KR101079292B1 - Bioconversion for genistein to 2’-Hydroxygenistein - Google Patents

Bioconversion for genistein to 2’-Hydroxygenistein Download PDF

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KR101079292B1
KR101079292B1 KR1020080084583A KR20080084583A KR101079292B1 KR 101079292 B1 KR101079292 B1 KR 101079292B1 KR 1020080084583 A KR1020080084583 A KR 1020080084583A KR 20080084583 A KR20080084583 A KR 20080084583A KR 101079292 B1 KR101079292 B1 KR 101079292B1
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genistein
yeast
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KR20100025858A (en
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이충환
최정남
김지영
이미연
임채성
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건국대학교 산학협력단
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
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    • C12Y114/13089—Isoflavone 2'-hydroxylase (1.14.13.89)

Abstract

본 발명은 기존에 존재하는 이소플라보노이드(Isoflavonoid)계 화합물 중 제니스틴(Genistein)을 수산화형태인 2′-하이드록시 제니스테인(2′-hydroxy genistein)으로 생물학적 변환(bioconversion) 시키는 기술에 관한 것이다.The present invention relates to a technology for bioconversion of genistein from 2'-hydroxy genistein, which is a hydroxyl form, among existing isoflavonoid compounds.

2′-하이드록시 제니스테인, 효모 2'-hydroxy Genistein, yeast

Description

생물학적 변환을 통해 제니스테인을 2-하이드록시 제니스테인으로 전환시키는 방법{Bioconversion for genistein to 2’-Hydroxygenistein}How to convert genistein to 2-hydroxy genistein via biological conversion {Bioconversion for genistein to 2’-Hydroxygenistein}

본 발명은 기존에 존재하는 이소플라보노이드(Isoflavonoid)계 화합물 중 제니스틴(Genistein)을 수산화형태인 2′-하이드록시 제니스테인(2′-hydroxy genistein)으로 생물학적 변환(bioconversion) 시키는 기술에 관한 것으로서, 더 자세하게는 효모(yeast)를 이용하여 배양액에 제니스테인(genistein)을 첨가시켜 생물학적 변환을 이용하여 화학식 1로 표시되는 수산화(hydroxylation)된 형태인 2′-하이드록시제니스테인을 에틸아세테이트(ethylacetate) 가용부를 분리하여 얻어내는 방법과 자체적으로 낮은 변환 효율을 증가시키는 방법에 관한 것이다.The present invention relates to a technology for bioconversion of genistein from a conventional isoflavonoid compound to 2'-hydroxy genistein, which is a hydroxide form, in more detail. Was added to the culture medium using yeast (genistein) to separate the soluble portion of the ethylacetate (ethylacetate) 2'-hydroxygenysteine of the hydroxylated form represented by the formula (1) using a biological transformation How to obtain and increase the low conversion efficiency by itself.

제니스테인(genistein)은 콩과식물에 많이 존재하는 대표적인 아이소플라본(isoflavone)화합물 중 하나로써 항산화 기능과 항암작용, 멜라닌 생합성 저해, 골다공증을 억제한다는 보고가 있으며, 특히 조직 안에서 자유 라디칼 손상효과를 방해하는 산화방지제로써의 항산화 기능이 탁월하다는 보고가 있다. 아 이소플라본을 비롯한 대부분의 플라보노이드(flavonoid)계 화합물의 이 같은 생물학적 활성, 특히 항산화 기능과 멜라닌 생합성 저해 활성은 주로 플라보노이드 화합물 구조에 결합해 있는 작용기인 수산화기(hydroxyl group)의 수와 결합 위치에 의해 좌우되며 (Kim et al. J. Agric. Food Chem. 2006, 54, 935-941) 이에 따라 기존의 제니스테인에 하나의 수산화기가 더 결합된 2′-하이드록시 제니스테인을 효모를 이용한 생물학적 변환을 통해 에틸아세테이트 가용부를 분리하여 얻어냄으로써 제니스테인의 생물학적 활성 보다 향상된 활성을 기대하게 된다. 실예로 본 발명자들은 참싸리에서 분리해 낸 2′-하이드록시 제니스테인과 유사 구조인 달버지오이딘(dalbergioidin)을 통해 탁월한 항산화 효과와 멜라닌 생합성 저해 활성을 확인하였다. Genistein is one of the most common isoflavone compounds in legumes. It has been reported to inhibit antioxidant function, anticancer activity, inhibit melanin biosynthesis and osteoporosis. It has been reported that the antioxidant function as an antioxidant is excellent. The biological activity of most flavonoid compounds, including isoflavones, in particular the antioxidant and melanin biosynthesis inhibitory activities, is mainly due to the number and location of hydroxyl groups, which are functional groups bound to the flavonoid compound structure. Kim et al. J. Agric. Food Chem. 2006, 54, 935-941) Accordingly, 2′-hydroxy genistein, which has one more hydroxyl group bonded to the existing genistein, is obtained by separating the ethyl acetate soluble part through the biological conversion using yeast, thereby improving the activity than the biological activity of genistein. Done. For example, the inventors of the present invention confirmed an excellent antioxidant effect and melanin biosynthesis inhibitory activity through dalbergioidin, which is a structure similar to 2′-hydroxygenysteine isolated from Chassam.

