Transcript Document

Biofuel Enzyme Kit
From Grass to Gas:
An Inquiry based study of enzymes
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.
[email protected]
Damon Tighe
[email protected]
Leigh Brown, M.A.
[email protected]
Biofuel
Enzyme Kit
Workshop
Timeline
• Introduction
• Review of enzymes
• Inquiry and collaboration using this kit
• Run control reaction and enzyme
reaction
• Measure absorbance values
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
Engineering
Technology
Math
Science
Inquiry
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
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
Firefly Luciferase – oxidizes
luciferin to produce oxyluciferin
and light
Transferase
Hexokinase – transfers a
phosphate group to glucose to
make glucose-6-phosphate
Hydrolase
Cellobiase – breaks down
cellobiose
Lyase
Histidine decarboxylase –
generates histimine from histidine
Isomerase
Glucose-6-Phosphate isomerase –
converts G-6-P to fructose-6phosphate
Ligase
DNA Ligase – covalently bonds two
pieces of DNA
(transfer of electrons)
(group-transfer
reactions)
(hydrolysis reactions)
(double bond
reactions)
(transfers to create a
new isomers)
(forms covalent bonds)
Substrate (S)
How do
enzymes
work?
Energy
considerations
Enzyme
Product (P)
S*
E
N
E
R
G
Y
S*enz
Eact Eact
S
P
REACTION COORDINATE
How do
enzymes
work?
Substrate free in
solution
Substrate binds to a
specific cleft or groove
in the enzyme
Physical
considerations
Activation energy
barrier is overcome and
reaction occurs
Product is released and
enzyme is free to catalyze
another reaction
What are
biofuels?
Fuels that are produced from a
biological source that was
recently living
• Biodiesel
• Syngas
• Ethanol from starches/sugars
• Cellulosic ethanol
Cellulosic
ethanol
production
A
B
C
D
Cellulose breakdown
1. Heat, acid,
ammonia or
other treatment
2. Enzyme
mixture added
Glucose
Endocellulases
Exocellulases
Cellobiase
Cellobiose
breakdowna closer look
4
1
4
6
5
3
+
Cellobiose + H2O
2
1
2 Glucose
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
Cellobiase
breakdown of pnitrophenyl
glucopyranoside
+
p-nitrophenyl glucopyranoside + H2O
glucose
+
p-nitrophenol
Basic
conditions
Clear
Yellow
Biofuels
Activity 1
Overview
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.
Measuring
Absorbance
Quantitatively
SmartSpec
Spectrophotometer
iMARK
Microplate reader
Biofuel Enzyme Kit
Procedure
Overview
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
Activities:
1. Reaction Rate & Std curve
2. Effect of Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of Substrate
Concentration
6. Bio-prospecting for
Celliobiase
S1
0
0
S2
12.5
0.2
S3
25
0.4
S4
50
0.8
S5
100
1.6
410 nm
Standard Curve
1.8
Absorbance at 410 nm
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Absorbance
Standard
Amount of
p-nitrophenol
(nmol)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Amount of p -nitrophenol (nmol)
120
Standard Curve
1.8
Absorbance at 410 nm
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Amount of p -nitrophenol (nmol)
120
Reaction Rate with Enzyme
Amount of p -nitrophenol
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
100
80
60
40
20
0
0
2
4
6
8
10
Time (min)
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
Activity 2 : Effect of Temp on Reaction Rate
rate p-nitrophenol produced
(nmol/min)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
100
90
80
70
60
50
40
30
20
10
0
Expon.
0
10
20
Temperature (C)
30
40
Initial reaction rate =
Time (min)
•This is the amount of p-nitrophenol produced in
2 minutes
Effect of pH on Initial Reaction Rate
20
Rate of p -nitrophenol
produced (nmol/min)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Amount of p-nitrophenol
produced (nmol)
18
16
14
12
10
8
6
4
2
0
4
5
6
7
pH
8
9
Amount of pnitrophenol formed
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
High enzyme
concentration
Low enzyme
concentration
Time (minutes)
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
Amount of pnitrophenol formed
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
1.5 mM
substrate
[High]
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
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Where can we find things
that break down cellulose?
T
s
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Where can we find things
that break down cellulose?
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).
Celliobiase Bio-Prospecting in Mushrooms (inquiry)
BioFuel Enzyme Kit - Activity 6: Reaction Rate for Mushroom Extracts
200
Wood
degrading
mushrooms
Wood Ear
160
Golden Chanterelle
140
p-nitropehnol (nmol)
Root
associating
mushrooms
180
120
Porcini
100
Chicken of the Woods
80
60
Oyster
40
Button
20
0
0
1
2
3
4
5
6
7
8
9
-20
Time (min)
•Ecological niches of each mushroom correlates with
celliobiase activity.
•Dried mushrooms work just as well as fresh ones and are
available at many stores
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
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)
pH/Temperature surface
Absorbance values
0-1
1-2
2-3
8.6
6.3
pH
5
3.5
0
22
37
Tem perature (°C)
50
80
Debate use of
cellulosic
ethanol as a
fuel source
•Get your social
sciences teacher
involved with the
debate and/or
argument research
papers on Biofuels
•Engineering
infrastructure
changes
•Competition
with food crops
CO2