Biofuel Enzyme Kit From Grass to Gas: An Inquiry based study of enzymes

Biofuel Enzyme KitFrom Grass to Gas:

An Inquiry based study of enzymes

Biofuel Enzyme Kit

Instructors

Stan HitomiCoordinator – Math & SciencePrincipal – Alamo SchoolSan Ramon Valley Unified School DistrictDanville, CA

Kirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High School and Delta College, Tracy, CA

Bio-Rad Curriculum and Training Specialists:Sherri Andrews, Ph.D.

[email protected]

Damon [email protected]

Leigh Brown, M.A. [email protected]

Biofuel Enzyme KitWorkshop 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

Science

Technology

EngineeringMath

Inquiry

• 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

Biofuel Enzyme Kit Advantages

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

Exocellulases

Endocellulases

Glucose

1. Heat, acid, ammonia or other

treatment2. Enzyme

mixture added

Cellulose breakdown

+

Cellobiose breakdown- a closer look

Cellobiose + H2O 2 Glucose

4

1

56

4 23

1

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 p-nitrophenyl glucopyranoside

+

p-nitrophenyl glucopyranoside + H2O glucose + p-nitrophenol

Basic conditions

Clear Yellow

BiofuelsActivity 1Overview

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

SmartSpecSpectrophotometer

iMARK Microplate reader

Biofuel Enzyme Kit Procedure Overview

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

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

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

1. Std curve / Std Reaction Rate


3. Effect of pH4. Effect of Enzyme

Concentration5. Effect of Substrate

Concentration6. Bio-prospecting for

Celliobiase

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






Celliobiase

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)






Celliobiase

Activity 2 : Effect of Temp on Reaction Rate

0102030405060708090

100

0 10 20 30 40

Temperature (C)

rate

p-n

itro

phen

ol p

rodu

ced

(nm

ol/m

in) Expon.






Celliobiase

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

)






Celliobiase

Am

oun

t of p-

nit

rophenol fo

rmed

(n

mol)

Time (minutes)

1. The initial reaction rate is faster when there is a higher enzyme concentration

High enzyme concentration

Low enzyme concentration

2. Given enough time, the same amount of product will be formed for both the high and low enzyme concentration reactions






Celliobiase

Am

oun

t of p-n

itro

phenol

form

ed (

nm

ol)

Time (minutes)

0.25 mM substrate

[Low]

1.5 mM substrate

[High]

1. Effect of substrate concentration on the initial rate

2. Final amount of product formed with varying substrate concentrations






Celliobiase

Where can we find things that break down cellulose?

Inquiry – find your own source of celliobiase.

Have students develop protocol for testing

activity

Art – document your source via photography

or drawings in a Lab Notebook

Technology – use GPS and mapping software to document

sources






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

Celliobiase Bio-Prospecting in Mushrooms (inquiry)

BioFuel Enzyme Kit - Activity 6: Reaction Rate for Mushroom Extracts

-20

0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5 6 7 8 9

Time (min)

p-ni

trop

ehno

l (nm

ol)

Wood Ear

Golden Chanterelle

Porcini

Chicken of the Woods

Oyster

Button

•Ecological niches of each mushroom correlates with celliobiase activity.

•Dried mushrooms work just as well as fresh ones and are available at many stores

Root associating mushrooms

Wood degrading

mushrooms

StudentInquiry: 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

StudentInquiry

• 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)

0 22 37 50 803.5

5

6.3

8.6

Temperature (°C)

pH

pH/Temperature surfaceAbsorbance values

0-1 1-2 2-3

Debate use of cellulosic ethanol as a fuel source

CO2

•Get your social sciences teacher involved with the debate and/or argument research papers on Biofuels

•Engineering infrastructure changes

•Competition with food crops

Documents

Biofuel Enzyme Kit From Grass to Gas: An Inquiry based study of enzymes