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

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. S* enz. E act. Substrate (S) Product (P).

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

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

  2. 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

  3. Enzyme S*enz Eact Substrate (S)Product (P) How do enzymes work?Energy considerations S* ENERGY Eact S P REACTION COORDINATE

  4. 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

  5. What are biofuels? Fuels that are produced from a biological source that was recently living • Biodiesel • Syngas • Ethanol from starches/sugars • Cellulosic ethanol

  6. Cellulosic ethanol production A B C D

  7. Cellobiase activity: Cellobiose glucose + glucose (substrate) cellobiase (enzyme) (products) Unfortunately, we can’t easily detect either the substrate or the products of this reaction.

  8. 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.

  9. 4 1 Cellobiose + H2O 2 Glucose + Cellobiose breakdown- a closer look 6 4 5 2 1 3

  10. p-nitrophenyl glucopyranoside + H2O glucose + p-nitrophenol + Basic conditions Clear Yellow Cellobiase breakdown of p-nitrophenyl glucopyranoside

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

  12. Biofuel Enzyme KitProcedure Overview

  13. Prepare and run reactions

  14. Example of Standards' Absorbance Readings

  15. 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

  16. 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

  17. Quantitative Determination of p-nitrophenol Amount

  18. Amount of p-nitrophenol produced (nmol) Initial reaction rate = Time (min) 50 nmol - 0 nmol Initial reaction rate = = 12.5 nmol/min 4 min - 0 min Calculating initial reaction rate with and without an enzyme present

  19. Conditions affecting reaction rate • pH • Temperature • Substrate Concentration • Enzyme Concentration

  20. Effects of pHPrepare and run reactions

  21. Amount of p-nitrophenol produced (nmol) Initial reaction rate = Time (min) Calculating initial reaction rate at different pH values • This is the amount of p-nitrophenol produced in 2 minutes

  22. 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

  23. 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

  24. Debate use of cellulosic ethanol as a fuel source CO2

  25. 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 explorer.bio-rad.comSupportWebinars

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