LAB 8 (Investigation on Enzyme Activity and

Kinetics)
Amirul Shahfrizan bin Mohamed (2021478478), Hanisah binti Mohamad Jaafar (2021861692), Nur Syafiqah binti
Mohamad Safri (2021478478), Nursyahirah binti Mohd Nazir (2021868142), Nur Azurah Zuraikha binti Mohd
Zulkarnain (2021482706)

CHE506 Reaction Engineering Laboratory

School of Chemical Engineering, College of Engineering Universiti Teknologi MARA, Selangor, Malaysia
29 January 2023

reactions, and each enzyme has a distinct personality that it uses

Abstract — The main objective of the experiment is to act on a substrate to produce a product.
to investigate the effect of pH and temperatures on enzyme
activity and study the relationship between substrate Determine the rate of the reaction and how it changes in
concentration and the maximum velocity of an enzyme. The
response to variations in factors like substrate concentration,
method of determining the Michaelis Constant, Km used in
the experiment is by Hanes - Woolf Method. After sitting in enzyme concentration, pH, and temperature. This method is
a specific temperature water bath for a period of time, the known as enzyme kinetics and is the primary strategy for
amylase solution is mixed with the tapioca starch solution. studying the mechanism of an enzyme-catalyzed reaction. One
The hydrolysis process is then allowed to take place. The of the crucial factors influencing a reaction's rate that an
spectrophotometer is used to determine the absorbance enzyme catalysis is the concentration of the substrate. But the
value. The optimum condition for the amylase enzyme is at fact that substrate changes during an in vitro reaction because
substrate concentration, [S] = 2 %. The Michaelis constant
substrate is converted to product elaborates on researching the
obtained by using the Hanes – Woolf Method is Km =
101.6746 min-1. The plotted graph of absorbance values effects of substrate concentration. This experiment
versus parameters has been accomplished. The standard demonstrates how temperature, pH, and substrate concentration
curve for concentration versus absorbance had also been all affect the activity of the enzyme.
plotted for glucose solution. The study of the optimum
conditions for the bio reaction is critical in order to obtain II. OBJECTIVES
the maximum output of the product and to determine the
The aim of this experiment is to determine the effect of pH and
limit of the enzyme.
temperature on enzyme activity. Next, this experiment is to
analyse the relationship between substrate concentration and
Keywords—amylase solution; starch solution;
the maximum velocity of an enzyme. Other than that, the
substrate; Michaelis-Menten equation objective of this experiment is to assess the Michaelis-Menten
Parameters and to evaluate the experimental and theoretical
I. INTRODUCTION data analysis of Michaelis–Menten kinetics model.
A family of enzymes called amylases breaks down starch
(polymers of glucose) into more manageable disaccharides III. THEORY
(maltose). During this reaction, a water molecule is also split,
and the OH- and H+ ions bond to the exposed ends of the An enzyme is a compound that functions as a catalyst in living
disintegrated starch polymer. The term "hydrolysis" refers to organisms, regulating the rate at which chemical reactions
the process (water splitting). Enzymes frequently use proceed without undergoing any behavioral alteration. Enzyme
assays are standardized techniques for determining the
hydrolysis to dissolve chemical bonds.
concentrations of certain enzymes in a sample. Enzyme activity
refers to the overall catalytic properties of an enzyme. Enzyme
Protein molecules called enzymes serve as biological catalysts. activity is measured in micromoles of substrate converted per
They speed up the rate of reactions without altering the overall minute, which represents the rate of reaction catalyzed by that
process. Long sequences of amino acids connected by peptide enzyme.
bonds make up enzymes. Additionally, they can be found in all
living cells and oversee the metabolic processes that turn food Each enzyme operates best at a specific pH and temperature.
This pH and temperature optimum can be identified in the lab
into energy and new cells. In addition, enzymes assist in the
by doing tests at various temperatures and solution
disintegration of dietary components into their most basic concentrations, or by performing the experiment in a buffer
forms. Substrates are the reactants in enzyme-catalyzed
with various pH ranges. The amount of substrate present also
has a significant effect on how quickly an enzyme-catalyzed Lineweaver-Burk Plot
reaction proceeds. As the concentration of the substrate rises,
the initial speed of the enzyme-substrate reaction increases.

