The ability of Zymomonas mobilis to produce almost stoichiometric amounts of ethanol from monosaccharides attracts this organism as a potential commercial producer of ethanol. Cloning of Zymomonas alcohol dehydrogenase (ZADH) gene is the first step of the study to understand the role of the gene in the production of ZADH which plays a key function in the ethanol fermentation. The ZADH gene was isolated from Zymomonas mobilis and cloned in E. coli in our laboratory. From the E. coli transformant, E. coli (pADS 93), harboring a recombinant plasmid (pADS 93) which contain the structural gene coding for ZADH was extracted and its characteristics were determined in this research. The same ZADH extracted from the gene donor (Z. mobilis) was also studied to compare with the enzyme produced by E. coli (pADS 93). The purification of ZADH was done using Affi-Gel Blue column and hydroxyl-apatite column chromatography. It was found that the ZADH produced by E. coli (pADS 93) had a single band at 40.000 dalton of molecular weight in the SDS-polyacrylamide gel electrophoresis indicating that it is ZADH II, one of the two Zymomonas native ADH isozymes. Analytical gel filtrations led to the conclusion that the molecule of ZADH II was composed of four subunits having the same molecular weight. The optimal pH of the ethanol oxidation was 10.0 and that of acetaldehyde reduction was about 8.0. Kinetic studies showed that reaction of ZADH II was ordered Bi system. Metal ion effects on activity of ZADH II was also examined. Zinc ions had no effect in restoring the EDTA-treated enzyme sample but cobaltous ion restored up to all of the original activity. Ferrous ions were most effective. In the presence of dithiothreitol, ferrous ions increased the enzyme activity drastically. From the observed results it was concluded that the ZADH produced by the E. coli transformant is identical to the ZADH II of Z. mobilis, and thus the alcohol dehydrogenase gene contained in the recombinant plasmid pADS 93 is that of ZADH II.
Zymomonas mobilis 의 알콜탈수소 효소 유전자가 cloning 된 대장균으로 부터 알콜탈수소 효소를 분리 정제하여 몇가지 특성을 연구하였다. 이들 대장균이 만들어 내는 효소는 Zymomonas 가 원래 가지고 있는 효소와 똑같았으며 이로서 대장균에 cloning 된 유전자는 Zymomonas 의 알콜탈수소 효소의 유전자 임이 확인되었다. 이들 효소는 분자량이 약 170,000 으로서 4개의 같은 단위로 이루어져 있으며, ZADH-II 임을 알게 되었다. 기질을 받아 들이는 범위가 좁아서 부탄올 이상의 큰 알콜은 이 효소에 의해서 작용을 받지 못하였다. 최적 pH 는 정반응인 경우 10.0, 역반응인 경우 8.0 가량 되었다. 또한 반응속도론적 연구를 규명한 결과 다른 알콜탈수소 효소처럼 ordered Bi Bi 기전으로 촉매됨을 알수 있었다. 이 알콜탈수소 효소는 기존의 다른 효소와는 달리 아연 이온에 의해서 활성화 되지 않았으며, 그와는 달리 철이나 코발트에 의해서 강하게 활성화됨을 관찰하였다.
