DIRECTED EVOLUTION OF A LOW pH TOLERANT PECTIN METHYLESTERASE

Abstract

The objective of this proposal is to facilitate the biofuel production from agricultural waste such as sugar beet pulp. Pectin methylesterase A (pmeA) is an enzyme that catalyzes the hydrolysis of the methylester groups of pectin to produce methanol and pectate; therefore, pme is able to demethylate the pectin (present in sugar beet pulp) and produce methanol which is a valuable commodity; in addition, pectinesterases work better on pectate than pectin itself, so demethylation facilitates the ethanol production reaction; however, pmes can not tolerate high temperature and low pH (the conditions for sugar beet processing). Using directed evolution techniques, researchers have evolved a thermostable pme, but thermostability alone will not suffice for biofuel application, the reason being that during the course of the pme reaction the pectate acidifies the solution to the point of inhibiting subsequent pme reaction. Therefore, not only should a pme be thermostable, but also it should be able to tolerate low pH (pH 3.0). Therefore, we would like to evolve a pme with tolerance to low pH for the biofuels application since previous studies have not addressed this requirement.