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Enzymes

Enzymes. Enzymes. What are enzymes? How do enzymes work (kinetics) How are enzymes used in reactors. Enzymes. Enzymes: Proteins with rare exceptions Catalysts for a variety of reactions Most common - hydrolysis – breakdown of chemical bond with addition of water

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Enzymes

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  1. Enzymes

  2. Enzymes • What are enzymes? • How do enzymes work (kinetics) • How are enzymes used in reactors

  3. Enzymes • Enzymes: • Proteins with rare exceptions • Catalysts for a variety of reactions • Most common - hydrolysis – breakdown of chemical bond with addition of water • Carboxylases “cut” C – 0 – C bond in sugar polymers • Proteases (aka Proteinases, Peptidases) cut N- C=O (amide) bond • Isomerases rearrange bonds in various molecules • xylose isomerase = glucose isomerase converts glucose to fructose • Various other types, kinases, decarboxylases etc.

  4. Industrially Important Corn milling (starch to sugar)

  5. Enzyme Visualization Penicillin Acylase 1FXV

  6. Enzyme with substrate Penicillin Acylase 1FXV

  7. ES complex S Reaction/hydrolysis Binding Product release Enzyme regeneration EP complex E General Enzymatic Catalysis

  8. Enzyme Assays • Enzymes are detected by their “activity” – the ability to catalyze a particular reaction • Often “artificial” or natural substrates which change color (absorbance) after the reaction are used to measure enzyme activity • The rate of an enzymatic reaction is proportional to the amount of enzyme present.

  9. Enzyme Activity 2 x enzyme Absorbance 1 x enzyme time

  10. Activity Relative rate of absorbance change Enzyme conc

  11. Specific Activity • The specific activity is the rate of change in absorbance per g of enzyme.

  12. Specific activity • 10 units/mg of protein • 1 unit hydrolyzes 1 umole of penicillin per minute at the conditions specified. • The specific activity is 10 umole of penicillin per minute per mg.

  13. Turnover # • 1 mg of protein is about 1.5 x 10-2 umole of protein (MW = 75000 daltons) • Turnover is 10 umole/min/1.5 x 10-2 umole = 6 x 102 per minute = 10/second. • For each molecule of enzyme 10 molecules of penicillin are reacted per second. • (typical turnovers range from 1 to 1000/sec)

  14. Michaelis-Menten Kinetics s-1 k1 (second order) k3 = kcat (first order) mM-1s-1 E + S ES EP E + P1 + P2 The reverse reactions are usually considered slow. k2 (first order) s-1 Rate of reaction = rate of appearance of product(s) = rate of disappearance of substrate units moles/min-L. Total (all forms) enzyme concentration mM Substrate conc mM mM

  15. kcatEo = Vmax Rate of reaction (mM/s) Km Substrate available (mM) Rate vs S

  16. M-M kinetics • Features • At high substrate concentration the rate of reaction becomes zero order. kapp = kcatEo (mM/s) • At low substrate concentration the rate of reaction becomes 1st order. kapp = kcatEo/Km (1/s) • Rate is proportional to the amount of enzyme used (Eo) • Values for kcat range widely, from 0 to 1000 s-1. • Km values from 10-3 mM to 10 mM.

  17. Substrate vs Time in Batch Reactor Starts out zero order Substrate Ends up first order time

  18. Reversible Reactions(Isomerizations) E + S ES EP E + P Equilibrium favors P in the case shown P curve S or P S curve Time (s)

  19. Inhibitors E + S ES E + P + I EI An inhibitor binds to the enzyme and reduces the amount of enzyme available ES complex S E EP complex EI complex

  20. Enzyme Deactivation • Proteins can deactivate with use • Unfold, oxidize, hydrolyze. • The reaction rate will slow because: Eo = Eoo exp (-kd t) Deactivation rate constant. Active enzyme at time t. Original active enzyme

  21. Continuous Reactors Plug Flow vs CSTR zero order regime (high substrate conc) low conversion –only one rate Rate puv is the same as in PFR. Rate puv is the same along reactor length. Volume of PFR is same as CSTR

  22. Continuous Reactors Plug Flow vs CSTR First order regime (low substrate conc) High conversion – rate depends on S conc Rate puv is rate for final (exit) concentra- tion. Rate puv starts out high gradually drops along reactor length. Volume of PFR is smaller than CSTR

  23. Approximating a PFR Multiple CSTR’s in series approximate a PFR (Easier to build); 3 – 5 usually close enough with similar total volume as PFR Concentration reduced gradually

  24. Immobilized Enzymes • Enzymes coupled to a solid surface or entrapped in a solid • Stay separate from the fluid being processed • Can be reused • Work well in a packed bed reactor

  25. Immobilized Enzyme Reactor Enzyme is physically tethered or physically entrapped in the solid matrix. The matrix may be (cross linked) agarose, polyacrylamide, dextran, cellulose (biocompatible)

  26. Problems/Limitations when Enzymes are Immobilized • Diffusion hinders substrate access to • enzyme (Theile modulus) 3. Not feasible with insoluble substrates. 2. pH may be different inside vs on the surface of the particle

  27. Summary • Enzymes are proteins that act as catalysts • The rate expression most often follows Michaelis-Menten kinetics (saturation kinetics) • Enzymes can be used as soluble catalysts in batch, CSTR and plug flow reactors. • Enzymes can be used in immobilized form – often in packed bed reactors.

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