DIRECTED EVOLUTION OF A THERMOSTABLE PECTIN METHYLESTERASE FOR USE IN A SUGAR BEET BIOREFINARY

Abstract

Worldwide demand for biofuel is increasing. Agricultural waste like sugar beet pulp is a sustainable, abundant, inexpensive, and readily saccharificable biomass feedstock. Pectin is a complex polymer abundant in plant cell walls. Pectin methylesterase (PME) catalyzes the hydrolysis of pectin methylester groups into methanol. Application of PME to sugar beet pectin yields biomethanol and enables further enzymatic conversion into other biofuels. The optimal temperature of PME is 50°C, yet sugar beet pulp exits processing at 60°C. To utilize this latent heat PME must be active at 60°C. This study uses directed evolution methods (site-directed mutagenesis and error-prone PCR) to combine known thermostable mutations to create one multiplymutant thermostable PME. The final clone, JL25, exhibited 100% activity at 65°C and enhanced activity on sugar beet pulp. These results confirm past research suggesting the additive effects of multiple mutations, and provide an example of an amino acid substitution with background-dependent phenotypic effects.