5-92: Enzyme Engineering of Glycoside Hydrolase-5 Endoglucanases Enzymes for Consolidated Bioprocessing

The enzymatic hydrolysis of cellulose into simpler sugars starts with the endoglucanases (3.2.1.4) and exoglucanases (3.2.1.91), which hydrolyze insoluble cellulosic polymers into smaller soluble sugars with degree of polymerization <6.  Our initial effort is focused on improving the activity of the endoglucanases; specifically, the endoglucanases that can be either used in consolidated bioprocessing by the addition of the enzymes into the pretreatment mix, or as a component of the ‘parts list’ for engineering into the three hosts- Saccahromyces cerevisiae, Escherichia coli and Sulfolobus acidocaldarius- for fuels production at JBEI. Therefore the selection of the endoglucanases for directed evolution is guided by the characteristics of the enzymes mapped onto the characteristics of the pretreatment method or the JBEI fuel synthesis host. Acidothermus cellulolyticus and Sulfolobus solfataricus enzymes were selected based on their activity, actual or predicted, in dilute acid pretreatment method and as an engineered part for S. acidocaldarius. The endoglucanases from Thermotoga maritima and the three Pyrococcus species were selected based on their predicted stability in temperatures > 80 oC, which would make these compatible with hydrothermolysis pretreatment method. Among these initial target genes, Pyrococcus horikoshii endoglucanase (Pho-EG) was the first candidate to be tested for directed evolution work, because our preliminary work have shown that Pho-EG has highest specific activity among the three Pyrococcus species that we selected. Endoglucanase assay was performed using azo-carboxymethyl cellulose (azoCMC) to determine the pH and temperature optima of the wild-type Pho-EG activity from the cell lysate.