KR100194075B1 - Purification Method of Cyclodextrin Glucanotransferase Using Aqueous Two-Phase System - Google Patents

Abstract

The present invention relates to a method for purifying cyclodextrin glucanotransferase (EC 2.4.1.19., Hereinafter referred to as 'CGTase') using an aqueous two-phase system. More specifically, the present invention uses an aqueous two-phase system in which phase separation is formed by polyethylene glycol and dextran, or salts of polyethylene glycol and salt. It relates to a method of purification. According to the CGTase purification method of the present invention, CGTase can be purified easily, efficiently and economically by mass production using an aqueous two-phase system.

Description

Method for Purification of Cyclodextrin Glucanotransferase Using Aqueous Two-phase System

The present invention relates to a method for purifying cyclodextrin glucanotransferase (EC 2.4.1.19) using an aqueous two-phase system. More specifically, the present invention provides a cyclodextrin glucanotransferase-producing microorganism using an aqueous two-phase system in which phase separation is formed by polyethylene glycol and dextran, or salt and polyethylene glycol. It relates to a method for purifying the electric enzyme from the culture solution of.

In general chemical processes, the material is separated from the mixture by various methods such as extraction, crystallization, distillation, dialysis, and drying, which are usually appropriate depending on the molecular nature of the material to be separated. For example, in the case of extraction, the organic solvent and water are mixed to collect the desired substance by concentration in one of two phases, and the impurities are separated by concentration in another phase. However, in the biotechnology field dealing with proteins such as physiologically active substances and enzymes, extraction methods using organic solvents are used only in special cases due to the decrease in physiological activity by contact with organic solvents. Therefore, in the biotechnology field, various methods for separating and purifying bioactive substances and proteins in a stable state without degeneration are being studied.

As one of them, an aqueous two-phase system is introduced, which means a phase separation formed due to incompatability between two polymer materials or between a polymer material and a salt.

Mattiasson et al. Applied a high yield of product by applying an aqueous two-phase system to remove the inhibition of microbial growth caused by acetone and butanol fermentation from Clostridium ace tobutylicum. (Mattiasson et al., Biotech, Bioeng., 7: 355-366 (1965)). In addition, the aqueous two-phase system is used for the separation of intracellular enzymes and cofactors, and Veide, etc. employs an aqueous two-phase system, and β-galactosi from E. coli. Β-galactosidase was isolated (see Veide et al., Biotech. Bioeng., 25: 1789-1800 (1983). Tjerneld is a glycosylation of cellulose and enzymatic bioconversion). Aqueous two-phase system (Tjerneld, Biotech. Bioeng., 27: 1036-1043 (1985)) and Menz et al., Bacillus subtilis producing α-amylase The same effect as immobilization of cells was obtained by culturing in an aqueous two-phase system while simultaneously increasing the productivity of the enzyme (Menge et al., J. Appl. Biochem., 5: 75-90 (1983).

On the other hand, cyclodextrin glucanotransferase (hereinafter referred to as 'CGTase') catalyzes the transglycosylation reaction (intramolecular transglycosylation, cyclization) in the same molecule to produce a cyclodextrin from the starch substrate. Electrolytic enzymes include Bacillus circulans, B. macerans, B. megaterium, B. ohbensis, B. stearothermophilus, It is known to be produced in microorganisms such as B. subtilis, Klebsiella oxytoga, Micrococcus sp., And Thermomoeroerobacter sp. CGTase produced therefrom has been used to prepare cyclodextrins, which are widely used for the purpose of stabilizing volatiles, eliminating bitumen, solubilizing insoluble substances and uniformizing colors in food, medicine, pesticides and flavorings.

Against this background, the process for separating and purifying the CGTase, an extracellular enzyme from an electric microorganism in an efficient and economic manner, has been a very important part in the industrial production of cyclodextrin.

Accordingly, the present inventors have made intensive studies to separate the electric enzymes from the CGTase producing microorganisms in an efficient and simple manner. As a result, the CGTase can be purified easily and efficiently and economically using an aqueous two-phase system. It has been found that the present invention has been completed.

After all, it is an object of the present invention to provide a method for purifying CGTase enzyme from a culture of CGTase producing microorganisms using an aqueous two-phase system.

1 is a graph showing the recovery of CGTase separated from the lower layer using an aqueous biphasic system formed of 10% PEG35000 and 15% salt.

2 is a graph showing the recovery of CGTase separated from the lower layer using an aqueous biphasic system formed of 10% PEG35000 and different concentrations of sodium sulfate.

