Mapúa University

School of Chemical, Biological, and Materials

Engineering and Sciences

Module 1 Problem Set

by
Aspuria, Aicee Julliane
Badillo, Allyssa Sophia
Macmac, Shaina
Sipat, Beatrice
Young, Javrielle

Submitted in Partial Fulfillment of the Requirements for

Biochemical Engineering (CH148)

Engr. Peter Matthew Paul Fowler

March 2022
QUESTION 1: Solve Exercise 1.4.1 again but using Eadie-Hofstee and Hanes-Woolf plots.

Determine the Michaelis-Menten parameters 𝑣𝑚𝑎𝑥 and 𝐾𝑀 for the reaction:

The rate of reaction is given as a function of urea concentration (𝐶𝑆 ):

Excel File (for the plots): CH148_PSET1_QUESTION1.xlsx

Solution and Answer:
a) Eadie-Hofstee
From Lineweaver-Burk equation:
1 𝐾𝑀 1 1
[ = + ] (𝑣 )(𝑣 )
𝑣 𝑣𝑚𝑎𝑥 [𝑆} 𝑣𝑚𝑎𝑥 𝑚𝑎𝑥
𝑣
𝑣𝑚𝑎𝑥 = 𝐾𝑀 +𝑣
[𝑆]
𝑣
𝑣𝑚𝑎𝑥 − 𝐾𝑀 =𝑣
[𝑆]
𝑣
𝑣 = 𝑣𝑚𝑎𝑥 − 𝐾𝑀
[𝑆]
𝑣
Plotting [𝑆] vs. 𝑣 using excel:

Eadie-Hofstee
1.2

0.8

0.6
v

0.4

0.2 y = -0.02443x + 1.22164

R² = 0.98239
0
0 10 20 30 40 50
v/[S]

𝑦 = −0.02443𝑥 + 1.22164

𝒌𝒎𝒐𝒍 𝒌𝒎𝒐𝒍
∴ 𝑲𝑴 = 𝟎. 𝟎𝟐𝟒𝟒𝟑 𝟑
, 𝒗𝒎𝒂𝒙 = 𝟏. 𝟐𝟐𝟏𝟔𝟒 𝟑
𝒎 𝒎 ⋅𝒔
 b) Hanes-Woolf
From Lineweaver-Burk equation:
1 𝐾𝑀 1 1
[ = + ] ([𝑆])
𝑣 𝑣𝑚𝑎𝑥 [𝑆] 𝑣𝑚𝑎𝑥
[𝑆] 𝐾𝑀 [𝑆]
= +
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥
[𝑆] 𝐾𝑀 1
= + [𝑆]
𝑣 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥
[𝑆]
Plotting [S] vs. 𝑣 using excel:

Hanes-Woolf
0.2
0.18
0.16
0.14
0.12
[S]/v

0.1
0.08
0.06 y = 0.82641x + 0.01985
0.04 R² = 0.99976
0.02
0
0 0.05 0.1 0.15 0.2 0.25
[S]

𝑦 = 0.82641𝑥 + 0.01985
1 𝑘𝑚𝑜𝑙
𝑣𝑚𝑎𝑥 = = 1.21 3
0.82641 𝑚 ⋅𝑠
𝑘𝑚𝑜𝑙
𝐾𝑀 = (0.01985)(1.21) = 0.024
𝑚3

𝒌𝒎𝒐𝒍 𝒌𝒎𝒐𝒍
∴ 𝑲𝑴 = 𝟎. 𝟎𝟐𝟒 𝟑
, 𝒗𝒎𝒂𝒙 = 𝟏. 𝟐𝟏 𝟑
𝒎 𝒎 ⋅𝒔
QUESTION 2: Vary the parameters 𝐾𝑀 and 𝑣𝑚𝑎𝑥 from the original Lineweaver-Burk
results of Exercise 1.4.1. Describe how varying either parameter affects the rate of
catalysis.
Excel File (for the plots): [Link]
Solution and Answer:

From the Lineweaver-Burk equation and given data in Exercise 1.4.1,

Table 1
v (kmol/m^3-s) [S] (kmol/m^3)
1.08 0.2
0.55 0.02
0.38 0.01
0.2 0.005
0.09 0.002

Km 0.0275
vmax 1.33

From Table 1, the obtained 𝐾𝑚 and 𝑣𝑚𝑎𝑥 are used as constants to solve for the rate of
catalysis 𝑣.

