Tuesday, May 1, 2007 - 7:25 PM

Customizing enzyme cocktails for biomass saccharification using parallel capillary electrophoresis and LC-MS analysis of oligosaccharide reaction products

Justin Stege1, Kevin Gray2, Julia Khandurina1, Hongjun Huang1, Thoas McClure1, Peter Domaille1, Abraham Anderson1, Ajit Botejue1, and Kelvin Wong1. (1) Diversa, 4955 Director's Place, San Diego, CA 92121, (2) Verenium Corp, 4955 Director's Place, San Diego, CA 92121

Effective saccharification of pretreated biomass requires the synergistic activities of multiple enzymes, particularly when significant hemicellulose structures remain.  With over 1000 plant cell wall-degrading enzymes in our collection, Diversa develops cocktails of enzymes that are customized for each particular feedstock, pretreatment and for different process conditions.  To identify the best enzyme cocktail for a given application, it is critical to have an array of analytical techniques to understand the structure and composition of the substrate, reaction products, and undigested residual material.  Combinatorial enzyme evaluation on insoluble biomass substrates requires both rapid screening methods to measure reducing sugars and more precise methods to accurately resolve and quantify the different reaction products.  In addition to HPLC, Diversa has developed a 96-well capillary electrophoresis system for profiling oligosaccharide and monosaccharide reaction products.  This high throughput CE technology enables us to rapidly screen hundreds of enzymes or enzyme combinations under different conditions with different substrates to select for desirable product profiles.  These customized cocktails can be further improved by analyzing the undigested carbohydrates remaining after enzyme digestion.  For many substrates accumulation of oligosaccharide products is often observed after extensive enzymatic digestion.  These “dead-end” products have structures or bonds that are resistant to digestion by the enzymes used in the saccharification cocktail, reducing the yield of fermentable sugars.   We have used HPLC fractionation to isolate undigested oligosaccharides for structure determination by LC-MS.  Using this compositional information, enzymes have been identified that saccharify the resistant material, greatly improving the yield of fermentable sugars.