KR20140123284A - Novel D-psicose-3-epimerase from Clostridium bolteae having production of functional rare sugar D-psicose and production method of D-psicose using thereof - Google Patents
Novel D-psicose-3-epimerase from Clostridium bolteae having production of functional rare sugar D-psicose and production method of D-psicose using thereof Download PDFInfo
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- KR20140123284A KR20140123284A KR1020130040400A KR20130040400A KR20140123284A KR 20140123284 A KR20140123284 A KR 20140123284A KR 1020130040400 A KR1020130040400 A KR 1020130040400A KR 20130040400 A KR20130040400 A KR 20130040400A KR 20140123284 A KR20140123284 A KR 20140123284A
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- South Korea
- Prior art keywords
- psicose
- epimerase
- leu
- glu
- ala
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Abstract
본 발명은 기능성 희귀당 사이코스의 생산능을 지닌 신규 클로스트리디움 볼티에(Clostridium bolteae) 유래의 사이코스-3-에피머화 효소 및 이를 이용한 사이코스의 생산방법에 관한 것으로서, 과당을 사이코스로 효율적으로 전환하여 사이코스를 생산할 수 있는 장점을 가지고 있다.The present invention relates to a novel cyclosporin- 3-epimerase derived from Clostridium bolteae having the ability to produce a functional rare saccharose , and a method for producing the same, It has the advantage of being able to efficiently convert and produce psicose.
Description
본 발명은 사이코스-3-에피머화 효소 및 이를 이용한 사이코스 생산방법에 관한 것으로서, 보다 상세하게는 기능성 희귀당 사이코스의 생산능을 지닌 신규 클로스트리디움 볼티에(Clostridium bolteae) 유래의 사이코스-3-에피머화 효소 및 이를 이용한 사이코스의 생산방법에 관한 것이다.
The present invention relates to a cyclosporin-3-epimerase and a method for producing the same. More particularly, the present invention relates to a cyclosporin-3-epimerase having the ability to produce a functional rare saccharose , -3-epimerase and a method for producing scicos using the same.
사이코스(D-psicose)는 과당(D-fructose)의 C-3 에피머(epimer)로서, 자연계에 매우 드물게 극소량이 존재하는 단당류이다. 배당체인 사이코푸라닌(psicofuranine)의 당부분에서 유래되었으며, 커피나 식탁용 소스, 무화과 등에도 소량 발견되기도 한다. 특히 우수한 감미질과 설탕 대비 70%의 당도를 지니면서도 설탕 대비 0.3%의 에너지를 내는 특징은 매우 중요한 장점 중의 하나이며 당알코올류인 자일리톨, 만니톨, 말티톨, 소르비톨 등과 달리 과량 섭취에 따른 설사와 같은 부작용도 없다(Food Sci Technol Res 12:137-143, 2006).
D-psicose is a C-3 epimer of D-fructose, a monosaccharide that is very rarely present in nature. It is derived from the sugar moiety of the glycoside psicofuranine and is found in small amounts in coffee, table sauces, figs, and the like. Especially, it has excellent sweetness and 70% sugar content compared to sugar, and it is one of the most important advantages of 0.3% energy compared to sugar. Unlike xylitol, mannitol, maltitol and sorbitol, which are sugar alcohols, side effects such as diarrhea ( Food Sci Technol Res 12: 137-143, 2006).
최근 이를 이용한 기능성에 관련한 보고에 비하여, 양산과 관련한 기술이 개발되지 않아서 이에 대한 관심이 증대되고 있는 실정이다. 기존의 사이코스 생산은 주로 화학적 기술에 기반하였다. Bilik 등(Chem Zvesti 28:106-109, 1973)은 몰리브산 이온의 촉매 작용을 통하여 과당으로 사이코스를 생산하였으며 McDonald(Carbohydr Res 5:106-108, 1967)는 화학적 기법을 이용하여 1,2:4,5-di-o-isopropylidene-beta-D-fructopyranose를 사이코스로 전환하여 생산하였고, Doner(Carbohydr Res 70:209-216, 1979)는 과당을 에탄올과 트리에틸아민(triethylamine)과 함께 끓여 사이코스의 생산을 시도하였다. 그러나 이러한 화학적 기술은 많은 생산비용과 정제비용이 발생되며 생산물의 안전성 등에서 문제가 있어 왔다.
Recently, the technology related to mass production has not been developed in comparison with the reports related to the use thereof, so that interest is increasing. The existing sci-cos production was mainly based on chemical technology. Bilik et al . ( Chem Zvesti 28: 106-109, 1973) produced synuclein with fructose through the catalytic action of molybdic acid ions. McDonald ( Carbohydr Res 5: 106-108, 1967) (Donbo, Carbohydr Res 70: 209-216, 1979) produced fructose with ethanol, triethylamine, and a mixture of fructose and isopropylidene-beta-D-fructopyranose I tried to produce Saikosu with boiling together. However, such a chemical technique has a problem in production cost, purification cost, and safety of the product.
이에 생물학적 방법 특히, 효소의 촉매 작용을 이용한 생산 기술들이 꾸준히 연구되어 왔다. Ishida 등(J Ferment Bioeng 83:529-534, 1997)은 슈도모나스 치코리(Pseudomonas cichorii) ST-24의 타가토스(tagatose) 에피머화 효소를 이용한 사이코스의 생산을 확인하였다. 대한민국 특허등록 제0744479호(발명의 명칭: 사이코스 에피머화 효소에 의한 사이코스의 생산방법)에는 아그로박테리움 투메패시엔스(Agrobacterium tumefaciens) 유래의 사이코스 에피머화 효소를 이용한 사이코스의 생산방법이 게시되어 있다. 대한민국 특허등록 제0832339호(발명의 명칭: 과당을 사이코스로 전환하는 신규한 시노리조비움 속 균주와 이를 이용한 사이코스 생산법)에는 시노리조비움 속(Sinorhizobium sp.) YB-58 균주의 배양 및 배양된 균체를 이용한 사이코스의 생산방법이 게시되어 있다. Zhang 등(Biotechnol Lett 31:857-862, 2009)은 로도박터 스패로이데스(Rhodobacter sphaeroides)의 D-타가토스-3-에피머화 효소를 이용한 사이코스의 생산을 보고하였으며, Mu 등(J Agric Food Chem 59:7785-7792, 2011)은 클로스트리디움 셀룰로라이티쿰(Clostridium cellulolyticum) H10 균주의 D-사이코스-3-에피머화 효소의 사이코스 생산과 관련한 특성을 보고하였다.
