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 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

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

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

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

Cellulosic ethanol production A B C D

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

p-Nitrophenyl glucopyranoside Glucose + p-Nitrophenol (substrate) cellobiase (enzyme) (products) Cellobiase also works on an artificial substrate: 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.

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

Cellobiase breakdown of p- nitrophenyl glucopyranoside + p-nitrophenyl glucopyranoside + H 2 O 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 Standard Amount of p-nitrophenol (nmol) Absorbance 410 nm S100 S S S S

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 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

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 Initial reaction rate = Amount of p-nitrophenol produced (nmol) Time (min) 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 High Heat Decomposition products

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

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

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

Mushroom extract enzymatic analysis

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

Michaelis- Menten Analysis

Combined pH and Temperature Effects

Debate use of cellulosic ethanol as a fuel source CO 2

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