Biofuel Enzyme Kit:

From Grass to Gas – A study of enzymes

Biofuel Enzyme
• Can be run qualitatively or quantitatively
Kit Advantages
• Construct and use a standard curve

• Determine the effects on the reaction rate

by changing:
– pH
– temperature
– enzyme/substrate concentration
What are Enzyme Class Example
enzymes? Oxidoreductase Firefly Luciferase – oxidizes
(transfer of electrons) luciferin to produce oxyluciferin
and light
Molecules, usually Transferase Hexokinase – transfers a
proteins, that speed (group-transfer
reactions)
phosphate group to glucose to
make glucose-6-phosphate
up the rate of a
reaction by Hydrolase Cellobiase – breaks down
decreasing the (hydrolysis reactions) cellobiose
activation energy
Lyase Histidine decarboxylase –
required without (double bond generates histimine from histidine
themselves being reactions)
altered or used up Isomerase Glucose-6-Phosphate isomerase –
(transfers to create a converts G-6-P to fructose-6-
new isomers) phosphate
Ligase DNA Ligase – covalently bonds two
(forms covalent bonds) pieces of DNA
 Enzyme
Substrate (S) Product (P)
How do
enzymes
work? S*

E
S*enz
Energy N
E Eact Eact
considerations R
G
Y
S

REACTION COORDINATE
How do Substrate free in
solution
enzymes
Substrate binds to a
work? 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
Cellulose breakdown Glucose
1. Heat, acid, Endocellulases
ammonia or
other treatment Exocellulases

2. Enzyme Cellobiase
mixture added
Cellobiose
breakdown-
a closer
look
4
1 +

Cellobiose + H2O 2 Glucose

6
4 5 2 1
3
 • Cellobiose and glucose are colorless when
dissolved
Protocol
Highlights: • Use of the artificial substrate p-nitrophenyl
glucopyranoside allows the reaction to be
Using a tracked by monitoring the appearance of
colorimetric yellow color
substrate to
track reaction
rate

cellobiose

p-nitrophenyl glucopyranoside
Cellobiase
breakdown of p-
nitrophenyl +
glucopyranoside

p-nitrophenyl glucopyranoside + H2O glucose + p-nitrophenol

p-nitrophenol is colorless to slightly

yellow at pH 5. However, under
basic conditions, the hydrogen ion of
the
hydroxyl group (OH– group) is
Basic removed, resulting in a negative
charge due to an extra pair of
conditions electrons on the remaining oxygen
group. This pair of electrons travels
along the nitrophenolate anion,
creating a resonance structure that
produces the yellow color.

Clear Yellow
How can this
enzymatic Basic solution (STOP SOLUTION):
reaction be - will develop color of any p-nitrophenol
easily present
quantified? - 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.
 Amount of Absorbance
Example of Standard
p-nitrophenol
(nmol)
410 nm
Standards'
Absorbance
S1 0 0

Readings
S2 12.5 0.2
S3 25 0.4
S4 50 0.8
S5 100 1.6

Standard Curve

1.8
Absorbance at 410 nm

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100 120

Amount of p -nitrophenol (nmol)

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
Quantitative
Determination of Standard Curve

p-nitrophenol 1.8
Amount

Absorbance at 410 nm
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100 120

Amount of p -nitrophenol (nmol)

Calculating
initial reaction
Reaction Rate with Enzyme

rate with and 100

Amount of p -nitrophenol
without an 80

enzyme
60

(nmol)
40
present 20

0
0 2 4 6 8 10
Time (min)

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
Conditions • pH
affecting
• Temperature
reaction rate
• Substrate Concentration

• Enzyme Concentration
Calculating Amount of p-nitrophenol
produced (nmol)
initial Initial reaction rate =

reaction rate
Time (min)

at different •This is the amount of p-nitrophenol produced in

pH values 2 minutes

Effect of pH on Initial Reaction Rate

20
18
Rate of p -nitrophenol
produced (nmol/min) 16
14
12
10
8
6
4
2
0
4 5 6 7 8 9

pH
Further • Effect of temperature on the reaction
rate
activities
• Effect of substrate concentration on the
included in reaction rate
the kit
• Effect of enzyme concentration on the
reaction rate

• Ability of a mushroom extract to

catalyze the breakdown of the
substrate