Biological processes, in particular, production techniques using catalysis of enzymes have been studied steadily. Ishida et al. ( J Ferment Bioeng 83: 529-534, 1997) confirmed the production of psicose using the tagatose epimerase of Pseudomonas cichorii ST-24. Korean Patent Registration No. 0744479 (the name of the invention: a method for producing a psicose by means of a psicose epimerase) discloses a method for producing psicose using a psicose epimerase derived from Agrobacterium tumefaciens This is posted. Korean Patent Registration No. 0832339 entitled Syntholizobium genus, which converts fructose into psicose, and a method for producing psicose using the same, comprises culturing a strain of Sinorhizobium sp. YB-58 A method for producing Sicos using cultured cells is disclosed. Zhang et al. ( Biotechnol Lett. 31: 857-862, 2009) reported the production of cyclosporin using D-tagatose-3-epimerase of Rhodobacter sphaeroides . Mu et al . ( J Agric Food Chem 59: 7785-7792, 2011) reported characteristics relating to the production of the D- cytosine -3-epimerase of Clostridium cellulolyticum strain H10 in relation to the production of the cytosine .
이에 본 발명자들은 생물학적 기술을 기반으로 하는 희귀당의 효율적 생산에 소용되는 효소 자원의 탐색을 통하여 기존에 알려진바 없는 클로스트리디움 볼티에(Clostridium bolteae) 유래의 사이코스 에피머화 효소를 통한 사이코스의 효율적 생산을 확인하였으며 이를 통하여 사이코스를 생산하는 새로운 효소와 생산방법을 발견하여 본 발명을 완성하였다.
Therefore, the inventors of the present invention have found through efficient search of enzymatic resources used for the efficient production of rare saccharides based on biological technology that the efficiency of cyclosporin is increased through the secretion of Clostridium bolteae derived from Clostridium bolteae The present inventors have discovered a new enzyme and a production method for producing the cytochrome, thereby completing the present invention.
본 발명은 기능성 희귀당 사이코스의 생산능을 지닌 신규 클로스트리디움 볼티에(Clostridium bolteae) 유래의 사이코스-3-에피머화 효소 및 이를 이용한 사이코스의 생산방법을 제공하는 것을 목적으로 한다.
The present invention aims at providing a novel cyclosporin- 3 epimerase derived from Clostridium bolteae having the ability to produce a functional rare saccharide, and a process for producing it.
상기와 같은 발명의 목적을 달성하기 위하여, 본 발명은 클로스트리디움 볼티에(Clostridium bolteae) 균주로부터 유래한 서열번호 1의 아미노산서열을 포함하는 사이코스(D-psicose) 전환능을 지닌 신규 사이코스-3-에피머화 효소(D-psicose-3-epimerase) 및 이를 이용하여 과당을 사이코스(D-psicose)로 전환하여 사이코스를 생산하는 방법을 제공하는 것을 특징으로 한다.
In order to accomplish the object of the present invention, the present invention relates to a novel cyclosporin D-psicose having an ability to convert D-psicose comprising the amino acid sequence of SEQ ID NO: 1 derived from a Clostridium bolteae strain, (D-psicose-3-epimerase) and D-psicose (D-psicose) by using the D-psicose-3-epimerase.
또한, 본 발명은 클로스트리디움 볼티에(Clostridium bolteae) 균주로부터 유래한 서열번호 2의 아미노산서열을 포함하는 사이코스(D-psicose) 전환능을 지닌 신규 사이코스-3-에피머화 효소(D-psicose-3-epimerase) 및 이를 이용하여 과당을 사이코스(D-psicose)로 전환하여 사이코스를 생산하는 방법을 제공하는 것을 특징으로 한다.
The present invention also relates to a novel cytosine -3-epimerase having the ability to convert D-psicose comprising the amino acid sequence of SEQ ID NO: 2 derived from a strain of Clostridium bolteae (D- psicose-3-epimerase) and a method for converting fructose into D-psicose by using the same.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 상세한 설명 중 사용되는 용어는 본 발명이 속하는 기술 분야의 통상적 지식을 가진 자가 이해하고 있는 통상적 의미를 가진다.The terms used in the description of the present invention have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.
한편, 종래와 동일한 기술적 구성 및 작용에 대한 반복되는 설명은 생략하였다.
In the meantime, repeated description of the same technical structure and operation as the conventional one is omitted.
본 발명은 현재 특성이 규명되지 않은 클로스트리디움 볼티에(Clostridium bolteae) 유래의 슈가 에피머화 효소(sugar epimerase) 가상 단백질(hypothetical protein)의 유전자(서열번호 1)를 클로닝하고 상기 유전자를 포함하는 단백질 발현벡터를 제작한 후 해당 발현벡터를 통하여 형질전환된 미생물을 배양하여 상기 슈가 에피머화 효소를 과발현시킨 후 효소의 특성 확인을 통하여 상기 슈가 에피머화 효소의 우수한 과당:사이코스 간의 전환 활성을 확인하였고, 상기 슈가 에피머화 효소의 사이코스-3-에피머화 활성을 통하여 사이코스를 생산하는 것을 특징으로 한다.
The present invention relates to a method for cloning a gene (SEQ ID NO: 1) of a sugar epimerase hypothetical protein derived from Clostridium bolteae which has not been characterized at present, After the expression vector was prepared, the transformed microorganism was cultured through the corresponding expression vector to overexpress the sugar epimerase. After the enzymatic characterization, the sucrose epimerase was found to have a superior fructose: , And is capable of producing psicose through the cytosine-3-epimerization activity of the sugar epimerase.
본 발명에서 그 기능이 규명된 사이코스-3-에피머화 효소는 종래의 사이코스-3-에피머화 기능성이 보고된 효소들의 아미노산 서열과는 매우 낮은 상동성을 보이고 있다. 즉, 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)의 사이코스-3-에피머화 효소(ZP 08527376)와는 49%, 클로스트리디움 셀룰로라이티쿰(Clostridium cellulolyticum)의 사이코스-3-에피머화 효소(YP 002505284)와는 53%, 슈도모나스 치코리(Pseudomonas cichorii)의 사이코스-3-에피머화 효소(O 50580)와는 38%, 로도박터 스페로이데스(Rhodobacter sphaeroides)의 타가토스-3-에피머화 효소(YP 355181)와는 32%의 상동성을 보이는데 불과하다.
The sarcosine-3-epimerase whose function has been confirmed in the present invention has very low homology with the amino acid sequence of the enzymes reported to have the conventional cyclic-3-epimerization function. That is, 49% of the gene encoding the cytosine-3-epimerase (ZP 08527376) of Agrobacterium tumefaciens , the cytosine-3-epimerase of Clostridium cellulolyticum YP 002505284), 38% of Cicos-3-epimerase (O 50580) of Pseudomonas cichorii , 38% of Rhizobacter sphaeroides tagatose-3-epimerase YP 355181), which is only 32% homologous.
본 발명의 사이코스-3-에피머화 효소의 특성 규명은 아래와 같이 이루어지는 것이 바람직하다. 사이코스의 생산에 사용 가능한 효소 단백질의 탐색 중에 본 사이코스-3-에피머화 효소의 유전자를 지닌 클로스트리디움 볼티에(Clostridium bolteae) 균주로부터 해당 유전자의 PCR(polymerase chain reaction)을 통하여 유전자의 증폭된 산물을 얻는다. 다음으로, 얻어진 유전자의 증폭 산물을 적절한 단백질 발현벡터에 삽입시켜 사이코스-3-에피머화 효소를 포함하는 재조합 단백질 발현벡터를 제작하고, 본 재조합 단백질 발현벡터로 적절한 미생물을 형질전환시키고 본 발명의 효소의 발현을 위한 발효 배지에서 배양 후 과발현된 효소를 정제하여 금속 조효소, 최적 pH, 최적 온도 등을 확인하는 데에 사용하는 것이 바람직하다.
