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Instability of Trp Operon Recombinant Plasmids in E. coli
김성훈,최영철,유두영,이세영,Kim, Sung-Hoon,Choi, Young-Chul,Ryu, Dewey D.Y.,Lee, Se-Yong 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.4
Plasmid pBR322와 ${\phi}80h$-pttrp190 혹은 ColEl-trp으로 부터 분리된 tryptophan operon을 T4 ligase에 의해 결합시켜 E. coil JA221 $r_k{^-}m_k{^+}$ recA ${\Delta}trpE5$에 도입해 ampicillin(50 ${\mu}g/ml$)과 tetracycline (10 ${\mu}g/ml$)을 함유한 Vogel Bonner minimal plate에서 그 recombinant를 선별하였다. 그러나 얻어진 transformant의 성장속도는 매우 느렸으며 그들의 $trp^+$ marker를 매우 빠른 속도로 상실하였다. MV12/pVH5 strain에서 tryptophan 생합성의 host repression sysyem이 indoleacrylic acid에 의해 부분적 derepression을 일으켰을 때 tryptophan operon의 효소 활성은, 가해준 indoleacrylie acid의 농도에 따라 증가하는 반면 pVH5의 안정성은 역으로 감소하였다. 또 이 strain을 glucose를 제한한 배지에서 연속 배양하였을 때 약 30세대가 지난 후부터 $trp^-$ variant가 나타나기 시작했으며 $trp^-$ 부분은 점차적으로 증가하였다. 그밖에 여러 다른 배양조건들로 부터 분리된 $trp^-$ 변종들은 원래의 pVH5에 비해 그 분자량이 증가되거나 감소된 형태의 plasmid를 가지고 있거나 plasmid 전체를 상실하였다. pBR322 and trp operon from ${\phi}80$ trp or ColEl-trp was covalently joined by T4 ligase and introduced into E. coli JA221 $r_k{^-}m_k{^+}$ ${\Delta}trpE5$recA, and the recombinant was selected on the VB minimal plate containing ampicillin (50 ${\mu}g/ml$) and tetracycline (10${\mu}g/ml$), The growth rate of transformants obtained was very low and they lost their $trp^+$ marker very rapidly. In MV12/ pVH5, when host repression system of the tryptophan biosynthesis was partially derepressed with indoleacrylic acid, the enzyme activity of trp operon was increased but the stability of pVH5 was inversely decreased with the concentration of indoleacrylic acid added. In the glucose limited continuous culture of this strain, $trp^-$ variant began to appear after about 30 generations and the $trp^-$ fraction increased gradually. $Trp^-$ cells isolated from cultures grown under various conditions have lost their entire plasmids or arbored modified plasmids which increased or decreased in molecular weight compared to original pVH5.
E . coli 의 Tryptophan 오페론 재조합 프라스미드의 불안정성에 관한 연구
김성훈,최영철,유두영,이세영 ( Sung Hoon Kim,Young Chul Choi,Dewey D . Y . Ryu,Se Yong Lee ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.4
pBR322 and trp operon from φ80 trp or ColEl-trp was covalently joined by T4 ligase and introduced into E. coli JA221 r_k^-m_k^+ ΔtrpE5 recA, and the recombinant was selected on the VB minimal plate containing ampicillin (50 ㎍/㎖) and tetracycline (10 ㎍/㎖), The growth rate of transformants obtained was very low and they lost their trp^+ marker very rapidly. In MV12/ pVH5, when host repression system of the tryptophan biosynthesis was partially derepressed with indoleacrylic acid, the enzyme activity of trp operon was increased but the stability of pVH5 was inversely decreased with the concentration of indoleacrylic acid added. In the glucose limited continuous culture of this strain, trp variant began to appear after about 30 generations and the trp fraction increased gradually. Trp cells isolated from cultures grown under various conditions have lost their entire plasmids or arbored modified plasmids which increased or decreased in molecular weight compared to original pVH5.
