KR920006399B1 - Purification of amino peptidase - Google Patents

Abstract

A method for purifying aminopeptidase from the fermented broth of Vibrio proteolyticus comprises: (a) filtering the fermented broth with membrane (pore size is 0.1 μm), concentrating and centrifuging to obtain the precipitate; (b) dissolving the precipitate in the zinc sulfate-contg. buffer soln. and heat-treating at 50-70 deg.C for 7-12 hrs.; (c) purifying the heat-treated soln. by gel filtration, anion exchange chromatography and hydrophobic chromatography to obtain the final product. The obtd. aminopeptidase is useful for the prodn. of foods, medicine etc..

Description

Method for Purifying Aminopeptidase from Vibrio Proteolyticus

1 shows the results of gel filtration chromatography according to the purification method of the present invention,

2 shows the results of anion exchange chromatography according to the purification method of the present invention,

3 shows the results of hydrophobic chromatography according to the purification method of the present invention,

FIG. 4 shows the results obtained by 15% sodium dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE).

The present invention relates to a method for purifying aminopeptidase from Vibrio proteolyticus, and more particularly, to purifying leucine-aminopeptidase from a culture medium of non-pathogenic bacterium Vibrio proteolyticus. It is related with the method of refine | purifying in a state.

Enzymes that hydrolyze proteins are largely divided into endopeptidase and exopeptidase according to their functions. Aminopeptidase belongs to exopeptidase and is an enzyme present in a variety of organisms, and has the property of sequentially releasing amino acids from the amino terminus of a substrate protein. There are many kinds of aminopeptidase in nature, and these enzymes show different affinity depending on the type of amino acid located in the amino terminal. In general, does the amino acid released from the amino terminal have hydrophobic properties or charge? Depending on the enzyme can be divided into two categories.

The aminopeptidase (EC. 3, 4, 11, 10) detected in fermentation broth in the culture of Vibrio proteolyticus was first isolated and purified by Prescott and Wilkes (Arch.

Biochem. Biophys., 1966, 117: 328; J. Biol. Chem., 1971, 246: 1756). This enzyme is generally weak in its specificity depending on the type of amino acid present at the amino terminus. However, when the free amino acid is a hydrophobic amino acid, the hydrolysis reaction proceeds more rapidly than the charged amino acid, which is called leucine-aminopeptidase (Eur. J. Biochem., 1973, 34: 459). In particular, if the amino acid in the second position from the protein, oligopeptide and amino terminus is negatively charged or proline, the reaction by this enzyme is further difficult to progress further (Methods Enzymol., 1976, 45B: 530). .

Protein drugs manufactured by genetic engineering techniques and expressed in yeast or bacteria can be appropriately applied for the removal of methionine groups located at the amino terminal, which requires mass production and purification of the enzyme.

The purification method of Vibrio proteolyticus used by Prescott and Wilkes is a method of increasing the purity by treating the fermentation medium with ammonium sulfate and then treating the organic solvent. Only Sephadex G-75 was used as a column (J. Biol. Chem., 1971, 246: 1756; Methods Enzymol., 1976, 45B: 530).

However, for industrial mass production, the use of organic solvents is not an efficient method due to the increase in volume during purification process, and the reaction with protein drugs requires high purity enzymes without contamination of foreign substances secreted from Vibrio bacteria. As a result, improvements in purification methods have been required.

Accordingly, the present inventors have intensively researched to solve the above problems, and have therefore developed a method capable of purifying aminopeptidase mass fermented from a fermentation medium with high purity.

That is, the present invention provides a method for mass purification of aminopeptidase with high purity from a fermentation medium of Vibrio proteolyticus.

Hereinafter, the present invention will be described in detail.

In the present invention, in purifying aminopeptidase from a fermentation broth of Vibrio proteolyticus, (A) the culture medium is filtered and concentrated to obtain a precipitate by centrifugation, (B) the precipitate is dissolved in a buffer solution, (C) It provides a method for purifying amidopeptidase from Vibrio proteolyticus, which is subjected to gel filtration chromatography, (D) anion exchange chromatography, and (E) hydrophobic chromatography.

Referring to the present invention in more detail as follows.

According to the purification method of the present invention, first, a Vibrio proteolyticus culture medium is passed through a hollow fiber of 0.1 μm pore size to obtain a cell filtrate from which cells are removed, and the filtrate is concentrated. Ammonium sulfate is added to a concentration of 65% and prevented at 4 ° C.

The aminopeptidase-containing solution precipitated by ammonium sulfate was centrifuged to obtain a precipitate, which was dissolved in pH 8.0 intris buffer solution and heat-treated at 50 to 70 ° C for several hours, at which time the protein concentration in the buffer solution 0.5 to 3 mg / ml.

It is then subjected to gel filtration chromatography to collect the required fractions followed by anion exchange chromatography and hydrophobic chromatography to obtain purified aminopeptidase.

In the purification process of the present invention, zinc sulfate is used as the buffer solution.

Hereinafter, the present invention will be described in more detail based on the following examples.

Example 1

Centrifuge 30 l of Vibrio proteolyticus (ATCC 15388) in a continuous centrifuge (cepa) to recover the culture and pass it through a hollow fiber (Amicon, H5MPO1-43) with a pore size of 0.1 μm. Collected. At this time, when the concentration of the cell filtrate that does not fall into 0.1μM pore was increased while diluting with buffer I (10mM Tris-HCl, pH 8.0, 100mM NaCl, 50μM zinc sulfate, 1mM PMSF). The cell solution was wiped to the extent that 5 L buffer I was used to treat 30 L cell culture fluid.