Moreover, 𝑣 is solved using the Michaelis-Menten equation,

𝑣𝑚𝑎𝑥 [𝑆]
𝑣= → (𝒆𝒒. 𝟏)
𝐾𝑚 + 𝑣𝑚𝑎𝑥

The resulting 𝑣 using the 𝐾𝑚 and 𝑣𝑚𝑎𝑥 with different [S] values are presented in Table
2.
v using michaelis
[S] (kmol/m^3) menten eq
0.2 1.16923077
0.02 0.56
0.01 0.35466667
0.005 0.20461538
0.002 0.09016949

The values of 𝑣 without varying the 𝐾𝑚 nor 𝑣𝑚𝑎𝑥 are obtained for comparison purposes
on the graph.

On one hand, in varying the 𝐾𝑚 and 𝑣𝑚𝑎𝑥 , the following values are assumed. In Table 3,
the original values as well as great and lower values of 𝐾𝑚 and 𝑣𝑚𝑎𝑥 are included. These
are the varied values plugged in (eq.1) in order to determine how varying the
parameters affect the rate of catalysis.
 Table 3
From
Lineweaver
-Burk Assumed Values
Km 0.0275 0.05 0.1 0.2 0.01 0.005 0.0025
vmax 1.33 1.5 2 3 1 0.5 0.05

a) In varying the 𝑲𝒎 parameter

𝑘𝑚𝑜𝑙
Table 4 shows the values when 𝐾𝑚 ( ) parameter is varied and 𝑣𝑚𝑎𝑥 is held
𝑚3
constant.
Table 4
[S] v (using assumed Km values)
(kmol/m^3) Km=0.05 Km=0.1 Km=0.2 Km=0.01 Km=0.005 Km=0.0025
0.2 1.064 0.88666667 0.665 1.26666667 1.29756098 1.31358025
0.02 0.38 0.22166667 0.12090909 0.88666667 1.064 1.18222222
0.01 0.22166667 0.12090909 0.06333333 0.665 0.88666667 1.064
0.005 0.12090909 0.06333333 0.03243902 0.44333333 0.665 0.88666667
0.002 0.05115385 0.02607843 0.01316832 0.22166667 0.38 0.59111111

Plotting the values in Table 4,

varying Km
1.4

1.2

0.8

0.6

0.4
0.2
0
0 0.05 0.1 0.15 0.2 0.25

In the figure above, the blue line represents the original experimental curve while the
green lines represent the lower 𝐾𝑚 values, and the orange lines represent the higher
𝐾𝑚 values.

From the figure, it can be denoted that lower 𝐾𝑚 values result to a steeper curve. This
entails that in order to reach the maximum velocity, lower substrate concentration is
needed. However, higher 𝐾𝑚 values result to a less steep curve which means that it
requires more substrate concentration in order to reach half-maximum or the
maximum velocity itself. With this, it can be accounted that lower 𝐾𝑚 means that the
enzyme has higher affinity for the substrate and vice versa.

