公开(公告)号：KR100257117B1

公开(公告)日：2000-05-15

申请号：KR1019970048802

申请日：1997-09-25

Applicant: 한국과학기술연구원

Inventor： 반재구 ,  이홍원 ,  염도영

IPC: C12N15/52

CPC classification number: C12N9/0006

Abstract: PURPOSE: A membrane bound gluconate dehydrogenase, its gene, and a method for producing 2-keto-D-gluconate using transformed recombinant E. coli are provided, thereby the 2-keto-D-gluconate can be converted from gluconate in higher yield. CONSTITUTION: It is revealed that the membrane bound gluconate dehydrogenase derived from Erwinia cypripedii consists of 65 kDa of first subunit, 45 kDa of second subunit and 20 kDa of third subunit, by SDS-polyacrylamide analysis, in which the amino terminal of the first subunit has the amino acid sequence of formula (I) of Ala-Asn-Glu-Leu-Lys-Lys-Lys-Val-Asp-Ala-Val-Val-Val-Gly-Phe-Gly, the amino terminal of the second subunit has the amino acid sequence of formula(II) of Asp-Asp-Gln-Ala-Asn-Asp-Ala-Leu-Val, the amino terminal of the third subunit has the amino acid sequence of formula (III) of Ala-Glu-Glu-Ser-Ser-Gly-Ser-Gln-Thr-Ala-Arg-Asp-Tyr-Gln-Pro, flavo adenine dinucleotide bond region of the first subunit has the amino acid sequence of formula (IV) of Asp-X-X-X-X-Gly-X-Gly-X-X-Gly-X-X-X-Ala-X-X-Leu-X-Glu-X-X-xX-X-X-Val-X-X-Glu-X-Gly, and heme bonds of the second subunit has the amino acid sequence of formula (V) of Cys-X-Y-Cys-His. The recombinant E. coli JM109(pGA313)(KCTC 8823BP) is produced by transforming with a vector pGA313 containing the membrane bound gluconate dehydrogenase, and 2-keto-D-gluconate is produced by incubating the recombinant E. coli JM109(pGA313)(KCTC 8823BP).

Abstract translation: 目的：提供膜结合的葡萄糖酸脱氢酶，其基因和使用转化的重组大肠杆菌生产2-酮-D-葡萄糖酸盐的方法，从而可以更高的产率将2-酮-D-葡萄糖酸盐从葡萄糖酸盐转化。 结论：通过SDS-聚丙烯酰胺分析，揭示了来自欧文氏卷曲酵母的膜结合的葡萄糖酸脱氢酶由第一个亚基65kDa，第二个亚基45kDa，第三个亚基20kDa组成，其中第一个亚基的氨基末端 具有Ala-Asn-Glu-Leu-Lys-Lys-Lys-Val-Asp-Ala-Val-Val-Val-Gly-Phe-Gly的式（I）的氨基酸序列，第二亚基的氨基末端 具有Asp-Asp-Gln-Ala-Asn-Asp-Ala-Leu-Val的式（II）的氨基酸序列，第三亚基的氨基末端具有式（III）的氨基酸序列的Ala-Glu -Glu-Ser-Ser-Gly-Ser-Gln-Thr-Ala-Arg-Asp-Tyr-Gln-Pro，第一亚基的黄素腺嘌呤二核苷酸键区具有Asp-XXXX的式（IV）的氨基酸序列 -Gly-X-Gly-XX-Gly-XXX-Ala-XX-Leu-X-Glu-XX-xX-XX-Val-XX-Glu-X-Gly，第二亚基的血红素键具有氨基酸 Cys-XY-Cys-His的式（Ⅴ）序列。 通过用含有膜结合的葡萄糖酸脱氢酶的载体pGA313转化来产生重组大肠杆菌JM109（pGA313）（KCTC8823BP），通过温育重组大肠杆菌JM109（pGA313）（pGA313）产生2-酮-D-葡萄糖酸 KCTC 8823BP）。

