Skript LSE Kapitel 3 Und 4

Bioprocess- and Bioreaction Engineering Prof. Dr.
Rainer Buchholz
3. Enzyme reactions
Enzymes are proteins, which act as catalysts in biochemical reactions. Their

specific characteristics and activity is usually superior to that of chemical
catalysts. The term enzyme was introduced in 1877, but already in earlier
times the presence of biological catalysts was suspected. The first general
theory of biochemical catalysis was published in 1835 by J. J. Berzelius.
Pasteur observed that alcohol fermentation is catalyzed by enzymes,
however in 1860 he claimed that those enzymes are integrated in viable
yeast-cells. In 1897 Eduard Buchner succeeded in extracting enzymes for
alcoholic fermentation from yeast cells. In 1927 J. B. Sumner isolated the
first enzyme (Urease) in pure crystalline form. Today there are more than
2000 enzymes known, of which 300 were purified and crystallized.
Enzymes are usually named by adding the syllable -ase to the name of
the converted substrate. E. g. the enzyme Urease is catalyzing the hydrolysis
of urea into ammonia and carbondioxide. But there also exists a relatively
great number of meaningless and trivial names such as Pepsin, Trypsin or
Katalase. As proposed by the International Enzyme Commission,
enzymes are divided into six main groups, which again are divided into
subgroups.
- 33 -
Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz
Table 3-1 Classification of enzymes
1. Oxidoreductases (redox reactions) 4. Lyases (additions at double bindings)

1.1 attacking >CH-OH 4.1 addition at >C=C<
1.2 >C=O 4.2 >C=O
1.3 >CH=CH- 4.3 >C=N-
1.4 >CH-NH2
1.5 >CH-NH-
1.6 NADH;NADPH
2. Tranferases (transfer of functional groups) 5. Isomerases (converting to isomers)

2.1 transferring C1-Groups 5.1 Racemases
2.2 aldehyde- or keto-groups
2.3 acyl groups
2.4 glycosyl groups
2.5 phosphate groups
2.6 S-containing groups
3. Hydrolases (hydrolytic reactions) 6. Ligases (forming bindings by using ATP splitting)

3.1 ester 6.1 C-O
3.2 glycoside bindings 6.2 C-S
3.3 peptide bindings 6.3 C-N
3.4 other C-N bindings 6.4 C-C
3.5 acid anhydride
Classification of Enzymes
Fig.: 3-1
- 34 -
Table 3-2 Industrial relevant enzymes
Enzyme microorganism reaction application

protease Bacillus unspecific endo- Washing enzymes,
Aspergillus hydrolysis of proteins, flour addition,
specific casein hydrolysis leather processing,
Mucor
cheese production
a-amylase Bacillus unspecific endo- starch
Aspergillus hydrolysis of starch to saccharification,
oligosaccharides textile and paper
processing, baking
and mash
processes
glycoamylases Aspergillus exo-hydrolysis of starch
oligosaccharides saccharification,
to glucose glucose and
ethanol production
glucose Streptomyces isomerisation of glucose starch syrup
isomerase Bacillus to fructose production
pectinase Aspergillus hydrolysis of processing of
polygalacturonic acid and juices, beer and
its methyl esters wine
cellulase Trichoderma hydrolysis of cellulose Drying of raw
resii plant material
lipase Candida sp. triglycerides, Fat hydrolysis,
Pseudomonas esterification of fatty acids improvement of
digestion,
Mucor
production of
emulsifiers and
special fats
lactase Aspergillus hydrolysis of lactose to Dairy waste water,
galactose and glucose improvement of
digestion
glucose oxidase Penicillium oxidation of glucose to glucose oxidase /
gluconic acid and katalase system for
hydrogenperoxide removal of oxygen
from canned fruit /
katalase Aspergillus removal of
vegetables and
hydrogenperoxide
beer
- 35 -
3.1 Kinetics of enzymatic reactions
In principle the above discussed reaction kinetics are directly valid for
enzymatic reactions. But there is one remarkable difference to non
enzymatic reactions:
Enzymatic reactions exhibit the phenomenon of substrate saturation.
Vmax
V0
1/2 Vmax
Km substrate concentration
Fig. 3-2: Influence of substrate concentration on rate of a simple enzymatic

reaction A P
a) For low substrate concentrations the rate of reaction is proportional to

substrate concentration, it is a first order reaction in substrate.
b) For rising substrate concentrations the growth of reaction rate slows
down, the reaction has a mixed order in this region.
c) For high substrate concentrations the reaction rate is constant, the
reaction is zero order, the enzyme is saturated by the substrate.
- 36 -
3.1.1 Derivation of the Michaelis-Menten-Equation
The general theory of enzyme kinetics was developed in 1913 by

L. Michaelis and M. L. Menten and later expanded by G. E. Briggs and
J. B. S. Haldane. For simplification, it is assumed, that the enzyme E and
the substrate S form a complex ES, which decomposes into enzyme E and
product P (in practical applications more than one transition stage occurs).
k1 k2
E S ES E P (3 1)
k 1 k 2
The formation rate of the enzyme-substrate complex (neglecting the back-

reaction from E and P) is:
dES
k1 ET ES S (3 2)
dt
[ET] = Total amount of enzyme (free and bound)

[ES] = Conc. of enzyme-substrate complex
[ET] - [ES] = Conc. of free enzyme
[S] = Substrate concentration
It is possible that [ES] is formed from E and P by a reverse reaction,

however this part can easily be neglected at start velocity V0, especially if [S]
is extremely high and [P] is near zero.
For the decomposition of the enzyme-substrate complex the following

equation applies:
dES
k 1 ES k 2 ES (3 3)
dt
- 37 -
If production- and decomposition rate of [ES] are equal, a steady state

exists:
k1 ET ES S k 1 ES k 2 ES (3 4)
For d[ES]/dt = 0 and [ES] = const.
Rearranging of the equation yields the Michaelis-Menten constant:

ET ES S k 1 k 2 KM (3 5)
ES k1
From eq. (3 5) the equilibrium concentration of the enzyme-substrate

complex follows:
ES Et S (3 6)
K M S
This steady-state concentration can be introduced in the initial production

rate:
V0 k 2 ES (3 7)
Putting eq. (3 7) into eq. (3 6) one gets:
V0 k 2
Et S (3 8)
K M S
If the total amount of enzyme is bound, the maximum initial production rate
follows:
Vmax k 2 ET (3 9)
Putting eq. (3 9) into eq. (3 8) the Michaelis-Menten equation follows:

