Biofuel Enzyme Kit:

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Biofuel Enzyme Kit From Grass to Gas A study
of enzymes
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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
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How do enzymes work?Energy considerations
Substrate (S) Product (P)
S
ENERGY
Eact
S
P
REACTION COORDINATE
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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
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What are biofuels?
Fuels that are produced from a biological source
that was recently living

• Biodiesel
• Syngas
• Ethanol from starches/sugars
• Cellulosic ethanol

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Cellulosic ethanol production
A
B
C
D
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Cellobiase activity
Cellobiose
glucose
glucose (substrate) cellobiase
(enzyme) (products)
Unfortunately, we cant easily detect either the
substrate or the products of this reaction.
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Cellobiase also works on an artificial substrate
p-Nitrophenyl glucopyranoside
Glucose p-Nitrophenol (substrate)
cellobiase (enzyme)
(products)

• A strong base will denature the enzyme and stop
  the reaction.
• p-Nitrophenol turns yellow in the presence of a
  base.
• More yellow means more p-Nitrophenol.
• More p-Nitrophenol means more of the substrate
  was broken apart.

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Cellobiose breakdown- a closer look
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4
5
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1
3
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Cellobiase breakdown of p-nitrophenyl
glucopyranoside
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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
• OR
• The amount of yellow color in the reaction
  solution can be quantified by measuring the
  absorbance at 410 nm using a spectrophotometer.

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Combined pH and Temperature Effects

• You will be producing line graphs

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Lab protocol overview
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Biofuel Enzyme Kit Procedure Overview
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Prepare and run reactions
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Example of Standards' Absorbance Readings
Standard Amount of p-nitrophenol (nmol) Absorbance 410 nm
S1 0 0
S2 12.5 0.2
S3 25 0.4
S4 50 0.8
S5 100 1.6
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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

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Quantitative Determination of p-nitrophenol
AmountRead SamplesAnalyze 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

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Quantitative Determination of p-nitrophenol
Amount
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Calculating initial reaction rate with and
without an enzyme present
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Conditions affecting reaction rate

• pH
• Temperature
• Substrate Concentration
• Enzyme Concentration

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Effects of pHPrepare and run reactions
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Calculating initial reaction rate at different pH
values

• This is the amount of p-nitrophenol produced in 2
  minutes

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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

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Effects of temperature
No enzyme
High Heat
Decomposition products
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Ways increasing temperature increases reaction
rate
A
B
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Effect of substrate concentration on the reaction
rate
1.5 mM substrate High

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
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Effect of enzyme concentration on the reaction
rate
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
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Mushroom extract enzymatic analysis
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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

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Michaelis-Menten Analysis
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Combined pH and Temperature Effects
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Debate use of cellulosic ethanol as a fuel source
CO2
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• explorer.bio-rad.com?Support?Webinars