POLYMERASE CHAIN REACTION-BASED RANDOM MUTAGENESIS - PRODUCTION AND CHARACTERIZATION OF THERMOSTABLE MUTANTS OF ZYMOMONAS-MOBILIS ALCOHOL DEHYDROGENASE-2

The adhB gene encoding alcohol dehydrogenase-2 from Zymomonas mobilis has been subjected to random mutagenesis to obtain more thermostable v ariants of the enzyme. Random mutagenesis was accomplished using the p olymerase chain reaction in mutagenic conditions. The optimum conditio ns involved restricting the concentration of one nucleotide to approxi mately one-tenth the normal amount. This introduced mutations at an av erage rate of 1 base in 600 in a 30-cycle PCR, sufficient to ensure th at the majority of encoding DNA sequences in the mutant library have a t least one base change from wild-type. Seven thermostable mutants wer e isolated from one library screening of 3000 colonies; two of these w ere selected for detailed study and purified using dye-ligand chromato graphy. Mutant TS-1 (F9S, V295A) was 3-degrees-C more stable than the wild-type and had altered kinetic characteristics, with reduced affini ty for ethanol and acetaldehyde and reduced ethanol oxidation V(max). 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 s election of mutants, 90% were transitions and 10% were transversions. Included were several T to C base changes which did not correspond wit h the nucleotide limitation used to create the mutant libraries. (C) 1 994 Academic Press, Inc.