Engineering melting temperatures of carbohydrate binding modules through site-directed mutagenesis

Abstract

Biofuels are a promising alternative to environmentally harmful fossil fuels. However, in order to displace fossil fuels, biofuels must be competitive in cost. Currently, the production process is the most significant issue regarding their cost, despite feedstock material being fairly inexpensive. One of the largest contributors to this cost is the use of enzymes to breakdown the biomass into simple sugars for subsequent use. If the enzymes used in this process could be easily recycled, the total cost of production would be significantly reduced. This project targets carbohydrate binding modules (CBMs) in an attempt to lower their melting temperature (Tm) through mutagenesis. These modules allow the holoenzyme (CBM with the catalytic domain) to bind to the substrate. Engineering a Tm of the CBM that was below the Tm of the catalytic domain would allow binding to be turned “off” for enzyme recovery, and later turned back “on” for future reactions. Tm values were established for three CBMs as well as refolding capabilities after heat treatment. Assays were developed for CBMs fused with green fluorescent protein by evaluating fluorescence signal on cellulose substrates in order to assess binding activity. Potential amino acid substitutions were identified in CBMs 11 and 44 from Ruminoclostridium thermocellum that were predicted to lower the Tm. Two of these mutations in CBM 44 that specifically interfere with Ca+ stabilization were created and sequenced. These mutants will be tested for changes in Tm and binding capabilities.