The characterization of the cytosine-3-epimerase of the present invention is preferably carried out as follows. During the search for an enzyme protein that can be used for the production of the cyclosporin , the amplification of the gene is carried out by PCR (polymerase chain reaction) of the gene from the Clostridium bolteae strain having the gene of the present cyclosporin- ≪ / RTI > Next, the amplification product of the obtained gene is inserted into an appropriate protein expression vector to prepare a recombinant protein expression vector containing the cytosine-3-epimerase, and the appropriate microorganism is transformed with the present recombinant protein expression vector, It is preferable to use the enzyme for purifying an overexpressed enzyme in a fermentation medium for expression of the enzyme to confirm the metal coenzyme, optimal pH, optimum temperature and the like.
또한, 본 발명의 사이코스 생성능을 지닌 사이코스-3-에피머화 효소는 서열번호 1의 아미노산 서열에 국한되지 않으며, 과당을 사이코스로 전환시켜줄 수 있는 경우 서열번호 1에서 일부 아미노산을 치환(substitution), 삽입(insertion), 소실(deletion)시킨 경우에도 사용이 가능하다.
In addition, the present invention is not limited to the amino acid sequence of SEQ ID NO: 1, and it is possible to substitute some amino acids in SEQ ID NO: 1 when it is possible to convert fructose into a cyucose ), Insertion, and deletion of the gene.
본 발명의 클로스트리디움 볼티에(Clostridium bolteae) 유래의 신규의 사이코스-3-에피머화 효소는 사이코스를 대량 생산하는 과정에 있어서 친환경적이고 생산성에 있어 유용성을 나타내며, 효소 개량을 위한 유용한 유전자원으로 이용될 수 있는 장점을 가지고 있다.
The novel cyclosporin- 3-epimerase derived from Clostridium bolteae of the present invention is environmentally friendly in the process of mass production of cyclosporin , exhibits usefulness in productivity, and is useful as a gene source for improving enzymes And the like.
도 1은 서열번호 1의 사이코스-3-에피머화 효소의 정제 과정 중에 분획들 중 이미다졸 농도가 각각 50mM(lane 3), 150mM(lane 4), 250mM(lane 5), 1000mM(lane 6)인 분획들을 SDS-PAGE로 분석한 것을 도식화한 것이다. lane 1은 정제전 세포파쇄액, lane 2는 정제전 세포파쇄액의 정제 레진 통과 후 용액(Flow-through)을 나타낸다.
도 2는 서열번호 1의 정제 분획을 이용한, 과당에서 사이코스로의 전환활성을 HPLC 크로마토그램으로 분석한 것이다.
도 3은 서열번호 1, 서열번호 2, 서열번호 3의 각각에 해당하는 사이코스-3-에피머화 효소를 대장균체에서 발현시킨 후, 해당 대장균을 파쇄시킨 용액과 과당을 반응시킨 반응액 상의 사이코스 생성 유무를 박층형크로마토그래피(TLC)를 이용하여 확인한 결과이다(a: 세포파쇄액, b: 세포파쇄액 중 원심분리 후 상층액, c: 세포파쇄액 중 원심분리 후 펠렛).
도 4는 서열번호 2의 사이코스-3-에피머화 효소의 정제 분획들을 SDS-PAGE로 분석한 것을 도식화한 것이다(M: 단백질 크기 마커, CL: 서열번호 2가 발현된 대장균 파쇄액의 원심분리 후 상등액, FT: CL의 컬럼 통과 후 용액, 50, 100, 250: 컬럼 세척(50mM) 및 목적 단백질의 용출(100mM, 250mM)에 사용된 이미다졸의 농도, f1~f8: 위에 표시된 이미다졸 농도에 따라 용출된 효소의 분획 번호).
도 5는 서열번호 2의 사이코스-3-에피머화 효소의 반응 온도 특성을 확인한 결과이다.
도 6은 서열번호 2의 사이코스-3-에피머화 효소의 반응 pH 특성을 확인한 결과이다.
도 7은 서열번호 2의 사이코스-3-에피머화 효소의 반응 금속이온 요구 특성을 확인한 결과이다.FIG. 1 shows that during the purification of the cyclosporin-3 epimerase of SEQ ID NO: 1, the concentration of imidazole in the fractions was 50 mM (lane 3), 150 mM (lane 4), 250 mM (lane 5), 1000 mM Lt; RTI ID = 0.0 > SDS-PAGE. ≪ / RTI >
2 is an HPLC chromatogram showing the conversion activity of fructose to psicose using the purified fraction of SEQ ID NO: 1.
FIG. 3 is a graph showing the results obtained by expressing the Escherichia coli strain of the present invention in the form of a mixture of Escherichia coli and Escherichia coli after the expression of the Escherichia coli strains corresponding to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: (A: cell lysate, b: supernatant after centrifugation in cell lysate, and c: pellet after centrifugation in cell lysate). ≪ tb >< TABLE >
4 is a schematic representation of the purified fractions of the cyclosporin-3 epimerase of SEQ ID NO: 2 analyzed by SDS-PAGE (M: protein size marker, CL: centrifugation of E. coli lysate expressing SEQ ID NO: 2 (50 mM) and elution of the target protein (100 mM, 250 mM), f1 to f8: Imidazole concentration as indicated above ≪ / RTI >
Fig. 5 shows the results of confirming the reaction temperature characteristics of the cyclosporin-3-epimerase of SEQ ID NO: 2.
6 shows the results of confirming the reaction pH characteristics of the cyclosporin-3-epimerase of SEQ ID NO: 2.
FIG. 7 shows the results of confirming the requirement for reactive metal ions of the cyclosporin-3-epimerase of SEQ ID NO: 2.
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 다음의 실시예는 본 발명의 범위를 한정하는 것은 아니며, 본 발명의 기술적 사상의 범위 내에서 당업자에 의한 통상적인 변화가 가능하다.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art within the scope of the technical idea of the present invention are possible.
