KR100262769B1 - Novel polyethylene glycol/levan aqueous two-phase system and protein partitioning method using thereof - Google Patents

Abstract

PURPOSE: Provided is an aqueous and hetero phase separation segregate system consisting of a high molecular aqueous solution of polyethylene glycol and levan, to separate proteins in large quantity with low cost. And a protein separation system using the same is also provided. CONSTITUTION: An aqueous and hetero phase separation segregate system consisting of a high molecular aqueous solution of polyethylene glycol and levan is composed of the following steps of: (a) preparing polyethylene glycol-levan hetero phase segregation system; (b) searching phase diagram of polyethylene glycol-levan; and (c) searching distribution coefficient according to the change of concentration of salt and pH value.

Description

Novel polyethylene glycol / levan aqueous two-phase system and protein partitioning method using conjugate

The present invention relates to an aqueous phase abnormality system of polyethylene glycol (PEG) and Leven, which can inexpensively separate a large amount of proteins, and a method for separating proteins using the same.

The aqueous phase separation system is a phenomenon in which two aqueous solutions cause layer separation as in the case of an organic solvent and a water-soluble solvent, and separates the target protein by the difference in protein distribution between the two aqueous layers. , Cell tissues, etc., and used for isolation and concentration or bioconversion (Skuse, DR et el., Enzyme Microb. Technol. , 14, 785, 1992; Venancio, A. et el., Bioseparation , 5, 253, 1995 ).

Separation of physiologically active substances using an aqueous phase separation system has the advantage of easy scale-up compared to conventional methods such as chromatography, filtration, and electrophoresis, and thus, large-scale purification of enzymes (Tjerneld, F., et el., Biotechnol Bioeng ., 30, 514, 1987), protein isolation using affinity (Chung, BH, et el., Biotechnol Letts ., 13, 615,1991; Chung, BH, et el., Methods in Enzymol ., 228 , 167, 1994), Isolation of Chiral Compounds (Albertsson, PA, Methods in Enzymol ., 228, 84, 1994) and Extracted Bioconversions (Zijlstra, GM, et el., Enzyme Microb.Technol ., 19, 2, 1996 ) And the like.

The aqueous phase abnormality system conventionally used is a system made of polyethylene glycol and dextran (hereinafter referred to as the aqueous phase abnormality system of polyethylene glycol-dextran), a system made of polyethylene glycol and a high concentration salt (hereinafter referred to as an aqueous phase abnormality of polyethylene glycol-salt) (Queiroz, JA, et el., Bioseparation , 5, 307, 1995) .However , aqueous phase abnormalities of polyethyleneglycol-salts require high concentrations of salt to produce an ideal system and high ionic strength. Due to the degeneration of the sensitive biological structure of the protein, as well as the disadvantage that can cause dissociation of most ligand-protein complexes. Therefore, the aqueous phase abnormality system of polyethyleneglycol-salts was not suitable for the process to be performed in the system containing the salt of low concentration. Thus, while the polyethylene-dextran aqueous phase system, which is a polymer-polymeric aqueous phase system, is more useful than the polyethylene glycol-salt phase system, its commercial use has been limited because dextran is expensive. As a result, there has been a need to develop a new, less expensive system of biphasic systems that can replace the polyethyleneglycol-dextran aqueous phase system, and instead of purified dextran (Kroner, KH, et. al., Biotechnol. Bioeng ., 24, 1015, 1982), starch derivatives (Venancio, A. et al., Biotechnol. Progr ., 9, 635, 1993; Tjerneld, F. et al., Enzyme Microb.Technol . , 8, 417, 1986), polyvinyl alcohol (Tjerneld, F., Separation using aqueous two-phase systems, Plenum Press, 429, 1989), maltodextrin (Szlag, DC et el., Biotechnol. Technique 2 (4) , 277, 1988) and cellulose derivatives (Skuse, DR et al., Enzyme Microb Technol ., 14, 785, 1992) have been used as polymer materials for making polyethylene glycol and aqueous phase separation systems.