2 단 연속식 발효 방식에 의한 솔비톨로 부터의 솔보즈 생산 : 조업 조건의 최적화 Optimization of Operating Condition
김영걸,구윤모,유두영 한국화학공학회 1980 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.18 No.5
Glucose를 starting material로 하는 vitamin C 생산에 있어서 중요한 중간 물질인 sorbose의 2단 연속식 발효 방식에 의한 생산에 대하여 연구하였다. Sorbitol의 탈수소 반응에 의한 sorbose의 생성은 화학적인 방법으로도 그 진행이 가능하지만 L-. D-형의 sorbose racemate가 생기기 때문에 그 수율면에서 미생물에 의한 발효에 뒤지고 있다. 회분식 발효에 있어서 기질 농도가 20% 일 때 90%의 전화율과 19mmole/ℓ/hr의 생산율을 얻었다. 회분식 실험에 의하여 sorbose의 생산은 발효액 중의 미생물 농도에 비례함을 알았고 미생물 농도를 증가시키기 위하여 연속식 발효 방식, 특히 여과장치를 갖춘 2단 연속식 발효 방식을 사용하여 생산율 증가를 시도하였다. 온도 29℃ pH 4.6, 기질농도 20% 등 최적 발효조건을 유지하며 90%의 전화율에서 45mmole/ℓ/hr의 생산율을 얻을 수 있었으며 이것은 회분식 최대 생산율의 2배 이상에 해당하는 값이다. Using a two-stage continuous culture system, the parameters that affect the productivity of sorbose were studied and the operating conditions which correspond to the maximal productivity of sorbose were determind. The maximum specific growth rate of Acetobacter surboxydans strain employed was 0.31hr and its optimal growth pH was determined as 4. 6. The maximum productivity of sorbose obtained employing a batch culture system was 19mmole/liter/hr at 90% conversion level. The enzymatic conversion of sorbose was found to be proportional to the cell concentration, and the cell concentration in the second-stage continuous bioreactor was increased by using a filter system installed at the effluent line. Under the optimal operating conditions, 29℃, pH 4.6, and sorbitol concentration 20%, the two-stage continuous fermentation system yielded the productivity of 45mmole sorbose/liter/hr while maintaining 90% or higher level of conversion, indicating that a significant improvement in the sorbose productivity could be achieved by employing the two-stage continous bioreactor system combined with the cell enrichment technique.
Review Articles-Biomolecular Engineering and Drug Development
Nam, Doo-Hyun,Ryu, Dewey D. Y. 영남대학교 약품개발연구소 1999 영남대학교 약품개발연구소 연구업적집 Vol.9 No.-
Biomolecular engineering is a technology to create novel structures of high-value bio-molecules for use in medicine and industry, through the directed alteration of proteins and/or biologically active molecules in living cells to produce a novel biometabolites as well as engineered protein itself. For the development of new drugs by biomolecular en-gineering, desired biomolecules have to be rationally designed based on their structure-stability/structure-activity relationship, and then screened through well-established mutation and selection program. Over the past decade, there has been significant pro-gress in mutation and selection methodology; DNA shuffling technology mimicking natural evolution for artificial DNA recombination and phage-displayed combinatorial peptide library for rapid selection of proteins expressed from mutated genes. Bioinfor-matic tools including functional genomics and proteomics have been also developed for the ready access to the information related to the protein-function and genome-Protein,leading to the design and identification of new drug targets. Throughout the use of an enormous amount of bioinformatic databases, many protein/peptide drugs and biome-tabolite molecules have been designed The candidates of new drugs are monoclonal an-tibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two human-ized monoclonal antibodies approved by FDA became the first tine of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, far the treatment of breast cancer and pediatric respiratory syncytial viral infection, respectively. Many more newly engineered biomolecules are under developing for medicinal application. Some clinical trials fer therapeutic applications are now in progress, and very positive results are already anticipated.
Cloning and Expression of Kluyveromyces fragilis β-Galactosidase Gene in Saccharomyces cerevisiae
BANG, JEONG HEE,NAM, DOO H.,KANG, DAE OOK,AHN, JONG SEOG,RYU, DEWEY D.Y. 영남대학교 약품개발연구소 1995 영남대학교 약품개발연구소 연구업적집 Vol.5 No.-
A gene coding for the β-galactosidase (lactase) of Kluyveromyces fragilis UCD 55-55 was isolated by complementation in Escherichia coli YMC9. From the plasmid library made from Sau3A-digested chromosomal DNA, one positive done was selected. The cloned gene for β-galactosidase was on 7.3 kilobase pair DNA fragment, and a slightly low level of β-galactosidase enzyme activity was detected in E. coli. It was also confirmed that the cloned gene comes from K. fragilis by DNA-DNA hybridization and immunochemical blotting experiments. In order to construct a new yeast strain having the metabolic ability for lactose, the cloned gene for K. fragilis β-galactosidase was inserted in yeast vector YEp24 and YRp17, and transformed into Saccharomyces cerevisiae YNN7 and M1-2B. The yeast transformants showed the nearly the same β-galactosidase productivity as level of K. fragilis when uninduced, but these could not utilize lactose as a sole carbon source, presumably due to the lack of lactose transport system. Nevertheless, a slightly higher ethanol productivity was achieved by these transformants than S. cerevisiae or K. fragilis, in the medium containing glucose and lactose.