BT 5121 – Bioprocess Engineering

Lab-2

Lab Manual

INSTRUCTORS:

Prof. Sathyanarayana N Gummadi

Department of Biotechnology
Indian Institute of Technology Madras
Chennai 600 036, INDIA

Page 1 of 42
Laboratory Guidelines:

• Total no. of students: 14

• The class is divided into 7 groups (2 each). There are seven experiments
(experiments 1-7) which runs in parallel. Each group will do a specific
experiment in each day and experiment rotates for each group. For
Experiemnts 8-11, the entire class will do the same experiment on a particular
day

• Each student is expected to maintain a laboratory record (80-page

notebook). After concluding the experiments, the lab records will be
signed by the respective TA.

• All students must submit a report on the experiments performed in the

succeeding week to respective TA. Late submissions will not be considered
for evaluation. Report should be submitted individually (hand written not
computergenerated).

• Follow the report format given below:

➢ Title Page (with name)

➢ Objective
➢ Principle of the experiment
➢ Materials
➢ Experimental Procedure
➢ Data collected
➢ Results and discussion
➢ Conclusions and recommendation
➢ Bibliography
➢ Appendix (sample calculation)

• Only handwritten reports and plots on graph sheets in pencil will be accepted.

• There will be a final laboratory exam (March 30-31) along with viva-voce
where student the needs to do experiment alone, analyze the data and do
the necessary calculations based on questions

• Wearing Laboratory coats and shoes is required for all laboratory classes,
without which a student will not be allowed to do experiments.

• In case of equipment problems or spillage, report immediately to Teaching

assistants (or) the instructors.

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 • After the experiment is over, clean the equipment/surrounding before handing
it over to the respective TA. Report to the respective TA’s before you leave the
lab.

• Marks distribution will be as follows:

Lab Reports - 40%

Final exam (experiment) - 40%

Final exam (viva) - 20%

• Eating, gum chewing, browsing net, chatting, unnecessary fiddling with

laboratory equipment, and any kind of frivolous activity is prohibited during the
laboratory class.

• Academic dishonesty and falsifying data are unacceptable. Students turning in

laboratory records with plagiarized or fudged data will be awarded a ‘U’ grade
in the course.

Groupwise Distribution

S. No. Name Roll No. Group

1 Ajith Kumar BT22M001
G1
2 Diksha Choudhary BT22M002
3 Hrithik Baradia BT22M003
G2
4 Khatravath Hreethika Rathode BT22M004
5 Muthuarunachalam S BT22M005
6 G3
Priya V BT22M006
7 Ritesh Suresh Kothawade BT22M007
8 BT22M008 G4
Shuvayan Dasgupta
9 Soma Prasad Sahoo BT22M009
G5
10 Subhrojyoti Ghosh BT22M010
11 Tejas Mahesh Kale BT22M011
G6
12 Trilochanaa M BT22M012
13 Yash Aggarwal BT22M013
G7
14 Ramshid N K BE19B028

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Lab timings: 2 pm – 5 pm (Wednesdays & Thursdays)

Expt. Jan Jan Jan Feb Feb Feb Feb

Experiments
No. 18 19 25 01 02 08 09

1 RTD G1 G2 G3 G4 G5 G6 G7

2 kLa G2 G3 G4 G5 G6 G7 G1

3 Mixing time G3 G4 G5 G6 G7 G1 G2

4 Conventional filtration G4 G5 G6 G7 G1 G2 G3

5 Tangential flow filtration G5 G6 G7 G1 G2 G3 G4

6 Cell lysis by sonication G6 G7 G1 G2 G3 G4 G5

Ammonium sulphate
7 G7 G1 G2 G3 G4 G5 G6
precipitation

8 Enzyme immobilization Feb 15-16

9 Enzyme kinetics Feb 22

Purification of recombinant
10 Feb 22-23
protein

11 Batch & Fed batch reactor Feb 25

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Experiments and TA incharge list

Expt. Experiments TA Incharge

No.