On the other hand, Levan is a fructose polymer linked by fructofuranoside bonds, and is produced with sucrose as a substrate by transproctylation reaction of levanskase, and a low molecular weight (molecular weight: <5,000) levan is a large number of plants. It is abundantly stored as carbohydrates in tissues, and when some microorganisms are cultured in sucrose medium, extracellular production produces levans of polymers. Levan is very soluble in cold water, very soluble in hot water, insoluble in pure ethanol and more soluble than inulin, which is fructan at room temperature. Levan is also non-reducing and not hydrolyzed by yeast invertase and amylase reactions but hydrolyzed by acids (Han, YW, Adv. Appl. Microbiol., 35, 171, 1990). Levan can be used like dextran, which is used as a plasma substitute because of its physical and chemical properties, and its hydrolyzate can be used as a sweetener. In addition, Levan has been applied to a wide range of fields such as emulsifiers, stabilizers and encapsulation reagents (Belghith, H. et el., Biotechnol. Letts , 18 (4), 467, 1996). Recently, Levan has been applied to cosmetics, food and pharmaceuticals as a new industrial gum.

The mass production of Levan depends mainly on the microbial fermentation process, but these methods have disadvantages such as low productivity and the generation of by-products and impurities. In order to overcome the above problems, the present inventors cloned the recombinant Levansucrease into E. coli, developed a new production process of Levan using this enzyme, and reported mass production of low-cost Levan (Belghith, H. et el. , Biotechnol.letts , 18 (4), 467, 1996).

Accordingly, an object of the present invention is to provide a novel aqueous phase separation system using polyethylene glycol and levane. Another object of the present invention is to provide a method for inexpensively separating and extracting a large amount of proteins using the novel aqueous phase abnormality system.

The above object of the present invention was achieved by investigating the novel aqueous phase abnormality formation conditions and investigating the partition coefficient of the standard protein using this system.

Hereinafter, the specific configuration and operation of the present invention.

1 is a photograph of a polyethylene glycol-levane water phase abnormality system.

2 is a phase diagram of polyethylene glycol-levane.

The present invention first examined the conditions for forming the aqueous phase abnormality system by adjusting the concentration of polyethylene glycol and levane. At this time, the composition of these components was mixed with 10.5% (w / w) polyethylene glycol 8,000 and 3.98% (w / w) of Levan's polymer aqueous solution. Under these conditions, the upper phase is mostly composed of polyethylene glycol, and the lower phase is an aqueous phase abnormality system mainly containing Levan (FIG. 1). To test the degree of protein separation using this new system, the distribution coefficients were examined using six model proteins, cytochrome-C from horse heart, hemoglobin, myoglobin, albumin from egg and lysozyme. The distribution of proteins in the aqueous phase system is due to van der Waals, hydrophobicity, hydrogen ion bonding, and ion correlation forces between the protein and the surrounding phases, and thus the hydrogen ions of the polyethylene glycol-levane ideal system obtained in the present invention. The distribution coefficient of each model protein was investigated by varying the concentration and salt concentration. When the pH was changed to 6, 7, 8, the five proteins tested (except for lysozyme only) also showed an increase in partition coefficient with increasing hydrogen ion concentration, but for lysozyme at 0.4 and 7 In 0.31 and 8, the partition coefficient of 0.32 was shown to be independent of the hydrogen ion concentration (Table 1).

The most important factor affecting the partition coefficient in the aqueous phase abnormality system is the composition of the salt. To investigate the distribution of protein according to the salt concentration change, the partition coefficient was obtained using lysozyme and albumin as model proteins. Sodium chloride was used as the salt and the concentration was varied from 0 to 0.5 mole. In the case of lysozyme, the distribution coefficient increased with increasing salt, but albumin decreased (Table 2).

Proteins separated into any one layer can be recovered by membrane filtration using only the molecular size difference and purified pure protein only. Hereinafter, the specific configuration and operation of the present invention will be described in detail by the following examples, but the scope of the present invention is not limited only to these examples.

The present invention comprises the steps of preparing a polyethylene glycol / Levan abnormal system; Examining the top diagram; And investigating the partition coefficient according to pH and salt concentration changes.

Example 1 Preparation of Polyethylene Glycol-Levan Abnormal System

A polymer aqueous solution of polyethylene glycol (average molecular weight 8,000) 60% (w / w) and 6.77% (w / w) of levan was prepared and used as a stock solution of polyethyleneglycol-levan. Meanwhile, polyethylene glycol and dextran storage solution were prepared by the same method as described above. Due to the polymer properties, polyethylene glycol and dextran solution were transparent liquids, but the levane was opaque white.