In addition, if the original experimental curve is referred to, it is shown that for the
same substrate amount, the 𝐾𝑚 value is directly proportional to the rate of catalysis.
This is to say that higher 𝐾𝑚 results to higher rate of catalysis and lower 𝐾𝑚 results to
lower rate of catalysis.

b) In varying the 𝒗𝒎𝒂𝒙 parameter

𝑘𝑚𝑜𝑙
Table 5 shows the values when 𝑣𝑚𝑎𝑥 ( 𝑚3 ∙𝑠 ) parameter is varied and 𝐾𝑚 is held
constant.
Table 5
[S] v (using assumed vmax values)
(kmol/m^3) vmax=1.5 vmax=2 vmax=3 vmax=1 vmax=0.5 vmax=0.05
0.2 1.31868132 1.75824176 2.63736264 0.87912088 0.43956044 0.04395604
0.02 0.63157895 0.84210526 1.26315789 0.42105263 0.21052632 0.02105263
0.01 0.4 0.53333333 0.8 0.26666667 0.13333333 0.01333333
0.005 0.23076923 0.30769231 0.46153846 0.15384615 0.07692308 0.00769231
0.002 0.10169492 0.13559322 0.20338983 0.06779661 0.03389831 0.00338983

Plotting the values in Table 5,

varying vmax
3

2.5

1.5

0.5

0
0 0.05 0.1 0.15 0.2 0.25
In the figure above, the blue line represents the original experimental curve while the
green lines represent the lower 𝑣𝑚𝑎𝑥 values, and the orange lines represent the higher
𝑣𝑚𝑎𝑥 values.

It is depicted from the figure above that there is a linear relationship between the value
of 𝑣𝑚𝑎𝑥 and maximum velocity achieved. This means that the higher the 𝑣𝑚𝑎𝑥 , the
higher the achieved maximum velocity is and vice versa.

With 𝐾𝑚 held constant,

QUESTION 3: Escherichia coli is used for production of recombinant porcine growth
hormone. The bacteria are grown aerobically in batch culture with glucose as the
growth-limiting substrate. Cell and substrate concentrations are measured as a
function of culture time with the following results.

Cell Substrate
concentration, concentration
𝒕𝒊𝒎𝒆(𝒉) 𝒌𝒈 𝒌𝒈
𝑿 ( 𝟑) 𝑺 ( 𝟑)
𝒎 𝒎
0.00 0.20 25.00
0.33 0.21 24.80
0.50 0.22 24.80
0.75 0.32 24.60
1.00 0.47 24.30
1.50 1.00 23.30
2.00 2.10 20.70
2.50 4.42 15.70
2.80 6.90 10.20
3.00 9.40 5.20
3.10 10.90 1.65
3.20 11.60 0.20
3.50 11.70 0.00
3.70 11.60 0.00
Excel File (for plots): M1_PS1_Q3.xlsx
Solution and Answer:

a) Plot 𝝁 as a function of time.

GIVEN PROCESSED DATA

𝒕𝒊𝒎𝒆(𝒉) 𝒌𝒈 𝒌𝒈 ∆𝒕 ∆𝑿 𝑿𝒂𝒗𝒆 ∆𝑿 𝟏 ∆𝑿
𝑿 ( 𝟑) 𝑺( )
𝒎 𝒎𝟑 ∆𝒕 𝑿𝒂𝒗𝒆 ∆𝒕
≈ 𝝁𝒈
0.00 0.20 25.00 0.00 0.00 0.0000 0.0000 0.0000
0.33 0.21 24.80 0.33 0.01 0.2050 0.0303 0.1478
0.50 0.22 24.80 0.17 0.01 0.2150 0.0588 0.2736
0.75 0.32 24.60 0.25 0.10 0.2700 0.4000 1.4815
1.00 0.47 24.30 0.25 0.15 0.3950 0.6000 1.5190
1.50 1.00 23.30 0.50 0.53 0.7350 1.0600 1.4422
2.00 2.10 20.70 0.50 1.10 1.5500 2.2000 1.4194
2.50 4.42 15.70 0.50 2.32 3.2600 4.6400 1.4233
2.80 6.90 10.20 0.30 2.48 5.6600 8.2667 1.4605
3.00 9.40 5.20 0.20 2.50 8.1500 12.5000 1.5337
3.10 10.90 1.65 0.10 1.50 10.1500 15.0000 1.4778
3.20 11.60 0.20 0.10 0.70 11.2500 7.0000 0.6222
3.50 11.70 0.00 0.30 0.10 11.6500 0.3333 0.0286
3.70 11.60 0.00 0.20 -0.10 11.6500 -0.5000 -0.0429