이에, 본 발명자들은 효모를 이용한 생물학적 변환을 통해 2′-하이드록시 제니스테인을 획득하는 방법을 연구하여 자체적 변환 효율을 높일 수 있는 방법을 연구하였고, 그 결과 효모의 생물학적 변환에 가장 적합한 조건을 찾아냄으로써 기존의 변환 효율보다 약 10% 향상 시킬 수 있는 방법을 발견하게 됨으로써 본 발명을 완성하게 되었다. Therefore, the present inventors studied a method of obtaining 2′-hydroxygenysteine through biotransformation using yeast to improve its own conversion efficiency, and as a result, by finding the most suitable conditions for the biotransformation of yeast, The present invention has been completed by discovering a method that can improve the conversion efficiency by about 10%.

따라서, 본 발명은 효모를 이용한 생물학적 변환을 통해 2′-하이드록시 제니스테인을 얻어 내는 방법을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a method for obtaining 2′-hydroxy genistein through biological transformation using yeast.

또한, 본 발명은 효모를 이용한 생물학적 변환을 통해 2′-하이드록시 제니스테인을 얻어 내는 방법의 낮은 변환 효율을 향상시킬 수 있는 방법을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide a method for improving the low conversion efficiency of the method for obtaining 2′-hydroxy genistein through biological conversion using yeast.

상기의 목적을 달성하기 위하여 본 발명은The present invention to achieve the above object

아이소플라본 2′-하이드록실레이즈(isoflavone 2`-hydroxylase) 유전자를 포함하는 발현벡터를 숙주 세포에서 발현시켜서 배양액에 첨가된 제니스테인(genistein)을 2′-하이드록시 제니스테인으로 변환시키는 방법을 제공한다.An expression vector comprising an isoflavone 2′-hydroxylase gene is expressed in a host cell, thereby providing a method of converting genistein added to the culture to 2′-hydroxy genistein.

본 발명의 일 실시예에 있어서, 상기의 방법은 상기 배양액을 용매 추출하고 정제하는 과정을 더욱 포함하는 것을 특징으로 한다.In one embodiment of the present invention, the method is characterized in that it further comprises the step of solvent extraction and purification of the culture solution.

본 발명에서 용매로서 C1 -5-알칸산 C1 -5-알킬 에스터를 사용하는 것이 바람직하고, C1-5-알칸산의 예는 포름산, 아세트산, 프로피온산 및 아이소프로피온산이며, C1-5-알킬 에스터의 예는 메틸, 에틸, 프로필, 아이소프로필, 뷰틸, 아이소뷰틸 및 3급-뷰틸 에스터이다. 본 발명의 바람직한 용매는 메틸아세테이트 및/또는 에틸아세테이트이다.As the solvent in the present invention C 1 -5 - alkanoic acid C 1 -5 - preferable to use an alkyl ester, for example of C1-5- alkanoic acid is formic acid, acetic acid, and propionic acid and isopropyl propionate, C1-5- alkyl Examples of esters are methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl esters. Preferred solvents of the present invention are methyl acetate and / or ethyl acetate.

