JPH0612992B2 - Method for producing immobilized protease - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an immobilized protease used as a catalyst when hydrolyzing a protein.

[Prior art] As a method of immobilizing an enzyme, 1) a covalent bond, an ionic bond, a physical adsorption, a carrier-bonding method of immobilizing it on an insoluble carrier by biochemical new power, etc. Cross-linking method of cross-linking with di (multi) functional reagent such as glutaraldehyde, 3) Polymerization or association of low molecular weight compounds, or transfer of high molecular weight compounds from soluble state to insoluble state Encapsulation method by encapsulating the enzyme in polymer gel (lattice type), microcapsule, liposome, hollow fiber (hollow fiber), ultrapermeabilization membrane, 4).
There is a composite method in which these are appropriately combined. Each of these immobilization methods has advantages and disadvantages, and it is necessary to use them according to the purpose and the type of enzyme.

Studies on protein hydrolysis by immobilized proteases have been conducted in a wide range for the purpose of, for example, preparing curd of cheese or preventing beer from becoming cloudy. At this time, since the protein has a surface charge, a covalent bond method in which the protease is difficult to detach is generally used as a method for immobilizing the protease.

As a conventional method for immobilizing a protease by a covalent bond method, a carrier (for example, silanized alumina) having a covalently bondable functional group (for example, an amino group or a carboxyl group) is attached to a polyfunctional crosslinking agent (for example, glutaraldehyde). After contacting with the solution to covalently bond, washing is performed to remove the free polyfunctional crosslinking agent, and then the carrier is contacted with the protease solution to covalently bond the protease to the activated functional group, and further to remove the salt. A method of removing immobilized protease with a solution (for example, a solution of sodium chloride) to obtain an immobilized protease is performed.

[Problems to be solved by the invention]

However, in the above-mentioned conventional immobilization method, since the degradation target is a high molecular weight protein having a surface charge, the protease is easily released from the immobilization carrier during the degradation, and the activity as the immobilized protease decreases. There was a problem.

[Means for solving problems]

Therefore, as a result of earnest research, the present inventors have found a method for obtaining a stable immobilized protease by eliminating elimination of the protease during hydrolysis of the protein.

That is, the present invention sequentially comprises: a) covalently bonding a cross-linking agent to a carrier having a covalently bondable functional group, b) covalently bonding a protease to the bonded cross-linking agent, and c) re-bonding to the immobilized protease. This is a method for producing an immobilized protease, which is characterized in that it is treated with a crosslinking agent.

That is, according to the present invention, the immobilized protease obtained by the above-mentioned conventional method is further treated with a crosslinking agent,
New cross-links occur between the immobilized proteases. Due to the new cross-linking between these proteases,
The elimination of protease during the hydrolysis of protein is prevented.

The carrier used in the present invention is an organic or inorganic carrier having a functional group capable of covalent bonding. Examples of the functional group capable of covalent bonding include an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group and the like, but a carrier having an amino group is particularly preferable. Specific examples of the carrier having these functional groups include aminosilane-containing inorganic carriers such as aminosilane derivatives of porous glass (eg, porous beads surface-treated with γ-aminopropyltriethoxysilane) or alumina. There is an aminosilane derivative, and an aminosilane derivative of alumina is preferably used. Specific examples of the organic carrier having an amino group include chito-san, polysaccharide derivatives (D-aminobenzyl cellulose, etc.), polyacrylamide derivatives, styrene resins (polyamino polystyrene, etc.), polyvinyl alcohol derivatives, etc. -Amino aminocellulose, aminoethyl polyacrylamide, amino derivatives of sepharose or polyvinyl alcohol derivatives are preferred. In addition, as a carrier having a carboxyl group, Amberlite XE-64, Amberlite IRC-50
There is an inorganic carrier such as, a carrier having a sulfhydryl group is an organic carrier such as enzacrylpolythiol, and a carrier having a hydroxyl group is an organic carrier such as polyvinyl alcohol.

Examples of polyfunctional crosslinking agents that can be used to carry out the method for producing the immobilized protease of the present invention include glyoxal, malonaldehyde, succinylaldehyde, glutaraldehyde,
There are polyaldehydes such as dialdehyde starch, imide esters such as diethylmalonimide or diethyladipinimide, diisocyanates or S-triazines, and glutaraldehyde is preferable.

Examples of the protease that can be used to carry out the method for producing the immobilized protease of the present invention include pepsin, trypsin, chymotrypsin, and protease of animal origin such as cathepsin, papain, bromelin, protease of plant origin such as fusin, basidiomycete protease, and A. niger. Acid protease, A.saitoi acid protease, A.oryzae neutral protease, B.subtilis neutral protease, B.licheniformis
Microbial proteases such as alkaline protease and S. griseus protease are used, but preferably pepsin is used.

In carrying out the method for producing the immobilized protease of the present invention,
After the immobilized protease is obtained in advance by the above-described conventional method, the polyfunctional crosslinking agent is then treated again to perform the crosslinking treatment between the immobilized proteases. Cross-linking between proteases is around pH 4.0 for acidic proteases and pH 7 for neutral and alkaline proteases.
It is preferable to carry out in a buffer solution of about 5 and the polyfunctional crosslinking agent is present in this buffer solution so as to have an appropriate concentration (for example, in the case of glutaraldehyde, about 10%). Then, a buffer solution containing 3 to 10 times the amount of the multifunctional cross-linking agent, which is prepared by the conventional method, relative to the wet weight of the immobilized protease is contacted with the immobilized protease or the column packed with the immobilized protease is contacted. Pass through the tower. The reaction time (contact time) may be about 1 hour at 4 ° C. After that, the unreacted polyfunctional crosslinking agent is removed by the above-mentioned buffer solution to obtain a more stabilized immobilized protease of the present invention.

