DIRECTED EVOLUTION OF PECTIN METHYLESTERASE A OF ERWINIA CHRYSANTHEMI TOWARD HIGHER THERMOSTABILITY

Abstract

Pectin methylesterase (PME) catalyzes the demethylesterification of galacturonic acid producing methanol, protons, and polygalacturonate. Directed evolution approaches were used to construct a thermally stabilized variant of Erwinia chrysanthemi PME A for potential use in the saccharification of sugar beet pulp in a biorefinery for biofuel. Using error-prone polymerase chain reaction a library of PME variants were generated. Out of a total of 3440 mutants screened, four thermostable PME variants harboring single amino acid substitutions at T273R, V251A, R253K and Y158H were discovered. The PME variant T273R was the most thermostable PME having a melting temperature (TO approximately 5.5 °C greater than that of the wild-type enzyme. PME variant R253 K showed activity comparable to that of wild-type but with a Tₘ that was 5 °C higher. The directed evolution approach found thermostabilizing mutations at sites not known for increasing thermostability and might have been missed by the rational design approach.