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

Among the CGTase-producing microorganisms of the genus Bacillus isolated from the soil, CGTase producing heat-resistant and high-productivity β-cyclodextrin was produced, and strains with high fermentation titer were selected. The selected strains were cultured (pH 8.0) consisting of 0.6% aqueous starch, 0.6% yeast extract, 0.6% bactopeptone, 0.16% NH ₄ Cl, 0.2% KH ₂ PO ₄ and 0.01% antifoam B suspension. The culture was carried out under the conditions of temperature 30 ℃, stirring speed 400rpm, aeration rate 1vvm.

In order to purify the CGTase enzyme from the previously cultured strain using an aqueous two-phase system, polyethylene glycol (PEG) and dextran or PEG and one salt are added to the strain culture solution, mixed and stirred, The enzyme was purified by standing still to form a phase separation and separating the CGTase present in the lower layer. In this case, PEG is a molecular weight of 4,000 or more, and salt is used as potassium phosphate, sodium phosphate or sodium sulfate. In addition, the aqueous two-phase system by electric PEG and dextran may further include a salt in addition to the two polymer materials.

For the aqueous biphasic system by PEG and dextran, 3 to 7% (w / v) PEG, most preferably 5% is added to the microbial culture, and 5 to 10% (w / v) dextran. ), Most preferably 7%. In addition, for the aqueous biphasic system with PEG and one salt, 7 to 12% (w / v) PEG, most preferably 10% is added to the microbial culture, and the salt is potassium phosphate or sodium phosphate Adds 10 to 18% (w / v), most preferably 15% of salt, and 3 to 10% (w / v), most preferably 10% of salt for sodium sulfate.

According to the CGTase purification method of the present invention as described above, mass production is easy, efficient and economical purification of CGTase using an aqueous two-phase system.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

[Isolation of CGTase Producing Strains]

To isolate CGTase-producing microorganisms from soil, 100 soil samples were cultured on phenolphthalein and starch-containing agar plates, and CGTase-producing strains were first selected by comparing their ability to form clear zones, and then selected. Strains were cultured in Erlenmeyer flasks and 36 strains were secondarily selected by comparing the titer of each isolated strain. The secondary screened strains were examined for temperature, heat resistance, composition and fermentation characteristics of the cyclodextrin produced by the enzyme in a 5 liter fermenter, and the productivity and fermentation potency of β-cyclodextrin were considered in consideration of these enzyme characteristics. Two strains of the genus Bacillus were selected.

Example 2

[Fermentation of CGTase Producing Strains]

In order to find the optimum fermentation conditions for the strains of the Bacillus genus isolated in Example 1, strains were fermented in 5 liters of fermenter while varying the cultivation conditions such as the composition and composition ratio of the culture medium and the culture pH, temperature, stirring speed, aeration rate, etc. Incubated. As a result, in a culture batch consisting of 0.6% water soluble starch, 0.6% yeast extract, 0.6% bactopeptone, 0.16% NH ₄ Cl, 0.2% KH ₂ PO ₄ and 0.01% antifoam B suspension, pH 8.0, It was confirmed that the strain cultured under the conditions of agitation speed 400rpm, aeration rate 1vvm grows best. Therefore, CGTase enzyme production was performed by culturing the strain in a 30 liter fermenter under fermentation optimization conditions as described above.

Example 3

Purification of CGTase Using Aqueous Two-phase System

Using a topographical chart of the various aqueous two-phase systems presented by Albertsson (Albertsson, Biochem. Biophys. Acta., 27: 378-395 (1958)), PEG and dextran, PEG and salts Several points on the same tie-line were selected for, and a distribution experiment of CGTase was performed at the composition ratio.

That is, PEG and dextran, or PEG and salt, are added to the strain fermentation broth in an amount corresponding to the composition ratio selected in the previous, mixed, stirred, and left to form a phase separation, and the CGTase present in the upper and lower layers of the separated phase. By measuring the titers of, the degree of purification of the CGTase enzyme was determined.

Example 3-1

[Purification of CGTase Using Aqueous Two-Phase System of PEG and Dextran]

To determine the phase separation form of PEG and dextran by aqueous two-phase system, 5% PEG 8000 was added 7% dextran T70 to the fermentation broth, and the phase separation form was investigated. As a result, it was confirmed that complete phase separation was formed into the upper layer containing a large amount of PEG and the lower layer containing a large amount of dextran, and the cells in the culture medium were formed in the middle of the upper layer and the lower layer to efficiently remove the cells at the same time as the phase separation. I could see that. In addition, the cell removal rates in the upper and lower layers were 98.3% and 97.5%, respectively. From these results, the aqueous two-phase system was found to be very effective in cell removal.