Hereinafter, the method for producing the immobilized protease of the present invention will be described in detail according to Examples, but the present invention is not limited to this.

Example 1 i) 2.5% glutaraldehyde was contacted with aminosilanized alumina (particle size 5 mm; manufactured by Sumitomo Alumina Co., Ltd.) under conditions of pH 7 to 8 and room temperature for about 1 hour, and then excess glutaraldehyde was distilled with distilled water. It was washed off. Next, 5 mg of pepsin (manufactured by Sigma, USA) was dissolved in 1 ml of 0.05N sodium acetate / hydrochloric acid buffer adjusted to pH 4.0, and this solution was wet weight of the aminosilane-treated alumina treated with the crosslinking agent. 2 ml was added to 1 g and the mixture was allowed to stand at 4 ° C. for 24 hours. Then, unfixed pepsin was washed off with a 1 M sodium chloride solution adjusted to pH 4.0, and then sodium chloride was washed off with the above-mentioned 0.05N sodium acetate / hydrochloric acid buffer solution to obtain immobilized pepsin.

ii) The immobilized pepsin prepared as described above was mixed in a weight ratio of 3
1 to 10 times glutaraldehyde (adjusted to pH 4.0 in 0.05N sodium acetate / hydrochloric acid buffer) 10 times to 10 times
Soak for hours. The immobilized pepsin was separated and washed with 0.05N sodium acetate / hydrochloric acid buffer (pH 4.0) to remove unreacted glutaraldehyde. Thus, the immobilized pepsin of the present invention was obtained.

Example 2 iii) In the same manner as in Example 1, glutaraldehyde was covalently bonded to aminosilanized alumina. Next, 5 mg of subtilisin A (Novo, Denmark) was dissolved in 1 ml of 0.05 N phosphate buffer adjusted to pH 7.5, and this solution was wetted with the aminosilane-modified alumina to which glutaraldehyde was covalently bonded. Add 2ml to 1g weight, 4 ℃
And allowed to stand for 24 hours. Next, unfixed subtilisin A was washed off with a 1 M sodium chloride solution adjusted to pH 7.5, and then sodium chloride was washed off with the above 0.05N phosphate buffer solution to obtain immobilized subtilisin A.

iv) The immobilized subtilisin A prepared as described above was used in an amount of 3 to 10 times the weight ratio of 10% glutaraldehyde (0.05N).
It was immersed in a phosphate buffer at pH 7.5) at 4 ° C for 1 hour. The immobilized pepsin was separated and washed with 0.05N phosphate buffer (pH 7.5) to remove excess glutaraldehyde, to obtain the immobilized subtilisin A of the present invention.

Evaluation of Immobilized Protease Activity A fixed amount of the conventional immobilized pepsin prepared only by the step i) of Example 1 and the immobilized pepsin of the present invention prepared by the steps i) + ii) were taken, and each was 1% The mixture was added to a casein solution (adjusted to pH 3.0), shaken at 30 ° C. for 30 minutes, filtered, and the filtrate was collected. A certain amount of this filtrate was sampled (this liquid is called X liquid),
The remaining filtrate was further shaken at 30 ° C. for 30 minutes to sample a fixed amount (referred to as solution Y).

A trichloroacetic acid solution was added to solution X and solution Y, and the soluble protein was colorimetrically determined by the phosphorin method, which was taken as the relative activity of pepsin over time. The results are shown in Table 1.

In addition, a fixed amount of the conventional immobilized subtilisin A prepared by only the step iii) of Example 2 and the immobilized subtilisin A of the present invention prepared by the step iii) + iv) is taken,
The relative activity over time was measured by the same method as that for the immobilized pepsin described above.

The results are shown in Table 2.

As is clear from Tables 1 and 2, the immobilized protease according to the present invention has little or no increase in the relative activity of the protease after filtration, as compared with the immobilized protease prepared by the conventional method. From this, it can be seen that the immobilized protease of the present invention suppresses the release of the protease from the carrier.

〔The invention's effect〕

As described in detail above, the immobilized protease of the present invention has an advantageous effect that the elimination of the protease from the carrier is effectively suppressed and the stability is improved as compared with the conventional method. is there.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Kengo Ogawa 356 Ishii, Sakado City, Saitama Prefecture (72) Inventor Takaki Yamagata 1-10-15 Sanmeicho, Abeno-ku, Osaka City, Osaka Prefecture (72) Inventor Toshio Tanaka 8-1-14 Ishii-cho, Hyogo-ku, Kobe-shi, Hyogo Prefecture (72) Inventor Atsushi Nishikawa 1-14-11 Makihama, Minoh-shi, Osaka (72) Inventor Jun Nakamura 8-5-5, Okusawa, Setagaya-ku, Tokyo (56) References JP-A-46-1837 (JP, A) JP-A-49-42875 (JP, A) JP-A-50-46890 (JP, A) JP-B-56-15231 (JP, B2)

Claims (1)

[Claims] 1. A method for producing an immobilized protease by a covalent bond method, which comprises: And c) a method for producing an immobilized protease, which comprises subjecting the immobilized protease to a cross-linking agent treatment again.