KR101099893B1

KR101099893B1 - method for chromatographic purification of phospholipase D from chinese cabbage

Info

Publication number: KR101099893B1
Application number: KR1020090028861A
Authority: KR
Inventors: 김예경; 서영석; 권형우; 최선윤; 이은희; 권유림; 김진희
Original assignee: 주식회사 고센바이오텍
Priority date: 2009-04-03
Filing date: 2009-04-03
Publication date: 2011-12-28
Also published as: KR20090047408A

Abstract

The technical field of the present invention is to provide a purification method by chromatography of domestic cabbage-derived phospholipase (Phospholipase D, abbreviation: PLD; EC 3.1.4.4).

The problem to be solved by the present invention is to purify the PLD with high purity. It is an object of the present invention to provide a production method in which PLD can be purified by chromatography with a purity that can be used for reagents or medicines.

The present invention selects a process phase and a fluidized bed of chromatography, controls the ionic strength of the fluidized bed (ionic strngth), and designs a manufacturing process to develop a high purity cabbage PLD production method. PLD was purified first from domestic cabbage and purified second by chromatography. Purified Chinese cabbage PLD was confirmed molecular weight and purity by SDS-PAGE, and enzyme activity and product were confirmed using HPLC.

The effect of the present invention is to purify high purity cabbage PLD with a purity of 80% or more by only one chromatography.

Health, lecithin, cabbage, phospholipids, chromatography, health, lecithin, chinese cabbage, phospholipid, chromatography

Description

Method for chromatographic purification of phospholipase D from chinese cabbage}

본 발명은 국내산 배추 유래의 포스포리파아제 (Phospholipase D, 약칭: PLD; EC 3.1.4.4) 를 고순도로 정제하는 방법을 제공하는 것이다. PLD는 생체내의 신호전달과 인지질 조성에 관련된 효소이다. 이 배추 PLD를 고순도로 정제하는 이유는 의약품이나 시약용의 제조에 경제적이고 안전한 기술이 필요하기 때문이다.The present invention provides a method for purifying phospholipase (Phospholipase D, abbreviation: PLD; EC 3.1.4.4) derived from domestic cabbage with high purity. PLDs are enzymes involved in signaling and phospholipid composition in vivo. The reason why this cabbage PLD is purified with high purity is that economical and safe technology is required for the manufacture of pharmaceuticals and reagents.

많은 연구 보고에서 크로마토그래피를 이용하여 정제하였고 순도가 높은 효소를 제조하는데 장점이 있다. 그러나 이런 공정들은 대개 여러 가지 크로마토그래피를 조합하여 사용하므로, 상업적 규모로 생산할 때 생산설비가 복잡하여 운전도 어렵고 생산 증대도 어렵다Many studies have reported the use of chromatography to produce purified enzymes with high purity. However, these processes usually use a combination of chromatographic combinations, which makes production difficult due to complex production facilities when producing on a commercial scale.

배추 PLD 에 대한 연구로서 배추 PLD를 배추의 아세톤 침전물로 효소 활성과 안정성, 그리고 유기용매 반응, 염기 치환 반응 등을 측정되었다. (이상영 등, 한국생물공학회지 (Kor. J. Biotechnol. Bioeng.), 5(2), pp.119-124, [1990]).As a study on Chinese cabbage PLD, enzyme activity and stability, organic solvent reaction, and base substitution reaction were measured as acetone precipitate of Chinese cabbage PLD. (Lee Sang-young et al., Korean Journal of Biotechnology and Bioengineering (Kor. J. Biotechnol. Bioeng.), 5 (2), pp. 119-124, [1990]).

양배추 PLD 에 대한 연구로서 사보이 양배추의 아세톤 침전물로부터 겔-여과 크로마토그래피와 이온-교환 크로마토그래피, 그리고 친화도 크로마토그래피 등을 모두 이용하여 정제하였다. 전기영동에 의해 분자량이 89킬로 돌턴으로 측정되었다. (이혜영 등, 한국생화학회지 (Biochemistry and Molecular Biology Reports), 26(7), pp.487-493, [1993]).As a study on the cabbage PLD, it was purified from acetone precipitates of savoy cabbage using both gel-filtration chromatography, ion-exchange chromatography, and affinity chromatography. The molecular weight was determined to be 89 kilo Daltons by electrophoresis. (Hye-young Lee et al., Biochemistry and Molecular Biology Reports, 26 (7), pp.487-493, [1993]).

그러나, 이 후로 미생물 유래 또는 재조합 PLD의 생산이나 이용이 일반화 되면서 식물 유래 PLD 의 기술적 연구는 미미하다.However, since the production and use of microorganism-derived or recombinant PLDs have become commonplace, the technical research of plant-derived PLDs is insignificant.