The pore size of the cell filtrate was concentrated only to hollow fiber (H10P10-20, manufactured by Amicon, H10P10-20) with a molecular weight of 10,000 Daltons to finally have a volume of 2 l. The amount of protein contained in this solution was measured by BCA measurement (Pierce) and found to be about 6.5 g.

Example 2

The solution 21 obtained in Example 1 was dissolved by adding 730 g (final concentration 65%) of ammonium sulfate at 4 ° C. and left for 16 hours. This was centrifuged at 8,000 rpm for 30 minutes (Beckman, J-10 rotor), and the precipitates were collected and dissolved in Buffer I to give a final protein concentration of 1-2 mg / ml. The protein solution was filtered through a membrane having a pore size of 0.45 μm and then heat-treated at 65 ° C. for 7-8 hours.

The solution was concentrated with an amicon filter membrane to have a protein concentration of about 10-20 mg / ml, and passed through a filtration membrane (cellulose-acetate, manufactured by Nalgene) having a pore size of 0.45 μm to remove insoluble substances precipitated during heat treatment. After frozen storage.

Example 3

700 mg of the heat-treated aminopeptidase obtained in Example 2 was passed through a Sephacryl S-300 (15 cm × 30 cm, Pharmacia) column equilibrated with buffer solution I, and the gel filtration chromatography was performed. The elution rate was about 500 ml /. It was time. The results are shown in FIG.

The activity of the aminopeptidase was in the fraction immediately following the void peak and the amount of protein when collected was about 140 mg.

In order to measure the activity of aminopeptidase, leucine-paranitroanilide (Leu-pNA) was used as a substrate, and 100 mM Tris-HCl, pH 8.0 was used as a buffer. The substrate was added to 1 ml of the buffer solution to a final concentration of 1.5 mM, and a small amount of the protein-containing solution eluted from the column containing the protein was added thereto and reacted at 37 ° C. for 1 minute. 200 μm of acetic acid solution (30% v / v) was added to stop the reaction, and then the absorbance was measured at 405 nm.

Fractions containing aminopeptidase have a yellow color reaction.

Example 4

The aminopeptidase-containing liquid partially purified by Example 3 was subjected to anion exchange chromatography on a DEAE-cellulose (Pharmacia, 2.5 cm x 10 cm) column. That is, after the protein-containing solution was injected into the column equilibrated with the buffer solution I, the protein was eluted by applying a linear gradient such that the salt concentration was 10 mM to 700 mM, respectively.

The results are shown in FIG. 2, and the protein having aminopeptidase activity was eluted at a salt concentration of about 200 mM.

This step yielded about 75 mg of purified protein.

Anion exchange chromatography using DEAE-Sepharose column showed the same results as in FIG.

Example 5

75 mg of the protein obtained in Example 4 was subjected to hydrophobic chromatography in a phenyl-sepharose (Ph-armacia) column. That is, equilibrate the solution I with 2M of salt and inject the protein-containing solution, and linearly gradient to 8 times the volume of the buffer in the direction of decreasing the salt concentration to 0M. Eluted.

The results are shown in FIG. 3, where a small amount of contaminating protein was eluted without adsorption on the phenyl-sepharose column, and the protein eluted at the linear gradient was almost pure aminopeptidase.

The amount of protein obtained was about 68 mg.

The aminopeptidase fraction obtained in each of the above purification steps was developed by 15% SDS-PAGE, and the results are shown in FIG.

In FIG. 4, the first column shows the molecular weights of 66.2K, 42.7K, 31.0K, 21.5K and 14.4K Daltons from above as standard proteins, and the second column shows the filtrate of the fermentation bath. The third column is the development of the concentrate of the fermentation batch, the fourth column is the result of fractionation of ammonium sulfate in the fermentation medium containing aminopeptidase, the fifth column is the result after heat treatment, the sixth column, Rows 7 and 8 show the results of gel filtration chromatography, anion exchange resin chromatography and hydrophobic chromatography, respectively.

As shown in FIG. 4, the purified protein according to the present invention was found to be about 29,500 Daltons on SDS-PAGE, and the Coomasubiru staining showed a purity of about 99%.

Claims (8)

In purifying aminopeptidase from a fermentation broth of Vibrio proteolyticus, (A) the culture solution is filtered and concentrated, centrifuged to obtain a precipitate, (B) the precipitate is dissolved in a buffer solution, and A method for purifying aminopeptidase from Vibrio proteolyticus, comprising gel filtration chromatography, (d) anion exchange chromatography, and (e) hydrophobic chromatography. The method according to claim 1, wherein the separation membrane having a pore size of 0.1 μm is used in the filtration step (a). The method of claim 1, wherein in step (b), the precipitate is dissolved in a buffer so that the protein concentration is 0.5 to 3 mg / ml. The method according to claim 1, wherein the precipitate is dissolved in a buffer in step (b) and then heat-treated at 50 to 70 ° C. for 7 to 12 hours. The method of claim 1, wherein the gel filtration chromatography of step (c) is performed using Sephacryl S-300 as a resin. The method of claim 1, wherein the anion exchange chromatography of step (d) is performed using DEAE-cellulose or DEAE-sepharose as the resin. The method according to claim 1, wherein the hydrophobic chromatography of step (e) is performed using phenyl-sepharose as a resin. The method according to claim 1, wherein the buffer solution contains zinc sulfate.

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