1 RTD Saivi

2 kLa Vishnu

3 Mixing time Mukunda

4 Conventional filtration Akilandaeswari

5 Tangential flow filtration Amit

6 Cell lysis by sonication Pranjali

7 Ammonium sulphate Sambita

precipitation

8 Enzyme kinetics Kavitha & Pranjali

9 Enzyme immobilization Arumugam N & Amit

10 Batch & Fed batch reactor Vishnu, Saivi

11 Purification of recombinant Mukunda, Akilandaeswari,

protein Sambita

Contact Details
S. No. Name of the TA Mobile

1 Akilandaeswari 9626410287

2 Amit 8917553859

3 Mukunda 9704897766

4 Pranjali 8888092249

5 Saivi 7003688783

6 Sambita 9040732411

7 Vishnu 8087384496

S. No. Name of the Technical Staff Mobile

1 Arumugam 8754513129

2 Kavitha 9600146167

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 1. Residence time distribution in CSTR and PFR (RTD)

Aim

To calculate the residence time distribution (RTD) of the given CSTR and PFR by
pulse input at flow rates and to study the effect of flow rates on RTD

Introduction

The elements of fluid flowing in a reactor take different times to pass through the
reactor. The distribution of these times for the stream of fluid leaving the vessel is
called the exit age distribution E, or residence time distribution, RTD of fluid. E has the
units of time-1.

By definition fraction of the exit stream of age between t and t+dt is Edt. Integrating
overall ages:
∞
∫ 𝐸(𝑡)𝑑𝑡 = 1
0

The fraction of the exit stream of age between t and t1 is:

𝑡1
∫ 𝐸(𝑡)𝑑𝑡
0

whereas the fraction of material older than t1 is:

∞ 𝑡1
∫ 𝐸(𝑡)𝑑𝑡 = 1 − ∫ 𝐸(𝑡)𝑑𝑡
𝑡1 0

Pulse input – C curve: An instantaneous pulse of tracer is injected into the stream
entering the reactor. The graph between concentration and time is called C curve. The
outlet response is normalized by dividing the measured concentration c by An, the area
under the concentration - time curve,

Thus:

∞ ∞ 𝑐
∫ 𝐶𝑑𝑡 = ∫ 𝑑𝑡 = 1

0 0 𝐴𝑛

In which:

∞
𝐴𝑛 = ∫ 𝑐𝑑𝑡
0

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Mathematically instantaneous pulse used here is known as direct delta function,
defined as f(t) = 0, t≠0

f(t) = M, t=0

Relation between C and E curves: For steady state flow in a closed vessel the RTD
for any batch of fluid entering must be the same as that of leaving.

Hence, the C-curve generated by injecting a pulse of tracer at the entrance must be
identical with E-curve.

i.e.C ≡ E

Mean of the residence time distribution (tE): It can be shown that for a reactor
operating at steady state the mean of the residence time distribution is:

∞
𝑡𝐸 = ∫ 𝑡𝐸(𝑡)𝑑𝑡
0

For an ideal reactor holding time or space time (τ)is equal to tE. i.e. τ = tE = V/ υ

Where, V = Volume of the reactor and υ = Volumetric flow rate.

Variance of the residence time distribution (σ2): Residence time distribution can be
compared by variance, that can be calculated by,

𝜎2 = ∫(𝑡 − 𝑡𝐸)2 𝐸(𝑡)𝑑𝑡

0

Reactor dimensions:
CSTR
Tank volume = 1.2 L;
PFR
Tube diameter inner diameter = 10 mm;
Tube length = 6 m;

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Procedure
RTD for CSTR
1. Fill the reservoir with the tap water
2. Adjust the inlet flowrate of water and allow the system to attain steady state
3. Set the agitator speed of the reactor at 500 rpm.
4. Pulse a 4 mL of a particular concentration of potassium permanganate into the
reactor and collect samples at the exit, every 20 seconds till 5 minutes and then
every 5 minutes till the sample becomes colorless.
5. Analyze the concentration of potassium permanganate in the samples by
measuring the OD at their respective λmax
6. Repeat the experiment with a different flow rate. The flow rate will be given by
the TA incharge on the day of the experiment.