배추 PLD를 여과, 침전, 원심 분리 등으로 조효소를 만들고, 아세톤이나 황산암모늄 침전법으로 정제하더라도 그 순도는 20％ 정도이다. 따라서 고순도 배추 PLD를 사용하려면 2차 정제가 필요하다.Even if cabbage PLD is produced by filtration, precipitation, centrifugation, etc., and purified by acetone or ammonium sulfate precipitation, the purity is about 20%. Therefore, the use of high purity cabbage PLD requires secondary purification.

본 발명의 과제는 배추 PLD를 가급적 간단히 크로마토그래피로 정제하는 것이다. 다수의 설비나 어려운 조작을 사용하지 않고 고순도 PLD를 제조하는 방법을 제공하는데 그 목적을 두고 있다.An object of the present invention is to purify Chinese cabbage PLD as simply as possible. Its purpose is to provide a method for producing high purity PLDs without the use of numerous facilities or difficult operations.

본 발명에서는 크로마토그래피의 공정상과 유동상을 선정하고, 유동상의 이온강도(ionic strength)를 조절하여 제조 공정을 설계하여, 고순도 배추 PLD 생산 방법을 개발하였다. 국산배추에서 PLD를 1차 정제된 것을 크로마토그래피로 2차 정제하였다. 정제된 배추 PLD를 SDS-PAGE로 분자량과 순도를 확인하고, HPLC 등을 이용하여 효소의 활성과 생산물 등을 확인하였다.In the present invention, the chromatographic process phase and the fluidized bed were selected, the manufacturing process was designed by adjusting the ionic strength of the fluidized bed, and a high purity cabbage PLD production method was developed. PLD was purified first from domestic cabbage and purified second by chromatography. Purified Chinese cabbage PLD was confirmed molecular weight and purity by SDS-PAGE, and enzyme activity and product were confirmed using HPLC.

배추 PLD를 2차 정제하기 위하여 DEAE-세라믹 레진이나, DEAE-아가 레진, DEAE-덱스트란 레진을 사용하여, 이온교환 크로마토그래피를 하되 버퍼의 농도와 염의 농도를 조절하여 이온 강도(ionic strength) 를 맞추어, PLD 분자의 흡착과 탈착을 조절하여 정제하였다.For secondary purification of Chinese cabbage PLD, ion exchange chromatography was performed using DEAE-ceramic resin, DEAE-agar resin, and DEAE-dextran resin, and the ionic strength was adjusted by adjusting the buffer concentration and salt concentration. In this way, the adsorption and desorption of PLD molecules were controlled and purified.

본 발명의 효과는, 고순도 배추 PLD를 단 한 번의 크로마토그래피로 80％ 이상의 순도로 정제하는 것이다.The effect of the present invention is to purify high purity cabbage PLD with a purity of 80% or more by only one chromatography.

본 발명을 구체적으로 설명하면 다음과 같다.The present invention will be described in detail as follows.

본 발명의 배추 포스포리파아제 D 의 크로마토그래피에 의해 고순도 정제 방법을 각 단계별로 설명하기로 한다.The method of purifying high purity by chromatography of Chinese cabbage phospholipase D will be described in each step.

제1단계: 효소 용해 공정Step 1: Enzyme Dissolution Process

1차 정제된 배추 PLD 를 중성 버퍼 (pH 6.5 ∼ 7.5 Tris-HCl 50 밀리몰 이온강도) 에 단백질 농도 5％ 정도로 용해한다.Primary purified Chinese cabbage PLD is dissolved in neutral buffer (pH 6.5 to 7.5 Tris-HCl 50 mmol ionic strength) at about 5% protein concentration.

제2단계: 효소 흡착 공정Second Step: Enzyme Adsorption Process

같은 조건의 버퍼로 충진한 크로마토그래피 칼럼에 위의 효소 혼합 용액을 통과시키면 효소가 흡착된다.Enzyme is adsorbed by passing the above enzyme mixture solution through a chromatography column filled with buffer under the same conditions.

제3단계: 부유물 제거 공정Step 3: Float Removal Process

효소 흡착 후, 칼럼부피의 약 10 배의 버퍼를 흘려주면 효소는 흡착이 유지되고 나머지 물질을 흘러 나간다.After enzymatic adsorption, about 10 times the buffer volume of the column is flowed, and the enzyme is maintained in adsorption and the remaining material flows out.

