Biofuel Enzyme Kit From Grass to Gas: An Inquiry based study of enzymes

2. Biofuel Enzyme Kit From Grass to Gas: An Inquiry based 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 Damon Tighe damon_tighe@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com Biofuel Enzyme KitInstructors

4. Biofuel Enzyme KitWorkshop Timeline • Introduction • Review of enzymes • Inquiry and collaboration using this kit • Run control reaction and enzyme reaction • Measure absorbance values

5. 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. Technology Engineering Math Science Inquiry

7. Biofuel Enzyme Kit Advantages • Aligns with current AP Biology AP Lab 2 and future AP Big Ideas 1 (Evolution), 2 (Cellular Processes), and 4 (Interactions) • Can be run qualitatively or quantitatively • Construct and use a standard curve (mathematics and technology) • Determine the effects on the reaction rate by changing: • pH • temperature • enzyme/substrate concentration • Mushroom extract activity for student run inquiry • Extension for Michaelis-Menten analysis

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. BiofuelsActivity 1Overview

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

22. Measuring Absorbance Quantitatively SmartSpec Spectrophotometer iMARK Microplate reader

23. Biofuel Enzyme Kit Procedure Overview • Activities: • Reaction Rate & Std curve • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase • Collaborative approach: • Each student group does activity 1 • Student groups do one activity each from 2-5 • Groups share data • All groups do activity 6 and share data

24. Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase

25. Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase

26. 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 • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase

27. Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase

28. Amount of p-nitrophenol produced (nmol) Initial reaction rate = Time (min) • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase • This is the amount of p-nitrophenol produced in 2 minutes

29. Amount of p-nitrophenol formed (nmol) Time (minutes) • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase High enzyme concentration Low enzyme concentration 1. The initial reaction rate is faster when there is a higher enzyme concentration 2. Given enough time, the same amount of product will be formed for both the high and low enzyme concentration reactions

30. 1.5 mM substrate [High] • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase Amount of p-nitrophenol formed (nmol) 0.25 mM substrate [Low] Time (minutes) 1. Effect of substrate concentration on the initial rate 2. Final amount of product formed with varying substrate concentrations

31. Inquiry – find your own source of celliobiase. Have students develop protocol for testing activity Where can we find things that break down cellulose? • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase Art – document your source via photography or drawings in a Lab Notebook Technology –use GPS and mapping software to document sources

32. Where can we find things that break down cellulose? • Std curve / Std Reaction Rate • Effect of Temperature • Effect of pH • Effect of Enzyme Concentration • Effect of Substrate Concentration • Bio-prospecting for Celliobiase Inquiry – find your own source of celliobiase! Have students develop protocol for testing activity based upon activity 1. Mushrooms are a great source of celliobiase and where the biofuels industry gets most of its enzymes currently, but there are many other potential sources out there….test them! Art – document your source via photography or drawings in a Lab Notebook Technology –use GPS and mapping software to document sources. Use excel or Vernier LoggerPro to analyze data. (Excel protocol available upon request).

33. Celliobiase Bio-Prospecting in Mushrooms (inquiry) Wood degrading mushrooms Root associating mushrooms • Ecological niches of each mushroom correlates with celliobiase activity. • Dried mushrooms work just as well as fresh ones and are available at many stores

34. StudentInquiry: A Stepwise Protocol approach • Questions to consider: • How important is each step in the lab protocol? • What part of the protocol can I manipulate to see a change in the results? Possible variables: ratio of enzyme to substrate, look at more temperatures – can you get failure at a high enough temperature?, look at more pH points – at what low pH does failure occur? • How do I insure the changes I make is what actually affected the out come? (Controls) • Write the protocol. After approval – do it

35. StudentInquiry • More Advanced Questions • How can I estimate the concentration of my novel celliobiase from activity 6? • Can I predict the activity of my novel celliobiase based upon the environment/organism I’m getting it from? • How does my novel celliobiase act under different pH and temperatures? • What is the optimal pH/temperature combination for my celliobiase? (Surface plots)

36. Debate use of cellulosic ethanol as a fuel source CO2 • Get your social sciences teacher involved with the debate and/or argument research papers on Biofuels • Engineering infrastructure changes • Competition with food crops