1. Biofuel Enzymes A Study of Enzyme Kinetics

2. Enzymes Speed up the rate of reactions Speed up the rate of reactions Generally proteins Generally proteins Have active site in cleft of protein where reactions take place Have active site in cleft of protein where reactions take place The charges and positions of the R groups (variable side chains) amino acids The charges and positions of the R groups (variable side chains) amino acids critical for activity of enzyme. critical for activity of enzyme. Active site is where reactants Active site is where reactants binds binds

3. Enzymes Enzymes position the substrate (reactants) in such a way as to lower the energy needed for the reaction to occur. Enzymes position the substrate (reactants) in such a way as to lower the energy needed for the reaction to occur.

4. Enzyme Factors Changes in salinity affect side chains in active site Changes in salinity affect side chains in active site Changes in pH affect side chains in active site Changes in pH affect side chains in active site Temperature affects the activity of an enzyme Temperature affects the activity of an enzyme Relative concentration of both substrates and enzymes affect the rate of reaction (Vmax). Relative concentration of both substrates and enzymes affect the rate of reaction (Vmax).

5. Cellulosic Ethanol Production Pretreat to remove non-cellulose biomolecules such as lignin Pretreat to remove non-cellulose biomolecules such as lignin Enzymatic Hydrolysis uses cellulases to produce six carbon sugars such as glucose Enzymatic Hydrolysis uses cellulases to produce six carbon sugars such as glucose Microbial fermentation converts sugars into ethanol. Microbial fermentation converts sugars into ethanol.

6. Cellulose vs Cellulase Cellulose makes up cell wall in plants Cellulose makes up cell wall in plants Cellulose in form of cellulose microfibrils and hemicellulose Cellulose in form of cellulose microfibrils and hemicellulose Cellulases break down cellulose into glucose Cellulases break down cellulose into glucose Animals do not make produce cellulases Animals do not make produce cellulases

7. Types of Cellulases Endocellulases – break down internal bonds within a long chain of glucose molecules Endocellulases – break down internal bonds within a long chain of glucose molecules Exocellulases – break the bonds on the ends of the cellulose to create two sugar cellobiose Exocellulases – break the bonds on the ends of the cellulose to create two sugar cellobiose Cellobiases- break the bonds in cellobiose to make two glucose sugars Cellobiases- break the bonds in cellobiose to make two glucose sugars

8. Studying Cellobiase Can’t quantitatively detect presence of glucose or absence of cellobiose Can’t quantitatively detect presence of glucose or absence of cellobiose Use artificial substrate of p-nitrophenyl glucopyranoside (PNPG) Use artificial substrate of p-nitrophenyl glucopyranoside (PNPG) When PNPG is cleaved by the cellobiase, it produces p-nitrophenol. If you add a base to p- nitrophenol, it will turn yellow. When PNPG is cleaved by the cellobiase, it produces p-nitrophenol. If you add a base to p- nitrophenol, it will turn yellow. The deepness of yellow color can be compared visually or by spectrophotometry to produce quantitative results The deepness of yellow color can be compared visually or by spectrophotometry to produce quantitative results

9. To study Enzyme Kinetics we will: 1. Determine the Rate of Reaction in the Presence or Absence of an Enzyme 2. Determine the Effect of Temperature on the Reaction Rate 3. Determine the Effect of pH on the Reaction Rate 4. Determine the Effect of Enzyme Concentration on the Reaction Rate 5. Determine the Effect of Substrate Concentration of Reaction Rate 6. Test the Ability of Mushroom Extracts to Increase the Reaction Rate