Purification and characterization of alcohol dehydrogenase encoded by zymomonas mobilis gene in escherichia coli transformant

Abstract

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 plasm...