<실시예 1>≪ Example 1 >
사이코스-3-에피머화 효소의 생산Production of cytosine-3-epimerase
사이코스-3-에피머화 효소(D-psicose-3-epimerase)의 확보를 위하여 클로스트리디움 볼티에(Clostridium bolteae) KCTC 5430 균주를 생명공학연구원 생명자원센터에서 분양받았다. 사이코스-3-에피머화 효소를 암호화하는 유전자를 확보하기 위하여 클로스트리디움 볼티에의 유전체(genomic DNA)를 토대로 클로스트리디움 볼티에 ATCC BAA-613의 슈가 에피머화 효소를 암호화하는 것으로 제안된 유전자의 염기서열(CLOBOL_00069)을 기본으로 프라이머(primer)를 설계하고 PCR을 통하여 증폭하였다. 상기 얻어진 증폭산물은 염기서열 제한효소(restriction enzyme)인 BamHI과 SacI을 이용하여 단백질 발현벡터인 pET-21a(+)(Novagen사)에 삽입되었다. 이렇게 얻어진 사이코스-3-에피머화 효소의 재조합 발현벡터인 pET21/CbDPE는 ㈜코스모진텍(서울)을 통하여 염기서열 시퀀싱(sequencing) 작업을 거쳐 예상되는 효소의 발현에 적합하게 제작되었음이 확인되었다. 상기 제작된 발현벡터 pET21/CbDPE는 통상적인 형질전환 방법을 통하여 대장균(Escherichia coli) BL21(DE3) 균주를 형질전환 시키는데 사용되었다. 형질전환된 BL21(DE3) 균주는 LB 배지 내에서 배양한 뒤 글리세롤을 이용하여 냉동보관하였다.
In order to obtain D-psicose-3-epimerase, KCTC 5430 strain of Clostridium bolteae was distributed at the Biotechnology Research Center. In order to secure a gene encoding the cytosine-3-epimerase, a gene proposed to encode a sugar-epimerase of ATCC BAA-613 on Clostridium bolt based on the genomic DNA of Clostridium volta (CLOBOL_00069) was designed and primers were amplified by PCR. The obtained amplification product was inserted into a protein expression vector pET-21a (+) (Novagen) using Bam HI and Sac I as restriction enzymes. It was confirmed that pET21 / CbDPE, which is a recombinant expression vector of the obtained Sicos-3-epimerase, was prepared for nucleotide sequence sequencing through COSMOGINTECH (Seoul, Korea) . The prepared expression vector pET21 / CbDPE was used to transform Escherichia coli strain BL21 (DE3) through a conventional transformation method. The transformed strain BL21 (DE3) was cultured in LB medium and stored frozen in glycerol.
사이코스-3-에피머화 효소를 생산하기 위하여 냉동 보관된 BL21(DE3) 균주는 LB 한천배지에 도말 및 배양한 후 적절한 집락을 선택하여 재조합 벡터의 단백질 발현이 자연유도(autoinduction)가 이루어지는 배지인 MagicMedia(Invitrogen사) 액체 배지에 접종되었다. MagicMedia의 사용설명서에 따라 30℃에서 250rpm의 교반 속도로 24시간 동안 배양하여 BL21(DE3) 균주의 생장과 단백질 대량 발현을 자연유도 하였으며 배양이 종료된 후 600nm에서의 흡광도 값은 5.8이었다. 배양이 끝난 후 사이코스-3-에피머화 효소의 과발현 여부를 SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 전기영동법을 이용하여 확인하였다.
The BL21 (DE3) strain, which was cryopreserved to produce the cytosine-3-epimerase, was plated on an LB agar medium and then cultured on an LB agar medium. The appropriate colony was selected and the protein expression of the recombinant vector was determined to be a medium And inoculated into a MagicMedia (Invitrogen) liquid medium. The culture of BL21 (DE3) was naturally induced and the protein mass expression was naturally induced at a stirring rate of 250 rpm at 30 ° C according to the manual of MagicMedia. The absorbance value at 600 nm after the completion of culture was 5.8. After incubation, the overexpression of cytosine-3-epimerase was confirmed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis.
<실시예 2> ≪ Example 2 >
사이코스-3-에피머화 효소의 정제Purification of cytosine-3-epimerase
원심분리를 통하여 상기 실시예 1의 배양된 BL21(DE3) 균체를 회수하고 20mM 트리스(tris) 완충용액을 이용하여 잔여 배지를 세척하였다. 세척된 균체를 재조합 대장균 균체의 파쇄완충용액인 벅버스터 마스터믹스(BugBuster Mastermix, Novagen사)에 현탁하고 상온에서 5분간 반응 후, 얼음에서 30분간 추가로 반응시켜 대장균 균체를 파쇄시켰다. 상기 세포 파쇄물을 4℃, 15,000 X g에서 10분간 원심분리한 후 상층액을 0.20㎛의 필터로 여과하고 단백질 결합완충용액(20mM TrisHCl, 500mM NaCl, 5 mM imidazole, pH 7.9)으로 평형된 컬럼에 통과시켜 정제 작업을 수행하였다. 이때 사용된 컬럼의 충진제는 니켈-나이트릴로트리아세틱산 아가로스(Ni-NTA agarose)였다. 상층액 내의 사이코스-3-에피머화 효소가 결합된 컬럼을 이미다졸(imidazole) 농도가 각각 50mM(lane 3), 150mM(lane 4), 250mM(lane 5), 1000mM(lane 6)로 조절된 단백질 결합완충용액을 이용하여 세척과 용출(elution)을 시도하였다. 컬럼을 통과한 후 모인 분획물의 정제 결과를 SDS-PAGE 전기영동법을 이용하여 확인하여 높은 순도의 사이코스-3-에피머화 효소가 포함된 분획을 수득하여 사용하였다.The cultured BL21 (DE3) cells of Example 1 were recovered by centrifugation and the remaining medium was washed with 20 mM tris buffer. The washed cells were suspended in a buckbuster master mix (BugBuster Mastermix, Novagen) which is a disruption buffer of recombinant E. coli cells, reacted at room temperature for 5 minutes, and then further reacted on ice for 30 minutes to disrupt the E. coli cells. The cell lysate was centrifuged at 15,000 × g for 10 minutes at 4 ° C., and the supernatant was filtered with a 0.20 μm filter and eluted with a protein binding buffer (20 mM TrisHCl, 500 mM NaCl, 5 mM imidazole, pH 7.9) And the purification operation was carried out. At this time, Ni-NTA agarose was nickel-nitrilotriacetic acid. The column in which the cyclosporin-3-epimerase was bound in the supernatant was adjusted to an imidazole concentration of 50 mM (lane 3), 150 mM (lane 4), 250 mM (lane 5), and 1000 mM Protein binding buffer was used for washing and elution. The purified fraction of the collected fractions after passing through the column was confirmed by SDS-PAGE electrophoresis to obtain a fraction containing high purity cyclosporin-3-epimerase.
그 결과를 도 1에 나타내었다.
The results are shown in Fig.
도 1에서 확인할 수 있는 바와 같이, 다단계의 정제를 하였으나 이미다졸(imidazole) 농도가 250mM로 조절된 단백질 결합완충용액으로 용출한 분획물의 SDS-PAGE의 결과 lane 5에서 화살표로 표시된 유사한 크기의 3종의 단백질이 발현됨을 확인할 수 있었다.
As can be seen from FIG. 1, SDS-PAGE of fractions eluted with protein binding buffer adjusted to imidazole concentration of 250 mM in the multistage purification resulted in three similar sizes Of the protein was expressed.
