KR930001385B1 - Method for purification of glucosyl transferase - Google Patents

Abstract

A process for the purification of alpha-Glucosyl transferase comprises (a) treating Erwinia sp. bacteria with triton X-100 as a biological surfactant and lysozyme, (b) adding ammonium sulfate (45-75 % of saturation degree) into the obtd. extracts at pH 5-8 for the selective precipitation, and (c) passing the obtd. enzyme part through agarose gel column (superose-6 column), anion exchange column (Mono Q column), and cation exchange column (Mono S column).

Description

Method for Purifying Glucosyl Transferase

1 is an elution pattern of alpha-glucosyl transferase by gel filtration.

FIG. 2 shows the elution pattern of alpha-glucosyl transferase by monocue column.

3 is the elution pattern of alpha-glucosyl transferase by the mono-s column.

4 is a molecular weight measurement result of alpha-glucosyl transferase.

5 is a diagram showing the results of HPLC analysis of the alpha-glucosyl transferase reaction product.

The present invention relates to a method for purifying alpha-glucosyl transferase, a production enzyme of isomaltulose, having a caries suppressing effect, from bacteria belonging to the genus Erwinia. Isomaltulose is a 6-0-alpha-di-glucopyranosyl-di-fructofuranose (6-0-α-D-glucopyranosyl-D-fructofuranose), a type of disaccharide, such as sugar, with a sweetness of sugar 1 It is a high value-added food material that can be used as a sweetener for various purposes such as confectionery and beverages because it has a property that does not cause tooth decay, but only about 2/2.

Alpha-glucosyl transferase involved in the production of isomaltulose is present in the plasma membrane space of bacteria and the substrate is sugar specific and undergoes a reaction to produce isomaltulose and trehalulose from sugar simultaneously. .

To date, isomaltulose-producing enzymes are found in the genus Protaminobacter (J. Jpn. Soc. Starch Sci., 35, 131, 1988), and in the Serratia genus (Biotechnol. Lett., 12, 667, 1990). ), Which was obtained from the crude enzyme extract (Biochem. J., 220, 213, 1984) and obtained from crude enzyme in the form of crude enzyme extract containing enzyme activity by osmotic shock or sonication. Therefore, the conventional extraction method is not only low recovery rate of enzyme but also contains a large number of other proteins and impurities, it is necessary to more effective extraction method and further it is necessary to obtain a pure purified enzyme.

Therefore, the present inventors obtained the enzyme extract in high yield by using a complex extraction method which treats microbial cells with a surfactant and cell membrane constituent polysaccharide hydrolysing enzyme and simultaneously applies osmotic impact, and newly fractionated by ammonium sulfate precipitation, gel filtration method. The enzyme was purified through a process such as anion exchange chromatography and cation exchange chromatography.

That is, an object of the present invention is to purify alpha-glucosyl transferase, which is an enzyme involved in the production of isomaltulose, which has a decay-inhibiting effect, by combining osmotic shock and treatment of a surfactant and a polysaccharide hydrolase. The purification method established by dissolving and extracting enzymes in the plasma membrane space of the cell in high yield and introducing new processes such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and cation exchange chromatography from the extracts To provide.

In the present invention, Erwinia rhapontici (ATCC 29283) was used as a strain.

Enzyme products can be easily obtained by separating cells from the medium containing enzymes from a medium containing enzymes from a medium containing enzymes by filtration, centrifugation, etc. However, enzymes produced in cells must separate enzymes from microbial cells. In order to perform such separation, the enzyme liquid may be obtained by separating the cell fragments by filtration after destroying the cells using a method such as sonication, grinding, and acid decomposition. In the present invention, since alpha-glucosyl transferase is present in the plasma membrane space, a surfactant and a cell membrane hydrolase are used for the purpose of extracting the membrane-bound protein. The surfactant mainly uses a cholic acid derivative or triton (trade name of a polyethylene glycol derivative) including a lipophilic chain capable of binding to a hydrophobic surface protein, and in the present invention, Triton X-100 (Triton) X-100) and lysozyme that hydrolyzes polysaccharides in the glycopeptide layer of the bacterial cell wall as cell membrane hydrolase.

The first step to selectively recover the desired enzyme from the enzyme stock solution extracted from the microorganism is mainly applied a precipitation method using an organic solvent or salt. Salts added to the enzyme solution include ammonium sulfate, sodium sulfate, magnesium sulfate. In the present invention, ammonium sulfate was used, and ammonium sulfate should be added at a saturation of more than 45% by the total weight. When the salt is added to the enzyme solution, the pH of the solution should be 5-8, usually about 7, and at this time, gently stirring Add. When the salt concentration is appropriate, the solution is stirred for another 5 hours, centrifuged to collect enzyme precipitate, and then re-dissolved.

