Sunday, April 29, 2007

Development of a thermostable pectin methylesterase

Chacko Chakiath1, Robert Kozak2, and Craig S. Laufer1. (1) Biology, Hood College, 401 Rosemont Ave., Frederick, MD 21701, (2) Atlantic Biomass Conversions, Inc., 507 N. Bentz St., Frederick, MD 21701

This research is directed toward the development of enzymes with properties suitable for treatment of sugar beet pulp to produce biofuels.  Specifically, we are developing pectin methylesterases (pme) to efficiently catalyze the demethylation of pectin found in sugar beet pulp.  This reaction directly liberates methanol and facilitates subsequent saccharification of the pectin.


The initial phenotypes we have addressed are enzyme thermostability and catalytic turn-over number.  Thermostable pme would be advantageous in the processing of sugar beet pulp in order to take advantage of the energy already invested in heating the pulp to extract sucrose.  We began with native bacterial pmes and performed random mutagenesis on pme genes to generate mutant libraries.  The libraries were screened for thermostability and activity and enzyme variants with desirable characteristics were selected for analysis and further mutation.


We have identified a number of amino acid substitutions that increase pme thermostability and activity.  The mutant enzymes have been characterized for their residual activity after heating and for their kinetic constants.  We are currently characterizing second generation enzymes that have combined several of the mutations identified in the initial selections.