<실시예 3> ≪ Example 3 >
사이코스-3-에피머화 효소를 이용한 과당의 사이코스로의 전환Conversion of fructose to cyclosaccharide with cyclosporin-3-epimerase
상기 실시예 2에서 정제된 3종의 사이코스-3-에피머화 효소의 과당-사이코스 간의 전환활성을 살펴보았다. 2중량%의 과당과 1mM의 염화망간, 75mM의 인산나트륨 버퍼(pH 7.4)로 구성된 반응액 500㎕에 상기 실시예 2의 정제된 3종의 사이코스 3-에피머화 효소 20㎕를 넣고 37℃에서 30분간 반응을 진행하였다. 반응물을 여과한 후, SP0810 컬럼(Shodex사)을 이용하여 굴절율 검출기(refractive index detector)가 장착된 고성능액체크로마토그래피(HPLC) 장치를 이용하여 분석하였다. 분석 조건은 컬럼오븐의 온도는 80℃이고, 증류수를 분당 1.0㎖의 속도로 흘려보내도록 설정하였다.The conversion activity of the three kinds of the cyclosporin-3-epimerase purified in Example 2 between fructose and psicose was examined. Twenty microliters of the purified 3 kinds of the cyclosporin 3-epimerase of Example 2 were added to 500 μl of a reaction solution consisting of 2% by weight of fructose, 1 mM of manganese chloride and 75 mM of sodium phosphate buffer (pH 7.4) For 30 minutes. The reaction products were filtered and analyzed using a high performance liquid chromatography (HPLC) apparatus equipped with a refractive index detector using an SP0810 column (Shodex). The analysis conditions were set so that the temperature of the column oven was 80 DEG C and the distilled water was flowed at a rate of 1.0 mL per minute.
그 결과를 도 2에 나타내었다.
The results are shown in Fig.
도 2에서 확인할 수 있는 바와 같이, 반응 전 과당만이 존재하였으나, 반응을 진행한 후 약 17%의 과당이 사이코스로 전환됨을 확인할 수 있었다.
As can be seen from FIG. 2, only fructose was present before the reaction. However, it was confirmed that about 17% of the fructose was converted to cyclosporin after the reaction.
<실시예 4> <Example 4>
사이코스-3-에피머화 효소의 N 말단 축소형 효소의 생산Production of N-terminal dimeric enzyme of psicose-3-epimerase
상기 실시예 2의 정제된 3종의 단백질로 발현되는 사이코스-3-에피머화 효소의 발현 양태를 단일화시키기 위하여 사이코스-3-에피머화 효소의 아미노산 서열(서열번호 1)을 분석한 결과(개시 아미노산인 Met 이후 아미노산 서열을 읽어가면서 개시 아미노산으로부터 멀지 않은 곳에 위치한 다른 Met들을 찾아보았다. 정확히 기전이 알려져 있지는 않으나, read-through를 통해서 두 번째 혹은 세 번째 Met에서도 아미노산 합성이 개시되거나, 두 번째 또는 세 번째 Met 앞 부분이 잘려나가지는 경우가 있어서 아미노산 서열을 차례로 읽어보았다)를 토대로 N 말단 부위를 축소한 두 가지 형태[서열번호 2:서열번호 1의 N말단에서 10개의 아미노산(MRYFKEEVAG)을 결실, 서열번호 3:서열번호 1의 N말단에서 31개의 아미노산(MRYFKEEVAGMKYGIYFAYWTKEWFADYKKY)을 결실]의 사이코스 3-에피머화 효소를 클로닝하고 발현시켰다.The amino acid sequence (SEQ ID NO: 1) of the cyclosporin-3-epimerase was analyzed to unify the expression pattern of the cyclosporin-3 epimerase expressed in the purified three proteins of Example 2 Other Mets located not far from the starting amino acid were searched while reading the amino acid sequence after the starting amino acid Met. Although the exact mechanism was not known, amino acid synthesis was initiated either in the second or third Met through the read-through, (SEQ ID NO: 2: deletion of 10 amino acids (MRYFKEEVAG) at the N-terminus of SEQ ID NO: 1). The amino acid sequences of the first and second Mets were cleaved , SEQ ID NO: 3: deletion of 31 amino acids at the N-terminus of SEQ ID NO: 1 (MRYFKEEVAGMKYGIYFAYWTKEWFADYKKY) The enzyme was cloned and expressed.
즉, 서열번호 1, 서열번호 2, 서열번호 3에 해당하는 각각의 사이코스-3-에피머화 효소를 상기 실시예 1과 동일한 방법으로 대장균체에서 발현시킨 후, 해당 대장균을 상기 실시예 2와 동일한 방법으로 파쇄시킨 용액과 2% 과당용액을 37℃에서 30분간 반응시키고 반응액 상의 사이코스 생성 유무를 박층형 크로마토그래피(TLC)를 이용하여 확인하였다.Namely, the respective cytosine-3-epimerase corresponding to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 was expressed in E. coli in the same manner as in Example 1, The same procedure was followed for the reaction of the 2% fructose solution and the 2% fructose solution at 37 ° C for 30 minutes, and the presence or absence of the formation of the psicose on the reaction solution was confirmed by thin layer chromatography (TLC).
그 결과를 도 3에 나타내었다.
The results are shown in Fig.
도 3에서 확인할 수 있는 바와 같이, 서열번호 1, 서열번호 2, 서열번호 3의 각각의 사이코스 3-에피머화 효소 모두 과당에서 사이코스로의 전환활성을 가짐을 알 수 있었다.
As can be seen from Fig. 3, it was found that all of the respective cytosine 3-epimerases of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 had a fucose-to-cicosan converting activity.
<실시예 5> ≪ Example 5 >
사이코스-3-에피머화 효소의 반응 온도 및 pH 특성 확인Identification of the reaction temperature and pH characteristics of psicose-3-epimerase
상기 실시예 4의 서열번호 2로 표시되는 사이코스 3-에피머화 효소가 단일 단백질 형태이고 발현 양상도 우수하여 상기 실시예 4의 서열번호 2의 사이코스-3-에피머화 효소의 반응 특성을 확인하기 위하여 온도 조건과 pH 조건을 달리하며 최적 반응 조건을 알아보았다.
The cyclosporin 3-epimerase shown in SEQ ID NO: 2 of Example 4 was in the form of a single protein and the expression pattern was excellent. Thus, the reaction characteristics of the cyclosporin-3-epimerase of SEQ ID NO: The optimum reaction conditions were investigated under different temperature and pH conditions.
5-1. 서열번호 2의 사이코스-3-에피머화 효소의 정제5-1. Purification of the cytosine-3-epimerase of SEQ ID NO: 2
효소특성 확인 작업을 위한 효소의 정제는 상기 실시예 2에서 언급한 것과 유사하게 분획별 이미다졸의 용출 농도차이를 이용하여 정제하였다.Purification of the enzyme for enzyme characterization was performed using the difference in the elution concentration of the fractionated imidazole similarly to that described in Example 2 above.