Example 2 Phase Diagram of Polyethyleneglycol 8000 and Levan

The composition of the polyethylene glycol-levane prepared in Example 1 was mixed with various compositions, stirred, and centrifuged at 4,000 rpm for 5 minutes to separate the phase and the bottom two phases. The concentrations of Polyethyleneglycol 8000 and Levan in each phase were quantified by HPLC. As a quantitative method, a Sodex ion exchange column (Shodex, Ionpak KS-802, USA) was used to pour distilled water at 0.5 ml / min and the temperature was 50 ° C to completely separate polyethylene glycol 8000 and levane and determine the respective concentration. It was. As a result, as shown in FIG. 2, the upper part of the binodal curve shows two phases where delamination occurs, and the lower part shows one phase where delamination does not occur.

Example 3 protein distribution according to the change of hydrogen ion concentration

Step 1

0.7 g of polyethylene glycol, 2.3 g of levane, and 1 g of protein solution were taken from the stock solution, and the mixture was mixed with 4 g of a total solution. Thus, 10.5% (w / w) of polyethylene glycol and 3.98% (w / w) of levane were prepared. It was. Standard proteins were dissolved in 10 mM sodium acetate buffer to prepare protein solutions, respectively. The protein solution was prepared by varying the type and pH of the standard protein. 5 mg / ml of cytochrome-C, hemoglobin and myoglobin, 10 mg / ml of albumin, lysozyme and BSA were used as standard protein, and pH 6, 7 and 8 were used for 10 mM sodium acetate buffer, respectively.

Step 2

After mixing the polymer solution of the polyethylene glycol-levane prepared in step 1 and the protein solution, the mixture was centrifuged at 4,000 rpm for 5 minutes to separate into two phases of the phase and the lower phase, and the proteins were quantified in each phase by spectrophotometer. Red heme proteins were measured at 410 nm wavelength and albumin, lysozyme and bovine serum albumin were quantified by the Bradford method.

Step 3

The distribution coefficient of the standard protein was defined as the ratio of the upper protein concentration and the lower protein concentration. The results are shown in Table 1. As a result, five proteins except lysozyme increased the distribution coefficient when hydrogen ion concentration was increased, and lysozyme showed 0.40, 0.31, and 0.32 partition coefficient at hydrogen ion concentrations of 6, 7, and 8, respectively. Indicated.

Partition coefficient of standard protein protein Distribution factor (K) pH 6 pH 7 pH 8 Cytochrome C 0.12 0.09 0.15 hemoglobin 0.04 0.04 0.32 Myoglobin 0.11 0.16 0.55 albumin 0.29 0.50 0.75 BSA 0.04 0.14 0.31 Lisozym 0.40 0.31 0.32

Example 4 Protein Distribution with Changing Concentrations of Sodium Chloride Salt

A polymer aqueous solution was prepared in the same composition as in Step 1 of Example 3, and lysozyme and albumin were dissolved in 10 mM sodium acetate buffer solution (pH 8) at a concentration of 10 mg / ml to prepare a protein aqueous solution. After separating into two phases of the phase and bottom phase in the same manner as in step 2 of Example 3, the protein in each phase was quantified at a wavelength of 595 nm according to the Bradford method, and the partition coefficient of the protein was calculated. The experimental results are shown in Table 2. As the salt concentration increased, the distribution coefficient of standard protein lysozyme increased but the albumin distribution coefficient decreased.

Distribution coefficient of protein (lysozyme, albumin) Final sodium chloride concentration (m) Distribution factor (K) Lisozym albumin 0 0.26 0.68 0.06 0.49 0.32 0.13 0.65 0.25 0.19 0.87 0.20 0.25 0.82 0.20 0.50 1.11 0.21

As can be seen from the above examples and experimental examples, the present invention has the effect of providing a novel aqueous phase separation system of polyethylene glycol and Levan, and the aqueous phase separation system of the polymer of the present invention is therefore a protein to be performed in a low salt system. There is an effect that can be used in the separation extraction process. In addition, the use of levan in place of dextran is a very useful invention in the bioindustry because it is possible to inexpensively separate a large amount of physiologically active substances by using the novel polyethylene glycol and levan aqueous phase separation system of the present invention.

Claims (2)

Aqueous phase separation system consisting of polyethylene glycol and Levan's polymer aqueous solution. A method for separating and extracting proteins using an aqueous phase separation system consisting of polyethylene glycol and a polymer solution of levane.

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