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

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. sherri_andrews@bio-rad.com Essy Levy, M.Sc. essy_levy@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

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

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

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 Oxidoreductases (transfer of electrons) Transferases (group-transfer reactions) Hydrolases (hydrolysis reactions or reverse condensation reactions) Lyases (adding a group to a double bond or removing of a group and adding a double bond) Isomerases (transfer of a group within a molecule to give a different isomer) Ligases (formation of C-C, C-S, C-O, C-N bonds)

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

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 Biodiesel - Product is released and enzyme is free to catalyze another reaction

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

Cellulosic ethanol production B C A- Plant material is collected. Preferred materials include fast growing poplar trees, switchgrasses, bagasse from sugar cane, corn stover. Mainly cell wall material – highest content of cellulose B – Plant products are processed using heat, mechanical means, acid, or ammonia to separate the cellulose and hemicellulose from lignin. Lignin inhibits further enzymatic reactions. Cellulose and hemicellulose can be broken down to create simple sugars. C- After removal of lignins, enzyme mixtures are added. Endocellulases cleave cellulose from the middle of chains. Exocellulases remove two-glucose units (cellobiose) from the ends of cellulose chains and cellobiase breaks apart cellobiose to glucose. End result is a predominantly glucose solution. D- Fermenting bacteria or yeast (think beer) are added to glucose solution to produce ethanol. Ethanol is further purified by distillation (removal of water) to produce fuel. Ethanol can also be added to gasoline to produce mixtures (E85 is 85% ethanol, 15% gasoline). D

1. Heat, acid, ammonia or other treatment Cellulose breakdown Glucose 1. Heat, acid, ammonia or other treatment Endocellulases Exocellulases 2. Enzyme mixture added Cellobiase Heat, acid, grinding (mechanical force), and/or ammonia used to break down plant material (switchgrasses, poplar trees, corn stover, sugar cane bagasse, etc). Also breaks up the hydrogen bonds between cellulose chains and within the cellulose chains. For ease of viewing, only showing one cellulose chain. Add enzymes to this. The endos work on internal beta 1-4 glucose bonds. Exos work on the reducing and non-reducing ends to cleave off cellobiose. When free cellobiose is present – cellobiase cleaves it to glucose. When all/most is glucose, can be fermented to ethanol

Cellobiose breakdown- a closer look + 4 1 PDB id: 1gnx Name: Hydrolase Title: B-glucosidase from streptomyces sp Structure: Beta-glucosidase. Chain: a, b. Engineered: yes Source: Streptomyces sp.. Organism_taxid: 1931. Expressed in: escherichia coli. Expression_system_taxid: 562 UniProt: Chains A, B: Q59976 (Q59976_STRSQ) Resolution: 1.68Å R-factor: 0.190 R-free: 0.210 Authors: A.Guasch,J.A.Perez-Pons,M.Vallmitjana,E.Querol,M.Coll Key ref: a.guasch et al. Beta-Glucosidase from Streptomyces. To be Published, . Date: 10-Oct-01 Release date: 17-Oct-02 Cellobiose + H2O 2 Glucose 6 4 5 2 1 3

p-nitrophenyl glucopyranoside • 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 Protocol Highlights: Using a colorimetric substrate to track reaction rate cellobiose p-nitrophenyl glucopyranoside

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

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.

Biofuel Enzyme Kit Procedure Overview

Prepare and run reactions

Example of Standards' Absorbance Readings Amount of p-nitrophenol (nmol) Absorbance 410 nm S1 S2 12.5 0.2 S3 25 0.4 S4 50 0.8 S5 100 1.6 Example of Standards' Absorbance Readings

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

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

Quantitative Determination of p-nitrophenol Amount

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

Conditions affecting reaction rate pH Temperature Substrate Concentration Enzyme Concentration

Effects of pH Prepare and run reactions

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

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

Effects of temperature No enzyme Effects of temperature High Heat Substrate is stored at 4C to prevent it from decomposing randomly due to heat. Decomposition products

Ways increasing temperature increases reaction rate ENERGY REACTION COORDINATE S P S* Eact Ways increasing temperature increases reaction rate A Heating adds energy to the substrate so that it can go over activation barrier to form product more quickly (too much can destroy substrate) Increases movement of the substrate and enzyme in solution so that there is a greater chance of them bumping into each other and binding B

Effect of substrate concentration on the reaction rate 1.5 mM substrate [High] Amount of p-nitrophenol formed (nmol) 0.25 mM substrate [Low] Time (minutes) Can do other concentrations of substrate (did make 3 mM in educator's prep) or also do different dilutions between 1.5 mM and 0.25 mM in order to do a full Michaelis-Menten analysis. Initially, the rate is controlled by how fast the enzyme can find and bind substrate. For the high concentration substrate – this is likely to happen quicker than for the lower concentration substrate. At very long times, there will be less and less product formed in each time unit. Each reaction can produce the same number of mols of product as amount of substrate put in the reaction so the high concentration substrate will produce 5 times more product than the low conc substrate. The final equilibrium is not affected assuming that each reaction has enough time to go to completion (ie 100% substrate going to 0% substrate and 100% product) only the speed at which this occurs changes. 1. Effect of substrate concentration on the initial rate 2. Final amount of product formed with varying substrate concentrations

Effect of enzyme concentration on the reaction rate Amount of p-nitrophenol formed (nmol) Time (minutes) High enzyme concentration Low enzyme concentration 1. The initial reaction rate is faster when there is a higher enzyme concentration 2. Given enough time, the same amount of product will be formed for both the high and low enzyme concentration reactions

Mushroom extract enzymatic analysis

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

Michaelis-Menten Analysis A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes.

Combined pH and Temperature Effects

Debate use of cellulosic ethanol as a fuel source CO2

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