Chapter V Enzyme

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Chapter V Enzyme
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Enzyme
Active protein acts as a biological catalyst.
Since it is a protein, enzyme consists of amino
acids. The molecular weight ranges from 15000 to
millions Dalton The enzyme reduces the activation
energy for the reaction in order to increase the
reaction kinetic constant (k). Arrhenius
equation
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Enzyme classification

1. Oxidoreductase transfer oxygen atoms or electron
2. Transferase transfer a group (amine, phosphate,
   aldehyde, oxo, sulphur, etc)
3. Hydrolase hydrolysis
4. Lyase transfer non-hydrolytic group from
   substrate
5. Isomerase isomerazion reactions
6. Ligase bonds synthesis, using energy from ATPs

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The example
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How do enzymes work?
Lock and key principle
?
? Substrate
enzyme substrate-enzyme
product enzyme
complex Many
enzymes need cofactor (apoenzymes) such as metal
ions or several organic compounds. However,
several metal ions and organics might inhibit
enzyme kinetics. For example, heavy metals at
high dose, EDTA, glucose, etc
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The mechanism of enzymatic reactions

• The simple schematic of enzymatic reaction
• S E ES E
  P
• Assumptions
• The enzyme total concentration is constant
• The amount of enzyme ltltlt substrate
• The product concentration is low
• 
  (1st order reaction)

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Michaelis-Menten kinetic

• The approaches
• Rapid equilibrium
• Quasy steady state
• V rate of enzymatic reaction
• Vm maximum rate of enzymatic reaction
• CS substrate concentration
• Km substrate affinity constant (Michaelis-Menten
  constant)

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How to obtain the kinetic parameter (Vm, Km)?

• Construct and obtain V at different CS. Use
  graphical methods
• Lineweaver-Burk plot (double reciprocal plot)
• Eadie-Hofstee plot
• Hanes-Woolf plot
• Batch kinetic plot
• Each plot has its own pros and cons. However, the
  most popular is double reciprocal plot.

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Allosteric enzyme
Enzyme with cooperative binding that has more
than one active site. Mostly is regulatory
enzyme.
n cooperative
coefficient
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The inhibitions on enzymatic reactions

• Inhibitors reduce enzyme activity to substrate.
  Four type of inhibitions
• Competitive inhibition
• Non competitive inhibition
• Uncompetitive inhibition
• Substrate inhibition
• Beside that, the temperature and pH control the
  enzymatic kinetics.
• The pH determines the changes in the ionic form
  of the enzymes active site and changes the
  enzyme activity. High T may give high rate of
  reaction, but enzyme thermal denaturation occurs.

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Immobilized enzymes

• The restriction of enzyme mobility in a fixed
  space
• Advantages
• Enzyme reutilization
• Elimination of enzyme recovery and purification
• May provide better environment for enzyme
  activity
• Disadvantages
• Problem in mass transfer
• Enzyme leakage into solution
• Reduced enzyme activity and stability
• Lack of controls on micro environmental
  conditions

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Methods of immobilization
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Chapter VIGrowth of microorganisms
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Batch culture

• Batch growth consists of
• Lag phase
• Acceleration phase
• Exponential growth phase
• Deceleration phase
• Stationary phase
• Death phase

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Lag phase
The lag phase occurs immediately after
innoculation and is a period of adaption of cells
to a new environment. Microorganisms reorganize
their molecular constituents when they are
transferred to a new medium. The internal
machinery of cells is adapted to the new
environmental conditions. During this phase,
cells mass may increase a little, without an
increase in cell number density. Pseudo-lag phase
may occur if the inoculum is small or poor
inoculum conditions. The long lag phase is
possible if the nutrient and growth factors
concentrations are low. Additionally, the lag
period increases with the age of inoculum. The
multiple lag phase may be observed when the
medium contains more than one carbon source ?
diauxic growth
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Log or exponential phase
In this phase, the cells have adjusted to the new
environment. Cell mass and cell number density
increase exponentially with time. This is the
period of balance growth in which all components
of cells grow at the same rate. The exponential
growth rate is first order
, CX cell concentration at
t t , ? specific growth rate CX0 cell
concentration at t 0 At this phase, mostly ?
?max.
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Stationary phase

• This phase starts at the end of deceleration
  phase, when the net growth rate is zero or when
  the growth rate is equal to the death rate. The
  cells are still metabolically active and produce
  secondary metabolites (nongrowth-related). During
  this phase
• Total cell mass concentration may stay constant,
  but the number of viable cells may decrease
• Cell lysis may occur, viable cell mass may drop
  ? causing cryptic growth (growth on lysis
  products of lysed cells)
• Secondary metabolite (some hormones,
  antibiotics) may be produced as a result of
  metabolite deregulation