즉, 원심분리를 통하여 상기 실시예 4의 배양된 서열번호 2의 사이코스-3-에피머화 효소로 형질전환된 BL21(DE3) 균체를 회수하고 20mM 트리스(tris) 완충용액을 이용하여 잔여 배지를 세척한 후, 세척된 균체를 재조합 대장균 균체의 파쇄완충용액인 벅버스터 마스터믹스(BugBuster Mastermix, Novagen사)에 현탁하고 상온에서 5분간 반응 후, 얼음에서 30분간 추가로 반응시켜 대장균 균체를 파쇄시켜 상기 세포 파쇄물을 4℃, 15,000 X g에서 10분간 원심분리한 후 상층액을 0.20㎛의 필터로 여과하고 단백질 결합완충용액(20mM TrisHCl, 500mM NaCl, 5mM imidazole, pH 7.9)으로 평형된 컬럼에 통과시켜 정제 작업을 수행하였다. 이때 사용된 컬럼의 충진제는 니켈-나이트릴로트리아세틱산 아가로스(Ni-NTA agarose)였다. 상층액 내의 서열번호 2의 사이코스-3-에피머화 효소가 결합된 컬럼을 이미다졸(imidazole) 농도가 각각 50mM, 150mM, 250mM, 1000mM로 조절된 단백질 결합완충용액을 이용하여 세척과 용출(elution)한 분획물을 SDS-PAGE 전기영동법을 통하여 확인하고, 높은 순도의 서열번호 2의 사이코스-3-에피머화 효소 분획의 정제도를 확인하고 이후 반응에 사용하였다.
Namely, the cultured BL21 (DE3) cells transformed with the cytosine-3-epimerase of SEQ ID NO: 2 of Example 4 were recovered by centrifugation, and the remaining medium was recovered using a 20 mM tris buffer solution After washing, the washed cells were suspended in a Buffer Mastermix (Novagen) which is a disruption buffer solution of recombinant E. coli cells, reacted at room temperature for 5 minutes, and then further reacted on ice for 30 minutes to break up E. coli cells The cell lysate was centrifuged at 15,000 × g for 10 minutes at 4 ° C. The supernatant was then filtered through a 0.20 μm filter and passed through a column equilibrated with protein binding buffer (20 mM TrisHCl, 500 mM NaCl, 5 mM imidazole, pH 7.9) The purification operation was performed. At this time, Ni-NTA agarose was nickel-nitrilotriacetic acid. The column bound to the cytosine-3-epimerase of SEQ ID NO: 2 in the supernatant was washed and eluted with a protein binding buffer adjusted to imidazole concentrations of 50 mM, 150 mM, 250 mM and 1000 mM, ) Fractions were confirmed by SDS-PAGE electrophoresis, and the purification degree of the high-purity cyclosporin-3-epimerase fractions of SEQ ID NO: 2 was confirmed and used in subsequent reactions.
그 결과를 도 4에 나타내었다.The results are shown in Fig.
도 4에서 확인할 수 있는 바와같이, 서열번호 2의 사이코스 3-에피머화 효소는 싸이즈 마커와의 비교를 통해서 목적하는 단백질이 32~35kDa의 위치에 나타나는 것을 알 수 있고, 단일 밴드로 발현이 되는 것을 확인할 수 있어 단일 단백질 형태이고 발현 양상도 우수하다는 사실을 알 수 있었다.
As can be seen from Fig. 4, it can be seen that the desired protein appears at a position of 32 to 35 kDa in comparison with the size marker in the case of the Saicos 3-epimerase of SEQ ID NO: 2, And it was found that it is a single protein form and has an excellent expression pattern.
5-2. 서열번호 2의 사이코스-3-에피머화 효소의 반응 온도 특성5-2. The reaction temperature characteristics of the cyclosporin-3-epimerase of SEQ ID NO: 2
특성을 확인하고자 하는 온도로 미리 맞춰 조정된 효소 반응액(2중량% 과당, 1 mM 코발트 2가 양이온, 50mM 인산나트륨 완충용액, pH 7.5) 995㎕에 서열번호 2의 사이코스-3-에피머화 효소 분획 5㎕를 넣고 30, 40, 50, 60, 70℃에서 각각 5분간 효소 반응을 수행하였으며, 반응 후 반응액을 10분간 끓여서 반응을 종료시켰다.2 was added to 995 쨉 l of an enzyme reaction solution (2 wt% fructose, 1 mM cobalt divalent cation, 50 mM sodium phosphate buffer, pH 7.5) adjusted in advance to the temperature at which the property was to be confirmed, The enzyme reaction was performed for 5 minutes at 30, 40, 50, 60, and 70 ° C, respectively, after adding 5 μl of the enzyme fraction. After completion of the reaction, the reaction solution was boiled for 10 minutes to terminate the reaction.
그 결과를 도 5에 나타내었다.
The results are shown in Fig.
도 5에서 확인할 수 있는 바와 같이, 최적 반응 온도는 60℃이며, 이는 비교적 고온에서 활성이 높게 나타난 것으로 대량 생산시 고온 반응에 따른 유동성 및 반응 평형의 측면에서 유의적으로 향상된 결과를 갖게 됨을 알 수 있었다.
As can be seen from FIG. 5, the optimum reaction temperature is 60 ° C, which indicates that the activity is relatively high at a relatively high temperature, and that it has a significantly improved result in terms of flowability and reaction equilibrium in a high- there was.
5-3. 서열번호 2의 사이코스-3-에피머화 효소의 반응 pH 특성5-3. Reaction pH characteristics of the cytosine-3-epimerase of SEQ ID NO: 2
반응 최적 pH 조건은 상기 온도 조건을 확인한 실험과 마찬가지의 조건에서 pH만 인산나트륨 완충용액과 트리스 완충용액을 이용하여 조정한 후 60℃에서 반응을 진행하였다.The optimum pH for the reaction was adjusted by using sodium phosphate buffer solution and Tris buffer solution under the same conditions as those for the above-mentioned temperature conditions, and then the reaction was carried out at 60 ° C.
그 결과를 도 6에 나타내었다.
The results are shown in Fig.
도 6에서 확인할 수 있는 바와 같이, 최적 pH는 7.5이나 pH 6.5~pH 8.0 사이에서 pH 7.5 대비 85% 전후의 활성을 보여주었다. 이러한 넓은 pH 적응성은 온도 변화나 첨가되는 기질에 따른 pH 변화에도 우월한 반응 적응성을 보여줌을 알 수 있었다.
As can be seen from FIG. 6, the optimal pH was about 7.5 or about 85% of the pH 7.5 between pH 6.5 and pH 8.0. This broad pH adaptability showed a superior response adaptability to the pH change depending on the temperature change and the added substrate.
<실시예 6> ≪ Example 6 >
사이코스-3-에피머화 효소의 금속이온 요구 특성 확인Determination of metal ion requirement of cyclosporin-3-epimerase
본 발명의 서열번호 2의 사이코스-3-에피머화 효소의 금속이온 요구 특성을 확인하기 위하여 1mM의 동일 농도로 금속이온을 바꾸어가며 실험을 진행하였다. 시험방법은 실시예 5에서 온도 조건을 탐색하기 위한 효소 반응액에 금속 이온만을 바꾸어가며 실험하였다. 바륨(Ba), 칼슘(Ca), 코발트(Co), 구리(Cu), 마그네슘(Mg), 망간(Mn), 니켈(Ni), 아연(Zn)과 같은 금속이온과 금속이온을 넣지 않은 반응액을 이용하여 실험을 진행하였다.In order to confirm the metal ion required characteristics of the cyclosporin-3-epimerase of SEQ ID NO: 2 of the present invention, the experiment was conducted by changing the metal ion to the same concentration of 1 mM. The test method was carried out by changing only the metal ion to the enzyme reaction solution for searching the temperature condition in Example 5. A reaction without a metal ion such as barium (Ba), calcium (Ca), cobalt (Co), copper (Cu), magnesium (Mg), manganese (Mn), nickel (Ni) The experiment was carried out using the solution.
