1. Biofuel Enzyme Kit Keith P. Madden 2010 Molecular Biology RET

2. Biofuel Enzyme Kit Bio-Rad 166-5035EDU $156.25 no refill options shown

4. Kit includes: enzyme substrate standard solution buffers disposable pipets tubes cuvettes, for 32 students

5. Student interest What are our options for future clean energy sources? How “Green” is each possible source? Can I use it as a gasoline substitute for my car?

6. Curriculum topics Environmental options Enzymology Reinforcement of chemical concepts (pH, concentration, catalysis Integration of math and science (quantitative data if spectrophotometer is used – time vs. conversion)

7. Concept: Cellulose is abundant, and cellulose is a sugar polymer. Use enzymes to break down the polymer. Break cellulose into smaller units with endocellulases. Break off 2 glucose units from the end of the smaller fragments with exocellulases. This two glucose fragment is cellobiose

8. Cellobiose

9. The reaction of cellobiase: 1 cellobiose + H2O gives:

10. Glucose can be fed to microbes Microbes ferment glucose to ethanol Ethanol recovered as fuel But for our purposes, we only need to look at glucose production – but it is invisible! So – use p-nitrophenyl glucopyranoside as a substitute substrate.

11. p-Nitrophenyl Glucopyranoside

12. Set-up 1. Label a 15 ml conical tube “1.5 mM Substrate”. Add 15 ml of 1.5 mM substrate to the tube. 2. Label a 15 ml conical tube “Enzyme”. Add 5 ml of low concentration enzyme to the tube. 3. Label a 15 ml conical tube “Stop Solution”. Add 15 ml of 1x stop solution to the tube. 4. Label a 15 ml conical tube “Buffer”. Add 8 ml of 1x resuspension buffer to the tube. 5. Label a cuvette S1. Add 1 ml of S1 standard to the cuvette. 6. Label a cuvette S2. Add 1 ml of S2 standard to the cuvette. 7. Label a cuvette S3. Add 1 ml of S3 standard to the cuvette. 8. Label a cuvette S4. Add 1 ml of S4 standard to the cuvette. 9. Label a cuvette S5. Add 1 ml of S5 standard to the cuvette.