typically, agar powder is reacted with an activator to impart highly reactive functional groups, such as epoxy or chloroformyl, to enable synthetic reactions with extension arms or chelating agents. Common activation methods include cyanogen bromide method, epoxidation method, diethylene method, etc. The most commonly used method is epoxidation. In a preferred embodiment of the present invention, the present invention uses epichlorohydrin as an activator for treating agar powder. Usually, about 0.45-0.85ml, preferably 0.55-0.75ml of activator is used per gram of extracted and drained wet agar powder. In a preferred embodiment, each gram of extracted and drained wet agar powder is treated with about 0.65 ml of activator. In the activated agar powder, the mass ratio of the agar powder to the activator is usually 800-1000:5-35, more usually 800-1000:10-30, more usually 1000:13-25.

The activator-treated agar powder can then be treated with a chelating agent. The use of chelating agents is mainly to assist metal ions to be fixed on solid supports in the form of coordination bonds, and chelating agents mainly form coordination bonds with subunits and metal ions, so they are called multi-subunit chelating agents, and the number of subunits is Three, four and five. Generally, transition metals have six coordination numbers, so after combining with the subunit of the chelating agent, the remaining coordination numbers can be combined with specific amino acids on the protein, and separated from proteins without specific amino acids. Common chelating agents include IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), TED [N,N,N-tris(carboxymethyl)ethylenediamine] and CM-Asp (carboxymethylated aspartic acid). Among them, the most commonly used chelating agent is IDA. After one nitrogen atom and two oxygen atoms on it are chelated with metal ions, the metal ions still have three empty orbitals to bind to proteins. IDA is small in size and less sterically hindered when combined with proteins, and IDA is hydrophilic, has two negative charges, and is electrically neutral after chelating with metal ions, which reduces non-specific adsorption between solid carrier proteins to a minimum. In a preferred embodiment of the present invention, IDA is used as a chelating agent to prepare the metal chelating affinity carrier of the present invention. Usually, 0.15-0.25 gram, preferably 0.18-0.22 gram of chelating agent is used per gram of activated agar powder. In a preferred embodiment, the chelating agent is IDA, and 0.18 grams of IDA is used per gram of activated agar powder.

The obtained agar powder can then be treated with a salt solution of metal ions. Usually the most used metal ions are transition metal ions Cu ²⁺ , Ni ²⁺ , Zn ²⁺ , Co ²⁺ in the first column, because they have more empty orbitals, the stability of the formed coordination compounds is better . The stability between metal ions and chelating agents is generally: Cu ²⁺ , Ni ²⁺ , Zn ²⁺ , Co ²⁺ , Fe ²⁺ , Ca ²⁺ or Mg ²⁺ . In a preferred embodiment of the present invention, the metal ion is Co ²⁺ . In another preferred embodiment, the metal ion is Ni ²⁺ . The salts of metal ions used in the present invention are also not particularly limited, for example, hydrochlorides, sulfates and the like of these metal ions can be used.

Another aspect of the present invention relates to the preparation of the metal chelating affinity carrier, comprising successively extracting agar powder with acetic acid buffer solution and sodium carbonate-sodium bicarbonate buffer solution, activating the extracted agar powder with an activator, and using a chelating agent Cross-linking, and then chelating the resulting product with metal ions. In the metal chelating affinity carrier of the present invention, the mass ratio of dry agar powder, chelating agent and metal ion salt in the carrier preparation process that is put into the material is 0.8-1.2: 0.45-1.62: 0.2-0.36; for the activation that is liquid itself In terms of activator, the amount of activator per 0.8-1.2 g of dry agar powder is about 1-10 milliliters, preferably about 2-5 milliliters. When the activator itself is non-liquid, it can be dissolved in a suitable solvent, with an amount of about 0.5-10 g, preferably about 1-5 g, per 0.8-1.2 g of dry agar powder.

In a preferred embodiment of the present invention, add pH4.0, 0.04mol/L acetic acid buffer solution (sodium acetate-acetic acid buffer solution) at the ratio of 1g agar powder plus 40-60ml buffer solution, stir and extract, every 2-5 Change the buffer solution every hour, filter the precipitate after the second extraction, wash with water several times (preferably 2-3 times), and place overnight. The next day, pH9.0, 0.05mol/L sodium carbonate-sodium bicarbonate buffer solution was added in the same proportion, stirred for 4-8 hours and left overnight. On the third day, filter the sediment, wash and drain, repeat the method of the previous day, wash with water until neutral, and drain.

Methods for the preparation of affinity carriers are also well known. Usually, extract the drained wet agar powder with acetic acid buffer solution and sodium carbonate-sodium bicarbonate buffer solution, and add 1.2-1.8mol/L hydroxide solution at a ratio of about 0.5-2 ml of sodium hydroxide solution to about 1 g of wet agar powder. Sodium solution (containing sodium borohydride), add acetone and epichlorohydrin while stirring at 23-28°C, and continue stirring. Based on 1 g of wet agar powder, about 0.1-0.5 milliliters of acetone is added per gram of wet agar powder, and about 0.1-0.3 milliliters of epichlorohydrin is added. When the reaction reaches about 2 hours, 4 hours and 8 hours, add epichlorohydrin again according to the above ratio respectively. After reacting for a total of 12 hours, wash with deionized water until neutral, and drain to obtain EPI-ARG. Add 190-220ml, 0.05-0.2mol/L sodium carbonate solution (containing 7-10% iminodiacetic acid) to every 100g of EPI-ARG, adjust the pH to 10.5-11.5 with sodium hydroxide, and stir at 25-28°C for 12 -14 hours, fully washed with deionized water, and drained to obtain EPI-ARG-IDA. Add 60-100ml, 30-70mmol/L, pH6.0 sodium phosphate buffer (containing 3-8mg/ml cobalt chloride, 0.5-2mol/L sodium chloride) for every 20g of EPI-ARG-IDA, 25-28℃ Stirring and reacting for 12-14 hours, and fully washing with deionized water, the carrier is obtained.

In the preparation method of the present invention, there is no special requirement on ambient temperature and pressure, and it is usually carried out at atmospheric pressure and room temperature.

The agar powder used to prepare the metal chelating affinity carrier of the present invention can be purchased from the market, such as Shanghai Bidirectional Sebas Technology Development Co., Ltd., China Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd. and so on.

Agar powder, as the raw material of agarose, has the advantages of agarose, uniform structure, stable physical and chemical properties, weak affinity with protein, a large number of OH functional groups, easy to modify, abundant sources, and cheap price. The price of agar powder on the market is more than 4 times lower than that of agarose. Therefore, the metal chelating affinity carrier of the present invention using agar powder as a matrix has the significant advantage of low cost. Moreover, the carrier prepared by the present invention can not only be used as a purification carrier, but also can be used as an immobilization carrier, and its performance as an affinity carrier is comparable to or even much better than that of commercially available carriers. In addition, the agar powder-based affinity carrier of the present invention that is cross-linked with a chelating agent but does not chelate metal ions is chelated with metal ions after a long period of storage, and its performance is the same as that of the non-placed, directly chelated metal ions of the present invention. Metal chelating affinity supports are basically the same. Therefore, the affinity carrier can be regarded as an intermediate of the metal-chelating affinity carrier.

The present invention will be described in detail below in the form of specific examples. It should be understood that the scope of the present invention is not limited to these preferred specific embodiments, and various modifications and changes can be made therein without departing from the spirit and scope of the present invention.

Example 1

Carrier preparation method: Weigh 30 grams of agar powder, add pH 4.0, 0.04mol/L acetate buffer solution (sodium acetate-acetate buffer solution) according to the ratio of 1g agar powder plus 50ml buffer solution, stir and extract, and replace it every 3 hours Buffer solution, after the second extraction, the precipitate was filtered, washed 3 times with water, and left overnight. The next day, it was extracted with pH 9.0, 0.05 mol/L sodium carbonate-sodium bicarbonate buffer according to the above ratio, stirred for 6 hours and left overnight. On the third day, filter the precipitate and wash and drain, then repeat the method of the previous day, wash to neutral, and drain to obtain 175g wet agar powder, then add 175ml, 1.6mol/L sodium hydroxide solution (containing 4g boron Sodium hydride), 57ml of acetone and 28.5ml of epichlorohydrin were added while stirring at 25°C, and the stirring reaction was continued until 2 hours, 4 hours and 8 hours, and then 28.5ml of epichlorohydrin were added respectively. After a total of 12 hours of reaction, it was washed with deionized water until neutral, and then drained to obtain 260 g of EPI-ARG. Add 520ml, 0.1mol/L sodium carbonate solution (containing 9% iminodiacetic acid) to 260g EPI-ARG, adjust the pH to 11 with 6mol/L sodium hydroxide, stir and react at 25°C for 12 hours, and wash thoroughly with deionized water Net, Lek EPI-ARG-IDA. Take 20g of EPI-ARG-IDA, add 80ml, 50mmol/L, pH6.0 sodium phosphate buffer solution (containing 5mg/ml cobalt chloride, 1mol/L sodium chloride), stir and react at 25°C for 12 hours, wash with deionized water After fully washing and draining, the carrier EPI-30-ARG-IDA-Co ²⁺ is obtained.

Using the same steps as the above method, but replacing the above cobalt chloride with nickel chloride, another carrier of the present invention, namely EPI-30-ARG-IDA-Ni ²⁺ , can be prepared.

Example 2

Comparison of immobilization efficiency of affinity carriers with different metal ion densities:

The preparation method of the affinity carrier with high metal density is the same as Example 1.

The preparation method of the low metal density affinity carrier EPI-5-ARG-IDA is as follows: the agar powder is swollen with buffer solution, washed and drained to obtain wet agar powder. Measure 100ml of wet agar powder, add 100ml of 0.4N NaOH (containing 2g of NaBH4), add 32ml of ethylene glycol dimethyl ether, mix well, add 16ml of epichlorohydrin (all need to be pre-cooled) while stirring, and stir at 25°C for reaction 4 Hours later, wash with deionized water to neutrality to obtain EPI-5-ARG 100ml, add 100ml 0.1M _Na2CO3 (containing iminodiacetic acid 9%) in _EPI -5-ARG, wash with 6N NaOH Adjust the pH to 11, stir and react at 25°C for 16 hours, wash thoroughly with deionized water, and drain to obtain EPI-5-ARG-IDA. Take 20g of EPI-5-ARG-IDA, add 80ml, 50mmol/L, pH6.0 sodium phosphate buffer solution (containing 5mg/ml cobalt chloride, 1mol/L sodium chloride), stir and react at 25°C for 12 hours, use The ionized water was fully washed and drained to obtain the carrier EPI-5-ARG-IDA.

carrier Carrier enzyme activity (U) EPI-30-ARG-IDA 16.3 EPI-5-ARG-IDA 2.14

Note: The enzyme used to detect the adsorption capacity of the prepared affinity carrier in this example is carbamoyl hydrolase, which is different from the enzymes used in other examples, and it can only be used as a reference to detect the adsorption capacity of the carrier.

Example 3

One-step purification and immobilization process of GL-7-ACA acylase: add 1.0 g of the carrier prepared in Example 1 to 28 ml (enzyme activity unit 21.5 U/ml) of GL-7-ACA acylase cell-free extract, fully Nitrogen seal, 18°C, 170 rpm, shake and keep warm for 24 hours, rinse with 20mmol/L sodium phosphate buffer solution pH8.0 (containing 0.2mol/L sodium chloride, 10% glycerol), and detect the outflow by Bradford colorimetry Solution, until OD595 is less than 0.06, wash with 0.1mol/L sodium phosphate buffer solution (pH7.0), vacuum remove the water, and measure the activity of the immobilized enzyme.

In this example, the cell-free extract of GL-7-ACA acylase was prepared with reference to the literature: Armise'n P, Mateo C, Corte's E, Barredo JL, Salto F, Di'ez B, Rode's L, Garci'a JL, Ferna'ndez-Lafuente R, Guisa'n JM, 1999, "Selective adsorption of poly-His-tagged glutaryl acylase on a custom-made metal-chelating support", J Chromatogr A, 848 : 61-70.

GL-7-ACA acylase immobilized enzyme activity assay was carried out according to the method described by K. Balasingham, D. Warburton, P. Dunnil, D. Lilly [Boichim. Biophys. Acta, (1972) 250-256].

According to the above method, the activity of the GL-7-ACA acylase immobilized enzyme was measured to be 100 U/g, and the immobilized yield was 16.7%.

Example 4

The immobilized acylase enzyme activity of the carrier prepared in Example 1 of the present invention is compared with the immobilized acylase enzyme activity of the commercially available carrier: 3ml (enzyme activity unit 17.5U/ml) GL-7-ACA acylase Three different carriers (i.e. EPI-30-ARG-IDA-Co ²⁺ , EPI-30-ARG-IDA-Ni ²⁺ and TALON metal affinity resin) were added to the cell-free extract respectively. 0.1g, the conditions for the combination of the enzyme and the carrier are the same, as follows. The mixture of carrier and enzyme was filled with nitrogen and sealed. After incubating at 18°C and 150 rpm for 24 hours, it was washed with 20mmol/L, pH8.0 sodium phosphate buffer solution (containing 0.2mol/L sodium chloride, 10% glycerol). Use the Bradford colorimetric method to detect the effluent, and when the _OD595 is less than 0.06, wash with 0.1mol/L, pH7.0 sodium phosphate buffer solution, vacuum out the water, and measure the enzyme activity of the immobilized enzyme according to the method described in Example 2. .

Results: The immobilized enzyme activity of EPI-30-ARG-IDA-Co ²⁺ : 95U/g;

EPI-30-ARG-IDA-Ni ²⁺ immobilized enzyme activity: 32U/g;

  Immobilized enzyme activity of Clontech TALON metal affinity resin: 30U/g;

The results show that the immobilized acylase enzyme activity of the present invention to prepare EPI-30-ARG-IDA-Co ²⁺ is 2-3 times higher than the immobilized enzyme activity of Clontech TALON metal affinity resin, EPI-30-ARG- The immobilized enzyme activity of IDA-Ni ²⁺ is equivalent to that of the Clontech TALON metal affinity resin.

Example 5

Continuous Transformation of Immobilized GL-7-ACA Acylase

Dissolve 3 grams of GL-7-ACA in 50mmol/L, pH7.0 sodium phosphate buffer solution, set the volume to 100ml, adjust the pH value to 8.0 with 6mol/L ammonia water, take 5ml of the solution and put it into 0.3g of Example 2 The prepared GL-7-ACA acylase immobilized enzyme was shaken in a water bath at 20°C, and the pH was adjusted to 8.0 during the 30 minutes of the 1st, 6th, 13th, 21st, 29th, 35th, 41st, 47th and 50th reactions , 60 minutes were sampled, HPLC determination. Continuously transform 50 times, the transformation efficiency is as follows:

Example 6

The carrier preparation method is the same as in Example 1.

Penicillin G acylase one-step purification and immobilization process: add 0.4g carrier to 40ml penicillin G acylase crude enzyme solution (20U/ml), incubate at 18°C and 160 rpm for 24 hours, then use 20mmol/L phosphate buffer pH8 .0 (containing 0.2mol/L sodium chloride, 10% glycerol) to wash, use the Bradford colorimetric method to detect the effluent, until the OD ₅₉₅ is less than 0.06, wash with 0.1mol/L sodium phosphate buffer (pH7.0), vacuum Drain off the water. The enzymatic activity of penicillin G acylase immobilized on the carrier was determined by p-dimethylaminobenzaldehyde (PDAB) method.

In this example, the penicillin G acylase crude enzyme was prepared according to the method disclosed in the following literature: Yong Wen, Xunlong Shi, Zhongyi Yuan and Pei Zhou, "His-tagged penicillin G acyl enzyme obtained from Kluyverella citricophilus Expression, purification and characterization of transferase in Escherichia coli", pp. 24-28, Protein Expression and Purification, 38(2004) 24-28.

The enzyme activity of immobilized penicillin G acylase is measured with reference to the p-dimethylaminobenzaldehyde (PDAB) method disclosed in the text (Biochim.Biophys.Acta.1972,276:250-256). Apparent enzymatic activity of G salt hydrolysis to prepare 6-APA.

According to the above method, the activity of immobilized penicillin G acylase was 400 U/g, and the immobilization yield was 20%.

Example 7

The carrier preparation method is the same as in Example 1.

Purification process of D-amino acid oxidase: 30ml D-amino acid oxidase crude enzyme solution was added with 1g carrier, incubated at 15°C and 140 rpm for 18 hours, and then loaded into the column. Wash with 50 times the volume of the column bed (containing 0.1mol/L Tris-HCl, 0.25mol/L sodium chloride, 5% glycerol, 5mmol/L imidazole, pH 8.0) to elute, and use Bradford colorimetric method to detect Effluent, until OD ₅₉₅ is less than 0.06, eluted with elution buffer (containing 0.1mol/LTris-HCl, 0.25mol/L sodium chloride, 5% glycerol, 250mmol/L imidazole, pH8.0), and collected pure enzyme .