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Stationary phase
The endogenous metabolism occurs where cells
catabolizes cellular reserves for new building
blocks and for energy-producing monomers. The
cells must always expend energy to maintain an
energized membrane (pmf), transport of nutrients,
and for essential metabolic functions such as
motility and repairing. This energy expenditure
is called maintenance energy. The reason for
termination of growth may be either exhaustion of
an essential nutrient or accumulation of toxic
products.
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Death phase
The death phase follows the stationary phase.
However, some cell death may start during
stationary phase, and a clear demarcation between
these two phases is not always possible. The rate
of death usually follows first-order kinetics

, NS concentration of
cells at the end of stationary phase N
concentration of cells at t time after stationary
phase kd 1st order death rate constant
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Monod kinetics

• Assumptions
• Only single chemical species is growth rate
  limiting
• No inhibitions either by substrates or products
• The maintenance coefficient is negligible
• ? specific growth rate, ?max maximum specific
  growth rate
• KS saturation constant, CS substrate
  concentration

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Monod kinetics
The Monod kinetic is similar to Michaelis-Menten
kinetic The double reciprocal plot (1/? vs. 1/CS)
is also used to determine KS and ?max. However,
usually ?max is obtained by plot CX vs.t.
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Other kinetics

• Several more complicated kinetics are available,
  e.q
• Blackman equation
• Tessier equation
• Moser equation
• Contois equation
• Multiple substrate equations
• Model with growth inhibitors
• Logistic equation, etc

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Rates yields
Specific rate (qi) ri rate of production/
consumption of component i Thus,
Yield is defined as the ration of production of
compound i to the consumption of the compound j
(Yji). Some literatures use Yi/j.
grams of cells produced /grams of substrate
consumed
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Maintenance coefficient
Cells consume substrate for growth, production,
and maintenance. Some part of substrate is used
for maintenance such as maintain an energized
membrane (pmf), transport of nutrients, and for
essential metabolic functions such as motility
and repairing.
The maintenance coefficient is independent to ?.
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Continuous culture in chemostat
The continuous culture provide constant
environment to cells in order to obtain prolonged
periods of growth and product formation. At
certain time, the system usually reaches steady
state where cells, products, and substrates
concentrations are constant.
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Chemostat dynamics
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The mass balances in chemostat
The cells mass balance
D dilution rate (hour-1) The substrate mass
balance Usually ? 1
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The mass balances in chemostat
The cells mass balance explains that the specific
growth rate is controlled by defining the feed
flow rate. Integrating with Monod equation
CS residual substrate concentration The biomass
concentration in gram dry weight/liter
Cso inlet substrate concentration Cxo 0
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Critical dilution rate
The dilution rate has the critical value, Dcrit ?
?max. It is slightly lower than ?max. If D gt
Dcrit, cell is washed out, CX 0. Then, the
substrate is not consumed, CS Cso.
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Critical dilution rate
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Comments on batch process

1. ?, YSX, qS are constant at ?max, YSXmax, qSmax
   (during log phase)
2. Microbial model parameters are easily determined
   by combining the exponential equation with CS and
   CX data vs. time.
3. ?, the most important rate, can not be controlled
   in batch culture

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Comments on continuous process

• All rates are defined. D ? is defined, thus
  qS, YSX, qP, qO2, etc are set.
• Residual substrate concentration is independent
  to CSo.
• A good experimental tool to study and apply
• Kinetics, to obtain YSXmax, mS, qSmax, KS.
• Physiological of microorganisms under defined
  steady state by changing substrate or electron
  acceptor or type of medium limitation
• Waste water treatment
• Industrial fermentation (not widely applied)
• Genetic and metabolic studies

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The comparison between batch and continuous
culture
Aspect batch chemostat
All rates at maximum during log phase, undefined defined and set
Tool for research moderate excellent
Fermenter down-time occurred avoided
Risk of contamination low moderate to high
Product and biomass concentration moderate to high low
Downstream processing moderate to easy difficult
Microbial selection for non producing mutant Not occur frequently occur
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Growth monitoring methods

• Can be direct or indirect
• Petroff-Hauser counting chamber. Rapid but cannot
  distinguish live and dead cells. Needs a high
  cell concentration
• Electronic cell counters. Use electrical
  resistance principle. Errors can occur due to
  cell clumping and the presence of particulate
  debris
• Plate counting technique. Counting cell colony,
  which is difficult
• Turbidity and spectrophotometric techniques.
  Measure the light scattered or absorbed by the
  cells. Most popular method, rapid, but not good
  for colored culture
• Dry weight estimation. Uses vacuum filtration and
  oven to obtain dry cells. Other suspended
  non-cellular material could give error
• On line estimation. Measuring O2, CO2 to estimate
  biomass concentration
• ATP bioluminometry. Measure cell concentration by
  measuring ATP because ATP is rapidly lost from
  dead cells.