公开(公告)号：KR1019990026601A

公开(公告)日：1999-04-15

申请号：KR1019970048802

申请日：1997-09-25

Applicant: 한국과학기술연구원

Inventor： 반재구 ,  이홍원 ,  염도영

IPC: C12N15/52

Abstract: 본 발명은 어위니아 시프리페디(Erwinia Cypripedii)로 부터 글루코네이트를 2-케토-디-글루코네이트로 전환하는 세포막결합 글루코네이트 탈수소효소(gluconate dehydrogenase), 글루코네이트탈수소 유전자 및 염기서열과 이 유전자를 함유하는 형질전환된 재조합 대장균을 이용하여 글루코네이트의 2-케토-디-글루코네이트로의 전환하는 방법을 제공한다.

公开(公告)号：KR1020000059736A

公开(公告)日：2000-10-05

申请号：KR1019990007559

申请日：1999-03-08

Applicant: 한국과학기술연구원

Inventor： 박영훈 ,  정준기 ,  이홍원

IPC: C12N15/53 ,  C12N15/81

CPC classification number: C12N9/0004 ,  C12N15/81 ,  C12R1/685

Abstract: PURPOSE: Provided is a method for mass producing glucose oxidase, from Aspergillus niger by that glucose oxidase is accumulated in high concentration, thereby, purification process is simplified and a production yield is promoted. CONSTITUTION: The mass production method of glucoseoxidase comprises following steps of: preparing an expression plasmid having galactosedehydroginase (GAL10) promoter and galactodehydroginase (GAL7) terminator from yeast, signal sequence and structural gene of Aspergillus niger ATCC-9029; obtaining transformed yeast pJKGo(designation No. KCTC-0581 BP) by introduction of the expression plasmid into Saccharomyces cerevisiae (ATCC-2805); preparing glucose oxidase by the transformed yeast; and then secreting glucose oxidase to outside of cell membrane.

Abstract translation: 目的：提供从葡萄糖氧化酶以高浓度积累葡萄糖氧化酶的方法，通过葡萄糖氧化酶积累葡萄糖氧化酶，从而简化了纯化过程，提高了产率。 构成：糖酵解酶的大量生产方法包括以下步骤：制备具有半乳糖脱氢酶（GAL10）启动子和来自酵母的半乳糖脱氢酶（GAL7）终止子的表达质粒，黑曲霉ATCC-9029的信号序列和结构基因; 通过将表达质粒引入酿酒酵母（ATCC-2805）中获得转化酵母pJKGo（命名号KCTC-0581BP）; 由转化酵母制备葡萄糖氧化酶; 然后将葡萄糖氧化酶分泌到细胞膜外。

公开(公告)号：KR100328639B1

公开(公告)日：2002-03-20

申请号：KR1019990007559

申请日：1999-03-08

Applicant: 한국과학기술연구원

Inventor： 박영훈 ,  정준기 ,  이홍원

IPC: C12N15/53 ,  C12N15/81

Abstract: 본발명은곰팡이의일종인아스퍼질러스나이거() 유래의글루코스옥시다제(glucose oxidase)를고발현하기위해재조합플라스미드 pJKGO-1을효모에도입하고상기형질전환된효모로부터글루코스옥시다제를세포막외부로분비시켜대량제조하는방법에관한것으로, 효모로부터유래한갈락토스디히드로지나제(GAL10) 프로모터와 GAL7 터미네이터에아스퍼질러스나이거 ATCC-9029호의글루코스옥시다제의신호서열(signal sequence)과그 구조유전자(structural gene)로구성되는발현플라스미드를효모균주사카로마이세스세레비제() (ATCC-2805호)에도입하여형질전환효모 pJKGO(수탁번호 KCTC-0581 BP호)를얻고상기형질전환효모를배양하여글루코스옥시다제를제조하며, 본발명의방법에의하면글루코스옥시다제가배양액에서보다고농도로축적되어정제과정을줄이고회수율을향상시켜글루코스옥시다제를대량제조할수 있다.