Figure 1: Graph velocity vs concentration in Figure 2: Lineweaver-Burk Plot

enzyme activities
Lineweaver and Burke simplified mathematical operations by
When the appropriate velocity is represented on the y-axis and showing the double inverse of substrate concentration and
the substrate concentration is plotted on the x-axis of the graph. reaction rate. Axis 1/Vo and 1/[S] have intercepts of 1/Vmax on
As seen in the graph, the Vo increases in direct proportion to the 1/Vo axis and -1/KM on the 1/[S] axis, respectively. This
the change in substrate concentration. But beyond a certain line has a slope of KM /Vmax. Another term for the double
substrate concentration, the velocity doesn't change any more; reciprocal presentation is the Lineweaver-Burk plot. The main
it stays the same. The enzyme-catalyzed reaction's maximum advantage of the Lineweaver-Burk plot is that it allows for a
speed when the substrate is saturated is referred to as the more precise estimation of the Vmax, which can only be
maximum velocity/speed. approximately determined from a straightforward Vo against S
graph.
Michaelis-Menten Equation IV. PROCEDURES

MATERIALS & APPPARATUS

Materials
1) Starch solution – Wheat flour
According to the Michaelis-Menten equation, since the second 2) pH buffer solution
stage is the rate-limiting phase, the rate of the overall reaction 3) DNSA Reagent
must be proportional to the concentration of the ES that reacts 4) Water bath
there. The relationship between substrate concentration, 5) Alpha Amylase enzyme
substrate, and initial enzyme velocity, Vo, is essentially the
same for most enzymes [1]. Considering the idea that the rate- Apparatus
limiting phase in enzymatic processes is the dissociation of the 1) Beaker
ES complex into free enzyme and product, the equation can be 2) Cylindrical test tube
written as follows: 3) Cuvette
4) Vortex Mixer
[𝑆]
𝑉𝑜 = 𝑉𝑚𝑎𝑥 5) Spectrophotometer
𝐾𝑚 + [𝑆] 6) Falcon tube
7) Falcon tube rack
All the values of Vo, Vmax, S and KM can be obtained from the 8) Micropipette and tips
experiment. 9) Hot plate
10) Thermometer

METHODOLOGY

I. Preparation of 2% Starch Solution

a) 4 g of soluble starch is mixed with approximately 50%
of cold water.
 b) The slurry is added to approximately 100 ml of gently VI. RESULTS AND DISCUSSIONS
boiling water in a large beaker while stirring. Results
c) 200 ml of final volume is top up and mixed well.

II. Effect of pH on the activity and stability of amylase Standard Curve

enzyme
a) Five test tubes with pH 5,6,7,8 and 9 are labelled. 1 ml Table 1: Standard Curve (Absorbance vs Concentration)
of 2% starch solution is placed in each test tube.
b) Get five additional clean test tubes and 2 ml of amylase
solution is put in each test tube.
Concentration(g/L) Absorbance value (nm)
c) All 10 test tubes are placed in the 37°C water bath for
about 5 minutes to allow the temperature to
equilibrate.
d) The content of each amylase test tube is poured into 400 1.106
each starch test tube and is mixed on a vortex mixer.
e) The test tubes are returned to the 37°C and water bath
and the hydrolysis reaction is let to proceed for exactly
10 minutes. 800 1.98
f) The amylase activity is determined using the method
given in Appendix 1.
g) Graph of pH vs. enzyme activity is plotted. 1600 2.843
III. Effect of temperature on the activity and stability of
amylase enzymes.
a) One test tube is labelled with 30°C. 1 mL of 2% starch 2000 3.231
solution and 1 mL of pH=7 buffer to the tubes is placed
in the test tubes.
b) Get additional clean test tubes and 2 ml of amylase
solution is put in the tube. The plotted graph of absorbance versus concentration only has
c) Both tubes are placed in the 30°C water bath for about four points (bold)
5 minutes to allow the temperature to equilibrate.
d) The content of the amylase is poured into a starch test
tube and mixed on a vortex mixer.
e) The tube is returned to a 30°C water bath and the
hydrolysis reaction proceeds for exactly 10 minutes.
f) The amylase activity is determined by using the
method given in Appendix 1.
g) Step a-f is repeated at different temperatures ranging
from 30°C - 70°C.
h) Graph of temperature vs. amylase activity is plotted.

