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

3. Biofuel Enzyme Kit Instructors
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.
Damon Tighe
Leigh Brown, M.A.

4. Biofuel Enzyme Kit Workshop 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
Enzyme Class
Example
Oxidoreductase
(transfer of electrons)
Firefly Luciferase – oxidizes luciferin to produce oxyluciferin and light
Transferase
(group-transfer reactions)
Hexokinase – transfers a phosphate group to glucose to make glucose-6-phosphate
Hydrolase
(hydrolysis reactions)
Cellobiase – breaks down cellobiose
Lyase
(double bond reactions)
Histidine decarboxylase – generates histimine from histidine
Isomerase
(transfers to create a new isomers)
Glucose-6-Phosphate isomerase – converts G-6-P to fructose-6-phosphate
Ligase
(forms covalent bonds)
DNA Ligase – covalently bonds two pieces of DNA
Oxidoreductases (transfer of electrons)
Transferases (group-transfer reactions)
Hydrolases (hydrolysis reactions or reverse condensation reactions)
Lyases (adding a group to a double bond or removing of a group and adding a double bond)
Isomerases (transfer of a group within a molecule to give a different isomer)
Ligases (formation of C-C, C-S, C-O, C-N bonds)

9. How do enzymes work? Energy considerations
S*enz
Eact
Enzyme
How do enzymes work? Energy considerations
Substrate (S) Product (P) 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
Biodiesel -
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 productionB C
A- Plant material is collected. Preferred materials include fast growing poplar trees, switchgrasses, bagasse from sugar cane, corn stover. Mainly cell wall material – highest content of cellulose
B – Plant products are processed using heat, mechanical means, acid, or ammonia to separate the cellulose and hemicellulose from lignin. Lignin inhibits further enzymatic reactions. Cellulose and hemicellulose can be broken down to create simple sugars.
C- After removal of lignins, enzyme mixtures are added. Endocellulases cleave cellulose from the middle of chains. Exocellulases remove two-glucose units (cellobiose) from the ends of cellulose chains and cellobiase breaks apart cellobiose to glucose. End result is a predominantly glucose solution.
D- Fermenting bacteria or yeast (think beer) are added to glucose solution to produce ethanol. Ethanol is further purified by distillation (removal of water) to produce fuel. Ethanol can also be added to gasoline to produce mixtures (E85 is 85% ethanol, 15% gasoline). D

13. 1. Heat, acid, ammonia or other treatment
Cellulose breakdown
Glucose
1. Heat, acid, ammonia or other treatment
Endocellulases
Exocellulases
2. Enzyme mixture added
Cellobiase
Heat, acid, grinding (mechanical force), and/or ammonia used to break down plant material (switchgrasses, poplar trees, corn stover, sugar cane bagasse, etc). Also breaks up the hydrogen bonds between cellulose chains and within the cellulose chains.
For ease of viewing, only showing one cellulose chain. Add enzymes to this. The endos work on internal beta 1-4 glucose bonds. Exos work on the reducing and non-reducing ends to cleave off cellobiose. When free cellobiose is present – cellobiase cleaves it to glucose.
When all/most is glucose, can be fermented to ethanol

14. Cellobiose breakdown- a closer look+ 4 1
PDB id: 1gnx Name: Hydrolase Title: B-glucosidase from streptomyces sp Structure: Beta-glucosidase. Chain: a, b. Engineered: yes Source: Streptomyces sp.. Organism_taxid: Expressed in: escherichia coli. Expression_system_taxid: UniProt: Chains A, B: Q59976 (Q59976_STRSQ)
Resolution: 1.68Å R-factor: R-free: Authors: A.Guasch,J.A.Perez-Pons,M.Vallmitjana,E.Querol,M.Coll Key ref: a.guasch et al. Beta-Glucosidase from Streptomyces. To be Published, . Date: 10-Oct Release date: 17-Oct-02
Cellobiose + H2O Glucose 6 4 5 2 1 3

15. p-nitrophenyl glucopyranoside
• 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
Protocol Highlights: Using a colorimetric substrate to track reaction rate
cellobiose
p-nitrophenyl glucopyranoside

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

17. Biofuels Activity 1 Overview

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. Amount of p-nitrophenol (nmol)
Standard
Amount of p-nitrophenol (nmol)
Absorbance
410 nm S1 S2
12.5
0.2 S3 25
0.4 S4 50
0.8 S5
100
1.6
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)
Std curve / Std Reaction Rate
Effect of Temperature
Effect of pH
Effect of Enzyme Concentration
Effect of Substrate Concentration
Bio-prospecting for Celliobiase
Initial reaction rate =
Amount of p-nitrophenol produced (nmol)
Time (min)
Initial reaction rate =
50 nmol - 0 nmol
4 min - 0 min
= 12.5 nmol/min

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 =
Amount of p-nitrophenol produced (nmol)
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. Amount of p-nitrophenol formed (nmol)
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)
Can do other concentrations of substrate (did make 3 mM in educator's prep) or also do different dilutions between 1.5 mM and 0.25 mM in order to do a full Michaelis-Menten analysis.
Initially, the rate is controlled by how fast the enzyme can find and bind substrate. For the high concentration substrate – this is likely to happen quicker than for the lower concentration substrate. At very long times, there will be less and less product formed in each time unit. Each reaction can produce the same number of mols of product as amount of substrate put in the reaction so the high concentration substrate will produce 5 times more product than the low conc substrate. The final equilibrium is not affected assuming that each reaction has enough time to go to completion (ie 100% substrate going to 0% substrate and 100% product) only the speed at which this occurs changes.
1. Effect of substrate concentration on the initial rate
2. Final amount of product formed with varying substrate concentrations

31. Where can we find things that break down cellulose?
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
A- Plant material is collected. Preferred materials include fast growing poplar trees, switchgrasses, bagasse from sugar cane, corn stover. Mainly cell wall material – highest content of cellulose
B – Plant products are processed using heat, mechanical means, acid, or ammonia to separate the cellulose and hemicellulose from lignin. Lignin inhibits further enzymatic reactions. Cellulose and hemicellulose can be broken down to create simple sugars.
C- After removal of lignins, enzyme mixtures are added. Endocellulases cleave cellulose from the middle of chains. Exocellulases remove two-glucose units (cellobiose) from the ends of cellulose chains and cellobiase breaks apart cellobiose to glucose. End result is a predominantly glucose solution.
D- Fermenting bacteria or yeast (think beer) are added to glucose solution to produce ethanol. Ethanol is further purified by distillation (removal of water) to produce fuel. Ethanol can also be added to gasoline to produce mixtures (E85 is 85% ethanol, 15% gasoline).
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
A- Plant material is collected. Preferred materials include fast growing poplar trees, switchgrasses, bagasse from sugar cane, corn stover. Mainly cell wall material – highest content of cellulose
B – Plant products are processed using heat, mechanical means, acid, or ammonia to separate the cellulose and hemicellulose from lignin. Lignin inhibits further enzymatic reactions. Cellulose and hemicellulose can be broken down to create simple sugars.
C- After removal of lignins, enzyme mixtures are added. Endocellulases cleave cellulose from the middle of chains. Exocellulases remove two-glucose units (cellobiose) from the ends of cellulose chains and cellobiase breaks apart cellobiose to glucose. End result is a predominantly glucose solution.
D- Fermenting bacteria or yeast (think beer) are added to glucose solution to produce ethanol. Ethanol is further purified by distillation (removal of water) to produce fuel. Ethanol can also be added to gasoline to produce mixtures (E85 is 85% ethanol, 15% gasoline).
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. Student Inquiry: 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. Student Inquiry 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