µ vs time
1.80
1.60
1.40
)

1.20
specific growth rate (

1.00
0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
-0.20
time (h)

Sample calculation:
∆𝑡 = 0.33 − 0.00 = 0.33
∆𝑋 = 0.21 − 0.20 = 0.01
0.20 + 0.21
𝑋𝑎𝑣𝑒 = = 0.2050
2
∆𝑋 0.01
= = 0.0303
∆𝑡 0.33
1 ∆𝑋 1
≈𝜇= (0.0303) = 0.1478
𝑋𝑎𝑣𝑒 ∆𝑡 0.2050

b) What is the value of 𝝁𝒎𝒂𝒙?

Plotting ln (X) as a function of time to determine the exponential growth

phase,
 ln(X) vs. time
3
2.5
2
1.5
1

ln(X)
0.5
0
-0.5
-1
-1.5
-2
0.00 1.00 2.00 3.00 4.00
time (h)

From the plot, it can be inferred that exponential growth occurs at t =

0.5 to t = 3.0.

Therefore, maximum growth prevails at such time interval:

Maximum Specific Growth

2.5
2
1.5
1
0.5
ln(X)

y = 1.4989x - 2.2585
0
-0.5
-1
-1.5
-2
0 1 2 3 4
time (h)

𝑙𝑛 (𝑋) = ln 𝑋0 + 𝜇𝑚𝑎𝑥 𝑡
𝒔𝒍𝒐𝒑𝒆 = 𝝁𝒎𝒂𝒙 = 𝟏. 𝟒𝟗𝟖𝟗 ≈ 𝟏. 𝟓𝟎 𝒉−𝟏

c) What is the observed (final) biomass yield 𝒀𝑿 from substrate?

𝑺
𝐺𝑖𝑣𝑒𝑛:
𝑋0 = 0.20; 𝑆0 = 25
𝑋𝑓 = 11.6; 𝑆𝑓 = 0

𝑡𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 ∆𝑋

𝑌𝑋⁄ = =−
𝑆 𝑡𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 ∆𝑆
 𝑋𝑓 − 𝑋0
𝑌𝑋⁄ =
𝑆 𝑆𝑓 − 𝑆0
11.6 − 0.20
𝑌𝑋⁄ =
𝑆 25.0 − 0
𝒀𝑿⁄ = 𝟎. 𝟒𝟓𝟔𝟎
𝑺

d) Is this observed 𝒀𝑿⁄ constant during the culture?

𝑺
The apparent or observed growth yield, 𝑌𝑋⁄𝑆 , represents the yield of cells based
on the amount of substrate consumed at the end of the batch growth period. In
lieu, this growth yield may only be constant throughout the culture if the
substrate is consumed solely for the purpose of growth. However, depending
on culture conditions, cells have energy expenditures other than growth (i.e.,
maintenance and other related activities). Therefore, this apparent growth
yield is not a true constant throughout the culture period. The figure below
illustrates the behavior of growth yield as a function of time, chiefly revealing
that the 𝑌𝑋⁄𝑆 is not constant during culture. The figure likewise indicates that
𝑌𝑋⁄𝑆 depends on the growth phases such that cells increase a little during the
first 30 minutes (lag phase) then exponentially grow after (exponential growth
phase) before yield graphically oscillates before stabilizing (stationary) into an
observed 0.4560 𝑔 𝐸. 𝑐𝑜𝑙𝑖 ⁄𝑔 𝑔𝑙𝑢𝑐𝑜𝑠𝑒. Beyond this point (not shown in graph)
is the start of decline phase where the priority of the culture shifts from growth
to survival, characterized by a decreasing growth yield as cells near death.

0.50
0.45
0.40
growth yield, Y(X/S)

0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.00 1.00 2.00 3.00 4.00
time, h