KR100323837B1 - Process for the preparation of low-molecular weight levan by using immobilized levansucrase or microorganism cells treated with an organic solvent - Google Patents

Abstract

The present invention relates to a method for producing a low molecular weight levan, which is a cell obtained by permeabilization of a levanschkrase immobilized on a magnetite bead or a cell expressing Levanschkrase with an organic solvent. According to the method of the present invention, which is used as an enzyme source of levanschkraze and prepares levan by using sugar as a substrate, it is possible to mass-produce low molecular weight levan economically and efficiently.

Description

PROCESS FOR THE PREPARATION OF LOW-MOLECULAR WEIGHT LEVAN BY USING IMMOBILIZED LEVANSUCRASE OR MICROORGANISM CELLS TREATED WITH AN ORGANIC SOLVENT}

The present invention relates to a method for mass production of low-molecular weight levan, and more specifically, to fix purified levansucrase (EC2.4.1.10) in magnetic beads, or cells containing levanschkrase The present invention relates to a method for mass production of low molecular weight levan by permeabilization with an organic solvent and using each as an enzyme source of levanschkrase.

Levan is a water soluble fructose polymer represented by Glu- (Fru) _n where Glu represents a glucose residue, Fru represents a fructose residue, and n represents a number of about 10 ¹ -10 ⁵ . Produced from sugar by reaction. It has similar physicochemical properties to dextran and can be used in place of dextran, such as separation of proteins using plasma substitutes or the formation of aqueous phase separation systems of polyethylene glycol / levan (Korean Patent Application Publication No. 99-54630), emulsifiers, It has been applied to a wide range of fields such as stabilizers, encapsulation reagents (Lwibovici et al., Anticancer Res. , 5 , 553 (1985); and Stark et al., Br. J. Exp. Path. , 67 , 141 (1986). )). Recently, Levan has been applied in industrial polymer food (Hatcher et al., Boprocess , technol. , 11 , 1 (1989)), cosmetics and pharmaceuticals (Whiting et al., J. Inst. Brew. , 73 , 422 ( 1961) and Han, Bioprocess Technol. , 12 , 1 (1990)).

In the industrial application of Levan, the structure and size of Levan serve as an important factor. That is, the low molecular weight uniform size of levan can be usefully used for protein fractionation using plasma substitute or polyethylene glycol / levane dendritic system formation, and is known to be excellent in antitumor activity or immunopotentiation. The characteristics of the enzyme synthesized Levan are changed by the degree of branching and the molecular weight of Levan and are affected by the enzyme reaction temperature and pH of the reaction solution during the reaction. For example, the molecular weight of the levan produced at a reaction temperature of 0-37 ^° C. is inversely proportional to the reaction temperature. Levan is also present as a low-molecular weight (molecular weight: <5,000) as a storage carbohydrate of some plants and can be produced as a relatively high polymer (> x10 ⁶ ) by medium containing sugar or extracellular synthesis of microorganisms in nature ( Han YW, Adv. Appl. Microbiol. , 35 , 171 (1990)).

A number of methods for preparing levans using levanschkrase have been reported. For example, US Pat. No. 4,879,228 and International Patent Publication No. 86-4091 disclose leban from sugar using microorganisms that act in metabolic processes. However, this method not only has low yield of levane but also has a lot of difficulty in purifying and producing a mixture of by-products and impurities, and has a high reaction temperature (37 ^o) to produce low molecular weight levane. As a result of applying C), the low molecular weight levane and oligosaccharide could be prepared, but the production yield of the levane to the substrate was reduced to 10 or less.

The method of preparing levan by batch method is disadvantageous in that it is economically disadvantageous because the enzyme reuse rate is mixed with substrate solution, and it is disadvantageous in the case of hydroxyapatite in a carrier such as hydroxyapatite. It is reported that the method of producing levane by immobilization of ethylene produces a higher molecular weight levane than the levane produced by a batch method (Chambert et al., Carbohydr. Res., 244 , 129 (1993)).

