108 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

If the complex of ES/ can be dissociated to product, the rate equation would result in mixed
competitive and non-competitive inhibitors:

EIS —~» EI +P (5.7.4.5)

app S
= Umax_ (5.7.4.6)
S+K*P

The competitive and non-competitive inhibitors are easily distinguished in a Lineweaver—

Burk plot. The competitive inhibitor intercepts on the 1/v axis whereas the non-competitive
inhibitor intercepts on the 1/S axis. The reaction of inhibitors with substrate can be assumed
as a parallel reaction while the undesired product is formed along with desired product. The
reactions are shown as:

A—*-»R_ desired (5.7.4.7)

A—2-+§ undesired (5.7.4.8)

Since enzyme is not shown in the reaction we assume an elementary rate equation may
explain the above reactions. The simple kinetics are discussed in most fermentation tech-
nology and chemical reaction engineering textbooks.*"!°

Example 1

An enzyme is produced for manufacturing a sun protection lotion. Given kinetic data for
1
the enzyme reaction with v,,,, = 2.5 a: Km = 8.9mM and Sy = 12 mM, what would be
ms
the time required for 95% conversion in a batch bioreactor?

Solution

3
Vi. =| 2.5 mmol \f 3600 s lm -~9 mmol
m°s h 1000 L hL

8.9mM, 12 (0.95)(12)
thatch —Q_,, na +t = 4.23h
9mM/h_ 0.6

Example 2

Calculate K,, and V,,,, for given substrate concentrations and rates. The inverse rate and
substrate concentrations are calculated in Table E.2.1.
 GROWTH KINETICS 109

TABLE E.2.1. Substrate concentration

and reaction rate

S (mol-1~') v (mol-1~! -min7!)

4.10 x 107-3 1.77 X 1074

9.50 x 10-4 1.73 X 10-4
5.20 X 1074 1.25 x 1074
1.03 x 1074 1.06 X 10-4
4.90 x 1075 8.00 x 10-5
1.06 x 1075 6.70 X 1075
5.10x 1076 4.30x 10-5

TABLE E.2.2.

1/S, 1-mol7! 1/v, 1-min-mol7!

243.9 5650
1052.6 5780
1923.0 8000
9708.7 9434
20408.1 12500
94339.6 14925
196078.4 23256

Solution

Let us inverse the substrate concentration and reaction rate as shown in Table E.2.2.
In evaluation of kinetic parameters, the double reciprocal method is used for linearisa-
tion of the Michaelis-Menten equation (5.7.3).
(a) Use the Lineweaver—Burk plot as defined in the following relation:

11, Kk, 1 (E.2.1)

Slope = 22500— 10000 0.077

ee 1.875X10° —2.5X10°

Vmax = 1.25 10* mol/-min

Am 0.077
vmax
(E.2.2)

K,, =9.6X10° mol/l

 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

25000

1/v, |. min. mol"! 20000 -

15000 +

10000 +

5000 7 1
Umax

0 q q q q

0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5

1/s, I/mol

Fic. E.2.1. Lineweaver—Burk plot.

TABLE E.2.3. Data collected and calculated for rate model

S (mol:17!) v, mol:1~!-min~! S/v, min v/S, min“!

4.10 x 10-3 1.77 X 10-4 23.00 0.044

9.50 xX 10-4 1.73 X 1074 5.50 0.182
5.20 X 10-4 1.25 x 1074 4.20 0.240
1.03 x 1074 1.06 x 10-4 0.97 1.030
4.90 x 1075 8.00 x 1075 0.60 1.670
1.06 X 10> 6.70 X 1075 0.16 6.250
5.10 x 107° 4.30 X 1075 0.12 8.330

(b) Use another form of linear graphical presentation to evaluate K,, and V,,,, based on
the following relation:

s Ky,
1 ¢ (E.2.3)
Vv Vv max
Vv. max

This method tends to create a cluster of data near the origin as shown in Figure E.2.2.

_ 22.5—6.0
Slope pF = 5500

Vmax = 1.82 X 107* mol/-min

 GROWTH KINETICS 111

25

SA, min

0 TT qT qT qT qT

0.000 0.001 0.002 0.003 0.004 0.005

S, mol/

Fic. E.2.2. Linear model for the Monod rate equation with populated data at the origin.

10
vw/S, min"

Q-

-2
9e5 4eS 6e5 B8e5 1e4 1e4 1e4 ed 2e-d 2e-4
v, mol. .min"'

Fic. E.2.3. Eadie—Hofstee plot.

0.5
K,, =~ =9.1X10-°mo
5500

For the Eadie—Hofstee plot, both coordinates contain rates that are subjected to the great-
est error, as indicated in Figure E.2.3.
112 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Y= Vmax Kn (E.2.3)

A plot of u/S versus v is presented in Figure E.2.3 for defining slope and intercept, K,, and
Urnaxs respectively.
A linearisation model is used to explain the equation of a simple straight line:?

y=bxt+a (E.2.4)

where

b=. —___ (E.2.5)

(E.2.6)

X,y are average values of x; and y;

Slope of the line = 1

K

K,, =6.5 X10 mol-I”’

Umax = 18 X10* moll” min“!

Example 3

In batch enzyme reaction kinetics, given K,,= 1073 M and substrate concentration S, =
3X 107° M, after 2 min, 5% of the substrate was converted. How much of the substrate was
consumed after 10, 20, 30 and 60 min?

Solution

For S,<<K,,, the rate model is reduced to a first-order rate equation:

The Michaelis—Menten rate equation is:

max (E.3.1)
 GROWTH KINETICS 113

The simplified first-order rate is

(E.3.2)

Carry out integrations:

(E.3.3)

(E.3.4)

The concentration profile is predicted by the following equation

S = Sye™" (E.3.5)

At time equal to 2 min:

S
(E.3.6)

(E.3.7)

(E.3.8)

(E.3.9)

= 0.2565 min

S _ 470.02
Y= 0.025651
So (E.3.10)
114 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Conversion

X,=1- = =] — @ 0.025654 (E.3.11)

oO

For t= 10 min, X, = 1 — ¢ °° = 0.226 or 22.6%.

For t= 20min, X, = 1 — e-?-56529) — 9.401 or 40.1%.
For t = 30min, X, = 1 — ¢°0-569 = 0.5367 or 53.67%.
For t= 60 min, X, = 1 — e~?-756569) — 9.785 or 78.5%.

Example 4

In a complex enzyme reaction, multiple substrate-enzyme complexes are formed. Assume

the following reaction mechanisms are taking place in three consecutive stages:

St+tE<*~5ES,<«©245 ES, —*» P+E (E.4.1)

Develop a suitable rate expression using the Michaelis—Menten rate equation and the quasi-
steady-state approximations for the intermediate complexes formed.

Solution

Equilibrium dissociation constants are defined as

1
_k, _ [EIS]
TEs (E.4.2)

k, — [ES,]
2 ==
i, [ES] ( E43 )

v= k.[ES] (E.4.4)

ES
LES, | = L651] (E.4.5)
2

[E]\[S]
[ES 1 ]-———=
K, ( E.4.6 )

y= “SES J= ‘sets
K, K,K> (E.4.7)
 GROWTH KINETICS 115

The total enzyme concentration is:

f= BES, es, ++ ENS). LEIS) _ pfs] 2 ! I (E.4.8)

1 KK, K, 12

E= é
(E.4.9)
(Ss
KK,

(E.4.10)

Umax = (E.4.11)

The final rate expression is:

k.
k5 s] kse,[S [Ae2 51
v= €, x [ _ 5€ol ] _
(E.4.12)
KK, Gerag K,K, +(K, +1)[S] [Ht hs
14 2

Example 5

Pesticide inhibition on an active enzyme has been reported, which caused enzyme activi-
ties to reduce. The collected data with and without inhibition are presented in Table E.5.1.
Determine the rate model with and without inhibitor (see Table E.5.1). Also define the type
of inhibition.

TABLE E.5.1. Substrate concentration and enzymatic rate calculation

with and without inhibition

S, mol-17! v (no inhibitor) v’ (inhibitor)

mol-1~!-min=! X 10® —s mol-17~!-min7! x 10°

3.30 x 1074 56 37
5.00 x 1074 71 47
6.70 X 1074 88 61
1.65 X 1073 129 103
2.21 X 1073 149 125
116 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

TABLE E.5.2. Inverse substrate concentration and inverse enzymatic rate calculation with and
without inhibition

1/S, 1-mol7! v (no inhibitor) 1/v v’ (inhibitor) 1h",

mol:1~'-min7! x 10° mol-1~!-min7! x 10° 1-min:mol7!

3030 56 17857 37 27027

2000 71 14085 47 21277
1492 88 11363 61 16393
606 129 7752 103 9709
452 149 6711 125 8000

Solution

Plot both sets of data as a Lineweaver—Burk plot for competitive inhibition (see Fig. E.5.1):

ke,S
v=
(E.5.1)
sexi)
k;

The mechanism of the enzyme with substrate in the present of inhibitor is:

E+S <4 ES (E.5.2)

E+IoEl (E.5.3)
ES
> E+P (E.5.4)
e, = E+[ES]+[EI] (E.5.5)
Without inhibition:

ke,s ke,S
v= =
(E.5.6)
S+K, S+84x10%

K*? = K, [+t (E.5.7)

ki

1.51073 =8.4x104 a (E.5.8)

I

+ = 0.786 (E.5.9)
k;

i 10°
,———= —— = 1.7310 pu (E.5.10)
0.786 0.786
 GROWTH KINETICS 117

Plug in a number

ke,X6.7X10*
88X10°° =
6.7X10 *+8.4X102 (E.5.11)

ke, =1.2X10? min’

1.21073 X6.7x10+
61X10 °= (E.5.12)
6.7X1074 +8.4x 1073 ( + i)
i

c = 0.49 (E.5.13)
1

The reading from the plot for the rate without inhibition:

{2IZSXW
—5)x10° _,
Slo
P 3000

kapp = 15X10
_ —3

For the data plotted with inhibition:

(18—5)X10° _
Slope 4.2
Pe 3100
35000

30000 - e@ Without inhibition

© with inhibition
25000 -
1A, |.min.mol'

20000 -

15000 +

10000 -

5000 +

-1000 0 4000 2000 3000

1/S, I/mol

Fic. E 5.1. Competitive inhibition based on the Lineweaver-Burk model.

118 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

As 42
Umax

K, =8.4X10* mol/L

Umax = 2*10~* mol/min

Based on the graphical presentation, this is a competitive inhibition.

Example 6

The respiratory quotient (RQ) is often used to estimate metabolic stoichiometry. Using
quasi-steady-state and by definition of RQ, develop a system of two linear equations with
two unknowns by solving a matrix under the following conditions: the coefficient of the
matrix with yeast growth (y = 4.14), ammonia (yy = 0) and glucose (y, = 4.0), where the
evolution of CO, and biosynthesis are very small (0 = 0.095). Calculate the stoichiometric
coefficient for RQ = 1.0 for the above biological processes:

Solution

At quasi-steady-state condition:
+ +
d[NADH H 12 xq—29- 3] v,—v,(1+a)— 2x05 |=0 (E.6.1)
dt n

RO= moles of CO, _ ato

+
(E.6.2)
moles of O, b

v,a 1 )
7 2b = 3 E — Uy +8)
a—b(RQ)=-a (E.6.3)

The mathematical solution for the above system is set as given by the following matrix:

]
= —2 f- > ¥p—¥s(1+0)—— vy (E.6.4)
1 —(rg)|-?! |-o
At singular point, let

2 =0 (E.6.5)