Zmomonas mobilis 알코올탈수소 효소 유전자의 분리와 분자생물학적 분석 및 zymomonas 에 쓰일 백타 개발

학위논문(박사) - 한국과학기술원 : 생물공학과, 1987.8, [ viii, 116 p. ]From the $\underline{Zymomonas}$ $\underline{mobilis}$, having potential value for ethanol fermentation, the structural gene encoding alcohol dehydrogenase (ADH; EC 1.1.1.1) responsible for the final step during alcoholic fermentation was cloned into $\underline{Escherichia}$ $\underline{coli}$ with plasmic pUC9 using allyl alcohol. Two recombinant plasmids were isolated from E.coli transformants showing ADH activity, which were sensitive to allyl alcohol, named pADS93 and pADL99. These plasmids was shown to share a common $\underline{Z.}$ $\underline{mobilis}$ chromosomal DNA of 2.6 kb. Electrophoretical analysis of total cell extrats on a nondenaturing polyacrylamide gel indicated that the two clones, $\underline{E.}$ $\underline{coli}$ (pADS93) and $\underline{E.}$ $\underline{coli}$ (pADL99), produced the identical enzyme displaying a same band of activity comigrating with one (ZADH-2) of the two $\underline{Z.}$ $\underline{mobilis}$ alcohol dehydrogenase isozymes (ZADH). In addition, the enzyme from crude extracts of $\underline{E.}$ $\underline{coli}$ (pADS93) was purified to compare with the ADH produced by $\underline{Z.}$ $\underline{mobilis}$. SDS-polyacrylamide gel electrophoresis of these final preparations revealed that ZADH-2 subunit purified from $\underline{E.}$ $\underline{coli}$ (pADS93) had a single band identical to the enzyme subunit from $\underline{Z.}$ $\underline{mobilis}$ at 40,000 dalton of molecular weight. Analytical gel filtration of Sephadex G-200 led to conclusion that this enzyme is a tetramer with molecular mass 170,000 dalton. Southern hybridization indicated that the structural gene (zadhII) for ZADH-2 was homologous between $\underline{Zymomonas}$ strains, though it did not have homology to that for other isozyme (ZADH 1). The complete nucleotide sequence of the zadhII was determined. The zadhII consists of an open reading frame, 1152 bp long, commencing from the ATG start codon encoding a polypeptide of 383 amino acid resi...한국과학기술원 : 생물공학과

Zymomonas 에 쓰일 plasmid vector 의 개발

학위논문(석사) - 한국과학기술원 : 생물공학과, 1984.2, [ vii, 46 p. ]For the ultimate purpose of introducing genes of cellulase or amylase into $\underline{Zymomonas}$ cells in order to make them able to produce ethanol from cellulose or starch, a suitable plasmid vector was attempted to develope. Plasmids were isolated from $\underline{Z}$. $\underline{anaerobia}$ and $\underline{Z}$. $\underline{mobilis}$. Among them a small plasmid isolated from $\underline{Z}$. $\underline{anaerobia}$ and designated as pZA2 was found to be most suitable. The size of plasmid pZA2 was estimated as 1.74 kilobase. It has one each cleavage site for Hind III and Bal I. Plasmids, pBR322 and pRK2501, have also been known to have one cleavage site each for Hind III. A hybrid plasmid was constructed using pZA2 and pBR322. The hybrid plasmid could be maintained in $\underline{E}$. $\underline{coli}$and be amplified by chloramphenicol. The hybrid plasmid could still be maintained in $\underline{E}$. $\underline{coli}$ even after the replicon portion of pBR322 was destroyed by treating with Hae II. The fact suggests that the replicon of pZA2 was not attacted by Hind III and was used for replication of the hybrid plasmid in E. coli. For the purpose of inserting selective markers another hybrid plasmid between pZA2 and pRK2501 was formed. Transformation of $\underline{Zymomonas}$ cells with this hybrid plasmid is now being tried.한국과학기술원 : 생물공학과