또한 본 발명의 숙주 효모 세포의 경우는 특정 유전자를 도입하여 발현시켜 특정 기질을 전화시키는 과정에서, 그에 대해 전환된 생산물의 양을 비교하였을 때 도 2에서 보시다시피 6시간이 지난 후 부터 증가하여서 9시간 발현 배양한 것이 가장 많은 생산물을 얻을수 있음을 확인하였고 12시간 발현 배양부터는 생산물이 줄어드는 현상을 확인할 수 있어서 본 발명에서 바람직한 배양 시간은 6시간∼12시간인 것을 특징으로 한다.In addition, in the case of the host yeast cell of the present invention, when a specific gene is introduced and expressed, the specific substrate is converted, and when the amount of the converted product is compared, as shown in FIG. Time expression culture was confirmed that the most product can be obtained from 12 hours of expression culture can be confirmed that the phenomenon that the product is reduced, characterized in that the preferred incubation time in the present invention is 6 hours to 12 hours.

또한 본 발명은 일반적으로 대부분의 효소 활성은 pH에 상당한 영향을 받는 것으로 알려져 있으므로, pH 변화를 최소화할 수 있는 버퍼를 이용하여 배양액에서 얻어낸 생산물보다 훨씬 많은 양의 생산물을 얻어 낼 수 있었다. 따라서 본 발명의 일 실시예에 있어서, 상기 방법은 변환 과정 중 버퍼에서 배양하는 것을 특징으로 하고, 본 발명의 일 실시예에 있어서, 상기 버퍼는 포타슘 포스페이트 버퍼인 것이 바람직하나 이에 한정되는 것은 아니다.In addition, since the present invention is generally known that most of the enzyme activity is significantly affected by the pH, it was possible to obtain a much larger amount of product than the product obtained from the culture medium using a buffer that can minimize the pH change. Therefore, in one embodiment of the present invention, the method is characterized in that the culture in a buffer during the conversion process, in one embodiment of the present invention, the buffer is preferably a potassium phosphate buffer, but is not limited thereto.

이하, 본 발명을 설명한다.Hereinafter, the present invention will be described.

본 발명은 기존에 존재하는 이소플라보노이드(Isoflavonoid)계 화합물 중 제니스틴(Genistein)을 수산화형태인 2′-하이드록시 제니스테인(2′-hydroxy genistein)으로 생물학적 변환(bioconversion) 시키는 기술에 관한 것으로서, 더 자세하게는 효모(yeast)를 이용하여 배양액에 제니스테인(genistein)을 첨가시켜 생물학적 변환시킴으로써 화학식 1로 표시되는 수산화(hydroxylation)된 형태인 2′-하이드록시제니스테인을 에틸아세테이트(ethylacetate) 가용부를 분리하여 얻어내는 방법과 그 방법에 대한 자체적으로 낮은 변환효율을 증가시키는 방법에 관한 것이다. 2~5%에 불과한 낮은 변환 효율을 12~16%까지 증대시킴으로써 산업적으로 이용될 가치가 있다.The present invention relates to a technology for bioconversion of genistein from a conventional isoflavonoid compound to 2'-hydroxy genistein, which is a hydroxide form, in more detail. Is obtained by separating the ethylacetate soluble portion of 2′-hydroxygenysteine, a hydroxylated form represented by Formula 1, by adding genistein to the culture medium using yeast for biological conversion. A method and method for increasing its own low conversion efficiency. Increasing the low conversion efficiency of only 2-5% to 12-16% makes it valuable for industrial use.

본 발명은 제니스테인과 비교하여 우수한 생물학적 활성을 지닐 것으로 기대되는 화학식 1로 표시되는 2′-하이드록시 제니스테인을 효모를 이용한 생물학적 변환을 통해 얻어 내는 방법을 제공한다. The present invention provides a method for obtaining 2′-hydroxy genisteine represented by Formula 1, which is expected to have good biological activity compared to genistein, through a biological transformation using yeast.

화학식 1로 표시 되는 물질에 대해서는 LC-ESI-MS (Liquid Chromatography electron spray ionization mass spectrometer), 핵자기 공명 스펙트럼 등의 방법을 이용하여 이화학적 특성을 분석하였고, 그 결과 다음과 같은 이화학적 특성을 갖는 것으로 확인되었다.For the material represented by Chemical Formula 1, physicochemical characteristics were analyzed using a method such as LC-ESI-MS (Liquid Chromatography electron spray ionization mass spectrometer) and nuclear magnetic resonance spectra. It was confirmed that.

