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    Biochemical Engineering: Week 10

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    Emmanuel
    This document discusses factors that affect enzyme kinetics, including temperature, substrate, pH, and enzyme concentration. It explains how pH can affect the shape and structure of enzymes by changing the ionization state of amino acids. The optimum pH range is described as the pH where each enzyme shows maximum activity. Extreme pH values outside this range can cause enzymes to denature and reduce their activity. Temperature is also discussed as another important factor, with higher temperatures generally increasing reaction rates up to a point, after which heat denaturation reduces activity.

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    Biochemical Engineering: Week 10

    Uploaded by

    Emmanuel
    30 views30 pages
    This document discusses factors that affect enzyme kinetics, including temperature, substrate, pH, and enzyme concentration. It explains how pH can affect the shape and structure of enzymes by changing the ionization state of amino acids. The optimum pH range is described as the pH where each enzyme shows maximum activity. Extreme pH values outside this range can cause enzymes to denature and reduce their activity. Temperature is also discussed as another important factor, with higher temperatures generally increasing reaction rates up to a point, after which heat denaturation reduces activity.

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    CEE3403 Final 19-20

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    This document discusses factors that affect enzyme kinetics, including temperature, substrate, pH, and enzyme concentration. It explains how pH can affect the shape and structure of enzymes by changing the ionization state of amino acids. The optimum pH range is described as the pH where each enzyme shows maximum activity. Extreme pH values outside this range can cause enzymes to denature and reduce their activity. Temperature is also discussed as another important factor, with higher temperatures generally increasing reaction rates up to a point, after which heat denaturation reduces activity.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    30 views30 pages

    Biochemical Engineering: Week 10

    Uploaded by

    Emmanuel
    This document discusses factors that affect enzyme kinetics, including temperature, substrate, pH, and enzyme concentration. It explains how pH can affect the shape and structure of enzymes by changing the ionization state of amino acids. The optimum pH range is described as the pH where each enzyme shows maximum activity. Extreme pH values outside this range can cause enzymes to denature and reduce their activity. Temperature is also discussed as another important factor, with higher temperatures generally increasing reaction rates up to a point, after which heat denaturation reduces activity.

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    BIOCHEMICAL ENGINEERING

    Week 10
    Factors affecting Enzyme kinetics

    ü  TEMPERATURE

    ü  SUBSTRATE

    pH

    Enzyme Concentration
    Effect of pH
    When the pH value of the reaction medium changes, the shape and structure of
    the enzyme will change. For example, pH can affect the ionization state of
    acidic or basic amino acids.

    There are carboxyl functional groups on the side chain of acidic amino acids.
    There are amine-containing functional groups in the side chain of basic amino
    acids.

    In the diagram below, the groups allowing ionic bonding are caused by the
    transfer of a hydrogen ion from a -COOH group in the side chain of one amino
    acid residue to an -NH2 group in the side chain of another.
    pH Effect
    Now think about what happens at a lower pH - in other words under acidic
    conditions. It won't affect the -NH3+ group, but the -COO- will pick up a
    hydrogen ion.

    What you will have will be this:

    You no longer have the ability to form ionic bonds between the substrate and
    the enzyme. If those bonds were necessary to attach the substrate and
    activate it in some way, then at this lower pH, the enzyme won't work.
    pH Effect
    What if you have a pH higher than 7 - in other words under alkaline conditions.
    This time, the -COO- group won't be affected, but the -NH3+ group will lose a
    hydrogen ion. That leaves…

    Again, there is no possibility of forming ionic bonds, and so the enzyme


    probably won't work this time either. At extreme pH's, something more drastic
    can happen. Remember that the tertiary structure of the protein is in part
    held together by ionic bonds just like those we've looked at between the
    enzyme and its substrate
    Optimum pH

    Each enzyme has an optimum pH range. Changing the pH outside of this range
    will slow enzyme activity. Extreme pH values can cause enzymes to denature.
    Optimum pH

    All enzymes have an ideal pH value, which is called optimal pH. Under the
    optimum pH conditions, each enzyme showed the maximum activity.

    For example, the optimum pH of an enzyme that works in the acidic environment
    of the human stomach is lower than that of an enzyme that works in a neutral
    environment of human blood.

    When the pH value deviates from the ideal conditions, the activity of the
    enzyme slows down and then stops.

    The enzyme has an active site at the substrate binding site, and the shape of
    the active site will change with the change of pH value.
    Cell Growth

    Cell Growth
    Cell Growth
    Growth
    • Increase in cellular constituents that may
    result in:
    – increase in cell number
    – increase in cell size
    • Growth refers to population growth rather
    than growth of individual cells
    How cells grow
    Growth curve
    The Growth Curve
    • Observed when microorganisms are cultivated in
    batch culture
    • Usually plotted as logarithm of cell number versus
    time
    • Has four distinct phases

    5
    Growth curve
    Lag Phase
    • Cell synthesizing new components
    – e.g., to replenish spent materials
    – e.g., to adapt to new medium or other
    conditions
    • Varies in length
    – in some cases can be very short or even
    absent
    – depends on harshness of medium
    • is it selective or enrichment medium?
    • what is the temperature of medium?
    Growth curve
    Exponential Phase
    • Also called log phase or log growth phase
    • Rate of growth and division is constant and
    maximal
    • Population is most uniform in terms of
    chemical and physical properties during this
    phase
    • Bacteria from this stage would be used for
    studies
    Growth curve
    Balanced Growth
    • During log phase, cells exhibit balanced
    growth
    – cellular constituents manufactured at constant
    rates relative to each other
    Growth curve