RTD for PFR

1. Fill the reservoir with the tap water
2. Adjust the inlet flowrate of water to 5 Lph and allow the system to attain steady
state
3. Inject 2ml of tracer and collect samples every 30 seconds till the sample
becomes colorless.
4. Analyze the concentration of potassium permanganate in the samples by
measuring the OD at their respective λmax
5. Repeat the experiment with a different flow rate. The flow rate will be given by
the TA incharge on the day of the experiment.

Observation
λmax of Potassium Permanganate = nm
Calibration of standard potassium permanganate or potassium dichromate solutions
with O.D:

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 Concentration of standard potassium Absorbance @ λmax
permanganate solution

0.02

0.04

0.06

0.08

0.10

Plot the calibration curve and fit it to a straight-line C=m* (O.D) + k

1. Plot the concentration versus time profile. This will give the concentration – time
curve

2. Calculate the area under the concentration – time curve, An numerically by

using Simpson’s 1/3rd rule

3. Divide each concentration by An, to get the various values of C ≡ E. i.e.

𝐶
𝑪≡ 𝐸 =
𝐴𝑛

4. Now, plot these values against time to get the E curve

5. Now calculate E(t) = t.E and plot E(t) =t.E against time to get E(t) versus t curve

6. Calculate the area under the E(t) versus t curve by numerical method in order
to get the mean residence time, tE of the reactor

7. Plot a graph of (t-tE)2.E(t) versus t.

Flow rate = Lph

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 Time, t OD Tracer conc. Area under C Normalized t.E(t) Mean RTD, tE =
(min) C=m*(O.D) versus t curve, conc. ≡ E(t) Area under t.
+k An = C/ An E(t) versus t
(g/L) (g-min/L) (min-1) curve.
(min)

Hence, Mean RTD, tE =

Standard deviation =

Time, t (min) (t-tE)2 (t-tE)2 E(t)

Results

Compare mean residence time and space time of CSTR and PFR at different
conditions

References

1. Levenspiel, “Chemical Reaction Engineering”,2nd Edition.(1999), John Wiley &

Sons
Coulson & Richardson, Volume-3., Chemical and Biochemical Reactors and Process
Control (2006) Elsevier.

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2. Determination of kLa in a bioreactor: estimation of kLaby static gassing out

Aim

To estimate the volumetric mass transfer coefficient (kLa) in a fermentation process using the
static gassing out method.

Introduction
In the static gassing out technique, oxygen from the media is scrubbed by sparging
nitrogen. The dissolved oxygen is measured with the help of a polarographic dissolved
oxygen probe. Once the dissolved oxygen reaches zero, nitrogen sparging is stopped,
and the air is resupplied to the reactor. The rise in dissolved oxygen over time is
recorded and used for the estimation of kLa. Since we are not using active culture in
the reactor, the respiration rate is not considered. The following equation gives the
change in dissolved oxygen concentration:

𝑑𝐶𝐿
= 𝑘𝐿 𝑎(𝐶 ∗ − 𝐶𝐿 )
𝑑𝑡

Here,

kL = liquid film oxygen transfer co-efficient (cm/hour)

a = Gas-liquid interfacial area /volume of liquid medium (cm2/cm3)
C* = saturation concentration of dissolved oxygen in the liquid medium
C = dissolved oxygen concentration in the liquid medium
L