Vmax S
V0 (3 10)
K M S
This rate equation for enzyme catalytic reactions connects the initial reaction
velocity, the maximum of reaction velocity and the initial concentration of
- 38 -
substrate with the constant of Michaelis-Menten. The concentration of

enzyme is found in the expression Vmax.
Replacing [S] in eq. (3 10) by KM , V0 = Vmax/2 results. Therefore, KM is

equal to the substrate concentration at which the reaction rate is half of the
maximum reaction rate. In the case of a single substrate reaction the value
for KM has the dimension of a concentration (mol/l) and is not affected by
the enzyme concentration.
The value for KM should not be mixed-up with the dissociation constant KS!
The only exemption is given in the case for k2 << k1. In this case you find:
KM
k 1
and K M K S
E S
k1 ES
Concentration-Time-Curve of an Enzyme- Substrate-Complex

c .c / mol L -1
E ES c S .c P / mol L -1
0.020
0.10
0.015
0.08
P
0.06
0.010
0.04
S
0.005 ES
0.02
E
0.000 0.00
0 50 100 150 200 250 300 350
time / s
Fig.: 3-3
- 39 -
[Product]
[E]
[Substrate] [ES]
[Product]
Time
[E] [ES]
Time
Fig. 3-4: Concentration-Time-Curve of an Enzyme-Substrate-Complex
3.1.2 Transformation of the Michaelis-Menten Equation

In order to simplify evaluation of experimental data, eq. (3 10) is
linearized by inverting and rearranging:
1 K S
M
V0 Vmax S
or
1

KM

S
V0 Vmax S Vmax S
1 K 1 1
M (3 11)
V0 Vmax S Vmax
By plotting 1/V0 versus 1/[S] the Lineweaver-Burk plot results, this yields for
Michaelis-Menten kinetics a straight line.
- 40 -
1
V0
slope Km
Vmax
1
Vmax
1
-1 S
Km
Fig. 3-5: Lineweaver-Burk plot
- 41 -
Other linearizations yield additional equations:
Eadie-Hofstee
V0
V0 Vmax K M (3 12)
S
Vmax
slope -Km
V0
Vmax
Km
V0
S
Fig. 3-6: Eadie-Hofstee plot
- 42 -
Hanes
S KM

S (3 13)
V0 Vmax Vmax
1
slope
S Vmax
V
-Ksm S
S
Fig. 3-7: Hanes plot
3.1.3 pH- and temperature influence on enzyme activity
The activity of enzymes is influenced by the pH-value and temperature. The

activity curve is bell-shaped in most cases, with a narrow optimum. This
optimum pH however, is not necessarily the pH-value within the cell. The
assumption seems reasonable that there is an influence of activity control of
the enzymes by the pH inside the cells.
The next figure shows possible curves of pH-activity of enzymes.
- 43 -
trypsin
pepsin
6 8 10 2 4 6
pH pH
papain (substrate =
cholinesterase benzoylargininamid)
6 8 10 4 6 8
pH pH
Fig. 3-8: Activity dependence on pH for various enzymes
The temperature dependence of enzymatic activity also exhibits an

optimum due to an increase at low temperature ranges like chemical
reactions followed by a clear decrease at a higher temperature. This can be
viewed as a result of two processes:
1) Increasing reaction rate from increase of temperature (c.f. chemical

reactions)
2) Decreasing activity caused by thermal denaturation of the enzyme
Most enzymes are deactivated at temperatures above 55 60 C, some are

quite resistant however, especially enzymes from thermophilic bacteria.
- 44 -
Some enzymes (e.g. ribonuclease) lose their activity during heating but
regain it after cooling.
3.2 Mechanisms of complex enzymatic reactions
Most of enzymatic reactions can be described with Michaelis-Menten

kinetics, but deviations from this model can be observed. Typical deviations
from the simple model are shown below:
a b
rmax rmax
r r
S S
Fig. 3-9: Substrate activation without (a) and with (b) substrate inhibition
c.f. a) Substrate activation: The binding of the first substrate molecule

helps binding more substrate molecules (cooperative effect which
occurs if the enzyme-molecule consists of protein-groups of the
same structure. Such an oligomer can bind several substrate
molecules)
c.f. b) Activation at low substrate concentration: For high substrate
concentrations the reaction rate decreases (inhibition).
- 45 -
Further examples for more complex enzymatic reactions are:
1) One substrate reacts to two products.

2) Two substrates react at the same enzyme to one product.
3) The enzyme has to be regenerated after formation of the product.
3.3 Enzyme inhibition
Enzyme inhibition can be reversible or irreversible. During irreversible

inhibition, the enzyme and the inhibitor form a stable complex; the
enzyme cannot be activated again. Such inhibitors are for example heavy
metals (e. g. lead, silver, mercury, cadmium) or ions which are capable of
forming stable metal complexes like the cyanide ion. This type of inhibition
is also called enzyme poisoning.
The reversible inhibition is characterized by an equilibrium for the
enzyme-inhibitor complex. Inhibitors can act on the enzyme, on the
enzyme-substrate complex, or both. Another possibility is a reaction of the
inhibitor with the substrate.
3.3.1 Competitive inhibition
Competitive inhibition means a competition of inhibitor and substrate for

the active center of the enzyme. It is likely to happen if the molecular
structure of substrate and inhibitor are similar. Additionally, the product
may also act as a competitive inhibitor. A simple reaction scheme for
competitive inhibition is shown below.
- 46 -
k1 k2
S ES E P
k 1
E (3 14)

ki
I EI
k i
ES: Active enzyme-substrate complex

EI: Inactive enzyme-inhibitor complex
For the dissociation of an enzyme-inhibitor complex EI into free enzyme E

and inhibitor I, the inhibition-constant KI is applied, which is comparable to
the dissociation-constant KS of the enzyme-substrate complex.
KI
E I (3 15)
EI
The reaction rate for conversion of substrate in the presence of an inhibitor I
is:
V0 Vmax
S (3 16)
K M 1 I / K I S
For I=0 the equation is equivalent to the Michaelis-Menten equation. By

increasing the substrate concentration, the competitive inhibition can be
decreased or completely suppressed. The linearized form of eq. (3 16) for
the Lineweaver-Burk plot (Fig. 3 10) is:
1 K 1 1
M 1 I / K I (3 17)
V0 Vmax S Vmax
- 47 -
3.3.2 Noncompetitive inhibition
Noncompetitive inhibition means no competition between substrate and

inhibitor. Both bind at different sites, but the inhibitor influences the
substrate activation. This type of inhibition cannot be suppressed by
increasing the substrate concentration. The inhibitor may bind at the
enzyme, at the enzyme-substrate complex, or at both.
k1
ES
k
ES
k
EP
1 2

