This research involves the development of an enzyme with properties suitable for the digestion of sugar beet pulp to produce alcohol-based biofuels. Specifically, directed evolution is used to optimize pectin methylesterase (pme) catyalization of the demethylation of pectin in sugar beet pulp, producing methanol. The phenotype addressed in this research is thermostability, which will make use of the energy already invested in heating the sugar beet during sugar processing.
    Random mutagenesis was performed on native bacterial pme to generate mutant libraries, which were then screened for thermostability and activity. Enzyme variants containing single amino acid substitutions were identified as conferring thermostability to pme.
    These mutations were then combined to make multiply-mutant enzymes. As each mutation was added, the mutated codon was randomized to look for novel substitutions. These libraries were screened for thermostability and activity. The mutations conferred thermostability in an additive fashion, resulting in a pme variant with an approximately 10C improvement over native pme.
    Currently, the mutant enzymes are being characterized for residual activity and for their kinetic constants. In addition, a random mutagenesis is being performed on the multiply-mutant pme variant, screening for further increases in thermostability and activity.