Figure 112008061484320-pat00001
Figure 112008061484320-pat00001

① 물질 성상 : 분말① Material Property: Powder

② 분자량: 286 ② molecular weight: 286

③ 분자식: C15H10O6 ③ Molecular Formula: C 15 H 10 O 6

④ 질량분석치 [M-H]-: 285(m/z)④ Mass Spec. [MH] - : 285 ( m / z )

또한, 본 발명은 상기 화학식 1로 표시되는 화합물로의 낮은 변환율을 약 10% 향상시킬 수 있는 방법을 제공한다.In addition, the present invention provides a method for improving a low conversion rate to about 10% to the compound represented by Chemical Formula 1.

이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 효모를 이용하여 생물학적 변환을 통해 상기 화학식 1로 표시되는 화합물을 얻는 방법을 제공한다. 이는 아이소플라본 2′-하이드록실레이즈(isoflavone 2`-hydroxylase)라는 특정 유전자를 포함하는 pYES2 I2`H라는 효모 발현 플라스미드(plasmid)를 효모 (Saccahromyces cerevisiae WAT11 strain,)에서 발현시켜 배양액에 첨가된 제니스테인(genistein)을 2′-하이드록시 제니스테인으로 생물학적 변환시키는 방법이다. The present invention provides a method for obtaining a compound represented by Chemical Formula 1 through biological transformation using yeast. It was expressed in yeast (Saccahromyces cerevisiae WAT11 strain) by expressing a yeast expression plasmid called pYES2 I2`H containing a specific gene called isoflavone 2′-hydroxylase. (genistein) is a biological conversion of 2'-hydroxy genistein.

본 발명의 상기 효모 발현 플라스미드는 일본 Nihon University의 Dr. Shin-ichi Ayabe로부터 제공받은 것이며 상기 유전자 또한 클로닝 되어 있는 상태로 플라스미드를 제공받은 것으로 그 제조방법 등에 대한 자세한 내용은 Tomoyoshi et al. Biochem. Biophys. Res. Commun. 1998. 251: 67-70에 기재되어 있다. The yeast expression plasmid of the present invention is Dr. Nihon University of Japan It was provided by Shin-ichi Ayabe and the plasmid was provided with the gene also cloned. Tomoyoshi et al. Biochem. Biophys. Res. Commun. 1998. 251: 67-70.

재조합된 플라스미드를 효모에 도입한 숙주 효모 세포를 12~48시간 가량 충분히 배양을 하고 그 후 원심분리를 이용해 세포만을 얻어낸 후 이를 다시 배양액이 아닌 버퍼에 resuspension 시켜 기질을 도입하고 갈락토스를 첨가하여 일정 시간, 바람직하게는 9시간 동안 유전자의 발현을 유도, 효소 반응을 하게 된다. 이 과정에서 기존의 방법은 배양액을 사용하여 갈락토스를 첨가하여 생산물을 얻었지만 효모의 배양 과정에서 배양액의 pH가 변함은 물론이고 효모 배양액 자체가 약 산성의 조건을 띄게 되기 때문에 발현되는 유전자가 영향을 받지 않을 것인가를 고려 완충용액인 버퍼를 이용하여 발현 되는 유전자가 최적의 조건으로 효소 활성을 지닐 수 있도록 시도하였다. 대부분의 효소 활성은 pH에 상당한 영향을 받는 것으로 알려져 있다. 그리하여 pH 변화를 최소화할 수 있는 버퍼를 이용하여 배양액에서 얻어낸 생산물보다 훨씬 많은 양의 생산물을 얻어 낼 수 있었다. 효모의 배양액 의 최적 pH는 3~6으로 알려져 있다. 그러나 약산성의 조건에서는 아무래도 외래 도입된 유전자의 발현이 영향을 받고 또한 발현된 유전자가 효소로서의 역할을 수행하는 데에도 영향을 미칠 것으로 사료되어 완충용액을 사용한 것이다.After culturing the host yeast cells in which the recombinant plasmid was introduced into the yeast for 12 to 48 hours, the cells were obtained by centrifugation, and then resuspensioned in the buffer, not in the culture medium, and the substrate was introduced and galactose was added for a predetermined time. Preferably, the expression of the gene for 9 hours, the enzyme reaction. In this process, the conventional method obtained a product by adding galactose using a culture medium, but in addition to changing the pH of the culture medium during the cultivation of the yeast, as the yeast culture itself is weakly acidic, the expressed gene is affected. Considering whether or not to receive the buffer The buffered gene was tried to have the enzyme activity in the optimal conditions. Most enzyme activities are known to be significantly affected by pH. Thus, using a buffer that can minimize the pH change yielded a much larger product than that obtained from the culture. The optimum pH of the yeast culture is known to be 3-6. However, under mildly acidic conditions, the expression of the introduced gene is influenced and the expressed gene is likely to affect the role of the enzyme.