제4단계: 효소 탈착 공정Step 4: enzyme desorption process

중성 염 버퍼 (pH 6.5 ∼ 7.5 Tris-HCl 50 밀리몰 이온강도와 NaCl 250 밀리몰 이온강도) 를 탈착 버퍼로 칼럼부피의 약 10 배의 버퍼를 흘려주면 효소는 탈착되고 나머지 물질은 남는다.Neutral salt buffer (pH 6.5 ~ 7.5 Tris-HCl 50 mmol ionic strength and NaCl 250 mmol ionic strength) is flowed into the desorption buffer about 10 times the volume of the column, the enzyme is desorbed and the remaining material remains.

제5단계: 칼럼 세척 공정Step 5: Column Wash Process

중성 염 버퍼 (pH 6.5 ∼ 7.5 Tris-HCl 50 밀리몰 이온강도와 NaCl 2 몰 이온강도) 를 세척 버퍼로 칼럼부피의 약 10 배의 버퍼를 흘려주면 잔류 물질이 탈착된다.Neutral salt buffer (pH 6.5 to 7.5 Tris-HCl 50 mmol ionic strength and NaCl 2 mol ionic strength) is washed with 10 times the volume of the column as a wash buffer to desorb the residual material.

제6단계: 칼럼 세척 공정Step 6: Column Wash Process

중성 버퍼 (pH 6.5 ∼ 7.5 Tris-HCl 50 밀리몰 이온강도) 를 칼럼부피의 약 10 배의 버퍼를 흘려주면 칼럼이 다음 효소 정제를 위해 재생된다.Neutral buffer (pH 6.5-7.5 Tris-HCl 50 mmol ionic strength) was passed about 10 times the buffer volume of the column and the column was regenerated for the next enzyme purification.

이렇게 2차 정제된 효소는 효소의 순도가 약 80％ 이상 된다.This second purified enzyme has an enzyme purity of about 80% or more.

이하, 실시 예와 비교 예에 의거 본 발명에 따른 배추 포스포리파아제 D 의 크로마토그래피 정제 방법을 더욱 구체적으로 설명하고자 한다. 그러나 이 실시 예는 발명을 이해시키기 위한 예시일 뿐 발명의 범위를 제한하지는 않는다.Hereinafter, the chromatographic purification method of Chinese cabbage phospholipase D according to the present invention will be described in more detail based on Examples and Comparative Examples. However, this embodiment is only an example for understanding the invention and does not limit the scope of the invention.

＃ 실시 예 1 : PLD 크로마토그래피 (DEAE-실리카 이온교환)Example 1: PLD chromatography (DEAE-silica ion exchange)

효소 순도를 높이기 위하여 크로마토그래피에 의한 효소 정제를 Pall Science 의 레진 (DEAE ceramic HyperD F)을 사용했다.In order to increase enzyme purity, enzyme purification by chromatography was performed using resin of Pall Science (DEAE ceramic HyperD F).

우선 50 밀리몰 이온강도 Tris-HCl (pH 7.0) 완충 용액으로 칼럼을 충진하고, 효소를 같은 중성 완충 용액으로 녹여서 칼럼에 통과 시켜 흡착시킨다. 같은 용액으로 칼럼 부피의 10배를 흘려보내면 부유물이 제거된다.First, the column is filled with 50 mmol ionic strength Tris-HCl (pH 7.0) buffer solution, and the enzyme is dissolved in the same neutral buffer solution and passed through the column for adsorption. Flowing 10 times the volume of the column with the same solution removes suspended solids.

이 후, 50 밀리몰 이온강도 Tris-HCl (pH 7.0) 이고 250 밀리몰 이온강도 NaCl 완충용액으로 PLD를 세척분리 (elution) 하였다.Thereafter, the PLD was washed with 50 mmol ionic strength Tris-HCl (pH 7.0) and 250 mmol ionic strength NaCl buffer.

칼럼 재생은 앞 [발명의 실시를 위한 구체적인 내용] 의 제5 ∼ 6 단계를 그대로 실시하였다.Column regeneration was carried out as described above in the fifth to sixth steps.

＃ 실시 예 2 : PLD 크로마토그래피 (DEAE-아가 이온교환)Example 2: PLD chromatography (DEAE-agar ion exchange)

Pall Science 의 레진 (Acrosep DEAE Sepharose CL-4B)을 사용하여 실시 예 1과 같이 했다.Example 1 was carried out using a resin of Pall Science (Acrosep DEAE Sepharose CL-4B).

＃ 실시 예 3 : PLD 크로마토그래피 (DEAE-덱스트란 이온교환)Example 3: PLD chromatography (DEAE-dextran ion exchange)

Sigma-Aldrich 의 레진 (Sephadex-DEAE A-50 )을 사용하여 실시 예 1과 같이 했다.It carried out like Example 1 using the resin of Sigma-Aldrich (Sephadex-DEAE A-50).