Therefore, the inventors of the present invention have produced levane using recombinant Levanschkrase that is expressed in E. coli transformed using the Levanschkrase gene of Zymomonas mobilis (Belghith, H. et al. ., Biotechnol. Letts , 18 , 467 (1996), Korean Patent Publication Nos. 176410 and 145946), continued research to produce low molecular weight levans in a more efficient and economical manner. The present invention has been found to be achieved by immobilizing Levanschkrase or permeabilization of cells containing Levanschkrase with organic solvents and using them as Levanschkrase enzyme sources, respectively. Completed.

It is an object of the present invention to provide a method for producing low molecular weight levans in high yield.

Figure 1 shows the result of the purification of levansucase produced in E. coli by metal affinity chromatography,

Figure 2 shows the result of measuring the molecular weight of Levan using a gel permeation chromatography column in HPLC.

In order to achieve the above object, in the present invention, the cells obtained by performing permeabilization of the microorganisms in which Levanschkrase immobilized on the magnetite beads or Levanschkrase are expressed by organic solvent treatment are used as the Levanschkrase enzyme source. It provides a method for producing levan by using sugar as a substrate.

Hereinafter, the present invention will be described in more detail.

Levanschkras used in the present invention is cultured strains designed for high expression of Levanschkrase, for example, recombinant Escherichia coli (KCTC 8661P) prepared in the Republic of Korea Patent Publication No. 145946 and then ultrasonically crushed and metal It can be obtained by purification by affinity chromatography (Fig. 1).

The method of immobilizing the separated and purified Levanschkrase on the surface of the immobilized carrier is as follows. First, 5 to 100 mg of magnetic iron oxide as an immobilization support was added to a mixed solution of TiCl ₄ and water (1:10 to 1:30 (v / v), preferably 1:20 (v / v)). After addition and stirring, treatment with 1M ammonia solution and stirring again. It is washed 1 to 5 times, preferably 3 or more times with acetic acid buffer solution of pH 3.0 to 7.0, preferably pH 4.0. Then, the prepared carrier and Levanschkrase 0.1 to 2 U, preferably 0.4 U are mixed in the same acetic acid buffer solution and stirred at a uniform rate at 4 ^o C for 30 minutes to 16 hours, preferably 1 hour. The enzyme is immobilized on the carrier. Unimmobilized enzyme is removed by, for example, washing three times or more until acetic acid buffer is not detected free enzyme activity.

In addition, the method of using the Levans-Curase enzyme source by performing the permeation of the cells expressing Levans-Chrase is as follows. In other words, the cells containing the Levanschkrase gene, for example, recombinant E. coli BL21 (DE3) / pEL12 (KCTC 8661P) incubated in a suitable medium, induces the expression of Levanschkrase and centrifuged and recovered the cells After washing with acetic acid buffer, preferably 50 mM acetic acid buffer (pH 6.0), and concentrating 10 to 20 times by adding the same acetic acid buffer solution, the organic solvent is 1:10 to 1: 100 (organic solvent: bacteria) ), Preferably in a ratio of 1:10 (organic solvent: bacteria) and vigorously stirred at room temperature for 3 to 10 minutes, preferably 5 minutes. Toluene, CTAB (cetyltrimethylammonium bromide), etc. can be used as an organic solvent, It is preferable to use toluene. Subsequently, the organic solvent is removed by centrifugation, and the cells are washed with acetic acid buffer and used as a Levanschkrase enzyme source (organic solvent treated cells).

The levan can be prepared by enzyme synthesis using sugar as a substrate using levanschkrase or organic solvent-treated cells immobilized on the magnetite beads thus obtained. The concentration of sugar as a substrate used for the enzyme reaction is 10 to 50 (w / v), preferably 10 to 20, the reaction pH is 4 to 6, preferably pH 4, the reaction temperature is usually 0 to 37 ^o C, preferably 0 to 20 ^° C., most preferably 4 ° C., is suitable because of low activity of the enzyme and extended life of the enzyme. At this time, the reaction time is carried out for 20 to 100 hours, preferably 70 hours.