In addition, the distribution pattern of CGTase according to the molecular weight change of 5% PEG and 7% dextran was investigated, and the results are shown in Tables 1 and 2 below.

As shown in Table 1, a large amount of CGTase was recovered from the lower layer than the upper layer, and the CGTase recovery rate in the lower layer had little effect on the molecular weight change of dextran, but was significantly affected by the molecular weight change of PEG. The higher the molecular weight of PEG, the higher the amount of CGTase separated into the lower layer. In addition, the CGTase recovery in the lower layer was the most effective (83.4%) when separating CGTase using PEG25000 and dextran T2000 aqueous phase system.

On the other hand, when 0.3 M sodium sulfate was added in small amounts to PEG35000 and dextran T2000, the CGTase recovery in the lower layer was increased to 85.8%. In addition, it was confirmed that phase separation of an aqueous two-phase system was not formed when the molecular weight of PEG was 4000 or less.

As shown in Table 2, it was found that the larger the molecular weight of PEG, the smaller the volume ratio and the CGTase partition coefficient. That is, it was confirmed that the volume increases with increasing CGTase recovery in the lower layer.

In Tables 1 and 2 above, when the aqueous two-phase system of 5% PEG35000 and 7% dextran T2000 was used for the separation of CGTase from the CGTase producing microorganism, it was found that the CGTase recovery in the lower layer was the highest.

Example 3-2

Purification of CGTase Using PEG and Aqueous Two-phase System

To determine the phase separation form of PEG and salts by aqueous two-phase system, 10% PEG35000 and 15% salt were added to the fermentation broth and the phase separation form was investigated. As a result, it was confirmed that complete phase separation was formed into an upper layer in which a large amount of PEG is present and a lower layer in which a large amount of salt is present.

In addition, the distribution patterns of 10% PEG35000 and CGTase according to the type and concentration of salts were investigated, and the results are shown in FIGS. 1 and 2. 1 is a graph showing the recovery of CGTase separated from the lower layer using an aqueous two-phase system formed with 10% PEG35000 and 15% salt. FIG. 2 is an aqueous two-phase system formed with 10% PEG35000 and sodium sulfate at different concentrations. It is a graph showing the recovery rate of CGTase separated from the lower layer by using.

As shown in FIG. 1, the recovery of CGTase in the lower layer was 90% in the aqueous two-phase system of 10% PEG35000 and 15% potassium phosphate, and the recovery of CGTase in the lower layer was 95.5 in the aqueous two-phase system of 10% PEG35000 and 15% sodium phosphate. It was found to be%. In addition, as shown in FIG. 2, in the aqueous two-phase system of 10% PEG35000 and 10% sodium sulfate, the recovery rate of CGTase in the lower layer was 93.2%. In addition, it was confirmed that phase separation is not formed when the concentration of sodium sulfate is 10% or more.

From the results of Examples 3-1 and 3-2, the PEG-salt system is more effective in terms of the recovery rate than the PEG-dextrin system, especially in the case of CGTase separation and purification using an aqueous two-phase system, in particular 10% PEG35000 and 15% Sodium phosphate was found to be the most effective.

As described and demonstrated in detail above, according to the CGTase purification method of the present invention, it is possible to purify CGTase easily and efficiently and economically using an aqueous two-phase system.

Claims (4)

In the culture medium of cyclodextrin glucanotransferase producing microorganisms, polyethylene glycol (polythylene gly col) and dextran having a molecular weight of 4,000 or more were 3 to 7% (w / v) and 5, respectively, for the electric culture medium. Adding 10% (w / v) to purify the cyclodextrin glucanotransferase using an aqueous two-phase system. At least one salt selected from the group consisting of polyethylene glycol (polythylene gly col) having a molecular weight of 4,000 or more and a phosphate salt and a sulfate salt in a culture medium of a cyclodextrin glucanotransferase producing microorganism is 7 to 7 days An aqueous two-phase comprising the addition of 12% (w / v) and phosphate 10-18% (w / v) or sulfate 3-10% (w / v) to form an aqueous two-phase system. purification of cyclodextrin glucanotransferase using a phase system. The microorganism according to claim 1 or 2, wherein the cyclodextrin glucanotransferase producing microorganisms are Bacillus sp., Klebsiella sp., Micrococcus sp. Purification method characterized in that it is selected from the group consisting of thermoanaerobacter sp. The tablet of claim 1, wherein the aqueous biphasic system further comprises one or more salts selected from the group consisting of phosphates and sulfates in addition to polyethylene glycol and dextran. Way.