V. Effect of substrate concentration on the activity of

amylase enzymes.
a) Starch solution is prepared by varying concentrations Figure 1: Graph of Standard Curve (Absorbance vs
(0.5, 1.5, 2.0, 2.5, and 3.0% w/v) as the substrate. Concentration)
b) Each test tube is labelled with starch concentration and
places 1 ml of each starch solution into the test tubes.
c) 1 mL of ph=7 buffer is added to the tubes.
d) Get five additional clean test tubes and 2 ml of amylase
solution is put in each tube.
e) The content of each amylase test tube is poured into
starch tubes and is mixed on a vortex mixer.
Effect of Substrate Concentration on Amylase Enzyme Effect of Temperature on Amylase Enzyme Activity
Activity
Table 3: Effect of Temperature on Amylase Enzyme Activity
Table 2: Effect of Substrate Concentration on
Amylase Enzyme Activity

Figure 4: Graph of Absorbance vs Temperature

Figure 2: Enzyme Activity, V vs Substrate Concentration [S]

Figure 5: Graph of Enzyme Activity vs Temperature

Figure 3: Graph of 1/[S] vs 1/V

Effect of pH on Amylase Enzyme Activity (Rheology Solutions, n.d.). Therefore, the purpose of this
experiment is to investigate the kinetics and activities of
Table 4: Effect of pH on Amylase Enzyme Activity enzymes dependent on a few factors, such as pH, temperature,
and substrate concentration. In this experiment, the amylase
enzyme was employed to measure enzyme activity along with
the DNSA reagent. The DNSA reagent is more precise and
easier to use than Benedict's reagent. When combined with a
colorimeter, it is ideal for monitoring the activity of enzymes
where reducing sugars are generated, such as invertase,
cellulase, and amylase (DNSA Reagent, 2016).

Standard Curve

In order to calculate the Michaelis constant, Km, the double

reciprocal technique is used. According to the standard curve,
the absorbance value increases along with the substrate
concentration. This implies that enzyme activity will increase
along with the concentration of substrate (Sohail et al., 2014).
Each parameter displays the maximum and minimum enzyme
activity levels. The enzyme exhibits its highest rate of activity
at its top limit. This denotes the highest rate of product
production as the synthesis process takes place. In accordance
with Beer's Law, the standard curve graph should, under ideal
circumstances, show a linearly increasing line with a clear
connection between the substance's concentration and
absorbance (Beer's Law, Chemistry LibreTexts). To put it
another way, as concentration increases, so does absorption. In
contrast, a solution with a lower concentration would have the
reverse effect, absorbing less light than a solution with a greater
Figure 6: Graph of Absorbance vs pH concentration. A spectrophotometer is used in this experiment
to determine the absorbance value. Figure 1 below shows a
graph of absorbance against concentration that is excellent. As
demonstrated in Figure 1, the standard curve graph for
absorbance against concentration follows the theoretical
prediction made by Beer's Law. The graph displays a rise in
concentration from 400 to 2000 g/L. Cleaning and drying the
cuvette before putting the sample concentration into it will yield
the correct spectrophotometer result.