Redissolved solutions should be desalted, either by dialysis or gel filtration. Among these, gel filtration is effective because it has a partial purification function to remove not only desalting but also some impurities. In the present invention, an agarose-based gel was used, and a specific gel is preferably a Super Rose-6 column (Pharmacia) manufactured by Pharmacia. Adsorbents that can be used to remove impurity proteins from the partially purified enzyme solution by gel filtration include strong basic anion exchange resins, medium basic anion exchange resins, and weak basic anion exchange resins, and strong acid cation exchange resins. There are acidic cation exchange resins and weakly acidic cation exchange resins, which may be ones that selectively adsorb the desired enzyme or do not selectively adsorb the desired enzyme. The resin used in the present invention is a mono-Q column, a strong basic anion exchange resin, and a Mono-S column, a strong acid cation exchange resin (more of which is a Pharmacia product). Adequately adjust the degree of adsorption, and separate the enzyme while increasing the concentration of salt in the eluent linearly or stepwise. A portion of the peak having enzymatic activity in the column fraction is collected, concentrated, and dialyzed against a buffer solution to obtain purified enzyme.

The alpha-glucosyl transferase purified by the above method had a single protein band when electrophoresed with polyacrylamide gel and had a molecular weight of 62,700 and an isoelectric point of 7.7. The maximum enzymatic activity was 30-35 ° C. and the optimum pH under citric acid-phosphate buffer was 6.0.

Enzyme activity was expressed in units, and 1 unit of alpha-glucosyl transferase refers to the amount of enzyme that transfers 1 μm of sugar to isomaltulose for 1 minute in a 55% sugar solution at 30 ° Cm pH5.5. Isomaltulose concentration is determined by quantifying reducing sugars by HPLC analysis or DNS (3.5-dinitro salysilic acid).

The present invention is explained in more detail in the following examples.

Example 1

[Step 1]

It is a process of extracting alpha-glucosyl transferase present in the plasma membrane space of cells from the bacterial cells of the genus Erwinia obtained through the culture. 100 g of the cells were taken, and 1 ml of 10% Triton X-100, 100 ml of beta-mercaptoethanol, and 2.5 ml of glycerol were added thereto, followed by vigorous stirring for 30 minutes. Add 300 ml of extraction buffer (200 mM phosphate buffer solution (pH7.0), 1 mM EDTA, 0.2 mgml ^-1 lysozyme, 10gml ^-1 deoxyribonuclease mixed urine solution), and stir vigorously for 30 minutes. After treatment, add 5 ml of PMSF solution (5 ml of acetone, 50 mg of phenylmethyl sulfonyl-fluoride, 1 mg of Pepstatin-A), and mix And cell fragments were separated to obtain a supernatant. The separated supernatant 310ml was used as enzyme extract.

[Step 2]

Inorganic salts are added to the enzyme extract to selectively precipitate the desired enzyme. 85.87 g of ammonium sulfate powder was slowly added to 310 ml of enzyme extract at 4 ° C while stirring to make the ammonium saturation of the solution 45%. The mixture was left at 4 ° C with stirring for at least 5 hours, followed by centrifugation. ML was obtained (AS-45 supernatant).

69.3 g of ammonium sulfate powder was slowly added while stirring so that the ammonium sulfate saturation of the AS-45 supernatant was 75%. The mixture was left at 4 ° C. for at least 5 hours and centrifuged to obtain a precipitate (AS-75 precipitate). AS-75 precipitates were dissolved in a small amount of 40 mM acetic acid buffer solution (pH 7.4) and centrifuged to remove insoluble materials. Thus, 54.8 ml of ammonium sulfate fractional precipitate solution was obtained.

[Step 3]

Ammonium sulfate fraction with alpha-glucosyl transferase activity is a process for purifying gel filtration, anion exchange chromatography, and cation exchange chromatography.

1. Gel Filtration

The Superrose-6 column (column volume 600 ml, separation range molecular weight 5,000-5,000,000) filled with agarose series gel was equilibrated with 40 mM acetic acid buffer solution and 17.5 ml of the solution obtained in step 2 was injected.

The flow rate was 4 mL / min and eluted with the same buffer solution. The eluate was collected in 10 mL fractions and the enzyme activity was measured. The elution pattern was the same as that in Fig. 1, and 51 ml of the peak portion having alpha-glucosyl transferase activity was obtained.

2. Anion Exchange Chromatography

Anion exchange chromatography was performed using a mono-Q column (column volume 20 ml), washed with 50 ml of 40 mM acetic acid buffer (pH 7.4) containing 1 M NaCl, and then equilibrated with 50 ml of 40 mM acetic acid buffer. 51 ml of the enzyme solution obtained by gel filtration was injected into the flask. Elution was carried out at a flow rate of 2.5 ml / min with increasing NaCl concentration as shown in Figure 2 using 40 mM acetic acid buffer solution containing 0M and 1M NaCl.

