Biofuel Prospecting and Assaying in the Lab!
Bio-Rad Biotechnology Explorer™ Biofuel Enzyme Kit
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Curriculum and Training Specialists
Damon Tighe
Tamica Stubbs
damon_tighe@bio-rad.com
tamica_stubbs@bio-rad.com
Sherri Andrews, Ph.D.
sherri_andrews@bio-rad.com
Leigh Brown
leigh_brown@bio-rad.com
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Objectives:
▪ Define biofuel and explain the need for
biofuels in our world.
▪ Describe the general workflow of biofuel
production.
▪ Define bioprospecting.
▪ Investigate the productivity of an enzyme
(extracted from an organism) required for
biofuel production.
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Background - Enzymes
What are
enzymes?
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)
Molecules, usually
proteins, that speed up
the rate of a reaction by
decreasing the activation
energy required without
themselves being altered
or used up
(hydrolysis reactions)
(double bond
reactions)
(transfers to create a
new isomers)
(forms covalent bonds)
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Background – Enzymes and Energy
Considerations!
Substrate (S)
Energy
considerations
Enzyme
Product (P)
S*
Energetics and
Structure
Energetics
Free energy of reaction
Activation energy
Reactant - Transition
state - Product
E
N
E
R
G
Y
S*enz
Eact Eact
S
P
REACTION COORDINATE
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Enzyme Superlative: Most Important Enzyme
to Life’s Existence?????
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Enzyme Superlative: Most Important Enzyme
to Life’s Existence?????
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What are biofuels and why are they needed?
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In Review: Biofuels production
Cellulose breakdown
1. Heat, acid,
ammonia or
other treatment
2. Enzyme
mixture added
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Glucose
Endocellulases
Exocellulases
Cellobiase
Catalyst of Interest: Cellobiase
Cellobiose 1,4 ß glycosidic link
Cellobiase
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Glucose
Cellobiase
Glucose
Cellobiase
Where can we find these enzymes in nature?
Bio-prospecting
Mushrooms:
Release Cellobiases
form hyphae to
access nutrients in
plants.
Ruminants: House
bacteria (Bacteroides
succinogenes)
release Cellobiases
to access nutrients in
plants.
Termite Guts: Bacteria (Rs-D17) residing in the protozoa (trychonympha)
that reside in the gut release cellobiases to access nutrients in plants.
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Mushroom Challenge: Which species is better suited for
improving the efficiency of biofuel production?
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Catalyst of Interest: In the lab!
P-nitorphenyl glucopyranoside
Cellobiase
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P-nitorphenyl
Cellobiase
Glucose
Cellobiase
Mushroom Challenge: Which species is better suited for
improving the efficiency of biofuel production?
▪ What do I need to test potential source for enzymes?
– Small scale model for testing enzyme activity
– Sources of enzymes
– Quick and easy detection
Cellobiose
P-nitrophenyl glucopyranoside
Basic
conditions
Clear
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Yellow
Enzyme Challenge: Guess that mushroom!!!
▪ Each group will test an extract from a “mushroom”.
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Typical Extraction Protocol
1. Obtain mushroom sample
2. Add ~ 0.25 g of mushroom to
microcentrifuge tube containing 750 µl
extraction buffer (EXB)
3. crush with blunted pipette tip
4. Spin down extract in microcentrifuge to
separate mushroom particles from liquid
fraction or filter and put liquid fraction in
new centrifuge tube (~250 µl)
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Mushroom Challenge: Which species is better suited for
improving the efficiency of biofuel production?
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Mushroom Challenge: Standards for Quantitative
Measurments?
Have Fun with Testing!
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Absorbance
Standard
Amount of
p-nitrophenol
(nmol)
S1
0
0
S2
12.5
0.2
S3
25
0.4
S4
50
0.8
S5
100
1.6
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410 nm
Wrapping Things Up: Questions?
Discoveries?
Question!
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Question!