Biofuel Enzyme Kit:
From Grass to Gas – A 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.
sherri_andrews@bio-rad.com
Essy Levy, M.Sc.
essy_levy@bio-rad.com
Leigh Brown, M.A.
leigh_brown@bio-rad.com
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
Biofuel Enzyme
Kit 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
Biofuel Enzyme
Kit
Workshop
Timeline
• Introduction
• Review of enzymes
• Run control reaction and enzyme
reaction
• Measure absorbance values
• Determine effect of pH on reaction rate
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
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.
Biofuel Enzyme Kit
Procedure Overview
Prepare and run reactions
Absorbance
Standard
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
Example of
Standards'
Absorbance
Readings
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
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
Quantitative
Determination of
p-nitrophenol
Amount
Read Samples
Analyze 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
Standard Curve
1.8
Absorbance at 410 nm
Quantitative
Determination
of p-nitrophenol
Amount
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)
Calculating
initial reaction
rate with and
without an
enzyme
present
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
Conditions
affecting
reaction rate
• pH
• Temperature
• Substrate Concentration
• Enzyme Concentration
Effects of pH
Prepare and
run reactions
Amount of p-nitrophenol
produced (nmol)
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)
Calculating
initial
reaction rate
at different
pH values
18
16
14
12
10
8
6
4
2
0
4
5
6
7
pH
8
9
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
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
Debate use of
cellulosic
ethanol as a
fuel source
CO2
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