    Stationary Phase
    • Closed system population growth eventually
    ceases, total number of viable cells remains
    constant
    – active cells stop reproducing or reproductive
    rate is balanced by death rate
    Growth curve

    Possible Reasons for Stationary


    Phase
    • Nutrient limitation
    • Limited oxygen availability
    • Toxic waste accumulation
    • Critical population density reached
    • Bacteria die off and liberate some nutrients
    Growth curve
    Stationary Phase and Starvation
    Response
    • Entry into stationary phase due to starvation
    and other stressful conditions activates
    survival strategy
    – morphological changes
    • e.g., endospore formation
    – decrease in size, protoplast shrinkage, and
    nucleoid condensation
    – RpoS protein assists RNA polymerase in
    transcribing genes for starvation proteins
    Growth curve
    Death Phase
    • Also called Log Death phase
    • Toxic waste build up, inadequate nutrients, oxygen
    depleted, etc.
    • Bacteria are dying off opposite to log growth phase
    – do not die all at once
    • Two alternative hypotheses
    – cells are Viable But Not Culturable (VBNC)
    • cells alive, but dormant, capable of new growth when
    conditions are right
    • Programmed cell death
    – fraction of the population genetically programmed to die
    (commit suicide)
    Growth curve
    Prolonged Decline in Growth
    and Survival
    • Bacterial population continually evolves
    • Process marked by successive waves of
    genetically distinct variants
    • Natural selection occurs
    • Bacteria that survive may not be genetically
    identical to the original population
    • May find mutations, endospores, VBNC bacteria (a
    bacterial culture will contain cellular debris at the
    bottom of the tube)
    Growth curve

    The Mathematics of Growth


    • Generation (doubling) time
    – time required for the population to double in
    size
    – varies depending on species of microorganism
    and environmental conditions
    – range is from 10 minutes for some bacteria to
    several days for some eukaryotic
    microorganisms
    – This is calculated during log growth phase
    Monod Equation
    Exercises

    Question (1)
    The aerobic production of acetic acid from ethanol proceeds as follows:

    C2 H 5OH + O2 → CH 3COOH + H 2O
    Acetobacter aceti bacteria are added to a vigorously-aerated medium
    containing 15 g/litre of ethanol. After some time, the ethanol
    concentration was found to be 1.88 g/litre, whilst the acetic acid
    concentration was found to be 10.4 g/litre.

    – How does the overall yield of acetic acid from ethanol compare with the
    theoretical yield?
    Exercises
    If the doubling time of a cell is 8 hours, what would the cell concentration be
    after 26 days (assuming all nutrients are in excess) if the initial concentration
    of cells is 0.5 x 10-5 cells/ml.

    Determine the amount of (NH4)2SO4 to be supplied in a fermentation medium


    where the final concentration is 40g/l in a 1000 liter culture volume. Assume
    that the cells are 12 % nitrogen by weight and (NH4)2SO4 is the only nitrogen
    source.
    Exercises

    Temperature (oC) Reaction Rate constant kd (min-1)


    65 0.0012
    75 0.012
    85 0.07
    100 1.5
    110 8.91
    Exercises
    Exercises
    Question (2)
    • The number of viable spores Time
    (min)
    Temperature (°°C)

    for a new genetically modified 85 90 110 120

    strain of E. coli is measured as 0.0 2.40 x 109 2.40 x 109 2.40 x 109 2.40 x 109

    a function of time at various 0.5 2.39 x 109 2.38 x 109 1.08 x 109 2.05 x 107

    temperatures. 1.0 2.37 x 109 2.30 x 109 4.80 x 108 1.75 x 105
    1.5 2.34 x 109 2.29 x 109 2.20 x 108 1.30 x 103
    2.0 2.33 x 109 2.23 x 109 1.04 x 108
    a) What are the specific death
    constants at the 3.0 2.32 x 109 2.17 x 109 9.85 x 107
    temperatures studied?
    b) Determine the time taken for 4.0 2.28 x 109 2.11 x 109 2.01 x 107
    the number of spores to fall
    by half at the temperatures 6.0 2.20 x 109 1.95 x 109 4.41 x 106
    used.
    c) What is the activation energy 8.0 2.19 x 109 1.87 x 109 1.62 x 105

    for thermal death of this new


    9.0 2.16 x 109 1.79 x 109 6.88 x 103
    bacteria strain?

    H82BOB 5
    Q(2) Hints
    a) Plot graph based on equation
    ln C A − ln C A0 = − kt
    – lnCA against t will give a straight line of slope –k

    b) Half life:
    ln 2
    half life, t1/ 2 =
    k

    c) Plot graph based on equation


    k − Ea
    ln =
    A RT
    – lnk against 1/T will have a slope equal to -Ea/R and an intercept of lnA

    H82BOB 6
    Answers
    T (°C) (a) (b) c) Slope, -Ea/R = -26,594.051
    R = 8.314 J/mol.K
    k (min-1) t1/2 (min)
    Ea = slope x R
    85 0.012 58.47 = 221,102.942 J
    90 0.032 21.50 = 221.103 kJ

    110 1.253 0.55


    120 9.610 0.07
    Problems

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