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

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

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

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

Biofuels Activity 1 Overview

How can this enzymatic reaction be easily quantified? Basic solution (STOP SOLUTION): - will develop color of any p-nitrophenol present - will stop the reaction Qualitative - Each reaction time point can be directly compared to a standard of known concentration of p-nitrophenol Quantitative- The amount of yellow color in the reaction solution can be quantified by measuring the absorbance at 410 nm using a spectrophotometer or microplate reader.

Measuring Absorbance Quantitatively SmartSpec Spectrophotometer iMARK Microplate reader

Biofuel Enzyme Kit Procedure Overview Activities: 1.Reaction Rate & Std curve 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase Collaborative approach : Each student group does activity 1 Student groups do one activity each from 2-5 Groups share data All groups do activity 6 and share data

Standard Amount of p-nitrophenol (nmol) Absorbance 410 nm S100 S S S S Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

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 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

Initial reaction rate = Amount of p-nitrophenol produced (nmol) Time (min) This is the amount of p-nitrophenol produced in 2 minutes 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

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 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

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 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

Where can we find things that break down cellulose? Inquiry – find your own source of celliobiase. Have students develop protocol for testing activity Art – document your source via photography or drawings in a Lab Notebook Technology – use GPS and mapping software to document sources 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

Where can we find things that break down cellulose? Inquiry – find your own source of celliobiase! Have students develop protocol for testing activity based upon activity 1. Mushrooms are a great source of celliobiase and where the biofuels industry gets most of its enzymes currently, but there are many other potential sources out there….test them! Art – document your source via photography or drawings in a Lab Notebook Technology – use GPS and mapping software to document sources. Use excel or Vernier LoggerPro to analyze data. (Excel protocol available upon request). 1.Std curve / Std Reaction Rate 2.Effect of Temperature 3.Effect of pH 4.Effect of Enzyme Concentration 5.Effect of Substrate Concentration 6.Bio-prospecting for Celliobiase

Celliobiase Bio-Prospecting in Mushrooms (inquiry) Ecological niches of each mushroom correlates with celliobiase activity. Dried mushrooms work just as well as fresh ones and are available at many stores Root associating mushrooms Wood degrading mushrooms

Student Inquiry: A Stepwise Protocol approach Questions to consider: –How important is each step in the lab protocol? –What part of the protocol can I manipulate to see a change in the results? Possible variables: ratio of enzyme to substrate, look at more temperatures – can you get failure at a high enough temperature?, look at more pH points – at what low pH does failure occur? –How do I insure the changes I make is what actually affected the out come? (Controls) –Write the protocol. After approval – do it

Student Inquiry More Advanced Questions –How can I estimate the concentration of my novel celliobiase from activity 6? –Can I predict the activity of my novel celliobiase based upon the environment/organism I’m getting it from? –How does my novel celliobiase act under different pH and temperatures? –What is the optimal pH/temperature combination for my celliobiase? (Surface plots)

Debate use of cellulosic ethanol as a fuel source CO 2 Get your social sciences teacher involved with the debate and/or argument research papers on Biofuels Engineering infrastructure changes Competition with food crops