위의 방법을 보다 상세히 설명하면If you explain the above method in more detail

1) pYES2 I2`H를 효모에 도입시켜 형질 전환된 효모에서 제니스테인을 생물학적변환 시키는 단계 (단계 1);1) introducing pYES2 I2′H into yeast to biotransform genistein in transformed yeast (step 1);

2) 생물학적 변환된 2′-하이드록시 제니스테인을 에틸아세테이트로 진탕 추출하고 그 추출액을 감압 농축하여 에틸아세테이트 추출 가용부를 얻는 단계 (단계 2); 2) shaking and extracting the biologically converted 2'-hydroxy genistein with ethyl acetate and concentrating the extract under reduced pressure to obtain an ethyl acetate extract soluble part (step 2);

3) 상기 가용부를 메탄올에 녹여 고압 액체 크로마토그래피를 통해 목적 조성물을 얻는 단계 (단계 3); 3) dissolving the soluble part in methanol to obtain the desired composition through high pressure liquid chromatography (step 3);

상기 단계 1을 구체적으로 설명하면, pYES2 I2`H를 Frozen-EZ Yeast TransformationⅡkit (Zymoresearch, Orange, CA)를 통해 효모에 도입 후 형질 전환된 효모를 글루코스(glucose)가 첨가된 Synthetic Drop Out 배지를 이용하여 30℃에서 24h 배양한 후 배양액을 원심분리하여 하단부 효모만을 취한 후 갈락토스(Galactose)를 첨가한 Synthetic Drop Out 배지를 이용하여 제니스테인을 첨가한후 이소플라본 2′-하이드록실레이즈를 발현시켜 12시간 배양한다. Referring to step 1 specifically, pYES2 I2`H was introduced into yeast through Frozen-EZ Yeast Transformation IIkit (Zymoresearch, Orange, CA) and transformed yeast using glucose-added Synthetic Drop Out medium. After incubation at 30 ° C. for 24 h, the culture medium was centrifuged to take only the yeast at the bottom, followed by the addition of genistein using Synthetic Drop Out medium containing galactose, and then isoflavone 2′-hydroxylase expression. Incubate.

상기 단계 2를 구체적으로 설명하면, 배양액을 원심분리 하여 상등액만을 취한 후 동량의 에틸아세테이트를 넣은 후 진탕 추출하여 얻은 상등액을 회수하여 30℃에서 감압 농축하여 에틸아세테이트 가용부를 얻는다. 동량의 에틸아세테이트 진 탕 추출을 3회 반복한다. Specifically, step 2, the culture was centrifuged to take only the supernatant, and then, the same amount of ethyl acetate was added, the supernatant obtained by shaking extraction was recovered, and concentrated under reduced pressure at 30 ℃ to obtain an ethyl acetate soluble part. The same amount of ethyl acetate shake extraction is repeated three times.

상기 단계 3을 구체적으로 설명하면, 농축된 에틸아세테이트 가용부를 소량의 메탄올에 용해한 후 YMC C18 Pack Pro 컬럼을 이용한 고압 액체 크로마토그래피(High Performance Liquid Chromatography)를 통하여 목적 조성물을 확인하고 활성 분액을 모은다. Specifically, step 3 is described. After dissolving the concentrated ethyl acetate soluble part in a small amount of methanol, the target composition is identified through high performance liquid chromatography using a YMC C18 Pack Pro column, and the active aliquots are collected.

