Biofuel Enzyme Kit: From Grass to Gas – A study of enzymes

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

How do enzymes work?

Energy considerations

Substrate (S) Product (P)

ENERGY

REACTION COORDINATE

S

P

S*

Eact

S*enz

Eact

Enzyme

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

What are biofuels?

• Biodiesel

• Syngas

• Ethanol from starches/sugars

• Cellulosic ethanol

Fuels that are produced from a biological source that was recently living

Cellulosic ethanol production

A

B

C

D

Cellobiase activity:

Cellobiose glucose + glucose(substrate) cellobiase (enzyme) (products)

Unfortunately, we can’t easily detect either the substrate or the products of this reaction.

p-Nitrophenyl glucopyranoside Glucose + p-Nitrophenol(substrate) cellobiase (enzyme) (products)

Cellobiase also works on an artificial substrate:

•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.

+

Cellobiose breakdown- a closer look

Cellobiose + H2O 2 Glucose

4

1

56

4 23

1

Cellobiase breakdown of p-nitrophenyl 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

OR

• The amount of yellow color in the reaction solution can be quantified by measuring the absorbance at 410 nm using a spectrophotometer.

Lab protocol overview

Biofuel Enzyme Kit Procedure Overview

Prepare and run reactions

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

Standard Curve

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 20 40 60 80 100 120

Amount of p -nitrophenol (nmol)

Ab

sorb

ance

at

410

nm

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 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

Quantitative Determination of p-nitrophenol Amount

Standard Curve

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 20 40 60 80 100 120

Amount of p -nitrophenol (nmol)

Ab

sorb

ance

at

410

nm

Calculating initial reaction rate with and without an enzyme present

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

Reaction Rate with Enzyme

0

20

40

60

80

100

0 2 4 6 8 10

Time (min)

Am

ou

nt

of p

-nit

rop

he

no

l (n

mo

l)

Conditions affecting reaction rate

• pH

• Temperature

• Substrate Concentration

• Enzyme Concentration

Effects of pH

Prepare and run reactions

Calculating initial reaction rate at different pH values

Initial reaction rate =

Amount of p-nitrophenol produced (nmol)

Time (min)

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

Effect of pH on Initial Reaction Rate

0

2

4

6

8

10

12

14

16

18

20

4 5 6 7 8 9

pH

Rat

e o

f p

-nit

rop

hen

ol

pro

du

ced

(n

mo

l/min

)

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

Webinars • Enzyme Kinetics — A Biofuels Case Study

• Real-Time PCR — What You Need To Know and Why You Should Teach It!

• Proteins — Where DNA Takes on Form and Function

• From plants to sequence: a six week college biology lab course

• From singleplex to multiplex: making the most out of your realtime experiments

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