When the levanschkrase or organic solvent treated cells immobilized on the magnetite beads of the present invention, the yield of the levan reaches about 12-30 g / l, and the molecular weight of the obtained levan is 2 million (toluene-treated cells). ) To 3 million (immobilized enzyme), which is significantly smaller than the molecular weight (more than 6 million) of the Levan produced by the untreated enzyme under the same conditions.

The enzyme reaction method can be carried out in a batch or semi-batch reaction.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Reference Example

(1) Protein Assay

Using ovalbumin as the standard protein, the protein was quantified as follows.

5 ml of Coomassie Blue solution (Bio-Rad protein assay, manufactured by Biorad) was added to 100 µl of the sample, mixed, and the mixture was left at room temperature for 10 minutes, and absorbance was measured at 595 nm to quantify the protein.

(2) Determination of Levanschkrase Activity

The activity of levanschkrase was measured by Omulan et al. (O'Mullan et al., Biotechnonol. Lett. , 13 , 137 (1991)) as follows.

After dissolving sugar in 50 mM acetate buffer (pH 5.0) to 1, 440 µl was taken, and 10 µl of coenzyme was added thereto and reacted at 37 ° C for 30 minutes. The reducing sugar produced in the reaction solution was quantified using a GOD-PAP kit (Berlinger Mahheim). The enzymatic activity of producing 1 μM of glucose for 1 minute was defined as 1 unit (U).

(3) Levan Assay

The enzyme reaction solution was filtered through a 0.45 μm filtration membrane, and then 20 μl of the filtrate was injected into HPLC (System Gold, Beckman Co., Ltd.) to quantify the amount of leban. HPLC analysis conditions were run by distilled water at a rate of 0.4 ml / min using a column (Ionpak KS-802, Shodex) maintained at 50 ^° C., was detected by the refractive index (refractive index).

(4) Molecular weight measurement of Levan

The enzyme reaction was filtered through a 0.45 μm filtration membrane, and then 100 μl of the filtrate was injected into HPLC (System Gold, Beckman) to compare the time for each Levan to pass through the column. In order to measure the passage time of the column according to the size of the molecular weight, polyethylene oxide (molecular weight 8x10 ⁶ ), dextran standards (molecular weight 8x10 ⁶ , 7.5x10 ⁵ , 1.7x10 ⁵ , 4x10 ⁴ ) and sucrose (molecular weight) were used as standards. 342). HPLC analysis conditions were the same as in (3), except that two connected columns (GPC columns 4,000-1,000, manufactured by Polymer Laboratories) were used. HPLC analysis conditions were run by distilled water at a rate of 0.4 mL / min using a column (Ionpak KS-802, Shodex Co., Ltd.) maintained at 50 ℃, it was detected by the refractive index (refractive index).

(5) Purification of Levanschkrase

Recombinant Escherichia coli BL21 (DE3) / pEL12 (KCTC8661P; Korean Patent Publication No. 145946) designed to express high level of Levanschkrase was added to M9-ZB medium (10 g of tryptone in 1 L, 5 g of NaCl, 1 g of ammonium chloride, potassium dihydrogen phosphate). Inoculate 3g, disodium phosphate 6g, glucose 4g, 1M magnesium sulfate 1ml) and shaken at 37 ° C, and absorbance at 600nm reaches 0.7, then add IPTG (from Sigma) to 1mM to make Levanschkra Expression of the agent was induced. After 3 hours, the culture solution was centrifuged to recover the cells, and then crushed by ultrasonic waves. The crushed solution was used as a column using a Ni-NTA resin column (Qiagen Co., Ltd.) and a metal affinity using 0.25M imidazole as eluent. Purification was carried out by metal affinity chromatography. Figure 1 shows the purified Levanschkrase, column M is the standard molecular weight marker, the first column is E. coli whole protein, the second column is levanschease purified by metal affinity chromatography.

