THERMOSTABLE VARIANTS OF ZYMOMONAS-MOBILIS ALCOHOL-DEHYDROGENASE OBTAINED USING PCR-MEDIATED RANDOM MUTAGENESIS

Using a random mutagenesis technique, the ferrous-ion-activated alcoho l dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant o ver 10 degrees C more stable at pH 8, with essentially unaltered kinet ic characteristics, was produced. However, the pH profile of thermosta bility of this variant was much altered compared with the wild-type, w ith a relatively small increase (4 degrees C) at pH 6. Sequencing of t he variants indicated five amino acids changes which contributed to th ermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were co ntained in the final stable variant, and the changes were partially ad ditive, with individual mutations causing between 2 and 3.5 degrees C stability increases (at pH 7.5). It is estimated that the most stable variant would have a half-life under physiological conditions at 70 de grees C of 15 min. (C) 1998 Academic Press.