Effect of Substrate Concentration on Amylase Enzyme

Activity

Based on Figure 2, it is indicated that when substrate

concentration rises, the relationship between enzyme activity
Figure 7: Graph of Enzyme Activity vs pH and substrate concentration increases (Apar & Zbek, 2005). The
amylase enzyme is shown in its ideal condition in Figure 2 at
DISCUSSION
the substrate concentration [S] = 3%, where it has the highest
percent activity. Although this is the enzyme's peak activity, it
The durability and high stability of enzymes in storage and becomes less active as the substrate concentration is raised.
application are still a major target of biotechnology. These This corroborates the idea that the reaction rate rises until it
enzymes can resist not just proteolytic attack and organic reaches saturation as the substrate concentration rises. The
solvents but also high temperatures (Eisenthal et al., 2006). effect of substrate concentration on the enzyme amylase is that
Enzyme reaction technology is also used in a wide range of as the concentration rises, the rate reaches a maximum rate, the
analytical applications, including those in the life sciences, the free enzyme ceases to interact with the substrate, and the rate
production of food and beverages, and agriculture. Along with rises until all of the enzyme's active sites are bound to the
factors like pH and enzyme concentration, temperature control substrate. (Free Essay: The Effect of Substrate Concentration
is one of the most important aspects of these reactions on the Enzyme Amylase | Studymode, n.d.) After this point,
increasing the concentration has no further impact. Typically, a consequently the rate of the reaction, can alter as a result of
Km value is used to express the maximum value for each variations in the pH of the medium. The enzyme's three-
enzyme (Michealis-Menten graph or Lineweaver-Burk plot) dimensional form is also altered by pH changes. Due to these
(Rodriguez et al., 2019). The rate constant, or substrate factors, enzymes are only active within a specific pH range. The
concentration, known as the Km value, determines the enzyme's maximum reaction rate and the enzyme's stability may be
maximal speed or its reciprocal, the velocity. The reaction's impacted by the medium's pH. While pH variations may not
greatest forward speed is indicated by Vmax. Vmax changes change the shape of an enzyme, they may alter the
when additional enzyme is supplied, but Vmax is unaffected by characteristics of the substrate, making it impossible for the
increased substrate. substrate to connect to the active site or conduct catalysis. The
enzyme is most active at the pH level that is considered to be
Effect of Temperature on Amylase Enzyme Activity the optimal pH. Figure 11 shows that from pH 5 to pH 6, the
enzyme activity decreased. The best pH for enzyme activity is
Typically, the graph of absorbance value versus temperature 6, which is. But the enzyme activity starts to rise at pH 6 to pH
will indicate the temperature that is best depending on the 7. At pH 8, the enzyme activity is then somewhat reduced, and
location of the curve's peak. However, according to the data in at pH 9, it increases once again. The outcomes show that there
Figure 4, the highest absorbance value was 3.386 nm at 30 °C are some variations. The absorbance reading may be impacted
and the lowest was 1.098 nm at 70 °C. Each enzyme has a range since the time for taking the reading of the absorbance for each
of temperatures where it reacts at its fastest pace. The maximum pH sample is not constant.
is defined as the temperature at which the enzyme performs
best. Around 98.6 °F (37 °C) is the optimal temperature for the CONCLUSION
majority of enzymes to function. Some enzymes also work The experiment was done successfully, with all the objectives
effectively at both low and high temperatures. For instance, achieved. The first objective is to determine how temperature
although animals in dry climates have enzymes adapted to affects enzyme activity. It shows that at a peak temperature of
greater temperatures, those in the Arctic have them adapted to 30°C, the value of amylase enzyme activity is 1.124 mol/min
lower optimal temperatures. However, enzymes are still before starting to fall until 70°C with 0.1471 mol/min.
proteins, and like all proteins, they begin to break down at Theoretically, when the temperature increases to its optimal
temperatures higher than 104 °F. The range of an enzyme's level, the enzyme activity of the substrate will also increase.
activity is therefore determined by the temperature at which an Next, the effect of pH on amylase enzyme activity may not
enzyme begins to work and the temperature at which a protein change the enzyme's shape, but it may alter the characteristics
begins to degrade (Temperature on Enzymatic Reaction, n.d.). of the substrate. At extremely low pH levels or high pH levels,
enzymes perform better. As the substrate concentration rises,
The temperature-dependent behaviour of enzymes is the enzyme-substrate reaction's initial speed increases. The best
