enzyme

• Fermentation


  1. Chemical breakdown of a substance (organic compound) by cells (bacteria, yeast etc.)

  2. Cells convert raw material into products of value

  3. Seed stock of cells put into growth media in shake flask

  4. Cell population grown in seed fermenter

  5. Cell population transferred to bioreactor with fresh media

  6. Fermentation conditions monitored (temperature, pressure, pH, oxygen, nutrients)

  
• Fermentation


  • Typical upstream process
  • Involves chemical breakdown of organic compounds by cells

  
• Bioreactor


  Vessel where cells continue to grow and manufacture desired product under well-monitored conditions
• Bioprocess flowsheet


  1. Phase 1: Choosing cell line for production

  2. Phase 2: Defining growth/kinetic activity of cellular system

  3. Phase 3: Bioreactor/fermenter design

  4. Phase 4: Downstream process to remove contaminants, isolate, purify and polish final bioproduct

  
• Enzymes


  • Largest class of proteins, over 2000 different kinds
  • Highly specific in function, have extraordinary catalytic power
• Advantages of using enzymes as catalysts


  • High specificity (fewer undesired side products)
  • Gentle reaction conditions (aqueous, ambient temperature)
  • Faster rate compared to non-biological catalysts
• Enzyme naming


  Add suffix -ase to substrate or reaction it catalyzes
• Common enzyme applications


  • Catalase in textile industry
  • Amylase in bread making
  • Xylanase in pulp treatment

  
• Commercial manufacture of high fructose corn syrup relies on 3 enzymes

  
• Enzyme active site


  Specific substrate is bound during catalysis
• Enzyme catalytic action


  Lowers activation energy by binding substrate and forming enzyme-substrate complex
• Michaelis-Menten model


  Mathematical model describing kinetics of single substrate enzyme-catalyzed reaction

  
• Michaelis-Menten reaction scheme


  1. Reversible enzyme-substrate complex formation

  2. Irreversible dissociation to yield product and regenerate enzyme
• Michaelis constant (Km)


  • Substrate concentration giving half-maximal reaction velocity
  • Measure of enzyme's affinity for substrate

  
• Maximum reaction rate (Vm)


  Changes with enzyme concentration, not substrate concentration

  
• Michaelis-Menten equation


  Rate of product formation = (Vm[S])/(Km + [S])
• Determining Michaelis-Menten parameters


  1. Measure [S] and rate v in batch reactor

  2. Plot 1/v vs 1/[S] (double reciprocal plot)

  3. Slope = Km/Vm, y-intercept = 1/Vm
• Km is an intrinsic parameter, Vm is not
• Plotting Michaelis-Menten data


  1. Plot hyperbolic curve of rate vs substrate concentration

  2. Plot double reciprocal (1/rate vs 1/substrate) to determine Km and Vm

  
• Enzyme inhibition


  • Certain compounds (inhibitors) bind to enzymes and reduce activity
  • Inhibition can be irreversible or reversible
• Competitive inhibition


  Inhibitor directly competes with substrate to bind active site
• Effect of competitive inhibition


  Maximum rate Vm is unchanged, but apparent Km (Km,app) increases
• Competitive inhibition can be linearized on double reciprocal plot
• Competitive inhibition


  1. Linearize rate equation

  2. Plot on double reciprocal plot
• Competitive inhibition


  • Maximum rate of reaction Vm is the same as uninhibited enzymatic reaction
  • Michaelis-Menten constant Km,app is larger than uninhibited reaction
• Km,app = Km[1 + [I]/KI]
• • Non-competitive inhibitors are not specifically substrate analogues
  • They may bind to the enzyme whether or not the substrate has already been bound
• Uncompetitive inhibitors bind to the ES complex only and have no affinity for the enzyme itself
• Derivations of enzyme kinetics for non-competitive and uncompetitive inhibition are not covered in this module and will not be assessed
• Solving a question on competitive inhibition

  Determine (i) value of KI (ii) maximum rate of reaction (iii) rate of reaction for initial substrate concentration of 0.001 M
• Microbial growth is an autocatalytic reaction
• Net specific growth rate


  μnet = 1/X * dX/dt = μg - kd
• Net specific replication rate


  μR = 1/N * dN/dt
• Batch growth


  1. Lag phase

  2. Exponential growth phase

  3. Deceleration phase

  4. Stationary phase

  5. Death phase
• Lag phase


  Period of adaptation of cells to a new environment
• Exponential growth phase


  Cells multiply rapidly, cell mass and number density increase exponentially, balanced growth
• Deceleration growth phase


  Growth decelerates due to depletion of nutrients or accumulation of toxic by-products, unbalanced growth
• Stationary phase


  Net growth rate is zero or growth rate equals death rate
• Death phase


  Rate of death follows first-order kinetics
• Calculating maximum net specific growth rate and yield coefficient YX/S

  Given data on batch growth of Penicillium chrysogenum