The crude enzyme solution of D-amino acid oxidase used in this example is prepared according to the literature: Jorge Alonso, Estrella Cortes, etc., "Genetic processing of D-amino acid oxidase obtained from trigonopsis variabilis to promote its overexpression in Escherichia coli and downstream processing using tailored metal-chelating carriers", Enzyme and Microbial Technology 25(1999) 88-95.

D-Amino Acid Oxidase Enzyme Activity Determination Method References: Tzann-Shun Hwang, Hui-Mei Fu, Long-Liu Lin and Wen-Hwei Hsu, "Trigonopsis variabilis D amino acid oxidase with lactose as inducer in Escherichia coli high-level expression", Biotechnology Letters, 22; 655-658, 2000.

The adsorption amount of the carrier for D-amino acid oxidase was measured according to the above method: 100 U/g.

The purification factor is about 6.4; the purification yield: 31.6%. See the table below for specific data.

Purification of histidine-tagged D-amino acid oxidase using the metal chelating affinity carrier prepared in the present invention

method Total protein (mg) Total activity (U) Specific activity (U/mg) Purification factor Purification rate (%) crude enzyme solution 2085 5943.7 2.85 1 100 IMAC 103.2 1880.2 18.25 6.4 31.6

Example 8

According to the steps described in Example 1, the following metal chelating affinity carriers were prepared with the following amounts of substances.

serial number Dry agar powder (g) Activator (ml) Chelating agent (g) Metal salt (species/gram) 1 30 48 13.5 Co²⁺, 6 2 30 80 20 Ni²⁺, 8 3 10 90 15 Co²⁺, 3.5 4 10 30 5 Ni²⁺, 3

The immobilization yield of these carriers was tested according to the procedure described in Example 3 to be about 15-25%.

Claims (10)

1. a metal chelating affinity carrier, is characterized in that, it is matrix with agar powder, and described agar powder is cross-linked with chelating agent, and described chelating agent is chelated with metal ion, and wherein, described agar powder matrix, The mass ratio of the chelating agent to the metal ion is 800-1200:10-45:1-15. 2. metal chelating affinity carrier as claimed in claim 1, is characterized in that, described chelating agent is selected from iminodiacetic acid, nitrilotriacetic acid, N, N, N-three (carboxymethyl) ethylenediamine or carboxymethylated aspartic acid. 3. metal chelating affinity carrier as claimed in claim 2, is characterized in that, described chelating agent is iminodiacetic acid. 4. The metal chelating affinity carrier as claimed in claim 1, wherein the metal ion is selected from Cu ²⁺ , Ni ²⁺ , Zn ²⁺ , Co ²⁺ , Fe ²⁺ , Ca ²⁺ or Mg ²⁺ . 5. an affinity carrier, it is characterized in that, it is matrix with agar powder, and described agar powder is cross-linked with chelating agent, and wherein, the mass ratio of described agar powder matrix and chelating agent is 800-1200: 10-45 . 6. a method for preparing the metal chelating affinity carrier claimed in claim 1, is characterized in that, described method comprises: (1) extract agar powder with the acid buffer solution of pH3.0-6.8, then extract agar powder with the alkaline buffer solution of pH8.0-10.0, drain, obtain wet agar powder; (2) the wet agar powder obtained in step (1) with the activator treatment step (1) that can make the solid carrier with the OH group have a reactive functional group; (3) cross-linking the agar powder obtained in step (2) with a chelating agent to obtain cross-linked agar powder with a chelating agent; and (4) chelating the chelating agent in the agar powder obtained in step (3) with metal ions to obtain the metal chelating affinity carrier. 7. The method of claim 6, wherein the activator is epichlorohydrin. 8. The method of claim 6, wherein the chelating agent is selected from iminodiacetic acid, nitrilotriacetic acid, N, N, N-tris (carboxymethyl) ethylenediamine or carboxymethylated Aspartic acid; the metal ion is selected from Cu ²⁺ , Ni ²⁺ , Zn ²⁺ , Co ²⁺ , Fe ²⁺ , Ca ²⁺ or Mg ²⁺ . 9. the method for claim 6 is characterized in that, in carrier preparation process, the mass ratio that the salt of dry agar powder, chelating agent and metal ion drops into material is 0.8-1.2: 0.45-1.62: 0.2-0.36, every Gram wet agar powder was treated with 0.45-0.85ml of activator. 10. The use of agar powder in the preparation of metal chelating affinity carriers.