Example 1 Immobilization of Levanschkraze and Generation of Levan

(Step 1)

25 μl of TiCl ₄ and 500 μl of water were added to 50 mg of magnetite oxide, followed by stirring at 4 ° C. for 1 hour. Then, 280 μl of 1M ammonia solution was added, followed by stirring at 4 ° C. for 1 hour. The carrier was recovered by centrifugation at 6,000 rpm, and then washed three times with 50 mM acetic acid buffer solution at pH 4.0.

The carrier and levanschkrase 0.4 U were mixed in acetic acid buffer solution at pH 4.0, stirred at 4 ^° C. for 1 hour, and washed with acetic acid buffer to obtain levanschkrase immobilized on magnetite with an enzyme recovery of 75. It was.

(Step 2)

The sugar was dissolved in 1 ml of 50 mM acetic acid buffer solution at pH 4.0 to a concentration of 10 (w / v), and the immobilized enzyme obtained in step 1 or 0.3 U of purified levanschkrase as described in (5) of Reference Example was added. Then, the temperature was adjusted to 4 ° C., 10 ° C., 20 ° C. and 37 ^° C. for 70 hours. The amount of leban produced after the reaction was measured as in Reference Example (3), and the results are shown in Table 1 below.

Enzyme Relative Leban Concentration According to Reaction Temperature () 4 ^o C 10 ^o C 20 ^o C 37 ^o C Immobilized Enzyme 100 97 84 61 Refined Levanschkrases 100 94 72 20

As shown in Table 1, when both enzymes were reacted at 4 ℃, the production of Levan was the most, and as the temperature increases, the yield of Levan was decreased. However, by the immobilization of enzymes, the stability of the temperature of the levanschkraase is greatly increased so that the levanschkrase is immobilized by the immobilized levanschkrase as compared to the levanschkrase which is not immobilized at a high reaction temperature, that is, 20 ° C or 37 ° C. It can be seen that the amount of production.

In addition, as a result of comparing the optimum pH of the two enzymes, it was observed that the optimum pH of the enzyme is changed to more acidic conditions by immobilization as pH 4.0 (immobilized enzyme) and pH 5.0 (purified levanschkrase), respectively. As a result of investigating the conversion to the levane product according to the substrate concentration, the two enzymes showed similar enzymatic properties. At 5% of the substrate concentration, there was no significant change in substrate conversion rate, so that the enzyme activity was not inhibited. Inhibition of enzymatic activity was observed.

Example 2 Preparation of Toluene or Acetone Cells and Production of Levan

(Step 1)

Recombinant Escherichia coli BL21 (DE3) / pEL12 (KCTC 8661P) containing the Levanschkrase gene was cultured as in (5) of the Reference Example, and the expression of Levanschkrase was induced, followed by centrifugation of 1 L of the culture solution. The cells were washed with 1/10 portions of 50 mM acetic acid buffer (pH 6.0), and 5 ml of 50 mM acetic acid buffer solution (pH 6.0) was further added to the recovered cells, thereby concentrating the Levanschkrase by 20 times. 0.5 ml and 0.05 ml of toluene and 0.5 ml of acetone were added to 5 ml of the concentrated cells, vigorously stirred at room temperature for 5 minutes, and then centrifuged to remove toluene and acetone, followed by 50 mM acetic acid buffer solution (pH 6.0) to wash the cells twice to obtain toluene or acetone-treated cells.

(Step 2)

Toluene or acetone-treated cells and 100 µl of untreated cells were added to 1 ml of 50 mM acetic acid buffer solution (pH 4.0) containing 10 sugars, respectively, and then reacted for 6 hours at 4 ^° C. The concentrations are shown in Table 2 below.

Processing method Relative Leban Concentration () Untreated cells 35 Toluene-treated cells (toluene: bacteria = 1: 100) 87 Toluene-treated cells (toluene: cells = 1:10) 100 Acetone-treated cells (acetone: cells = 1:10) 29

As shown in Table 2, the cells treated with toluene at a ratio of 1:10 (toluene: cells) had about three times higher enzymatic activity than untreated cells, and those treated with toluene were treated with acetone. The enzyme activity was 3.5 times higher. On the other hand, in the acetone-treated group, the leban production rate was lower than that in the acetone-treated group, indicating that acetone treatment was not appropriate for the levanschkrase.

