Abstract 2052 A Lab Exercise in Degradation of Polysaccharides to Illustrate Enzyme Specificity and Relevance to Biofuel Production

Carbohydrate chemistry is an important topic in biochemistry courses. In this laboratory exercise, students use carbohydrate chemistry to compare the specificities of enzymes that cleave different polysaccharides into monosaccharides. The enzymes are derived from natural sources: amylase from students' saliva and cellulase and lignin-degrading enzymes from a wood rot fungus. For topical relevance, the lab handout highlights current efforts to harness fungal enzymes to process woody waste for biofuel production. The exercise detects the enzymatic cleavage of starch and cellulose polysaccharides by the release of glucose in an assay that detects reducing sugars through a reaction with dintrosalicyclic acid (DNSA). This reaction results in a color change quantified by absorbance. The polysaccharides tested are food-grade starch, cellulose (carboxymethyl cellulose), and maple wood sawdust. The cellulose in wood cannot be enzymatically cleaved without the degradation of its co-complexed lignin, a major impediment to using woody cellulose for biofuel production. For the enzymes, students use their own saliva as a source of amylase and the wood rot fungus Trametes veriscolor (Turkey Tail fungus) as a source of cellulase and lignin degrading enzymes. The fungus was purchased commercially and cultivated on maple sawdust. Amylase is specific for cleaving α linkages (glycosidic bonds) as found in starch while cellulase is specific for cleaving β linkages as found in cellulose. A protocol was optimized to enable the detection of glucose as a reducing sugar following incubation of saliva with starch and of fungus with cellulose or sawdust. Positive reactions gave strong signals comparable to a 2 mM glucose standard. The extracts were found to be specific in that saliva showed little to no reaction with cellulose and sawdust, and fungus showed little to no reaction with starch. This correlated with the expected substrate specificities of amylase and cellulase. The reducing sugar release from sawdust caused by the fungus showed that the fungus contained lignin-degrading enzymes in addition to cellulase. When the exercise was implemented in a biochemistry course as a 3-hour lab, 73% of the students saw an absorbance signal for amylase and starch that was larger than the 2 mM glucose standard and 87% of the students saw absorbance signal for fungus and cellulose that was larger than the 2 mM glucose standard. For the fungus and sawdust samples, 87% of the students saw an absorbance signal close to or greater than the 2 mM glucose standard. Instances of apparent cross substrate reactions were limited. The exercise was effective in demonstrating enzyme specificities for different polysaccharides while relating how natural enzymes can be harnessed to help degrade woody waste into a renewable energy source.