상기 방법을 통하여 얻어진 2′-하이드록시 제니스테인의 낮은 생물학적 변화율을 고려하여 이를 향상 시킬 수 있는 최적 조건을 탐색한 결과 본 발명자들은 기존의 2~5%에 달하는 변화율을 12~16% 까지 향상시킬 수 있는 방법을 완성하였다. Considering the low biological change rate of 2′-hydroxy genistein obtained through the above method, the present inventors searched for the optimum conditions to improve the present invention. As a result, the present inventors can improve the change rate of 2 to 5% to 12 to 16%. The method was completed.

이상에서 설명한 바와 같이, 본 발명에 따른 기존의 제니스테인의 생물학적 활성의 향상성을 위한 수산화 반응에서 자체적인 낮은 반응 효율을 약 10% 높일 수 있는 방법을 발명함으로써 2′-하이드록시 제니스테인의 다량 확보에 용이하게 사용될 수 있다.As described above, by inventing a method that can increase its own low reaction efficiency by about 10% in the hydroxide reaction for improving the biological activity of the existing genistein according to the present invention, a large amount of 2′-hydroxy genistein can be secured. It can be used easily.

이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.

1) 제니스테인을 2′-하이드록시 제니스테인으로 변화시킬 수 있는 효모의 최적 반응 시간을 탐색하였다.1) The optimum reaction time of yeast capable of changing genistein to 2'-hydroxy genistein was investigated.

2) 제니스테인을 2′-하이드록시 제니스테인으로 변화시킬 수 있는 효모의 최적 반응 환경을 탐색하였다.2) The optimum reaction environment of yeast for changing genistein to 2'-hydroxy genistein was investigated.

<< 실시예Example 1> 효모의 최적 반응 시간 탐색 1> Exploration of optimum reaction time of yeast

상기 1)의 발명을 구체적으로 설명하면, 형질 전환 된 효모내의 아이소플라본 2′-하이드록실레이즈를 발현시킨 후 제니스테인을 첨가하여 반응 시간을 측정한 결과 첨가 후 9시간까지는 2′-하이드록시 제니스테인의 생성이 증가하였으나 9시간후부터는 급격히 감소함을 보였다, 이에 효모의 생물학적 변환의 적정 반응시간은 9시간임을 밝히고 이에 따른 결과는 도 2에 포함하여 나타내었다. Specifically, the invention of 1) is expressed in the isoflavone 2'-hydroxylase in the transformed yeast and the reaction time was measured by the addition of Genistein and as a result 9 hours after the addition of 2'-hydroxy genistein The production increased but showed a sharp decrease after 9 hours. The optimum reaction time of the biological conversion of the yeast was found to be 9 hours, and the result is shown in FIG. 2.

<< 실시예Example 2> 효모의 최적 반응 환경 탐색 2> Exploration of the optimal reaction environment of yeast

상기 2)의 발명을 구체적으로 설명하면, 제니스테인 첨가 9시간 후 급격히 감소되는 2′-하이드록시 제니스테인의 양을 감안하여 분해되는 현상이 효모의 생장에 따른 pH의 변화에 기인할 것으로 착안, pH의 보정과 변환과정 중에서의 생장 중지를 위해 배지 대신 포타슘 포스페이트 버퍼 (potassium phosphate buffer, pH7.4)를 사용하여 기존의 Synthetic Drop-Out media 사용보다 많은 양의 2′-하이드록시 제니스테인을 확보하게 되었다. 이는 도 2 에 포함하여 나타내었다. In detail describing the invention of 2), considering that the amount of 2'-hydroxy genistein is sharply reduced after 9 hours of the addition of genistein, the decomposition is due to the change of pH according to the growth of yeast. Potassium phosphate buffer (pH7.4) was used instead of the medium to stop growth during the calibration and conversion process, resulting in higher amounts of 2'-hydroxygenysteine than conventional Synthetic Drop-Out media. This is shown in Figure 2 included.