crucial to several areas of biotechnology. This has long been pH for enzyme activity is pH 5, which is 1.15773 mol/min.
explained in terms of catalytic activation energy and enzyme Overall the amylase enzyme performs best when the pH is 5,
stability. The amount of product produced over a specific the temperature is 30°C, and the substrate is 3.0% Km = 6.2584
amount of time, or in some cases, the amount of substrate min-1.
consumed, should be the same thing in order to calculate an
enzyme's activity. In order to measure either product or
substrate while the other is present, it would be ideal (Scopes, RECOMMENDATIONS
n.d.). According to the plotted graph in Figure 5, the enzyme
activity peaked at 30 °C with 1.124 mol/min before starting to There are several precautions and recommendations to consider
fall until 70 °C with 0.1471 mol/min. Theoretically, when the carefully while handling the experiments to obtain the most
temperature rises to its ideal level, the substrate's enzyme precise data for the experiment. First, gloves and safety goggles
activity will rise along with it. The results of this experiment
must be worn when handling hazardous materials, chemicals,
may be affected by a variety of factors, including the frequency
of enzyme-substrate collisions or the capacity of the enzyme and boiling materials to prevent anything bad from happening
and substrate to interact (Enzyme Activity n.d.). Less flexibility and to avoid touching the cylindrical tube with bare hands
occurs as the temperature is lowered because molecules and because this experiment involves a heating process. The
atoms move more slowly at lower temperatures. Each enzyme experiment requires the device to run for around 15 minutes to
has an ideal temperature range, or "comfort zone," where it maintain a steady state and prevent inaccuracy. The instrument
functions at its best (Enzyme Activity, n.d.) must be calibrated before measuring the absorbance with a
blank value of distilled water. Also, ensure the sample cells in
Effect of pH on Amylase Enzyme Activity
the cuvette must be cleaned entirely and ensure the transparent
The active areas of some enzymes contain ionic groups, which sides of the cuvette are facing the light source of the
must be in the correct form (an acid or a basic) to work. The spectrophotometer to avoid parallax error during reading.
ionic form of the active sites and the activity of the enzyme, and
 REFERENCES January20,2023,from
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reaction-temperature-with-circulator-baths/
Towns, “Michaelis-Menten Graphs, Lineweaver-Burk Plots,
and Reaction Schemes: Investigating Introductory
Biochemistry Students’ Conceptions of Representations in
[3]
Enzyme Kinetics,” J Chem Educ, vol. 96, no. 9, pp. 1833–1845,
Sep. 2019, doi:
10.1021/ACS.JCHEMED.9B00396/ASSET/IMAGES/LARG
E/ED9B00396_0009.JPEG.
[2] “1.2: Beer’s Law - Chemistry LibreTexts.”
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry
/Molecular_and_Atomic_Spectroscopy_(Wenzel)/1%3A_Gee
ral_Background_on_Molecular_Spectroscopy/1.2%3A_Beers
_Law (accessed Nov. 17, 2022).
[3] Apar, D. K., & Özbek, B. (2005). α-Amylase
inactivation during rice starch hydrolysis. Process
Biochemistry, 40(3), 1367–1379.
https://doi.org/10.1016/j.procbio.2004.06.006
[4] DNSA reagent Instructions for preparation and use.
(2016)
https://www.gov.uk/government/uploads/system/uploads/attac
hment_data/ Effect of Temperature on Enzymatic Reaction -
Creative Enzymes. (n.d.). Retrieved January 20, 2023, from
https://www.creative-enzymes.com/resource/effect-of-
temperature-on-enzymatic-reaction_50.html
[5] Eisenthal, R., Peterson, M. E., Daniel, R. M., &
Danson, M. J. (2006). The thermal behaviour of enzyme
activity: implications for biotechnology. Trends in
Biotechnology,24(7),289–292.
https://doi.org/10.1016/j.tibtech.2006.05.004
[6] Enzyme Activity | BioNinja. (n.d.). Retrieved January
20,2023,from https://ib.bioninja.com.au/standard-level/topic-
2-molecular-biology/25-enzymes/enzyme-activity.html
Free Essay: The Effect of Substrate Concentration on the
Enzyme Amylase | Studymode. (n.d.). Retrieved January 20,
2023,from https://www.studymode.com/essays/The-Effect-Of-
Substrate-Concentration-On-1107058.html
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Towns, M. H. (2019). Michaelis–Menten Graphs, Lineweaver–
Burk Plots, and Reaction Schemes: Investigating Introductory
Biochemistry Students’ Conceptions of Representations in
Enzyme Kinetics. Journal of Chemical Education,96(9),1833–
1845. https://doi.org/10.1021/acs.jchemed.9b00396
[8] Sohail, M., Aqueel, M., Afzal, M., Raza, A., Karman,
M., Khalil, M., & Rehman, F. ur. (2014). Biochemical studies
on the amylase of Mango Mealybug (Drosicha Stebbingi
Green). Turkish Journal of Entomology, 38.
https://doi.org/10.16970/ted.82875
[9] Scopes, R. K. (n.d.). Enzyme Activity and Assays.
www.els.net
[10] Controlling Enzyme Reaction Temperature with
Circulator Baths | Rheology Solutions. (n.d.). Retrieved
 APPENDIX