Example 3 Leban Formation According to Reaction Temperature of Toluene Treated Cells

Toluene-treated cells were used at a ratio of 1:10 (toluene: cells), and the reaction temperatures were the same as those of Example 2 except that the reaction temperatures were 4 ° C, 10 ° C, 20 ° C, and 37 ° C as shown in Table 3 below. By measuring the concentration of the leban produced according to the temperature and the results are shown in Table 3 below.

Enzyme Relative Leban Concentration According to Reaction Temperature () 4 ^o C 10 ^o C 20 ^o C 37 ^o C Toluene Treated Cells 100 100 83 43 Refined Levanschkrases 100 94 72 20

As shown in Table 3, the optimum leban production temperature by the toluene-treated cells under the reaction conditions was 4 ℃, compared to the purified levansuckrase, the stability to temperature is greatly increased, that is, high reaction temperature At 20 ° C. or 37 ° C., it can be seen that the amount of levane produced is greater than that of purified levane sukrases.

In addition, as a result of examining the conversion rate to the levane product according to the substrate concentration, the two enzymes showed similar enzymatic properties. At 5% substrate concentration, there was no significant change in substrate conversion rate, so that the enzyme activity was not inhibited. Inhibition of enzyme activity was observed at the concentration.

Example 4 Measurement of the Molecular Weight of the Prepared Levan

Example 2 at a reaction temperature of 4 ° C. with unimmobilized Levanschkrase ((5) of Reference Example), immobilized Levanschkrase (Step 1 of Example 1) and toluene treated cells (Example 3) The molecular weight of the Levan produced by the method was analyzed by gel permeation chromatography as in (4) of the Reference Example.

As a result, the molecular weight of the levan synthesized by the free enzyme that was not immobilized under the same reaction conditions was observed to be 6 million or less, and the leban produced from the toluene-treated cells was measured to about 2 million, and the levan produced by the immobilized enzyme. The molecular weight of was measured to about 3 million, it can be seen that the leban produced by the toluene-treated cells or immobilized enzyme is lower molecular weight than the leban produced from the free enzyme (Fig. 2).

Example 5 Reuse Test of Immobilized Enzyme

The reusability of the immobilized enzyme was investigated in the following manner. In 1 ml of 50 mM acetic acid buffer (pH 4.0), sugar was dissolved to a concentration of 10 (w / v), 0.3 U of the immobilized enzyme prepared in Step 2 of Example 1 was added, and then reacted at 4 ^° C. for 6 hours. After the reaction was terminated by centrifugation to recover the immobilized enzyme. Subsequently, the enzyme solution was added to the recovered immobilized enzyme and the enzyme solution was added to 1 ml of 50 mM acetic acid buffer (pH 4.0) at a concentration of 10 (w / v). Was investigated and the procedure was repeated.

Frequency of reuse of immobilized enzyme 1 time Episode 2 3rd time 4 times Remaining enzyme activity () 100 84 52 15

As shown in Table 4, there were more than 50 activities until three times of repeated use and only about 15 of the initial enzyme activity remained after four times of repeated use. Therefore, when the levanschkrase immobilized on the surface of the magnetite bead (magnetite bead), the enzyme reuse was low because the enzyme reaction product is a large molecule Levan was possible about 3 times the enzyme reuse.

In this way, by immobilizing Levanschkrase to magnetite or treating the cells expressing Levanschkrase with organic solvent and using them as Levanschkrase enzyme source, it is possible to increase the stability of the enzyme and enable repeated use. Levan can be prepared, and can be usefully used in medicine, food, cosmetics, and the like.

Claims (4)

A method of producing levan by using sugar as a substrate by using levanschkrase immobilized on magnetite beads. A method of producing levan by using sugar as a substrate using cells subjected to permeabilization by treating organic cells with Levanschkrase expressed cells. The method of claim 2, The organic solvent is toluene. The method according to claim 1 or 2, Characterized in that it is carried out at a reaction temperature of 4 ℃ to 37 ℃.