도 1a는 제니스테인(genistein) 화합물에 대한 액체 크로마토그래피-질량분석기(LC-ESI/MS) 스펙트럼(spectrum)을 나타낸것이고,FIG. 1A shows a liquid chromatography-mass spectrometer (LC-ESI / MS) spectrum for genistein compounds,

도 1b는 형질 전환 되지 않은 효모의 에틸아세테이트 가용부에 대한 액체크로마토그래피-질량분석기(LC-ESI/MS) 스펙트럼(spectrum)을 나타낸것이고, Figure 1b shows a liquid chromatography-mass spectrometer (LC-ESI / MS) spectrum of the ethyl acetate soluble portion of untransformed yeast,

도 1c는 형질 전환된 효모의 에틸아세테이트 가용부에서 화학식 1로 표시되는 화합물의 액체크로마토그래피-질량분석기(LC-ESI/MS) 스펙트럼(spectrum)을 나타낸 것이다.Figure 1c shows a liquid chromatography-mass spectrometer (LC-ESI / MS) spectrum of the compound represented by Formula 1 in the ethyl acetate soluble portion of the transformed yeast.

도 2는 화학식 1로 표시되는 화합물의 효모를 통한 변환율 증가를 두 번에 걸친 연구 수행 결과 그래프로 나타낸 것이다. 일반적인 배지를 이용한 생물학적 변환의 낮은 변환 효율도 포함하였다.Figure 2 is a graph showing the results of two studies of the increase in the conversion rate through the yeast of the compound represented by the formula (1). It also included low conversion efficiencies of biological conversion with conventional media.

도 3은 화학식 1로 표시되는 화합물과 기질로 사용된 제니스테인의 수소 핵자기공명(1H-NMR)의 데이터를 표로 나타낸 것이다.3 is a table showing the data of the hydrogen nuclear magnetic resonance ( 1 H-NMR) of the genistein used as a compound and a substrate represented by the formula ( 1 ).

Claims (7)

a)아이소플라본 2′-하이드록실레이즈(isoflavone 2`-hydroxylase) 유전자를 포함하는 발현벡터를 효모 세포에서 배양하는 단계;a) culturing an expression vector comprising isoflavone 2′-hydroxylase gene in yeast cells; b)상기 배양액으로부터 효모 세포만을 분리하는 단계;c) 상기 분리한 세포를 배양액에서 배양하지 아니하고, 포타슘 포스페이트 버퍼에서 배양하며, 이 단계에서 제니스테인(genistein)을 첨가하여 9시간 동안 배양하는 단계를 포함하는 제니스테인을 2′-하이드록시 제니스테인으로 변환시키는 방법.b) separating only the yeast cells from the culture medium; c) culturing the isolated cells in the culture medium in potassium phosphate buffer, without culturing in the culture medium, and in this step, culturing for 9 hours by adding genistein. Converting genistein to 2'-hydroxy genistein. 삭제delete 제 1항에 있어서, 상기의 방법은 상기 c)단계 후 발현 버퍼액을 용매 추출하고 정제하는 과정을 더욱 포함하는 것을 특징으로 하는 제니스테인(genistein)을 2′-하이드록시 제니스테인으로 변환시키는 방법.The method of claim 1, wherein the method further comprises solvent extraction and purification of the expression buffer solution after step c) to genistein to 2'-hydroxy genistein. 제 3항에 있어서, 상기 용매는 메틸아세테이트 또는 에틸아세테이트인 것을 특징으로 하는 제니스테인(genistein)을 2′-하이드록시 제니스테인으로 변환시키는 방법.4. The method of claim 3, wherein the solvent is methyl acetate or ethyl acetate. 삭제delete 삭제delete 삭제delete
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037652A2 (en) 1998-12-21 2000-06-29 E.I. Du Pont De Nemours And Company Flavonoid biosynthetic enzymes
US20050172354A1 (en) 2003-10-29 2005-08-04 The Samuel Robert Noble Foundation Plant isoflavonoid hydroxylases and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037652A2 (en) 1998-12-21 2000-06-29 E.I. Du Pont De Nemours And Company Flavonoid biosynthetic enzymes
US20050172354A1 (en) 2003-10-29 2005-08-04 The Samuel Robert Noble Foundation Plant isoflavonoid hydroxylases and methods of use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Biochem. Biophys. Res. Commun.(1998) Vol.251, pp.67-70*

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