(3 18)
I I
kI k I
k I k I
EI ESI
For the reaction rate holds:
1
Vmax S
1 I / K I
V0 (3 19)
KM S
In this case, the Michaelis-Menten constant remains unchanged, but the

maximum reaction rate is decreased. For graphical representation, eq. (3
19) is linearized (c.f. Fig. 3 10 b):
1 K 1 1
M 1 I / K I 1 I / K I (3 20)
V0 Vmax S Vmax
- 48 -
3.3.3 Uncompetitive inhibition
Uncompetitive inhibition means that the inhibitor may only react with the
enzyme-substrate complex, not with the free enzyme.
k1 k2
E S ES E P
k 1
(3 21)
kI
I ESI
k I
The reaction rate can be expressed by:

1
Vmax S
1 I / K I
V0 (3 22)
KM
S
1 I / K I
The linearized form of eq.(3 22) is:

1 K 1 1
M 1 I / K I (3 23)
V0 Vmax S Vmax
In this case the Michaelis-Menten constant as well as the maximum reaction

rate are reduced.
- 49 -
competitive non-competitive
inhibition inhibition
1 Slope of the 1 Slope of the

inhibited reaction inhibited reaction
V0 V0
without inhibitor without inhibitor

slope slope
1 1
[S] [S]
uncompetitive substrate
inhibition inhibition
Slope of the Slope of the
1 inhibited reaction 1 inhibited reaction
V0 V0
without inhibitor without inhibitor

slope slope
1 1
[S] [S]
Fig. 3-10 a-d: Lineweaver-Burk plots for different types of inhibition
3.3.4 Substrate inhibition
Substrate inhibition is the inhibitory influence of high substrate

concentrations on the reaction. In this case the substrate binds at the ES
complex, forming an ESn complex, which cannot decompose into enzyme
and product.
- 50 -
k1 k2
E S ES E P
k 1
(3 24)
k3
S ES2
k 3
The reaction rate for substrate inhibition is:
V0 Vmax
S (3 25)
K M S S2 / K 3
whereby
KM
E S k 1 k 2
ES k1
K3
ES S k 3
ES2 k 3
Linearization of eq.(3 25) results in (c.f. Fig 3 11 d):
1 K 1 1 1
M S (3 26)
V0 Vmax S Vmax K 3 Vmax
Although this will not be a straight line in the Lineweaver-Burk plot, there
are two parts of the curve. For low substrate concentrations it follows:
1 K 1 1
M Michaelis Menten (3 27)
V0 Vmax S Vmax
For high substrate concentrations:
1 1 1
S (3 28)
V0 Vmax K 3 Vmax
- 51 -
reaction system reaction

pH, temp., enzyme and design
substrate concentration
properties of
thermodynamics kinetics
biocatalyst
Keq Mr, mechanical fragility, Vm, Km, Ki
activity, stability
process reactor
Tau, XA, productivity, design
enzyme consumption
Fig 3-11
Definitions
(R)-Product
(R,S)-
Substrate (S)-Product
molS
Turn Over Frequency (TOF) =
molcat * time
1
Deactivation Number (kde) =
time
TOF
Total Turnover Number (TTN ) =
kde
- 52 -
Definitions
(R)-Product
(R,S)-
Substrate (S)-Product
S
conversion (X) = 1-
S0
(R)-Product
Yield (Y) =
S0
(R)-Product
Space-Time-Yield (STY) =
Tau
(R)-Product
Selectivity =
S0 - S
Enantioselectivity (ee) = (R)-Product - (S)-Product

S0 - S
S0 = initial substrate concentration
Tau = residence time
Enantioselectivity as Function of (Chemical) Selectivity

100 100
80
enantiomeric excess (ee) / %
(R)-product
(R,S)-substrate
(R) 50 (S)-product 10
ratio R/S / -
90
0 1
(R)-product
(S) 50 selectivity =
S0 - S 0.1
(R)-Product - (S)-Product
ee =
S0 - S
100 0.01
0 20 40 60 80 100
chemical selectivity / %
- 53 -
4. Growth Kinetic
4.1 Biomass
Target of a quantitative description of the growth rate of any kind of
organisms or cells is the estimation of the functional correlation of biomass
increase during time.
dX g (biomass) 1
dt lfermentation broth time

Due to the complexity of biological systems often there is only a rough

approximation possible. Furthermore, numerous parameters are not
detectable and an experimental proof of a model description is only rarely
possible. Whether there is a sensible use of expert systems and
mathematical models for the process description depends on the given
cultivation system. In some cases it was possible to develop the whole
process control by using process models.
For measuring the biomass in a biological culture two different methods can
be applied.
1) Biomass (dry weight)

2) Cell number (cell counting, optical density)
A schematic correlation between biomass concentration, number and

weight of the cells and some ingredients of the cells is given in Fig. 4-1.
- 54 -
10
3
5
2
2
1 1
time
Fig 4 - 1: Schematic scope of growth parameters in time correlation
For biomass estimation the measuring of dry-weight appears to be the most

accurate method. One of the noticeable disadvantages of this method is the
fact, that it is non on-line. Furthermore, it is impossible to distinguish
between vital and non-vital cells, however, this method will fail, provided
substrates of solid consistency are used.
In order to determine the amount of yeast and bacteria the cell count as
well as the dry-weight can be used. For pellet or mycel forming organisms
(e.g. fungies, streptomyces etc.) the method of dry-weight estimation is the
most dependable procedure.
For the biomass estimation the DNA and RNA concentration can also be
applied. During the lag phase the RNA content increases slightly and the
DNA content decreases, but during the exponential growth the relation
between both is constant. At the end of the growth phase, the stationary
phase, the RNA content decreases and the DNA content increases.
- 55 -
4.1.1 Batch culture

Exponential growth
The growth of microorganisms in batch culture exhibits similar features for

different species.
V
IV
VI
III
II
I
time
Fig. 4 -2: Schematic growth curve
4.1.2 Formal kinetic of batch mode growth
I. Lag phase
N N 0 konst (4 1)
II. Acceleration phase

