Biofuel Enzyme Kit: From Grass to Gas – A study of enzymes

2. Biofuel Enzyme Kit: From Grass to Gas – A study of enzymes

3. Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Essy Levy, M.Sc. essy_levy@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com Biofuel Enzyme KitInstructors

4. Why Teach about enzymes? • Powerful teaching tool • Real-world connections • Link to careers and industry • Tangible results • Laboratory extensions • Interdisciplinary – connects physics, chemistry, biology and environmental science • Standards based

6. Biofuel EnzymeKit Advantages • Aligns with AP Biology AP Lab 2 • Can be run qualitatively or quantitatively • Construct and use a standard curve • Determine the effects on the reaction rate by changing: • pH • temperature • enzyme/substrate concentration • Mushroom extract activity for independent study • Extension for Michaelis-Menten analysis

7. Biofuel Enzyme KitWorkshop Timeline • Introduction • Review of enzymes • Run control reaction and enzyme reaction • Measure absorbance values • Determine effect of pH on reaction rate

8. What are enzymes?Molecules, usually proteins, that speed up the rate of a reaction by decreasing the activation energy required without themselves being altered or used up

9. Enzyme S*enz Eact Substrate (S)Product (P) How do enzymes work?Energy considerations S* ENERGY Eact S P REACTION COORDINATE

10. How do enzymes work?Physical considerations Substrate free in solution Substrate binds to a specific cleft or groove in the enzyme Activation energy barrier is overcome and reaction occurs Product is released and enzyme is free to catalyze another reaction

11. What are biofuels? Fuels that are produced from a biological source that was recently living • Biodiesel • Syngas • Ethanol from starches/sugars • Cellulosic ethanol

12. Cellulosic ethanol production A B C D

13. Glucose Cellulose breakdown 1. Heat, acid, ammonia or other treatment Endocellulases Exocellulases 2. Enzyme mixture added Cellobiase

14. 4 1 Cellobiose + H2O 2 Glucose + Cellobiose breakdown- a closer look 6 4 5 2 1 3

15. Protocol Highlights:Using a colorimetric substrate to track reaction rate • Cellobiose and glucose are colorless when dissolved • Use of the artificial substrate p-nitrophenyl glucopyranoside allows the reaction to be tracked by monitoring the appearance of yellow color cellobiose p-nitrophenyl glucopyranoside

16. p-nitrophenyl glucopyranoside + H2O glucose + p-nitrophenol + Basic conditions Clear Yellow Cellobiase breakdown of p-nitrophenyl glucopyranoside

17. How can this enzymatic reaction be easily quantified? Basic solution (STOP SOLUTION): - will develop color of any p-nitrophenol present - will stop the reaction • Each reaction time point can be directly compared to a standard of known concentration of p-nitrophenol • The amount of yellow color in the reaction solution can be quantified by measuring the absorbance at 410 nm using a spectrophotometer.

18. Biofuel Enzyme KitProcedure Overview

19. Prepare and run reactions

20. Example of Standards' Absorbance Readings

21. Qualitative Determination of Amount of Product Formed • Visually compare the color of the reaction time points E1-E5 and the controls Start and End against the standards of known amount • Plot the amount of p-nitrophenol formed at each time point to generate a reaction curve

22. Quantitative Determination of p-nitrophenol AmountRead SamplesAnalyze Results • Read the absorbance at 410 nm for each standard and generate a standard curve • Determine the amount of product for each reaction time point using the standard curve

23. Quantitative Determination of p-nitrophenol Amount

24. Amount of p-nitrophenol produced (nmol) Initial reaction rate = Time (min) 50 nmol - 0 nmol Initial reaction rate = = 12.5 nmol/min 4 min - 0 min Calculating initial reaction rate with and without an enzyme present

25. Conditions affecting reaction rate • pH • Temperature • Substrate Concentration • Enzyme Concentration

26. Effects of pHPrepare and run reactions

27. Amount of p-nitrophenol produced (nmol) Initial reaction rate = Time (min) Calculating initial reaction rate at different pH values • This is the amount of p-nitrophenol produced in 2 minutes

28. Further activities included in the kit • Effect of temperature on the reaction rate • Effect of substrate concentration on the reaction rate • Effect of enzyme concentration on the reaction rate • Ability of a mushroom extract to catalyze the breakdown of the substrate

29. Extensions • Perform a complete Michaelis-Menten analysis and determine the Vmax and Km for the cellobiase in this kit • Determine the optimum pH and temperature for the enzyme by preparing a temperature/pH surface plot • Debate use of crops for cellulosic ethanol production

30. Debate use of cellulosic ethanol as a fuel source CO2

31. Webinars • Enzyme Kinetics — A Biofuels Case Study • Real-Time PCR — What You Need To Know and Why You Should Teach It! • Proteins — Where DNA Takes on Form and Function • From plants to sequence: a six week college biology lab course • From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars