Chemical Reaction Engineering

Enzymatically catalyzed reactions

• An enzyme, E, is a protein or proteinlike substance with
catalytic properties.
• A substrate, S, is the substance that chemically transformed at
an accelerated rate because of the action of the enzyme on it.
• One enzyme can catalyze only one reaction. Unwanted
products are easily controlled.
• Enzymes are produced only by living organisms (bacteria, for
example).
• Enzymes usually work under mild conditions (pH 4~9, 75 ~ 160 F).
 Enzymes
• Most enzymes are named in terms of the reactions
they catalyze (***ase), for example:
• urease: the enzyme that catalyzes the decomposition of urea
• tyrosinase: the enzyme that attacks tyrosine
• Three major types of enzyme reactions:
– soluble enzyme - insoluble substrate (e.g. laundry detergents)
– insoluble enzyme - soluble substrate (similar to packed
catalytic bed rxn)
– soluble enzyme - soluble substrate (e.g. many biological rxns)
our interest
 Enzymatically catalyzed rxn example
The proposed mechanisms of the catalytic action of urease which causes urea
to decompose into ammonia and carbon dioxide : (Levine and LaCourse, 1967)

1.The enzyme urease reacts with the substrate urea to form an enzyme-substrate complex, E•S:
S E E•S
NH
2 
CONH
2 


k
1

urease
[NH
2CON
2
*
urea
]
2.This complex can decompose back to urea and urease:

[
NH 
CONH
2 2 
*

k
2

urease
] NH
CON
2 2ure
3.Or, it can react with water to give ammonia, carbon dioxide, and urease:
W P
[
NH 
CONH
2 2 
*
2 


k3
urease
]HO 2
NH
3 
CO
2ur
 SE
k
1
ES
ES

k2
ES

ES
W 

k
3

PE
The rate of disappearance of the substrate is: 
rSk
1(
E)(
S
)k
2(
E 
S)

The net rate of formation of the E•S complex is: 

r

E
Sk(
E
1 
)(
S
)k
2(
E
S
)k
3(
W 
)(
E
S)

t)
The enzyme is not consumed by the reactions: (E )
(E (ES)
PSSH  rES  0

k(
E )(
S
)
(
E 
S) 1t
k
(
1S
) k
2k
3(
W )

kk (
W )(
Et)(
S)

r  13
 
S
k
1(
S) k2 k3(
W )
 kk (
W )(
Et)(
S)

r  13
 
S
k
1(
S) k2 k3(
W ) k’3
k k Since the reaction of urea and urease is carried out
Km  3 2
k1 in aqueous solution (water): (W) ~ constant
Vmax 
k3(E S) This is the form of the “Michaelis-Menten Equation”
t)(
r
S
(S)Km
and Km is call the Michaelis constant

At low substrate concentration: (S) << Km -rS

V (S) Vmax
rS  max
Km
At high substrate concentration: (S) >> Km Vmax/2

rS Vmax
Km (S)
V
In a special case, when rS  max KmS|rsVmax
/2
2
Km is equal to the substrate concentration at which
the rate of reaction is equal to one-half the maximum rate.
 Michaelis-Menten equation
Vmax(S)
rS 
(S) Km

Vmax k3 (Et )

Vmax and Km characterise the enzymatic reactions described by Michaelis-Menten kinetics.

Vmax is dependent on total enzyme concentration.
Km is independent of total enzyme concentration.
 Michaelis-Menten kinetics example
SE
k
1
ES ES

k2
E 
SES
W 

P
E k
3

3
C
ur
e
a (
kmo
l
/
m ) 0.
2 0
.
020
.
010
.
0050
.
00
2
-
r
ur
e
a (
kmo
l
/
m 
3
s)1
.
08 0
.
550
.
380
.
20 .
09

V (S) 1(S
)
Km 1 K
rS  max   m
(S)Km 
r
S V(
S)V
max V
max(S
)
max

Km
Slope = 0.02 =
1/-rs Vmax 1.33
C
r urea

S
0
.0266C
1 ure
Intercept = 0.75 =
Vmax

(1/S) (i.e. 1/Curea)

 Batch enzymatically catalyzed rxn
Artificial kidney design
dC V C
A batch reactor in liquid phase: 
r 
 
urea
max
urea

urea
dtC K
urea
m

KC 0 C C
1
ln
1 Vmax tmln 
ureaurea
0 urea

t 1 X Intercept = V C
max
urea V
max
Km
CC 1X)
0(
Curea0
Slope =  1 1 V C X
Km ln  
max
urea
0

t 1XKm Kt
m

X
t
 Inhibition of enzyme reactions
• The rate of enzyme-catalyzed reactions is affected by
pH and inhibitors.
• Three most common types of reversible inhibition:
– Competitive
• Substrate and inhibitor are usually similar molecules that compete for the
same site on the enzyme.
– Uncompetitive
• The inhibitor deactivetes the enzyme-substrate complex, usually by attaching
itself to both the substrate and enzyme molecules of the complex.
– Noncompetitive
• Enzymes containing at least two different types of sites. The inhibitor
attaches to only one type of site and the substrate only to the other.
 Bioreactors
• Microorganisms and mammalian cells are used to produce a
variety of products, such as insulin, most antibiotics, and
polymers.
• Advantages:
– mild reaction conditions
– high yields
– can catalyze successive steps in a reaction for organisms
contain several enzymes
– stereospecific catalyst (single desired isomer can be formed)
In general, the growth of an aerobic organism follows:

[ 
cells
]
[ [ 
carbon
source
]
nitrog
sourc
]
[
oxyg
sou
]
[
ph 
so
]
.








,



culture
media
 
conditions
(pH

temperatur
e
,
etc
)
[
CO
]
[
2H
O
2]
[
pro
]
[
moce
]



cell

Substrate
More
cells
Pro
duct

The rate of this reaction is proportional to the cell concentration and

the reaction is autocatalytic.
Four phases are included in cell growth:
Log cell concentration
2 3
1 4

time

• (1) Lag phase

– little increase in cell concentration
– synthesizing transport proteins for moving the
substrate into the cell
– synthesizing enzymes for utilizing the new substrate
– beginning the work for replicating the cell’s genetic
material
• (2) Exponential growth phase
– the cell’s growth rate is proportional to the cell
concentration
– the cells are able to use the nutrients most efficiently
• (3) Stationary phase
– the cell reach a minimum biological space where the
lack of one or more nutrients limits cell growth.
– many important fermentation products, including
most antibiotics, are produced in the stationary phase
• (4) Dead phase
– result of either the toxic by-product and/or the
depletion of nutrient supply
 Rate laws of bioreactors
 


 
conditions
Cells
Substrate
More
cells
Pro
duct

The most commonly used expression is the Monod equation for exponential growth:

rg  Cc

Cell concentration
Cell growth rate
Substrate concentration
C
Specific growth rate =  s

s
max
K Cs

Maximum specific growth reaction rate

Monod constant
 p 
n
C C  C  max
g
r  c s account for inhibition
r
1
C
cCs
max
Ks C (one model here) g
 C*
s
s
 p K Cs
Monod equation
where Cp* = product concentration at which all metabolism ceases and n = experimental constant

Other cell growth rates:

 C 
s
Tessier equation r
g
1
max 

exp Cc
  k


max
C
Moser equation rg  
c

(1kC
s )

The cell death rate is:

d
r d
(k k
tCt)
C c
Specific toxic death rate constant
Specific natural death rate constant
Concentration of a substrate toxic to the cell
 Stoichiometry for cell growth
 


 
conditions
Cells
Substrate
More
cells
Pro
duc
• Very complex : vary with microorganism / nutrient
system; vary with environmental conditions; even
more complex when more than one nutrient contributes
to cell growth.
• May be simplified as: S 
cells
Yc C
/s 
Y p P
/s

mass
of
new
cells
for
where Yc/s is the yield coefficient : Y
c
/
s
mass
of
substr
consu
to
pro
ne
c
mass
of
prod
form
where Yp/s is the product coefficient : Y
p
/
s
mass
of
substr
cons
to
for
pr
 Produce new cells
Substrate consumption
Maintain a cell’s daily activities

mass
of
substrate
consumed
for
m
aintena
ce

m typical value = 0.05 h-1
of
mass
cells
time

Therefore, the rate of substrate consumption for maintenance: rsm  mCC

The yield coefficient, Y’c/s , account for substrate consumption for maintenance:

mass
of
new
cells
formed

Y
c
/s
mass
of
substrate
consumed
 During the growth phase rp Yp/crg

Product formation

During the stationary phase rp Yp/s(rs)

The net rate of substrate consumption:


rs
Ys
/cr
gY
s
/pr
pmC
c

Consumption rate for maintenance

Consumption rate to form product

Consumption rate by cell growths

 
rs
Ys
/cr
gY
s
/pr
pmC
c

If the product if formed

during the growth phase
s
r Y
s/crgmC
c rp Yp/ crg

If the product if formed

during the stationary phase
r
snY
snr
/p pmC
c rp 
kpC C
sn c

KsnCsn
where Csn is the concentration of the secondary nutrient
 Mass balance on cells
For a CSTR, a mass balance on the microorganism gives :

Rate of accumulation of cells

dC
Vc
vC
0c
0 vC
c(
rgr
)
dV
dt

Rate of net generation of live cells

Rate of cells leaving

Rate of cells entering

 Mass balance on substrates
For a CSTR, a mass balance on the substrate gives :

Rate of accumulation of substrate

dC
V s
vC
0 s
0 
vCs
rV
s
dt

Rate of net generation of substrates

Rate of substrate leaving

Rate of substrate entering

 Mass balance on cells
For a batch system, a mass balance on the microorganism gives :

Rate of accumulation of cells

dC
V c g
(r r
d)V
dt

Rate of net generation of live cells

 Mass balance on substrates
For a batch system, a mass balance on the substrate gives :

Rate of accumulation of substrate Rate of substrate used for cell growth

dC
In the growth phase Vs
r
sV
Ys
/(
cr
g)
VmC
cV
dt

Rate of substrate used for maintenance

dC
In the stationary phase Vs
Ys (
/pr
p)
VmC
V
c
dt

dC
V   
p
Rate of product formation r
pVYp
/s(r)
sV
dt
Example: bacteria growth in a batch reactor
A fermentation process is carried out in a batch reactor. Plot the concentrations of cells,
substrate, and product and growth rates as functions of time. The initial concentration is
1.0 g/dm3 and the substrate concentration is 250 g/dm3.

Values of the parameters:

C*p 93 3
g/dm Yc / s  0.08 g / g
n0.52 Yp / s  0.45 g / g
max0.33
h1 Yp / c  5.6 g / g
Ks 1.7g/dm
3
k d  0.01h 1
m0.03
(gsubstrate h)
)/(gcells
Mass balances Rate laws Stoichiometry
 C 
0
.
52
rp Yp/crg
Cells:
dC
V c g
(r r
d)V r*
1
 C
p
 K
CC
c s

 s
g max
dt C
 p s

dC
Substrate: V s
Ys
/c
(r)
gV
r Vr  k C
sm d d c
dt

dC
V  rsm  mCc
p
Product Y
p/c(
rg)
V
dt
0
.
52
C C
dC
c
 
1


max
dt  C

p

*

c
K
C
s

C
k
C
dc
 s s
p


0
.
52
dC C CC
s
Y 
1
p cs
mC
dt
s/ 
cmax
C
*

KC
c
 p
 s s
dC
Yp/crg
p

dt
 Chemostats
• Chemostats are essentially CSTRs that contain
microorganisms.
• One of the most important features of the
chemostat is that is allows the operator to
control the cell growth rate.
– By adjusting the volumetric feed rate