Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2

Abstract

The adhB gene encoding alcohol dehydrogenase-2 from Zymomonas mobilis has been subjected to random mutagenesis to obtain more thermostable variants of the enzyme. Random mutagenesis was accomplished using the polymerase chain reaction in mutagenic conditions. The optimum conditions involved restricting the concentration of one nucleotide to approximately one-tenth the normal amount. This introduced mutations at an average rate of 1 base in 600 in a 30-cycle PCR, sufficient to ensure that the majority of encoding DNA sequences in the mutant library have at least one base change from wild-type. Seven thermostable mutants were isolated from one library screening of 3000 colonies; two of these were selected for detailed study and purified using dye-ligand chromatography. Mutant TS-1 (F9S, V295A) was 3 degrees C more stable than the wild-type and had altered kinetic characteristics, with reduced affinity for ethanol and acetaldehyde and reduced ethanol oxidation Vmax. Mutant TS-2 (M13I, E19K, M192I) also had increased thermostability of 3 to 4 degrees C, but its kinetic characteristics were similar to that of the wild-type. Of the base changes found after sequencing a wide selection of mutants, 90% were transitions and 10% were transversions. Included were several T to C base changes which did not correspond with the nucleotide limitation used to create the mutant libraries.