Biofuel Prospecting and Assaying in the Lab!

Transcription

1 Biofuel Prospecting and Assaying in the Lab! Bio-Rad Biotechnology Explorer Biofuel Enzyme Kit 1

2 Curriculum and Training Specialists Damon Tighe Tamica Stubbs Sherri Andrews, Ph.D. Leigh Brown 2

3 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. 3

4 Background - 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 Oxidoreductase (transfer of electrons) Transferase (group-transfer reactions) Hydrolase (hydrolysis reactions) Lyase (double bond reactions) Isomerase (transfers to create a new isomers) Ligase (forms covalent bonds) Example Firefly Luciferase oxidizes luciferin to produce oxyluciferin and light Hexokinase transfers a phosphate group to glucose to make glucose-6-phosphate Cellobiase breaks down cellobiose Histidine decarboxylase generates histimine from histidine Glucose-6-Phosphate isomerase converts G-6-P to fructose-6- phosphate DNA Ligase covalently bonds two pieces of DNA 4

5 Background Enzymes and Energy Considerations! Energy considerations Substrate (S) Enzyme Product (P) Energetics and Structure Energetics Free energy of reaction Activation energy Reactant - Transition state - Product E N E R G Y S S* S* enz E act E act P 5 REACTION COORDINATE

6 Enzyme Superlative: Most Important Enzyme to Life s Existence????? 6

7 Enzyme Superlative: Most Important Enzyme to Life s Existence????? 7

8 What are biofuels and why are they needed? 8

9 In Review: Biofuels production Cellulose breakdown 1. Heat, acid, ammonia or other treatment 2. Enzyme mixture added Glucose Endocellulases Exocellulases Cellobiase 9

10 Catalyst of Interest: Cellobiase Cellobiose 1,4 ß glycosidic link Glucose Glucose Cellobiase Cellobiase Cellobiase 10

11 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. 11

12 Mushroom Challenge: Which species is better suited for improving the efficiency of biofuel production? 12

13 Catalyst of Interest: In the lab! P-nitorphenyl glucopyranoside P-nitorphenyl Glucose Cellobiase Cellobiase Cellobiase 13

14 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 Yellow 14

15 Enzyme Challenge: Guess that mushroom!!! Each group will test an extract from a mushroom. 15

16 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) 16

17 Mushroom Challenge: Which species is better suited for improving the efficiency of biofuel production? 17

18 Mushroom Challenge: Standards for Quantitative Measurments? Have Fun with Testing! Standard Amount of p-nitrophenol (nmol) Absorbance 410 nm S1 0 0 S S S S

19 Wrapping Things Up: Questions? Discoveries? Question! Question! 19