2
t t0
N N 0 N L N 0 (4 2)
tL t0
III. Exponential phase

Cells proliferate by division, for the generation time g, the number of
generations n and time t holds:
- 56 -
t
g (4 3)
n
Is the initial number of cells N0, for n divisions it follows:
N N 0 2n (4 4)
The number of divisions per hour is defined as division rate v:
n log N log N 0 1
(4 5)
t log 2 t t 0 g
Considering the biomass concentration X instead of the cell number N, the

change of biomass is proportional to the amount of biomass. Defining the
specific growth rate , the growth rate increases after the lag phase and
reaches the value max, which is constant during the exponential phase.
Consequently, the growth is a first order reaction. Therefore from the mass
balance follows:
dX
max X (4 6)
dt
Integration with the initial condition X = X0 at t = t0 :
X
ln max t (4 7)
X0
X X 0 e max t (4 8)
The time for doubling the amount of biomass is called doubling time td .
It may be calculated by inserting X=2 X0 into eq. (4-8):
2X 0 X 0 e max t (4 9)
ln 2 0.693
td (4 10)
max max
The specific growth rate and the specific rate of division v are not
necessarily equal. Only in the case of a constant cell size and the division of
one cell into two cells the are values equal.
- 57 -
Table 4-1: Maximum growth rates for different microorganisms (substrate: glucose)
Organism T [C] m [ h 1 ] Doubling time [h]
Aspergillus niger 30 0.20 3.46

Aspergillus nidulans 20 0.09 7.72
25 0.148 4.68
30 0.215 3.23
37 0.360 1.96
Penicillium
25 0.123 5.65
chrysogenum
Mucor hiemalis 25 0.17 4.1
Fusarium avanaceum 25 0.18 3.8
Fusarium
30 0.28 2.48
graminearum
Verticillium
25 0.24 2.9
agaricinum
Geotrichum candidum 25 0.41 1.7
Geotrichum candidum 30 0.61 1.1
Neurospora sitophila 30 0.40 1.73
Candida tropicalis 30 0.60 1.1
Bacillus
60 0.18 3.8
stearothermophilus
Escherichia coli 40 0.35 2.0
Bacillus subtilis 40 0.43 1.6
- 58 -
4.1.3 Influence of substrate concentration on biological growth

(Monod equation)
The substrate concentration influences the growth.
1.0
(5)
0.8
(4)
0.6
(3)
0.4
(2)
0.2
(1)
1 2 3 4
time t [h]
Fig. 4-3: Growth of E. coli under various initial glucose concentrations
(conc. In g/l: (1)=0, (2)=15, (3)=25, (4)=35, (5)=45)
In this simple case, the maximum specific growth max rate is independent of
a substrate concentration. The maximum biomass concentration at the end
of the process however, is a function of initial substrate concentration. This
leads to the definition of the yield coefficient YX/S :
X max X 0 g Biomass
YX / S g Substrate (4 11)
S0
In the preceding example the yield coefficient is constant at about 0.45, but
influences by other substrates or products may change the linear relation
between biomass produced and substrates consumed:
- 59 -
dX
dt
YX / S is not necessarily constant ! (4 12)
dS
dt
The experimentally observed dependence of the specific growth rate of

the substrate concentration is shown in Fig. 4 8.
max
1/2 max
Km=0.22 10-4 mol/l
1 2 3 4 5
-4
glucose concentration [10 mol/l]
Fig. 4-4: Specific growth rate vs. substrate concentration (E. Coli
cultivation using glucose as carbon source)
The observations are similar to the reaction rate of the Michaelis Menten
equation. Therefore, in analogy the equation is set up:
max
S (4 13)
K S S
The analogy between enzyme kinetics and microbial growth was first
described by Monod (1942). Provided there are two limiting substrates, the
specific growth rate is formulated accordingly:
- 60 -
max
S1 S2 (4 14)
K S S1 K S S2
1 2
Setting up the mass balances for microbial growth in a batch reactor using
the simple Monod equation, two coupled differential equations result:
dX
max
S X (4 15)
dt K S S

dS

1
max
S X (4 16)
dt YX / S K S S
These equations can be solved by numerical methods. Computer

simulations may be used to predict the time course of bioprocesses and to
control the optimal conditions during cultivation.
4.1.4 Substrate inhibition kinetics
Especially due to biodegradation in the field of industrial waste water

treatment, the problem of inhibitory or toxic substrates arises. In analogy to
substrate inhibition, Haldane expanded the Monod equation to include
such effects in the description of microbial growth:
max
S (4 17)
K S S
S
2
K1
The differential equations for modelling a batch process are:
dX
max
S X (4 18)
dt
K S S
S
2
K1
dS

1
max
S X (4 19)
dt YX / S
K S S
S
2
K1
- 61 -
Depending on the values of KS and KI , computer simulations show, that the

maximum growth rate is only reached at the end of cultivation in a batch
process. Moreover, only at the end of the batch process the substrate
concentration is below the inhibitory level. It is therefore preferable to use
fed-batch or continuous processes to degrade inhibitory substrates.
During the cultivation of Lactobacillus delbrueckii on a glucose/yeast extract

media several exponential growth phases were found. Conclusively, more
than one substance from the yeast extract serves as a substrate and those
substrates were utilized after each other. Consequently, the Monod model
was extended by Tsao and Hanson.
dX 1l S1l 1 jS1 j
X .....
dt K S K1 j S1 j
1l 1l
2l S2l 2 j S2 j
..... (4 20)
K S K 2 j S2 j
2 l 2 l
il Sil ij Sij
.... .....
K S K ij Sij
il il
with:
ij specific growth velocity with Sij
Sij substrate concentration
Kij Monod constant at given substrate
Several groups of essential substrates (i = 1 ... i) can exist containing

different growth increasing substances j (j = 1 ... j). Those substrates j
appear as summands at the different Monod terms.
The cultivation of Proteus vulgaris on the substrate mix of glucose and Na

(sodium)-citrate is formulated very well using this model (i =1; j = 2):
- 62 -
dX S S
X 1 1 21 2 (4 21)
dt K1 S1 K 2 S2
The substrates S1 and S2 are consumed mainly one after the other.
mg/l
2,0
1,5
1,0
ln X
0,5 ln S1
10
ln S2
10
0 10 20 30 40 50 h
Fig. 4-5: Growth of Proteus vulgaris with glucose (S1) and Citrat (S2)
- 63 -
4.1.5 Extended growth models for batch fermentations
In many cases the Monod equation is unable to describe the experimental

data received from a batch fermentation process correctly. For this reason
several empirical modified equations are found in literature. Most of them
can be reduced to the following power equation:
dX
K max X
p
(4 22)
dt
with
K = constant
p = exponent
In the case of fast growth an adaptation of the Monod equation compared
to experimental data is often not possible. Here the following equations are
used:
max
S (4 23)
K So S0 S
or
max
S (4 24)
K S K So S0 S
with S0 = start concentration of the limiting substrate