＃ 실시 예 4 : 전기영동법Example 4 Electrophoresis

크로마토그래피에서 얻은 효소에 대한 SDS-PAGE 분석하였다. 배추 PLD 분자량은 약 91kDa으로 알려져 있는 양배추 PLD와 비슷한 약 89kDa이고, 오차 한계는 5 kDa 임을 확인하였다.SDS-PAGE analysis on the enzyme obtained by chromatography. The cabbage PLD molecular weight was about 89kDa, similar to the cabbage PLD known as about 91kDa, and the margin of error was 5 kDa.

＃ 실시 예 5 : 배추 PLD 정제효소 포스파티딜세린(PS) 제조 실험Example 5: Chinese cabbage PLD Purifying Enzyme Phosphatidylserine (PS) Preparation Experiment

배추 PLD를 이용하여 포스파티딜세린(PS)을 제조하였다. 아세트산 버퍼에서 난황 레시틴 (egg yolk lecithin)을 농도별로 녹이고 실리카 겔 (silica gel) 반응액의 20％를 넣고, 세린 (serine)을 반응액의 30％를 넣은 후, 배추 PLD 효소를 반응액의 0.1％를 넣어 반응시킨 결과 아래와 같은 포스파티딜세린 수율 (PS yield) 을 보였다.Phosphatidylserine (PS) was prepared using Chinese cabbage PLD. Dissolve egg yolk lecithin in acetic acid buffer at different concentrations, add 20% of the silica gel reaction solution, add serine to 30% of the reaction solution, and then add cabbage PLD enzyme to 0.1% of the reaction solution. The reaction was carried out with% to give the following phosphatidylserine yield (PS yield).

표 1. 실시 예별 포스파티딜세린 수율 (PS yield)Table 1.PS yield of phosphatidylserine by example

본 발명은 산업상 이용가능성이 매우 높다. 국산 배추를 이용하므로 식품 제품의 안전성이 높아 본 발명의 방법을 이용하여 인지질의 제조가 용이하다 할 수 있다. 그리고 건강에도 매우 유익하므로 건강 기능성 식품 제조 등의 응용 범위가 매우 넓다.The present invention has a very high industrial applicability. Since domestic cabbage is used, the safety of food products may be high, and thus the production of phospholipids may be facilitated using the method of the present invention. In addition, since it is very beneficial to health, there is a wide range of applications, such as manufacturing health functional food.

도 1 은 1차 정제된 배추로 PLD 로 고순도 배추 PLD의 제조 순서를 도시한 제조 순서도이다. 공정 (표기: 정사각형, 마름모) 별로 투입되는 재료 (표기: 6각형)와 부산물 (표기: 사다리꼴), 주산물(표기: 타원형)을 표시한다.FIG. 1 is a manufacturing flowchart illustrating a manufacturing procedure of high purity cabbage PLD with PLD as primary purified cabbage. Indicate the materials (notation: hexagonal), by-products (notation: trapezoid), and the main product (notation: oval) for each process (notation: square, rhombus).

Claims

As a high-purity purification method by chromatography of Chinese cabbage, Phospholipase D (abbreviated as: PLD; EC 3.1.4.4)

1) enzyme dissolution process

Primary purified cabbage PLD is dissolved in neutral buffer (pH 6.5-7.5 Tris-HCl 20-80 mmol ionic strength).

2) enzyme adsorption process

The enzyme solution is adsorbed by passing the above enzyme solution through a chromatography column having ion exchange resin as a fixed bed.

3) float removal process

After the adsorption of the enzyme, a neutral buffer of 5-20 times the column volume is further flowed to remove suspended matter and impurities while maintaining the adsorption of the enzyme.

4) Enzyme Desorption Process

Neutral salt buffer (pH 6.5-7.5 Tris-HCl 50 mmol ionic strength and NaCl 200-300 mmol ionic strength) is passed through 5-20 times the column volume with desorption buffer to desorb and collect the adsorbed enzyme.

5) column washing process

Neutral salt buffer (pH 6.5-7.5 Tris-HCl 50 mmol ionic strength and NaCl 1-3 mol ionic strength) is flowed at least 10 times of the column volume into the wash buffer to desorb all remaining proteins.

6) Column Regeneration Process

Neutral buffer (pH 6.5-7.5 Tris-HCl 50 mmol ionic strength) is flowed at least 10 times the column volume to regenerate the ion exchange resin of the column.

Purification method by chromatography of Chinese cabbage phospholipase D characterized by the above method

The method of claim 1, wherein the stationary phase for chromatography can be used for ion exchange resins based on silica, agar, dextran, ceramic or polymer synthetic resins. Chromatography Purification Method