t = time (s)
On integrating the above equation with boundary conditions as CL(0)=0;
CL(t)=CL
𝑙𝑛(𝐶∗ − 𝐶𝐿) = −𝑘𝐿𝑎 ∗ 𝑡
From the plot of ln(C* - CL) versus t, kLa can be estimated.
Procedure
1. Fill 1L of water in the fermentor.
2. Check all the connections and fittings before starting the experiment.
3. Set the impeller at 300 rpm.
4. Start sparging nitrogen into the fermenter. Keep sparging till the dissolved
oxygen reaches a minimum level. Calibrate at this point as zero.
5. Clamp the inlet from nitrogen and supply the fermenter with air.
6. Once the dissolved oxygen probe stabilizes, calibrate the probe to 100%.
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 7. Strip the oxygen from the liquid medium by sparging in nitrogen till it reaches
zero.
8. Start sparging air by adjusting the aeration to 1 vvm.
9. Record the dissolved oxygen readings at 10 seconds interval till it reaches
100%.
10. Repeat the procedure with different aeration, agitation and liquid media. The
TA in charge will inform the conditions for the experiment on the day of the
experiment.
Observations
Time(s) Dissolved oxygen (%)

Plot a graph of ln(C*-CL) versus t. Estimate kLa from the slope.

Result
Find the kLa value and discuss the effect of aeration, agitation, and media on kLa.

References
1. Fermentation and Enzyme Technology Daniel I.C Wang, Charles L. Cooney, Arthur
E. Humphrey, John Wiley Sons Inc., (1979)
2. Principles of Fermentation Technology P.F. Stanbury, A. Whitaker and S.J. Hall,
Second Edition (1995), Butterworth Heinem3.

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 3. Determination of mixing time
Aim

To measure mixing time in stirred vessel with both Newtonian and Non-Newtonian
fluid and relate it to Reynolds number

Procedure

1. Fill the vessel to 2/3rd of the total capacity with the distilled water and start the
stirrer
2. Add 250 µL of 5N NaOH solution to make the solution alkaline (pH =9.2)
3. Set the speed of the stirrer to 50 rpm
4. Inject an appropriate amount of Phenolphthalein tracer into the vessel
5. Start the stop watch as soon as the tracer is added and note the time when the
solution in the vessel seems homogenous. This way mixing time is obtained as
function of stirrer speed
6. Repeat step 1 to 5 for 5 different stirrer speeds
7. Repeat the steps 1 to 6 for high viscous and low viscous CMC
8. Obtain a correlation of mixing time, for the given stirred vessel by plotting
/
dimensionless mixing factor ft versus impeller Reynolds number N Re.

Observation

For the given stirred vessel,

Diameter of the impeller, Da =
Vessel diameter, Dt =
Height of liquid level in the vessel, H =

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Type of liquid Density of Viscosity of Power Law Impeller Dimensionless mixing Impeller Reynolds

factor f t = tT (NDa )1/ 2 g 3 / 2 D a

used in the the liquid, ρ Newtonian liquid, constants for speed 2 2/31/ 6 1/ 2 number
vessel (kg/m3) µ (kg/m.s) H Dt
Pseudoplastic (s-1) D 2 N
N/ = a
fluid Re

K N or
D 2
N (2−n ) 
N = / a

11(n−1) K
Re

Newtonian
Fluid (water)

High viscous
CMC solution

Low

viscous CMC -----------

solution

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 /
Plot dimensionless mixing factor (ft) vs. Impeller Reynolds number ( N Re) from the

experimentally obtained data.

Values of the power law constants (both K and n) will be given to you by the TA
incharge while performing the experiment.

Results

Compare the effect of agitation and medium viscosity on mixing time.

Reference

1. C. J. Geankoplis, “Transport Processes and Unit Operations”, 3rd edition, (1993).

Prentice Hall.

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 4. Conventional filtration
Aim

To determine the specific cake resistance and the medium resistance.