KSo and KS = constants
Using these models the experimental behaviour of the deviation from the
exponential growth by increasing start concentration (S0) of the substrate
instead of approaching max can be described.
- 64 -
Logistic growth law
This model starts out of the fact that the population is regulated by the
restricted space available. It is borrowing from the animal growth in nature.
In contrast to the pure exponential growth a mathematical function is
introduced which reduces the growth by increasing cell density. This
enhanced model is given by:
dX X
max X 1 (4 25)
dt X max
whereby Xmax is given by the maximum cell density in the stationary phase.
With increasing cell density the expression in the bracket decreases until the
change dX/dt becomes 0 by the maximum cell density. The equation (4
25) can be read as a combination of a growth term in
the 1st order and a dying term in the 2nd order.
dX
max X max X 2 (4 26)
dt X max
X max
exponential
growth evironment resistance
dX
= max X actual growth
dt
dX X
= max X 1-
dt X max
time t
Fig. 4-6: Schematic diagram of the logistic growth law

- 65 -
The integration of the equation (4 25) within the boundaries between 0

and t and X0 and X leads to:
X0
max t
X max 1 e X max
X (4 27)
The logistic model itself describes only the part of the growth curve between
the exponential and the stationary phase.
Other empiric expressions

1
Moser, H.: max (4 28)
1 K S B
Teissier, G.: max 1 e k

S
(4 29)

S
Contois, D. E.: max (4 30)
BX S
Model introduced by Fujimoto
Fujimoto describes the growth of microorganisms like a catalytic reaction

using a transition complex, meaning the biological growth behaves similar
to the enzyme kinetic.
k1 k2
X S X...S XS P (4 31)
k 1
with:
(X...S) = transition complex
[XS] = new biomass
It is also possible to define the reaction between substrate and enzyme:

k1 k2
E S / X EXS XS E P (4 32)
k 1
The dimension of E is hereby [g enzyme / g cell mass].

- 66 -
The fundamental of this particular model is:

The conversion of substrate is carried out in three steps.
1) Interaction between substrate and surface of the microorganism
2) Transport into the inside of a cell
3) Enzymatic reaction
Only the substrate which has direct contact with the cell can react. The
temporary change of the transient complex is given by:
dEXS S
k1 E k 1 k 2 EXS (4 33)
dt X
- 67 -
For the equilibrium d[EXS]/dt = 0 results:

E S k 1 k 2 K (4 34)
EXS X k1
or:
EXS E S
KX
The specific growth rate is given by:
1 dX
k 2 EXS (4 35)
X dt
The maximum growth rate can be obtained, if the total amount of enzymes
are available as an EXS-complex:
max k 2 E t k 2 E EXS

k 2 E
E S (4 36)

KX
1 S
k 2 E 1
K X
Dividing by max leads to:

k2
E S/ X
(4 37)
max K k 2 E 1 S / X 1 / K
That means:
S/ X
max
K S/ X
or:
S
max (4 38)
KX S
This equation is formally equal to the Monod equation containing a
substrate limiting constant KS which depends on the cell density X .
K S f X (4 39)
- 68 -
This model can describe the influence of the initial cell density during the
lag phase in a certain qualified sense.
The decline phase is not describable because never becomes negative.
4.1.6 Growth model for mycel forming organisms
The Monod model can not be used without restrictions for fungi cultures,
due to the different rules compared to bacteria or yeast in growth
behaviour. E.g. the fungi Penicillium chrysogenum (belonging to the group
of Plectomycete) is growing filamenteously. The hyphes are equipped with
multiple branches. The active growth of this type of fungi is restricted to the
top of the hyphes. The growing area is small compared to the entire
biomass of mycelia, and for each top of the hyphes this area remains
constant. In literature one can find three to five stages of cell distinctions
with different nutrient consumptions, age distributions and metabolite
productions.
In submerged culture two extremes of morphology can be observed: the
filamenteous and the pellet form. If pellet behaviour is found, numerous
spherical compact cell clusters are formed. These clusters are only growing
on the outer surface. If filamentuous growth is observed a mycelia of low
density is formed. Ideally, in the case of filamenteous growth all hyphes are
homogeneously supplied with nutrients. Only then a kind of exponential
growth can be observed.
The growth of mycel-forming organisms is shown in the next graph.
- 69 -
pherific growth zone

apex
septa with closed porosity septa with open porosity
t0
X
t1
X
t2
X0 X1 X2
Fig. 4-11: Growth behaviour of filamenteous mycelia
The hyphes consist of the cell membrane and the cytoplasma with its
inclusions. The hyphes grow in one dimension only at the top (apex), also
named apecal growth. Some organisms form so-called septa (Mycomycetes
like Aspergillus spec. and Penicillium spec.). Others grow without forming
septa (Phycomycetes like Mucor spec. and Rhizopus spec.).
The pellet growth can not be described by the Monod equation (Emmerson:
J. Bacteriol. 60, 221 (1950)), therefore the following expression was
developed:
dX 2
Rx X 3 (4 40)
dt
- 70 -
X can be obtained by the integration of Eq. (5 - 40):
1
X X 03 t
3
(4 41)
2
6 3
k g const.

with
= pellet density
Kg = dRP / dt
RP = radius of pellet
Pirt found the same relations using the assumption that oxygen is
transported only by diffusion into the pellet. Consequently, growth can only
take place in a zone at the surface due to the lack of oxygen in the centre of
the pellet. It is often found that the pellets with a diameter of more than one
millimeter are hollow spheres.
- 71 -
4.2 Product formation

4.2.1 Fundamentals of the product formation in batch processes
Regarding the formed products, fermentations can be divided into two

groups:
1) Production of biomass
2) Production of metabolites
The following tables give an incomplete summary on the spectra of
fermentation processes.
Table 4-2: Cell substance as fermentation products
Cell Substance as Fermentation Products
Product of Microorganisms Main Production Fermentation

fermentation substrates condition type
1. SCP Candida spec. molasses, whey, non sterile aerobic

fodder-, nutrient and other pulp, paraffin,
yeast yeasts sulfite liquors
bakers yeast S. cerevisiae molasses reduced germs aerobic
bacterial protein Methylomonas ethanol reduced germs aerobic
2. human pathogenic various bacteria sugar/ protein sterile aerobic

bacteria species hydrolysate anaerobic
3. insect pathogenic Bac. thuringiensis sugar/ protein sterile aerobic

bacteria Bac. popilliae hydrolysate
4. nodule bacteria Rizobium spec. sugar/ protein sterile aerobic