그 결과를 도 7에 나타내었다.
The results are shown in Fig.
도 7에서 확인할 수 있는 바와 같이, 코발트에서 가장 높은 활성(무처리 대비 8.1배)을 나타냄을 확인할 수 있었다.As can be seen from Fig. 7, it was confirmed that cobalt exhibited the highest activity (8.1 times as compared with no treatment).
<110> KOREA YAKULT CO., LTD. <120> Novel D-psicose-3-epimerase from Clostridium bolteae having production of functional rare sugar D-psicose and production method of D-psicose using thereof <130> P13-10 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 301 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 1 Met Arg Tyr Phe Lys Glu Glu Val Ala Gly Met Lys Tyr Gly Ile Tyr 1 5 10 15 Phe Ala Tyr Trp Thr Lys Glu Trp Phe Ala Asp Tyr Lys Lys Tyr Met 20 25 30 Asp Lys Val Ser Ala Leu Gly Phe Asp Val Leu Glu Ile Ser Cys Ala 35 40 45 Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu Gln Leu Ile Glu Leu Arg 50 55 60 Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu Thr Ala Gly Tyr Gly Pro 65 70 75 80 Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp Pro Glu Ala Val Arg Arg 85 90 95 Ala Met Thr Phe Phe Lys Asp Leu Leu Pro Lys Leu Gln Leu Met Asp 100 105 110 Ile His Ile Leu Gly Gly Gly Leu Tyr Ser Tyr Trp Pro Val Asp Phe 115 120 125 Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg Ala Arg Ala Val Arg Asn 130 135 140 Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu Cys Asp Val Val Leu Gly 145 150 155 160 Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr Ile Leu Asn Thr Cys Glu 165 170 175 Glu Ala Ile Asp Phe Val Asp Glu Ile Gly Ser Ser His Val Lys Ile 180 185 190 Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Thr Asn Met Ala Asp 195 200 205 Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly His Leu His Leu Gly Glu 210 215 220 Gln Asn Arg Leu Val Pro Gly Lys Gly Ser Leu Pro Trp Ala Glu Ile 225 230 235 240 Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln Gly Ala Ala Val Met Glu 245 250 255 Pro Phe Val Met Gln Gly Gly Thr Ile Gly Ser Glu Ile Lys Val Trp 260 265 270 Arg Asp Met Val Pro Asp Leu Ser Glu Glu Ala Leu Asp Arg Asp Ala 275 280 285 Lys Gly Ala Leu Glu Phe Cys Arg His Val Phe Gly Ile 290 295 300 <210> 2 <211> 291 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 2 Met Lys Tyr Gly Ile Tyr Phe Ala Tyr Trp Thr Lys Glu Trp Phe Ala 1 5 10 15 Asp Tyr Lys Lys Tyr Met Asp Lys Val Ser Ala Leu Gly Phe Asp Val 20 25 30 Leu Glu Ile Ser Cys Ala Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu 35 40 45 Gln Leu Ile Glu Leu Arg Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu 50 55 60 Thr Ala Gly Tyr Gly Pro Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp 65 70 75 80 Pro Glu Ala Val Arg Arg Ala Met Thr Phe Phe Lys Asp Leu Leu Pro 85 90 95 Lys Leu Gln Leu Met Asp Ile His Ile Leu Gly Gly Gly Leu Tyr Ser 100 105 110 Tyr Trp Pro Val Asp Phe Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg 115 120 125 Ala Arg Ala Val Arg Asn Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu 130 135 140 Cys Asp Val Val Leu Gly Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr 145 150 155 160 Ile Leu Asn Thr Cys Glu Glu Ala Ile Asp Phe Val Asp Glu Ile Gly 165 170 175 Ser Ser His Val Lys Ile Met Leu Asp Thr Phe His Met Asn Ile Glu 180 185 190 Glu Thr Asn Met Ala Asp Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly 195 200 205 His Leu His Leu Gly Glu Gln Asn Arg Leu Val Pro Gly Lys Gly Ser 210 215 220 Leu Pro Trp Ala Glu Ile Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln 225 230 235 240 Gly Ala Ala Val Met Glu Pro Phe Val Met Gln Gly Gly Thr Ile Gly 245 250 255 Ser Glu Ile Lys Val Trp Arg Asp Met Val Pro Asp Leu Ser Glu Glu 260 265 270 Ala Leu Asp Arg Asp Ala Lys Gly Ala Leu Glu Phe Cys Arg His Val 275 280 285 Phe Gly Ile 290 <210> 3 <211> 270 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 3 Met Asp Lys Val Ser Ala Leu Gly Phe Asp Val Leu Glu Ile Ser Cys 1 5 10 15 Ala Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu Gln Leu Ile Glu Leu 20 25 30 Arg Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu Thr Ala Gly Tyr Gly 35 40 45 Pro Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp Pro Glu Ala Val Arg 50 55 60 Arg Ala Met Thr Phe Phe Lys Asp Leu Leu Pro Lys Leu Gln Leu Met 65 70 75 80 Asp Ile His Ile Leu Gly Gly Gly Leu Tyr Ser Tyr Trp Pro Val Asp 85 90 95 Phe Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg Ala Arg Ala Val Arg 100 105 110 Asn Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu Cys Asp Val Val Leu 115 120 125 Gly Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr Ile Leu Asn Thr Cys 130 135 140 Glu Glu Ala Ile Asp Phe Val Asp Glu Ile Gly Ser Ser His Val Lys 145 150 155 160 Ile Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Thr Asn Met Ala 165 170 175 Asp Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly His Leu His Leu Gly 180 185 190 Glu Gln Asn Arg Leu Val Pro Gly Lys Gly Ser Leu Pro Trp Ala Glu 195 200 205 Ile Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln Gly Ala Ala Val Met 210 215 220 Glu Pro Phe Val Met Gln Gly Gly Thr Ile Gly Ser Glu Ile Lys Val 225 230 235 240 Trp Arg Asp Met Val Pro Asp Leu Ser Glu Glu Ala Leu Asp Arg Asp 245 250 255 Ala Lys Gly Ala Leu Glu Phe Cys Arg His Val Phe Gly Ile 260 265 270 <110> KOREA YAKULT CO., LTD. <120> Novel D-psicose-3-epimerase from Clostridium bolteae having production of functional rare sugar D-psicose and production method of D-psicose using <130> P13-10 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 301 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 