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Enzyme Optimization for Cell-Free Ethanol Production

Enzyme Optimization for Cell-Free Ethanol Production. Eric J. Allain Assistant Professor Dept. of Chemistry Appalachian State University. The Fuel Crisis. Ethanol as a fuel solution. Renewable Greenhouse gas neutral Works with existing systems. CO 2. Enzymes. Hammer mill. Jet Cooker.

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Enzyme Optimization for Cell-Free Ethanol Production

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  1. Enzyme Optimization for Cell-Free Ethanol Production Eric J. Allain Assistant Professor Dept. of Chemistry Appalachian State University

  2. The Fuel Crisis

  3. Ethanol as a fuel solution • Renewable • Greenhouse gas neutral • Works with existing systems

  4. CO2 Enzymes Hammer mill Jet Cooker Liquef action Slurry tank Whole corn Mash cooling Fermentation Fresh water & 4 recycled water sources Yeast 5% Gasoline Molecular sieves 200 proof Denatured ethanol 200 proof ethanol Beer 190 Proof Ethanol Drum dryer DDG Wet grain Three column distillation system Evaporators Syrup Thin stillage Whole Stillage Centrifuge Condensate Fuel Alcohol Production Process Flow

  5. Glucoamylase Hydrolysis of Dextrins to Glucose a-amylase a-Amylase Breakdown of Gelatinized Starch glucoamylase Amylose Amylopectin Glucose

  6. Yeast Uses the Glucose for Energy and Makes Ethanol as a Byproduct

  7. What about places that can’t grow corn? Wheat, barley, rye, etc. (starch containing plants) Sugar cane Cellulose – over half of the carbon in the biosphere!

  8. Glucose Ethanol Biomass Yeast Improving the economy of ethanol production Increase the rate of ethanol production The Cell-Free Ethanol Concept If yeast is the limiting factor then why not get rid of it and use only the enzymes needed to convert glucose to ethanol?

  9. Cell-Free Ethanol Production • An idea as old as biochemistry! • Viability no longer a concern • We can change enzyme levels to whatever we need them to be • Ethanol toxicity not a problem • Enzyme engineering to tailor enzymes for max ethanol production • Greater process flexibility (ie higher temperature) • Higher yield (no carbon used for yeast growth)

  10. kcat [Enz] [S] V = KM + [S] ATP ATPase ADP Modeling Ethanol Glycolysis 50 mM Glc ATP HK ADP G6P PGI F6P ATP PFK Teusink et al Eur. J. Biochem.267, 5313-5329 (2000) ADP F16BP Ald Pi DHAP GAP TPI NAD+ GAPDH NADH BPG ADP PGK ATP 3PG PGM 2PG Eno PEP ADP PK ATP Pyr PDC AcAl NADH ADH NAD+ EtOH 50 mM

  11. Ethanol production rate = 2.2 mM/min

  12. 1.7 mM/min Model Predicted Steady State Ethanol Production Rate = 8.9 mM/min Cellular enzyme level = 13035 mg /L of Cytoplasm For consideration of Cell-Free Ethanol Production use total enzyme level of 2742 mg/L 0.2 L cytoplasm / L Fermentation Adjusted Rate = 1.78 mM/min Can we adjust these enzyme levels to increase rate of ethanol production?

  13. Glc ATP HK ADP G6P PGI F6P ATP PFK ADP F16BP Ald Pi DHAP GAP TPI NAD+ GAPDH Dln Flux NADH BPG = Control Coefficient ADP Dln [Enz] PGK ATP 3PG PGM 2PG Eno PEP ADP ATP PK ATPase ATP Pyr ADP PDC AcAl NADH ADH NAD+ EtOH Optimization of Enzyme Levels for Cell Free Ethanol Production • Find steady state flux • Increase the level of one enzyme by a small amount • Find new steady state flux Use metabolic control analysis for optimization • Enzymes with high control coefficients are exerting a greater amount of control on the flux through the pathway

  14. Optimization Algorithm Calculate control coefficients for each enzyme Raise the level of each enzyme by 10mg/L x the control coefficient for that enzyme Rescale so total enzyme level is still 2742 mg/L Initial rate = 1.86 mM/min After 1st iteration rate = 1.88 mM/min Calculate new rate of ethanol production

  15. Optimized Rate of Ethanol Production = 3.66 mM/min

  16. Is the optimum a true optimum?

  17. Other possibilities Enzyme engineering (PFK deregulation) Using the ATP Current Focus Validation of model predictions in the lab Recycling the enzyme ideas • Immobilization • Altering the process – high temperature reaction

  18. Acknowledgements Collaborators: Dr. Eric Marland Dr. Rene Salinas Allain Lab: Kristi Bilotti Diana Dardugno Andrew Madison Brittany Overfield Laurin Robertson Benjamin Shepperd Russell Vegh Patrick Williams Funding: ASU URC

  19. Funding…

  20. Converting Biomass to Ethanol Corn 72% Starch 10% Cellulose/Hemicellulose 9% Protein 4.5% Oil 3.5% Other Yield = 114 gallons ethanol/dry ton = 18 lbs. corn grain/gallon Acid pretreated corn stover 56% Cellulose 5% Hemicellulose 28% Lignin 13% Other Corn stover 38% Cellulose 32% Hemicellulose 17% Lignin 13% Other Yield = 72 gallons ethanol/dry ton = 30 lbs. corn stover/gallon

  21. Converting Biomass to Ethanol Corn 72% Starch 10% Cellulose/Hemicellulose 9% Protein 4.5% Oil 3.5% Other Yield = 114 gallons ethanol/dry ton = 18 lbs. corn grain/gallon Enzyme usage ~ 1 g protein/gallon Acid pretreated corn stover 56% Cellulose 5% Hemicellulose 28% Lignin 13% Other Corn stover 38% Cellulose 32% Hemicellulose 17% Lignin 13% Other Yield = 72 gallons ethanol/dry ton = 30 lbs. corn stover/gallon Enzyme usage ~100 g protein/gallon

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