Buc 052-1
hydrolysate
- 72 -
Table 4-3: Catabolic metabolites as fermentation products
Catabolic Metabolites as Fermentation Products

1. ethanol S. cerevisiae molasses, starch reduced germs anaerobic

hydrolysate
2. butanol/ acetone Clostridium molasses, protein sterile anaerobic

acetobutylicum hydrolysate
3. lactic acid Lactobac. spec. molasses, whey non sterile anaerobic
4. acetic acid Acetobacter sp. ethanol reduced germs aerobic
5. gluconic acid Aspergillus sp. glucose reduced germs aerobic
6. citric acid Aspergillus sp. molasses, starch reduced germs aerobic

hydrolysate
7. dihydroxyacetone Acetobacter sp. glycerol sterile aerobic
8. sorbose Acetobacter sp. sorbitol sterile aerobic

Buc 051
Table 4-4: Biosynthetic conversions as fermentation products
Biosynthetic Metabolic Products

1. dextran Leuconst. spec. saccharose non sterile anaerobic
2. L-glutamic acid Micrococc. spec. glucose, sterile aerobic

Brevibacter spec. molasses
3. 5- inosine acid a.o. mutants of sugar/ mineral sterile aerobic

5- ribonucleotide various bacteria salts + certain
purines
4. antibiotics Penicillum spec. sugar containing sterile aerobic

ca. 50 different Streptomyces sp. complex substrates
products Bacillus sp. a.o.
5. secale alkaloids Claviceps spec. sugar sterile aerobic

polyol Buc 049
- 73 -
Biosynthetic Metabolic Products

6. -carotene a.o. Blakeslea sugar sterile aerobic

carotenoids trispora a.o.
7. vitamine B12 Propionbacter glucose / protein sterile aerobic

shermanii hydrolysate
8. vitamine B2 Ashbya gossypii sugar/ protein sterile aerobic

hydrolysate
9. gibberellin Gibberelo sugar/ protein sterile aerobic

fujik. hydrolysate
10. 1,1--hydroxyl. Aspergillus non hydroxylic sterile aerobic

steroids ochraceus a.o. steroids
11. 1-dehydrate Bacillus lentus steroids without sterile aerobic

steroids a.o. appropriate
double bond
Buc 0501
Table 4-5: Enzymes as fermentation products
Enzymes as Fermentation Products

1. -amylase Aspergillus sp. starch sterile, aerobic

Bac. subtilis reduced germs
2. amyloglucosidase Aspergillus sp. dextrine sterile, aerobic

reduced germs
3. pectinase Aspergillus sp. bran, pulp reduced germs aerobic
4. cellulase Aspergillus sp. plant products non sterile aerobic

(mixtures) Trichoderma a.o.
5. glucose oxidase Aspergillus sp. glucose non sterile aerobic
6.
-galactosidase Aspergillus sp. lactose non sterile, aerobic
Saccharomyces sp. reduced germs
Buc 047
- 74 -
Enzymes as Fermentation Products

7. microb. rennet Mucor spec. casein sterile, aerobic

reduced germs
8. acetic & neutral various fungi plant protein non sterile aerobic
protease various bacteria
9. alkaline protease Bac. subtilis plant protein non sterile aerobic

(cleaning enzyme)
10. lipase fungi, bacteria plant oil, train oil reduced germs aerobic
11. penicillinase Bac. subtilis sugar/protein sterile aerobic

and other hydrolysate
12. asparaginase E. coli a.o. protein sterile anaerobic

and other hydrolysate semi aerobic
Buc 048
Table 4-6: Food as fermentation products
Food as Fermentation Products

1. alcoholic Saccharomyces sp. sugar containing non sterile, anaerobic

drinks raw materials, reduced germs
starch
2. fermentative Lactobacillus sp. milk, reduced germs generally

milk products a.o. milk protein anaerobic
3. fermentative various raw plant non sterile aerobic/

plant products microorganisms material anaerobic
Buc 043
- 75 -
The following tables show the compositions of the mainly used carbon
sources. For being competitive waste materials are often used.
Carbohydrates are the traditional substrates of the fermentation industry.
Pure glucose or saccharose are seldom used as substrate due to its price.
The only exception is the demand on pure and exactly defined substrates
for product validation.
Molasses
This carbon source is a by-product of sugar production and is one of the

most inexpensive substrates. Other valuable compounds and tracers are
contained under a high concentration of saccharose. The main
disadvantage is a remarkable change in quality depending on the raw
material, the region, the climatic conditions, and last but not least on the
production processes of the sugar companies.
Hydrol-molasses, a by-product of the hydrolysis of starch, is also often
used in fermentations.
Malt extract
The aqueous extract of malted barley is suitable for many microorganisms

as the only carbon source. Carbohydrate, accounting for more than 90%,
contains hexoses like glucose and fructose, di-saccharides like maltose and
saccharose, tri-saccharides like maltotriose, and several dextrines.
Additionally, various proteines such as peptides, amino acids and vitamines
are contained in malt extract.
Other suitable raw materials for fermentations are starch, dextrines, and the
sugar containing wastes of the cellulose industry. Also vegetable oils,
alcohols and alkanes are used as substrates.
- 76 -
Table 4-7: Comparison of the composition of molasses from sugar beets

and sugar cane
Comparison of the Composition:

Sugar Beet and Sugar Cane
(Rhodes and Fletscher, 1966; Imrie, 1969)
Composition Sugar beet Sugar cane
Dry substance % 78 - 85 77 - 84
Saccharose 48.5 33.4
Raffinose 1.0
Invert sugar 1.0 21.2
Organic non sugar 20.7 19.6
N 0.2 - 2.8 0.4 - 1.5
P2O5 0.02 - 0.7 0.6 - 2.0
CaO 0.15 - 0.15 0.1 - 1.1
MgO 0.01 - 0.1 0.03 - 0.1
K2O 2.2 - 4.5 2.6 - 5.0
SiO2 0.1 - 0.5
Al2O3 0.005 - 0.06
Fe2O3 0.001 - 0.02
Ash 4 -8 7 -11 Buc 045
Composition Sugar beet Sugar cane

g /100 g dry weight
Thiamine 130 830
Riboflavin 41 250
Pyridoxine 540
Buc 046
- 77 -
Table 4-8: Typical composition of dry malt extract
Typical Composition of Malt Extract
(dry)
Components %-Share of dry weight
Maltose 52.2
Hexoses (glucose, fructose) 19.1
Saccharose 1.8
Dextrin 15.0
Other carbohydrates 3.8
Substances containing nitrogen 4.6
Ash 1.5
Water content 2.0

pH (10% solution) = 5.5 Buc 042
4.2.2 Rates and productivity
The productivity is defined as:

complete concentration of product
P
fermentation time
The volumetric rate is given by the change in concentration in time [mass
per volume and time]. The specific rate is the volumetric rate divided by
the biomass concentration [mass per time and biomass].
- 78 -
max.
productivity
overall productivity
production rate = dP/dt
productivity P/t
cleaning preparation lag phase production phase time
Fig. 4 - 8: Overall productivity and maximum of productivity
4.2.3 Kinds of fermentations
Examples for the classifications of fermentation processes are developed by

