12.

4 DETERMINING ENZYME KINETIC CONSTANTS FROM BATCH DATA 621

12.4 DETERMINING ENZYME KINETIC CONSTANTS

FROM BATCH DATA

To specify fully the kinetics of MichaelisMenten reactions (see Section 12.3.3), two rate
constants, vmax and Km, must be evaluated. Estimating kinetic parameters for
MichaelisMenten reactions is not as straightforward as for zero- and first-order reactions.
Several graphical methods are available; unfortunately some do not give accurate results.
The first step in kinetic analysis of enzyme reactions is to obtain data for the rate of reac-
tion v as a function of substrate concentration s. Rates of reaction can be determined from
batch concentration data as described in Section 12.2. Typically, only initial rate data are
used. This means that several batch experiments are carried out with different initial sub-
strate concentrations; from each set of data the reaction rate is evaluated at time zero. The
initial rates and corresponding initial substrate concentrations are used as (v, s) pairs that
can then be plotted in various ways to determine vmax and Km. Initial rate data are preferred
for analysis of enzyme reactions because experimental conditions, such as the enzyme and
substrate concentrations, are known most accurately at the beginning of reactions.

12.4.1 MichaelisMenten Plot

This simple procedure involves plotting (v, s) values directly as shown in Figure 12.7.
vmax and Km can be estimated roughly from this graph; vmax is the rate as s-N and Km is
the value of s at v 5 vmax/2. The accuracy of this method is usually poor because of the dif-
ficulty of extrapolating to vmax.

12.4.2 LineweaverBurk Plot

This method uses a linearisation procedure to give a straight-line plot from which vmax
and Km can be determined. Inverting Eq. (12.37) gives:
1 Km 1
5 1 ð12:43Þ
v vmax s vmax
Therefore, a plot of 1/v versus 1/s should give a straight line with slope Km/vmax and
intercept 1/vmax. This double-reciprocal plot is known as the LineweaverBurk plot and is
found frequently in the literature on enzyme kinetics. However, the linearisation process
used in this method distorts the experimental error in v (Section 3.3.4) so that these errors
are amplified at low substrate concentrations. As a consequence, the LineweaverBurk
plot often gives inaccurate results and is therefore not recommended [3].

12.4.3 EadieHofstee Plot

If Eq. (12.43) is multiplied by v ðvKmax
m
Þ and then rearranged, another linearised form of the
MichaelisMenten equation is obtained:
v vmax v
5 2 ð12:44Þ
s Km Km

4. REACTIONS AND REACTORS

622 12. HOMOGENEOUS REACTIONS

According to Eq. (12.44), a plot of v/s versus v gives a straight line with slope 21/Km and
intercept vmax/Km. This is called the EadieHofstee plot. As with the LineweaverBurk
plot, the EadieHofstee linearisation distorts errors in the data so that the method has
reduced accuracy.

12.4.4 Langmuir Plot

Multiplying Eq. (12.43) by s produces the linearised form of the MichaelisMenten
equation according to Langmuir:
s Km s
5 1 ð12:45Þ
v vmax vmax
Therefore, a Langmuir plot of s/v versus s should give a straight line with slope 1/vmax and
intercept Km/vmax. Linearisation of data for the Langmuir plot minimises distortions in
experimental error. Accordingly, its use for evaluation of vmax and Km is recommended
[6]. The Langmuir plot is also known as the HanesWoolf plot.

12.4.5 Direct Linear Plot

A different method for plotting enzyme kinetic data has been proposed by Eisenthal
and Cornish-Bowden [7]. For each observation, the reaction rate v is plotted on the vertical
axis against s on the negative horizontal axis. This is shown in Figure 12.11 for four pairs
of (v, s) data. A straight line is then drawn to join corresponding (2s, v) points.
In the absence of experimental error, lines for each (2s, v) pair intersect at a unique point,

ν FIGURE 12.11 Direct linear plot for deter-

mination of enzyme kinetic parameters.
From R. Eisenthal and A. Cornish-Bowden,
1974, The direct linear plot: a new graphical
νmax procedure for estimating enzyme kinetic para-
meters. Biochem. J. 139, 715720.

ν4

ν1

–s4 –s3 –s2 –s1

Km

4. REACTIONS AND REACTORS

822 14. REACTOR ENGINEERING

advantages associated with batch culture. The risk of contamination is lower in batch sys-
tems than in continuous flow reactors; equipment and control failures during long-term
continuous operation are also potential problems. Continuous fermentation is feasible only
when the cells are genetically stable; if developed strains revert to more rapidly growing
mutants, the culture can become dominated over time by the revertant cells. Because
freshly produced inocula are used in batch fermentations, closer control over the genetic
characteristics of the culture is achieved. Continuous culture is not suitable for the produc-
tion of metabolites normally formed near stationary phase when the culture growth rate is
low; as mentioned above, the productivity in a batch reactor is likely to be greater than in
a CSTR under these conditions. Production can be much more flexible using batch proces-
sing; for example, different products each with small market volumes can be made in dif-
ferent batches. In contrast, continuous fermentations must be operated for lengthy periods
to reap the full benefits of their high productivity.

14.5.10 Evaluation of Kinetic and Yield Parameters in Chemostat Culture

In a steady-state chemostat with sterile feed and negligible cell death, as indicated in
Eq. (14.57), the specific growth rate μ is equal to the dilution rate D. This relationship is
useful for determining the kinetic and yield parameters that characterise cellular reactions.
Combining the Monod expression of Eq. (12.91) with Eq. (14.57) gives an equation for D in
chemostat cultures:
μmax s
D5 ð14:89Þ
KS 1 s
where μmax is the maximum specific growth rate, KS is the substrate constant, and s is the
steady-state substrate concentration in the reactor.
Equation (14.89) is analogous mathematically to the Michaelis
Menten expression for
enzyme kinetics, Eq. (12.37). If s is measured at various dilution rates, the same techniques
described in Section 12.4 for determining vmax and Km can be applied for evaluation of
μmax and KS. Rearrangement of Eq. (14.89) gives the following linearised equations that
can be used for Lineweaver
Burk, Eadie
Hofstee, and Langmuir plots, respectively:
1 KS 1
5 1 ð14:90Þ
D μmax s μmax
D μmax D
5 2 ð14:91Þ
s KS KS
and
s KS s
5 1 ð14:92Þ
D μmax μmax

For example, according to Eq. (14.90), μmax and KS can be determined from the slope and
intercept of a plot of 1/D versus 1/s. The comments made in Sections 12.4.2 through
12.4.4 about the distortion of experimental error with linearisation of measured data apply
also to Eqs. (14.90) through (14.92).

4. REACTIONS AND REACTORS

 14.6 STERILISATION 823
1 FIGURE 14.35 Graphical determination of the maintenance
Y'XS coefficient mS and true biomass yield YXS using data from chemo-
stat culture.

Slope = mS
1
YXS

1
D

Chemostat operation is also convenient for determining true yields and maintenance
coefficients for cell cultures. An expression relating these parameters to the specific growth
rate is given by Eq. (12.112). In chemostat culture with μ 5 D, Eq. (12.112) becomes:
1 1 mS
0 5 1 ð14:93Þ
YXS YXS D

where Y0XS is the observed biomass yield from substrate, YXS is the true biomass yield from
substrate, and mS is the maintenance coefficient. Therefore, as shown in Figure 14.35, a plot
of 1=Y0XS versus 1/D gives a straight line with slope mS and intercept 1/YXS. In a chemostat
with sterile feed, the observed biomass yield from substrate Y0XS is obtained as follows:
x
Y0XS 5 ð14:94Þ
si 2 s
where x and s are measured steady-state cell and substrate concentrations, respectively,
and si is the inlet substrate concentration.

14.6 STERILISATION

Commercial fermentations typically require thousands of litres of liquid medium and

millions of litres of air. For processes operated with axenic cultures, these raw materials
must be provided free of contaminating organisms. Of all the methods available for sterili-
sation, including chemical treatment, exposure to ultraviolet, gamma, and X-ray radiation,
sonication, filtration, and heating, only the last two are used in large-scale operations.
Aspects of fermenter design and construction for aseptic operation were described in
Sections 14.3.1 and 14.3.2. Here, we consider the design of sterilisation systems for liquids
and gases.

14.6.1 Batch Heat Sterilisation of Liquids

Liquid medium is most commonly sterilised in batch in the vessel where it will be
used. The liquid is heated to the sterilisation temperature by introducing steam into the

4. REACTIONS AND REACTORS