Thermostable variants of Zymomonas mobilis alcohol dehydrogenase obtained using PCR-mediated random mutagenesis

Using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant over 10 degrees C more stable at pH 8, with essentially unaltered kinetic characteristics, was produced. However, the pH profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees C) at pH 6. Sequencing of the variants indicated five amino acids changes which contributed to thermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were contained in the final stable variant, and the changes were partially additive, 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 degrees C of 15 min.