Maxon (Appl. Microbiol. 3, 110 (1955)); Deindoerfer (Adv. Appl.
Microbiol. 2, 321 (1960)); Gaden (Chem. & Ind. London, p 154 (1955)
and J. Biochem. Microbiol. Technol. Eng. 1, 413 (1959).
For the kinetic considerations the classification given by Gaden is preferred.
Classification by Gaden:
Type of fermentation I:
This type is issued if the product is deduced directly from the primary
metabolism. For example, ethanol, lactic acid and gluconic acid are some
potential products next to pure biomass. In this case, growth, substrate
consumption and product formation are running under nearly identical
conditions. Trophophase (exponential growth phase) and Idiophase
(production phase) can not be separated from each other.
Example: alcohol production with Saccaromyces cerevisiae.
- 79 -
6 30 60 X Biomass dry wt, g/l

a) Cell mass- and product concentrations 5 25 50
P Alcohol
S Substrate used
and substrate uptake,
4 20 40
b) Growth-, product formation- and X P S X
substrate uptake rates and 3 15 30 P
c) specific rates as function of time 2 10 20 S
1 5 10
0 0 0
a)
0 2 4 6 8 10 12 14
RX Growth, g/(lh)
RP Alcohol synthesis, g/(lh) 0.25 1.2 2.5
RS Sugar utilization, g/(l
h)
5 10 0.20 1.0 2.0
1
4 8 0.8
0.8 0.15 1.5
RP,RS 0.6 ,
0.6 3 6
RX 0.10 1.0
RX RP RS
2 4 0.4
0.4 Growth, g/(g h)
0.05 0.2 0.5 Alcohol synthesis, g/(g
h)
0.2 1 2 Sugar utilization, g/(g
h)
0 0 0 0
0 0
0 2 4 6 8 10 12 14
b) 0 2 4 6 8 10 12 14 c)
Time [t]
Buc 036
Fig. 4-9: Type of Fermentation I (alcohol production)
These fermentation products are the result of dissimulation reactions with a

negative free energy.
A products
A B C products
It is typical for these types to have only one maximum in all rates. These
maxima coincide roughly.
Type of fermentation II:

Also, in this case the product is deduced from the primary metabolism for
energy uptake. The biochemical reaction is carried out by bypassing the
metabolism. It is then separated from the primary metabolism.
substrate A B C D primary metabolism
E F product
- 80 -
This type leads to two maxima in all volumetric rates during a batch
fermentation. In the beginning there is only growth in biomass, a high
consumption of substrates, and no or only a small product forming. Later
the growth is reduced, but by using a high consumption of substrate,
product formation is induced. Trophophase (exponential growth phase) and
Idiophase (production phase) are separated from each other.
Examples for this type of fermentation are the production of citric acid,
itaconic acid and some amino acids like glutaminic acid.
24 240 S
X Biomass dry wt, g/l
20 200 P Citric acid, g/l
Titrable acidity
Citric acid
a) Cell mass- and product concentrations 16 160 S Substrate
X
used
and utilized substrate,
X 12 P,S 120
b) Growth-, production and substrate
uptake rates and 8 80 P
c) specific rates as function of time 4 40

a)
0 0
0 40 80 120 160 200 240 280
Time [t]
0.14 0.28
0.10 10 RX Growth, g/(lh) 0.12 0.24
RP Acid synthesis, g/(lh) Growth, g/(g h)
RS Sugar utilization, g/(l
h) Acid synthesis, g/(gh)
0.10 0.20 Sugar utilization, g/(g
h)
0.08 1.6
RX RP ,RS
0.08 0.16
0.06 1.2
,
0.06 0.12
0.04 0.8 RP
RX RS
0.04 0.08
0.02 0.4
0.02 0.04
0 0 b) c)
0 40 80 120 160 200 240 280 0 0
0 40 80 120 160 200 240 280
Time [t] Time [t]
Buc 037
Fig. 4-10: Type of Fermentation II (citric acid production)
- 81 -
Type of fermentation III:

Here the primary metabolism is also separated from the product formation.
Contrary to type II the product is not deduced from the primary metabolism
for energy uptake, but from the amphibolism (secondary metabolism). In
the beginning of these fermentations, a growth of biomass is found (primary
metabolism), while later on the product is formed by secondary metabolism.
Examples of this type of fermentations are the antibiotic productions such as
penicillin, streptomycin, etc. Also synthesis of vitamines, fats and some
biopolymers belong to this group.
25 1.25
S
20 1.00
a) Cell mass- and product concentrations
and utilized substrate, 15 0.75 X
X,S P
P
b) Growths-, production- and substrate 10 0.50
conversion rates and
X Biomass dry wt, g/l
c) specific rates as function of time 5 .25 P Penicillin, g/l
S Substrate us ed, g/l
0
a) 0 20 40 60
Time [t]
80 100 120 140
0.5 0.020 1.0 RX Growth, g/(lh)

RP Penicillin synthesis, 0.25 0.0012 0.12 Growth, g/(g h)
g/(l
h) penicillin synthesis,
0.4 0.015 0.8 RS Sugar utilization,
0.10 g/(g
h)
g/(l
h) 0.20 0.0010 Sugar utilization,
QO2 Oxygen uptake, g/(l h) g/(g
h)
0.3 0.6 0.0008 0.08 Q2X-1 Oxygen
0.010 0.15 uptake, g/(g h)
, Q2,X-1
0.2 0.4 0.0006 0.06
RX ,RS RP QO2
0.005 QO2 0.10
0.1 0.2 RS 0.0004 0.04
RX RP Q2X-1
0 0 0 0.05 0.0002 0.02
-0.1 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
b) Time [t] c) Time [t]
Buc 038-1
Fig. 4-11: Type of Fermentation III (penicillin production)
Significant for this type of fermentation is the appearance of a maximum of

growth- and substrate consumption rate, and also another maximum of
product formation which is moved in time.
- 82 -
The three types of fermentation processes are represented as

follows:
Type I Type II Type III
Time Time
growth substrate consumption product formation
Fig. 4 - 12: Schematic types of fermentation processes
4.3 Kinetic models of product formation