As a result of enzyme activity, alpha-glucosyl transferase was eluted at 0M NaCl concentration without adsorption on the column, and other unnecessary protein was eluted as the NaCl concentration increased. The elution pattern was shown in FIG. 2, and 83 ml of the eluate of the peak part containing enzyme activity were collected, and pH was adjusted to 5.0.

3. Cation Exchange Chromatography

The mono-Q column (1 mL column volume) was washed with 4 mL of 40 mM acetic acid buffer (pH 5.0) containing 1 M NaCl, equilibrated with 4 mL of 40 mM acetic acid buffer (pH 5.0) and then obtained from the mono-Q column. Injection into an 83 ml column. 40 mM acetic acid buffer solution (pH 5.0) containing 0 M and 1 M NaCl was eluted at a flow rate of 0.3 ml / min while increasing the NaCl concentration as shown in FIG. 3. As a result of measuring the enzymatic activity of the eluate, it can be seen that the alpha-glucosyl transferase was eluted at a concentration of 80-90 mM NaCl as shown in the elution pattern of FIG. 3. The peak fractions were collected, concentrated, and dialyzed against 40 mM acetate buffer. 3.1 ml of purified alpha-glucose transferase was obtained.

Purification diagram showing the enzyme activity and purification degree for each purification step is shown in Table 1 below.

Table 1. Purification Chart

Finally, the obtained enzyme had a specific activity of 650 units, and the recovery rate was 16.4% of the total activity, which was purified 90 times.

Example 2

Extraction of alpha-glucosyl transferase from the bacteria of the genus Uronia, pulverization by homogenizer (H method), sonication (S method), osmotic impact treatment (O method), surfactant treatment (D method) And the like. H method is to add 10 g of the cells to a homogenizer tube, homogenize for 5-30 minutes by adding 10 ml of 40 mM acetic acid buffer solution, and centrifuge the solution to obtain a supernatant. V) A suspension is maintained at 0-4 ° C, sonicated for 30 minutes-3 hours, centrifuged to obtain a supernatant. O method suspended 2 g of cells in 100 ml of cooled deionized water, stirred for 30 minutes-3 hours, left to stand, and centrifuged to obtain a supernatant. D method suspended 1 g of cells in 20 ml of Triton X-100 solution. After treatment for 1-5 hours, the supernatant is obtained by centrifugation. For each supernatant obtained by the various methods, the enzyme activity and the amount of protein were measured to compare the extraction degree, and the results are shown in Table 2.

[Table 2. Enzyme Extraction Method Comparison]

As shown in Table 2, the extraction method by O method is good as the extraction yield and the amount of impure protein is small, so that it can be used in a good way, but it is applied to the method of the present invention which is subjected to an osmotic impact with the treatment with a surfactant. In comparison, the extraction yield decreased and the amount of impure protein was found to be high.

Example 3

Ammonium sulfate is used to selectively precipitate the desired enzyme from the enzyme stock extracted from the microorganism, and the saturation of the ammonium sulfate is 30%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, respectively. The precipitate was obtained, dissolved in 40 mM acetic acid buffer, and the enzyme activities were compared. The results are shown in Table 3.

Table 3. Precipitation Comparison of Alpha-Glucosyl Transferase According to Ammonium Sulfate Concentration]

Purified alpha-glucosyl transferase in the above example had the following characteristics.

[One. Molecular Weight]

Purified alpha-glucosyl transferase was subjected to SDS-polymerized acyl amide gel electrophoresis with a concentration gradient of 10% to 15%, and the molecular weight was measured. As shown in FIG. 4, the molecular weight of 62,700 was found. Could.

[2. Isoelectric point]

The isoelectric point of alpha-glucosyl transferase was pH7.7 when the isoelectric point was measured with Phast gel IEP3-9 using the Pharm System (Phast System, Pharmacia).

[3. Optimal temperature]

The enzyme activity was measured after the reaction was carried out at 20 to 60 ℃, the temperature showing the maximum activity was 30-35 ℃.

[4. Optimal pH]

Each reaction was carried out at pH 3-8 to measure the enzyme activity.

[5. Reaction product]

Purified alpha-glucosyl transferase was reacted with 55% sugar solution at optimum condition and HPLC analysis of the product showed that most of the sugar was converted to isomaltulose and trehalulose as shown in FIG. Can be.

Claims (1)

Erwinia bacteria are treated with Triton X-100, a biological surfactant, and lysozyme, a cell membrane hydrolase, and extracted by applying an osmotic shock simultaneously to the extract. While maintaining the pH at about 5-8, ammonium sulfate was added at a saturation of 45% to 75% to obtain an enzyme fraction by selective precipitation, followed by gel filtration with an agarose gel (Superrose-6 column, pharmacia). The purified enzyme was purified by sequentially performing ion exchange chromatography with a mono-Q column (Mono Q column, pharmacia), an anion exchange resin, and a mono-s column (Mono S column, pharmacia), a cation exchange resin. Purification method of alpha-glucosyl transferase (α-Gulcosyl transferase) characterized by the above-mentioned.