1 Met Arg Tyr Phe Lys Glu Glu Val Ala Gly Met Lys Tyr Gly Ile Tyr 1 5 10 15 Phe Ala Tyr Trp Thr Lys Glu Trp Phe Ala Asp Tyr Lys Lys Tyr Met 20 25 30 Asp Lys Val Ser Ala Leu Gly Phe Asp Val Leu Glu Ile Ser Cys Ala 35 40 45 Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu Gln Leu Ile Glu Leu Arg 50 55 60 Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu Thr Ala Gly Tyr Gly Pro 65 70 75 80 Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp Pro Glu Ala Val Arg Arg 85 90 95 Ala Met Thr Phe Phe Lys Asp Leu Leu Pro Lys Leu Gln Leu Met Asp 100 105 110 Ile His Ile Leu Gly Gly Gly Leu Tyr Ser Tyr Trp Pro Val Asp Phe 115 120 125 Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg Ala Arg Ala Val Arg Asn 130 135 140 Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu Cys Asp Val Val Leu Gly 145 150 155 160 Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr Ile Leu Asn Thr Cys Glu 165 170 175 Glu Ala Ile Asp Phe Val Asp Glu Ile Gly Ser Ser His Val Lys Ile 180 185 190 Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Thr Asn Met Ala Asp 195 200 205 Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly His Leu His Leu Gly Glu 210 215 220 Gln Asn Arg Leu Val Pro Gly Lys Gly Ser Leu Pro Trp Ala Glu Ile 225 230 235 240 Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln Gly Ala Ala Val Met Glu 245 250 255 Pro Phe Val Met Gln Gly Gly Thr Ile Gly Ser Glu Ile Lys Val Trp 260 265 270 Arg Asp Met Val Pro Asp Leu Ser Glu Glu Ala Leu Asp Arg Asp Ala 275 280 285 Lys Gly Ala Leu Glu Phe Cys Arg His Val Phe Gly Ile 290 295 300 <210> 2 <211> 291 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 2 Met Lys Tyr Gly Ile Tyr Phe Ala Tyr Trp Thr Lys Glu Trp Phe Ala 1 5 10 15 Asp Tyr Lys Lys Tyr Met Asp Lys Val Ser Ala Leu Gly Phe Asp Val 20 25 30 Leu Glu Ile Ser Cys Ala Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu 35 40 45 Gln Leu Ile Glu Leu Arg Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu 50 55 60 Thr Ala Gly Tyr Gly Pro Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp 65 70 75 80 Pro Glu Ala Val Arg Arg Ala Met Thr Phe Phe Lys Asp Leu Leu Pro 85 90 95 Lys Leu Gln Leu Met Asp Ile His Ile Leu Gly Gly Gly Leu Tyr Ser 100 105 110 Tyr Trp Pro Val Asp Phe Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg 115 120 125 Ala Arg Ala Val Arg Asn Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu 130 135 140 Cys Asp Val Val Leu Gly Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr 145 150 155 160 Ile Leu Asn Thr Cys Glu Glu Ala Ile Asp Phe Val Asp Glu Ile Gly 165 170 175 Ser Ser His Val Lys Ile Met Leu Asp Thr Phe His Met Asn Ile Glu 180 185 190 Glu Thr Asn Met Ala Asp Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly 195 200 205 His Leu His Leu Gly Glu Gln Asn Arg Leu Val Pro Gly Lys Gly Ser 210 215 220 Leu Pro Trp Ala Glu Ile Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln 225 230 235 240 Gly Ala Ala Val Met Glu Pro Phe Val Met Gln Gly Gly Thr Ile Gly 245 250 255 Ser Glu Ile Lys Val Trp Arg Asp Met Val Pro Asp Leu Ser Glu Glu 260 265 270 Ala Leu Asp Arg Asp Ala Lys Gly Ala Leu Glu Phe Cys Arg His Val 275 280 285 Phe Gly Ile 290 <210> 3 <211> 270 <212> PRT <213> Unknown <220> <223> D-psicose-3-epimerase from Clostridium bolteae <400> 3 Met Asp Lys Val Ser Ala Leu Gly Phe Asp Val Leu Glu Ile Ser Cys 1 5 10 15 Ala Ala Leu Arg Asp Val Tyr Thr Thr Lys Glu Gln Leu Ile Glu Leu 20 25 30 Arg Glu Tyr Ala Lys Glu Lys Gly Leu Val Leu Thr Ala Gly Tyr Gly 35 40 45 Pro Thr Lys Ala Glu Asn Leu Cys Ser Glu Asp Pro Glu Ala Val Arg 50 55 60 Arg Ala Met Thr Phe Phe Lys Asp Leu Leu Pro Lys Leu Gln Leu Met 65 70 75 80 Asp Ile His Ile Leu Gly Gly Gly Leu Tyr Ser Tyr Trp Pro Val Asp 85 90 95 Phe Thr Ile Asn Asn Asp Lys Gln Gly Asp Arg Ala Arg Ala Val Arg 100 105 110 Asn Leu Arg Glu Leu Ser Lys Thr Ala Glu Glu Cys Asp Val Val Leu 115 120 125 Gly Met Glu Val Leu Asn Arg Tyr Glu Gly Tyr Ile Leu Asn Thr Cys 130 135 140 Glu Glu Ala Ile Asp Phe Val Asp Glu Ile Gly Ser Ser His Val Lys 145 150 155 160 Ile Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Thr Asn Met Ala 165 170 175 Asp Ala Ile Arg Lys Ala Gly Asp Arg Leu Gly His Leu His Leu Gly 180 185 190 Glu Gln Asn Arg Leu Val Pro Gly Lys Gly Ser Leu Pro Trp Ala Glu 195 200 205 Ile Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln Gly Ala Ala Val Met 210 215 220 Glu Pro Phe Val Met Gln Gly Gly Thr Ile Gly Ser Glu Ile Lys Val 225 230 235 240 Trp Arg Asp Met Val Pro Asp Leu Ser Glu Glu Ala Leu Asp Arg Asp 245 250 255 Ala Lys Gly Ala Leu Glu Phe Cys Arg His Val Phe Gly Ile 260 265 270
Claims (5)
D-psicose-3-epimerase having a D-psicose converting ability comprising the amino acid sequence of SEQ ID NO: 1.
상기 사이코스-3-에피머화 효소는 클로스트리디움 볼티에(Clostridium bolteae) 균주로부터 유래한 것을 특징으로 하는 사이코스-3-에피머화 효소(D-psicose-3-epimerase).
The method according to claim 1,
3. The D-psicose-3-epimerase according to claim 1, wherein the cyclosporin -3 epimerase is derived from a strain of Clostridium bolteae .
A process for producing sarcosine characterized by converting sarcosine to D-psicose by using the sarcosine-3-epimerase of claim 1 or 2.
상기 사이코스-3-에피머화 효소는 서열번호 2의 아미노산서열을 포함하는 사이코스(D-psicose) 전환능을 지닌 신규 사이코스-3-에피머화 효소(D-psicose-3-epimerase).
3. The method according to claim 1 or 2,
3-epimerase having the ability to convert D-psicose comprising the amino acid sequence of SEQ ID NO: 2. The D-psicose-3-epimerase has a D-psicose converting ability.
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