Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling

Previously it is suggested that a single mutation (Q102R) in the lactate dehydrogenase (LDH) gene from Bacillus stearothermophillus (bs) could switch the substrate specificity by 3 orders of magnitude from lactate to malate and produce a highly efficient malate dehydrogenase (MDH). In order to examine if random mutation and screening could improve this, a DNA-shuffling method would be used to generate a mutant LDH and recombinant LDH genes which will be later used for transforming Escherichia coli. The recombinant colonies are blotted and screened for their ability to catalyse the oxidation of malate. The most active MDH produced by this method is only slightly more efficient than the rationally designed Q102R variant. In addition to this mutation, the shuffled version incorporates further seven residue changes which are chemically conservative. These experiments demonstrate that the blind shuffling can achieve a huge shift in specificity which was a known, highly effective single-site mutation designed using structural knowledge.