Sample Calculation for the Glucose Concentration, X

From the standard curve graph, equation of the tangent line is y = 0.0013x + 0.7561

x = Substrate Concentration
y = Absorbance Value

𝑦−0.7561
X=
0.0013

Y at pH5 = 3.465

3.465−0.7561
X=
0.0013
X= 2083.7692g/mL

Sample Calculation for the Mole Of Glucose Produce

MW of Glucose = 180.16 g/mol

Volume of amylase enzyme = 2 mL

Moles of Glucose produced (mol) =

𝑔
𝐶𝑜𝑛𝑐 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 (𝑚𝐿) × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑚𝑦𝑙𝑎𝑠𝑒 𝑒𝑛𝑧𝑦𝑚𝑒(𝑚𝐿)
𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 (𝑚𝑜𝑙 ) = 𝑔
𝑀𝑊 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (𝑚𝑜𝑙 )
𝑔
2083.7692 (𝑚𝐿) × 2(𝑚𝐿)
𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 (𝑚𝑜𝑙 ) = 𝑔
180.16 (𝑚𝑜𝑙)

𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 (𝑚𝑜𝑙 ) = 11.5773 𝑚𝑜𝑙

Sample Calculation of the Enzyme Activity, V

Time for hydrolysis reaction = 10 Minutes

𝑚𝑜𝑙 𝑚𝑜𝑙 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑒(𝑚𝑜𝑙)

𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( )=
min ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 (𝑚𝑖𝑛)

𝑚𝑜𝑙 11.5773 (𝑚𝑜𝑙)

𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( )=
min 10 (𝑚𝑖𝑛)

𝑚𝑜𝑙
𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( ) = 1.15773 𝑚𝑜𝑙/𝑚𝑖𝑛
min
Sample Calculation of Enzyme Maximum Velocity, Vmax

By using Michaelis Constant:

𝑉𝑚𝑎𝑥 (𝑠)
𝑉=
𝐾𝑚 + (𝑠)

Double Reciprocal Method,

1 𝐾𝑚 1
= +
𝑉 𝑉𝑚𝑎𝑥[𝑆] 𝑉𝑚𝑎𝑥

From graph substrate concentration,

y = 0.833x + 0.1331
From the y-intercept, Vm can be obtained,

1
=0.1331
𝑉𝑚

Vm = 7.5131 mol/min

Find value of Km from slope,

𝐾𝑚
=0.833
𝑉𝑚

Km = (0.833) (7.5131)

Km = 6.2584
RESULT