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

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Presentation on theme: "Biofuel Enzyme Kit: From Grass to Gas – A study of enzymes."— Presentation transcript:

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

3 Biofuel Enzyme Kit Instructors Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. Essy Levy, M.Sc. Leigh Brown, M.A.

4 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

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6 Aligns with AP Biology AP Lab 2 Can be run qualitatively or quantitatively Construct and use a standard curve Determine the effects on the reaction rate by changing: –pH –temperature –enzyme/substrate concentration Mushroom extract activity for independent study Extension for Michaelis-Menten analysis Biofuel Enzyme Kit Advantages

7 Biofuel Enzyme Kit Workshop Timeline Introduction Review of enzymes Run control reaction and enzyme reaction Measure absorbance values Determine effect of pH on reaction rate

8 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 ClassExample 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

9 How do enzymes work? Energy considerations Substrate (S) Product (P) ENERGYENERGY REACTION COORDINATE S P S* E act S* enz E act Enzyme

10 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

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

12 Cellulosic ethanol production A B C D

13 Cellobiase Exocellulases Endocellulases Glucose 1. Heat, acid, ammonia or other treatment 2. Enzyme mixture added Cellulose breakdown

14 + Cellobiose breakdown- a closer look Cellobiose + H 2 O 2 Glucose

15 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

16 Cellobiase breakdown of p- nitrophenyl glucopyranoside + p-nitrophenyl glucopyranoside + H 2 O glucose + p-nitrophenol Basic conditions Clear Yellow

17 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.

18 Biofuel Enzyme Kit Procedure Overview

19 Prepare and run reactions

20 Example of Standards' Absorbance Readings Standard Amount of p-nitrophenol (nmol) Absorbance 410 nm S100 S S S S

21 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

22 Quantitative Determination of p-nitrophenol Amount Read Samples Analyze 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

23 Quantitative Determination of p-nitrophenol Amount

24 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

25 Conditions affecting reaction rate pH Temperature Substrate Concentration Enzyme Concentration

26 Effects of pH Prepare and run reactions

27 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

28 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

29 Effects of temperature No enzyme High Heat Decomposition products

30 Ways increasing temperature increases reaction rate ENERGYENERGY REACTION COORDINATE S P S* E act A B

31 Effect of substrate concentration on the reaction rate Amount of p- nitrophenol formed (nmol) 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

32 Effect of enzyme concentration on the reaction rate Amount of p- nitrophenol formed (nmol) 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

33 Mushroom extract enzymatic analysis

34 Extensions Perform a complete Michaelis-Menten analysis and determine the V max and K m 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

35 Michaelis- Menten Analysis

36 Combined pH and Temperature Effects

37 Debate use of cellulosic ethanol as a fuel source CO 2

38 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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