4.3.1 Model developed by Luedeking and Priret
(Lit.:R.Luedeking; E.L.Piret; J.Biochem. Mikrobiol. Technol., 1, 393 (1959)
This model distinguishes between a growth related production rate and a

non growth related production rate . It is also based on the classification
given by Gaden.
The production rate is demonstrated by the following equation:
dP dX
X (4 42)
dt dt
divided by the biomass the following equation is valid:
1 dP
(4 43)
X dt
The coefficients and can be obtained by the graphic (5-14).
- 83 -
= 1/X dX/dt
Fig 4 - 13: Estimation of the production rates and
First, this model was used to estimate the kinetic of the production rate of
lactic acid. It was shown that the production is neither alone proportional to
the growth rate nor to the biomass concentration. Using equation (4-42) the
following types of fermentation can be distinguished.
Type A: The production rate is not related to the growth rate, that means
=0
dP
X (4 44)
dt
Type B: The production rate is only related to the growth rate, that
means = 0
dP dX
(4 45)
dt dt
An example of this particular type is the fermentation of sorbose with
Acetobacter suboxidans.
Type C: The production rate is related to the growth rate as well as to

the cell density
dP dX
X (4 42)
dt dt
- 84 -
An example of this type is the fermentation of lactic acid with Lactobacillus

delbrckii.
Type C
Type B
Type A

1/XdX/dt
Fig 4 -14: Graphic representation of production types as defined by

Luedeking & Piret
Transforming Eq. (4 - 42) into:

dP 1 dX

dt dt
it is evident, that for = max the following expression is valid:
dP 1 dX

dt max dt
that means
dP dX
const
dt dt
In some cases the formed product is not stable, e.g. penicillin production.
Here the product hydrolysis is in an aqueous solution identical to a 1st order
reaction. This phenomena can be taken into account by using a destruction
term:
- 85 -
dP dX
X P (4 46)
dt dt
4.3.2 Influence of the product on the growth of organisms
The influences of the product on growth behavior can be described in

analogy to the enzyme kinetics.
Competitive inhibition:
dX 1
max X (4 47)
dt P
K S 1 S
K P
Noncompetitive inhibition:
dX S KP
max X (4 48)
dt KS S K P P
Furthermore, some empiric assumptions are proven in practice like the

linear equation defined by Hinshelwood:
dX
X 1 a P (4 49)
dt
a: empiric proportional constant
- 86 -

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Bioprocess- and Bioreaction Engineering Prof. Dr.

Enzymes are proteins, which act as catalysts in biochemical reactions. Their

Table 3-1 Classification of enzymes

1. Oxidoreductases (redox reactions) 4. Lyases (additions at double bindings)

2. Tranferases (transfer of functional groups) 5. Isomerases (converting to isomers)

3. Hydrolases (hydrolytic reactions) 6. Ligases (forming bindings by using ATP splitting)

Table 3-2 Industrial relevant enzymes

Enzyme microorganism reaction application

3.1 Kinetics of enzymatic reactions

Fig. 3-2: Influence of substrate concentration on rate of a simple enzymatic

a) For low substrate concentrations the rate of reaction is proportional to

3.1.1 Derivation of the Michaelis-Menten-Equation

The general theory of enzyme kinetics was developed in 1913 by

The formation rate of the enzyme-substrate complex (neglecting the back-

[ET] = Total amount of enzyme (free and bound)

It is possible that [ES] is formed from E and P by a reverse reaction,

For the decomposition of the enzyme-substrate complex the following

If production- and decomposition rate of [ES] are equal, a steady state

For d[ES]/dt = 0 and [ES] = const.

Rearranging of the equation yields the Michaelis-Menten constant:

From eq. (3 5) the equilibrium concentration of the enzyme-substrate

This steady-state concentration can be introduced in the initial production

Putting eq. (3 9) into eq. (3 8) the Michaelis-Menten equation follows:

substrate with the constant of Michaelis-Menten. The concentration of

Replacing [S] in eq. (3 10) by KM , V0 = Vmax/2 results. Therefore, KM is

Concentration-Time-Curve of an Enzyme- Substrate-Complex

Fig. 3-4: Concentration-Time-Curve of an Enzyme-Substrate-Complex

3.1.2 Transformation of the Michaelis-Menten Equation

Fig. 3-5: Lineweaver-Burk plot

Other linearizations yield additional equations:

Fig. 3-7: Hanes plot

3.1.3 pH- and temperature influence on enzyme activity

The activity of enzymes is influenced by the pH-value and temperature. The

The next figure shows possible curves of pH-activity of enzymes.

Fig. 3-8: Activity dependence on pH for various enzymes

The temperature dependence of enzymatic activity also exhibits an

1) Increasing reaction rate from increase of temperature (c.f. chemical

Most enzymes are deactivated at temperatures above 55 60 C, some are

3.2 Mechanisms of complex enzymatic reactions

Most of enzymatic reactions can be described with Michaelis-Menten

c.f. a) Substrate activation: The binding of the first substrate molecule

Further examples for more complex enzymatic reactions are:

1) One substrate reacts to two products.

3.3 Enzyme inhibition

Enzyme inhibition can be reversible or irreversible. During irreversible

3.3.1 Competitive inhibition

Competitive inhibition means a competition of inhibitor and substrate for

ES: Active enzyme-substrate complex

For the dissociation of an enzyme-inhibitor complex EI into free enzyme E

For I=0 the equation is equivalent to the Michaelis-Menten equation. By

3.3.2 Noncompetitive inhibition

Noncompetitive inhibition means no competition between substrate and

In this case, the Michaelis-Menten constant remains unchanged, but the

3.3.3 Uncompetitive inhibition

The reaction rate can be expressed by:

The linearized form of eq.(3 22) is:

In this case the Michaelis-Menten constant as well as the maximum reaction

1 Slope of the 1 Slope of the

without inhibitor without inhibitor

without inhibitor without inhibitor

Fig. 3-10 a-d: Lineweaver-Burk plots for different types of inhibition

3.3.4 Substrate inhibition

Substrate inhibition is the inhibitory influence of high substrate

The reaction rate for substrate inhibition is: