Sample For Official Solution Manual For Bioprocess Engineering 3rd Edition - Michael Shuler, Fikret Kargi

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Chapter 3
Problem 3.1
S  E  k1  ES 1  k 3 (ES) 2 

k k k5
P  E
2 4
rate  k5  ES 2
a.) Equilibrium approach

* r values are the same as k values all are rate constants
r1  S  E   r2  ES 1
(1)
r3  ES 1  r4  ES 2
(2)
 E 0    E    ES 1   ES 2 (3)
r
 ES 1  4  ES 2
from (2) r3 (4)
r2 r4  ES  2
 E 
r1r3  S
from (1) + (4) (5)
Substitute (4) and (5) into (3):
 r2 r4  r1 r4  S   r1 r3  S  
 E 0      ES 2
 r1 r3  S 
r1 r3  E 0   S 
  ES 2 
r2 r4  r1 r4  S   r1 r3  S 
r5 E 0 S r2 r
rate  where Km1  , Km 2  4
Km 2  Km1  S   S r1 r3
b.) Quasi–steady–state approach
d  ES 1
 r1  E   S  r2  ES 1  r4  ES 2  r3  ES  1  0
dt (6)
d  ES 2
 r3  ES 1  r4  ES 2  r5  ES  2  0
dt (7)
r r
 ES 1  4 5  ES 2
from (7) r3 (8)
 r2 r4  r2 r5  r3 r5 
 E      ES 2
 r1 r3  S 
from (6) + (8): (9)
substitute (8) and (9) into (3):
1
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 r2 r4  r2 r5  r3 r5  r1 r4  S  r1 r5  S   r1 r3  S  
 E 0      ES 2
 r1 r3  S 
 r1 r3  E 0   S 
 ES 2   
 r2 r4  r2 r5  r3 r5  r1 r4  S   r1 r5  S   r1 r3  S  
r5 E 0 S
 rate 
 Km2  r5 r3   Km1  S   r5 r1  S
r2 r
Km1  Km 2  4
where r1 and r3
Problem 3.2
* r values are the same as k values all are rate constants
E  S  1  ES   2 E  P
r r
r1 r2
d  ES
 r1  E   S  r2  E   P   r1  ES   r2  ES   0
dt (1)
d  P
rate   r2  ES  r2  E   P 
dt (2)
 E 0    E    ES ,   E    E 0    ES 
(3)
r1  r2
 E   ES
r1  S   r2  P 
from (1): (4)
 r1  S   r2  P   r1  r2 
 E 0      ES
 r1  S   r2  P  
Combine (3) + (4):
E 0  r1  S   r2  P  
  ES 
r1  S   r2  P   r1  r2
(5)
Substitute (3) and (5) into (2):
r1 r2 E 0  S  r2 r2 E 0  P 
rate   r  2  E 0   ES    P 
r1  r2  r1  S   r2  P 
Substituting (ES) in terms of E0:
r1 r2 E  S   r1 r2 E 0  P 
rate 
r1  r2  r1  S   r2  P 
LetVS = r2 E0 and VP = r-1 E0
2
r1 Vs  S   r2 VP  P 
rate 
r1  r2  r1  S   r2  P 
Divide top and bottom by (r-1 + r2) and let

1 r 1 r
 1  2
K m r1  r2 and K P r1  r2
1
 rate 
V s K1m  S   VP K P  P
S P
1 1 
K m KP
Problem 3.3
k 1  k 2
Km   4.5 105 M
a) k1
E 0  106 M
S  103 M
V S
V  m1
b)
Km  S
but Vm  r2 E 0
 V  9.58 104 M sec 1
Problem 3.4.
At low substrate concentrations So< 150 mg/l substrate inhibition is negligible.

V0 = Vm0 So / (Km + So) or 1/V0 = 1/ Vm + Km/Vm (1/S0)
For S0 < 150 mg/l Plot 1/V0 versus 1/S0
Slope = Km/Vm0 = 13.8 y-intercept = 1/ Vm = 0.023
Then, Vm = 43.5 mg/l-h and Km = 600 mg/l
At high substrate concentrations above 150 mg/l , substrate inhibition is significant.

V0 = Vm0/ (1+ S0/Ksı) or 1/V0 = 1/Vm0 + S0/Vm Ksı
For S0 > 150 mg/l plot 1/V0 versus S0
Slope = 1/ Vm0Ksı = 2.59x 10-3 Then, Ksı = 8.9 mg/l
Low Ksı indicates severe substrate inhibition.
Problem 3.5.
Plot 1/V versus 1/S at different inhibitor concentrations

Since the lines intercept at the same point on y-axis inhibition is competitive (Constant Vm,
increased Km).
For I = 0 , No inhibitor : From the intercept on y axis , 1/Vm = 0.2 and Vm = 5 mM/h
3
And from the intercept on X-axis, - 1/Km = -1.2 and Km = 0.83 mM

From 1/V versus 1/S plot for I = 1.3 mM and S0 = 0.50 mM V = 1.3 mM/h
Then, V = Vm S/ (Km(1+I/Ksı)) + S , 1.3 = 5(0.5)/ (0.83(1+1.3/Kı) + 0.5 )
Then Kı = 1.82 mM
Problem 3.6 .
a. V = Vm (1 + A/KA) S /(Ks + S) = Vm (1+A/KA) / (1+Ks/S)

Define Vm(1+ A/KA) = Vapp and plot Vapp versus activator (A or M0) concentration
Slope = 9.2x 10-3 = Vm/KA and y –intercept = Vm = 0.04 ml/h
Then, KA = 4.35 ug/l
b. V= Vm (1+ KA) = 0.04(1+60/4.35) = 0.59 ml/h
Problem 3.7.
a. V = KL (S0 - Ss) = Vm Ss / (Km+ Ss)

Ss = S0 – V/KL Calculate Ss at different RPMs
RPM 25 50 100 200 300 400
Ss(mg/l) 33.3 50 78.8 101.2 150 180
b. V = Vm Ss/ (Km+ Ss) or 1/V = 1/Vm + Km/Vm (1/Ss)
Plot 1/V versus 1/Ss at different RPMs
1/Ss 0.03 0.02 0.0127 0.0099 0.0067 0.005
1/V 0.033 0.0277 0.025 0.024 0.0227 0.022
y- intercept: 1/Vm = 0.02 Vm = 50 mg/l
Slope : Km/Vm = 0.42 Then, Km = 21 mg/l
Problem 3.8
E
H 2 O  CO 2    HCO3  H 
Plot 1/V versus 1/S (Lineweaver – Burk plot)
1 1 1

y – intercept = Vm k 2 E 0 , x – intercept = K m
1 40
  4  103
V  Co 2 
Hydration:
1 V  2.50 104 M sec 1

 4000M 1 sec  m
Vm r2  8.93 10 4 sec 1
1 4000
  100M 1  K m  0.01M
Km 40
1 163.15
  1.31104
V  HCO3 

Dehydration:
4
1 V  7.61105 M sec 1
 1.31104 M 1 sec  m
Vm r2  2.72 10 4 sec 1
1 1.31104 1
 M  K m  1.24  102 M
Km 163.15
Problem 3.9
1 1
 9.525  0.60
a.) Plot 1/V versus 1/S: V S
1 0.60
  0.063M 1  K m,app  16.0 g S / L
K m,app 9.525
SinceKm,app> Km the inhibitor is competitive.
b.)
5
  I 
K m,app  K m 1  
 KI 
KI 
 I  1.37g I L
K m,app K m  1
Problem 3.10
Plot 1/V versus 1/S
a.) For E0 = 1.6 g/L

Km = 0.0246 mmol/ml at 30°C
Km = 0.0238 mmol/mlat 49.6°C
1 mmol
Vm   3.31
b.) 0.302 ml.min
c.) The inhibitor is competitive.

  I   I   0.6 mmol mL
K m,app  K m 1  
 KI  K m,app  0.052 mmol mL
Problem 3.11
MW ATPase  5  104 g mol, K m  110 4 mol L

molecules ATP
S  0.02 mol L , r2  1 10 4
min molecule enzyme
r
ATP 
ATPase
 ADP  Pi or E  S  r 1 ES 
r2
E  P
1
Enzyme inactivation kinetics: E = E0e-rt, r = 0.1 min-1

V S
V m
Michaelis-Menten Kinetics: Km  S
6
 V  Vm  r2 E
But S K m ,
dp
  r2 E 0e  rt
dt
0.002 

0
dp  r2 E 0  e rt dt
0
 r2 E 0
0.002 M   1 , E 0  2.0 10 8 M
r
mol  g   6 g 
2.0 108   110 3 L    5 10 4   110   1.0g
L  mol   g 
1.0 g
  0.1
Fraction of pure enzyme 10  g
 10% of the crude protein was ATPase.
Problem 3.12
At steady – state: reaction rate = mass transfer rate
Vm1  Ss 
V  rL   Sb    Ss    J
S 
2
K m   Ss   s
K SI
Solving graphically,
a.) For [Sb] when the enzyme is inhibited and kL has an intermediate value, multiple steady
states are possible.
Multiple steady – states occur as the enzyme changes from being reaction controlled to
being diffusionally controlled.
7
b.) Yes. Diffusional limitations can decrease the substrate concentration such that it is no
longer inhibitory. Thus the apparent reaction rate will be greater than the intrinsic reaction
rate for [SS] less than [Sb].
Problem 3.13
By inspection,Vmax = 125 μmol/min, Km = 20 μmol/L

Plot V versus V/[S](Eadie- Hofstee) where y-int = Vm, x-int. = Vm/Km and slope = - Km.
Plotting the date and using the data points for high substrate concentration,
Vm = 130 μmol/min, Km = 8.24 μmol/L
From the plot it is obvious that the data do not fit into Michaelis – Menten kinetics at low
substrate concentrations.
Some of the error may be attributed to the method since both axes contain v. However, there
are two possible explanations for the deviations from Michaelis – Menten Kinetics:
(i) the enzyme is immobilized, thus diffusional effects become important

(ii) there might be unspecific binding of substrate to the enzyme thus requiring a critical
substrate concentration for the reaction to follow Michaelis – Menten Kinetics.
Problem 3.14
a.) Plot [P] versus t data for both cases.
8
Obtain rates by taking tangents at specific time points.
d  P
v  slope of tangent
dt
Obtain the amount of substrate present for both cases by a mass balance.
H 2O  Starch 
 dextrose
MW 18 180
amount of dextrose formed (0.1) = amount of H2O used

(180) (0.1) = 18
 starch remaining = S0 – (dextrose formed – H2O used)

= S0 – 0.9 ∆ P
Using initial time data, a plot of 1/V versus 1/S can be made.
9
Soluble:
1
 0.16
Vm
 1 mg   1 
Vm    
 0.16 mL  min   11600 units 
mg
 5.39 104
mL  min unit enzyme
1 mg
Km   575
0.00174 mL
Immobilized:
1  1 mg   1  4 mg
 0.12, Vm      1.80 10
Vm  0.12 mL  min   46 400 units  mL  min unit of enzyme
1
Km   575 mg mL
0.00174
b.) Plot In Vm versus 1/T for both cases.
Vm  r2 E 0  E 0 Ae  Ea RT
Ea Ea
ln Vm  ln E 0  ln A   slope 
RT R
Soluble:lnVm = 41.18 – 8299 (1/T)
 cal  Kcal
 E a  8299K 1.987   16.5
 mol  K  mol
10
Immobilized: lnVm = 35.65 – 6582 (1/T)

 cal  Kcal
E a  6582 K 1.987   13.1
 mol  K  mol
c.) Since Km (soluble) = Km (apparent) there is no diffusional limitation.
11

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S  E  k1  ES 1  k 3 (ES) 2 

a.) Equilibrium approach

Substitute (4) and (5) into (3):

b.) Quasi–steady–state approach

substitute (8) and (9) into (3):

Substitute (3) and (5) into (2):

Substituting (ES) in terms of E0:

LetVS = r2 E0 and VP = r-1 E0

Divide top and bottom by (r-1 + r2) and let

At low substrate concentrations So< 150 mg/l substrate inhibition is negligible.

At high substrate concentrations above 150 mg/l , substrate inhibition is significant.

Low Ksı indicates severe substrate inhibition.

Plot 1/V versus 1/S at different inhibitor concentrations

And from the intercept on X-axis, - 1/Km = -1.2 and Km = 0.83 mM

a. V = Vm (1 + A/KA) S /(Ks + S) = Vm (1+A/KA) / (1+Ks/S)

a. V = KL (S0 - Ss) = Vm Ss / (Km+ Ss)

1 V  2.50 104 M sec 1

Plot 1/V versus 1/S

a.) For E0 = 1.6 g/L

c.) The inhibitor is competitive.

MW ATPase  5  104 g mol, K m  110 4 mol L

Enzyme inactivation kinetics: E = E0e-rt, r = 0.1 min-1

 10% of the crude protein was ATPase.

At steady – state: reaction rate = mass transfer rate

By inspection,Vmax = 125 μmol/min, Km = 20 μmol/L

(i) the enzyme is immobilized, thus diffusional effects become important

a.) Plot [P] versus t data for both cases.

Obtain rates by taking tangents at specific time points.

amount of dextrose formed (0.1) = amount of H2O used

 starch remaining = S0 – (dextrose formed – H2O used)

b.) Plot In Vm versus 1/T for both cases.

Soluble:lnVm = 41.18 – 8299 (1/T)

Immobilized: lnVm = 35.65 – 6582 (1/T)

c.) Since Km (soluble) = Km (apparent) there is no diffusional limitation.

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