Principle
Filtration is an operation used to separate particulate or solute components in a fluid
suspension by flowing under a pressure differential through a porous medium. The
two broad categories of filtration are conventional filtration and cross flow filtration. In
conventional filtration, the flow of fluid is perpendicular to the filter medium. In cross
flow filtration the flow is parallel to filter medium. We would be working on conventional
filtration.
In conventional filtration, when slurry flows against a filter medium by application of
pressure gradient, solids begin to build up. The built up solids is called cake. Darcy’s
law, which describes flow of fluid through a porous bed, can be applied to this case-
1 𝑑𝑣 𝛥𝑃
=
𝐴 𝑑𝑡 µ𝑅
Where,
v is the volume of the filtrate
t is the time

A is cross sectional area exposed to filter medium

µ is the viscosity of filtrate
R is the resistance of porous bed (Combination of filter medium resistance and
cake resistance,Rc).
R= Rm + Rc
Rc= αρc (V/A)

ρc is mass of the dry cake per volume of the filtrate

The equations can be combined and expressed as
𝑡 µ𝛼𝜌𝑐 µ𝑅𝑚
= (𝑉⁄𝐴) +
(𝑉⁄𝐴) 2𝛥𝑃 𝛥𝑃

A plot of t/(V/A) with V/A would yield a straight line. The specific cake resistance and
filter medium resistance can be calculated using the intercept and the slope obtained.

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Procedure
1. Note down the weight of dry membrane
2. Test the filtration equipment with water initially, and check for leaks
3. Prepare feed slurry with a defined concentration.
4. Send the slurry inside the filter by switching the pump on
5. Note the volume of filtrate collected at regular intervals using a stop clock until no
filtrate is formed
6. Dry the solids collected at the end of the process and determine its weight
7. Make appropriate plots and determine the specific cake resistance and filter medium
resistance (Assume the density of cell slurry to be equal to that of water).
Observations and calculations

Time (s) Volume of filtrate collected (mL)

∆P= .............. Pa

Effective diameter of filter membrane =………….

Area, A= ...................... m2

Weight of filter paper before filtration=…………….

Weight of filter paper after filtration=…………….

Weight of cake formed on the filter membrane=…………..

Viscosity of water= ................. Pa.s

Calculate the Rm and α from the plot of t/(V/A) versus V/A.

Draw the plot of flux versus time

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 5. Tangential flow filtration

Aim

To determine the resistance due to membrane (media) and due to cake formation.

Principle
Filtration is used to separate the solute components in a solution according to their
size by flowing under a pressure difference through a porous medium. The substance
lesser than the cut off of the membrane will pass through the membrane and that
above the cut-off will be retained by the membrane
There are two broad categories of filtration, (1) Dead end filtration and (2) cross flow
filtration. Based on the pore size of the membranes they CFF is categorized as UF
and MF. The pore size of UF membrane are of 0.001 to 0.1 µm range and that of MF
is between 0.1 to 10 µm. Batch concentration, diafiltraion, purification can be
performed by CFF technique.

Procedure
1. Before starting the experiment check the filtration assembly for any leakage and
wash with distilled water. While washing leave both the retentate and permeate
line in the drain.
2. Note down the filtration area given on the filtration capsule.
3. For at least three different P values find the flow rate and record the values.
This is to find out the membrane resistance before the filtration of the sample.
4. Pour 100 mL of the sample in the reservoir vessel of the filtration assembly. Fix
a definite P value. Connect the retentate line to the reservoir and permeate
line to a measuring cylinder. Start the pump and record the time taken to collect
every 10 mL of permeate in a measuring jar till you collect about 90 mL of
permeate. Record the exact volume of the retentate and permeate. Find the
concentration of both the two constituents by appropriate method
5. Wash the membrane with about 100 mL distilled water. While washing both the
retentate and permeate line should be in drain.
6. Again for at least three different P values find the flow rate and record the
values. This is to find out the membrane resistance after the filtration of the
sample.

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 7. Finally wash the membrane with about 50 mL of 0.5 M NaCl and then with 0.1
M NaOH. Store the membrane in 0.1M NaOH.
Calculation
Resistance before filtration:

Table: A Area:50…..cm2  = …0.01. g/cm/s

s.no P, PSi Volume, mL Time, s Flow rate, mL s-1 Flux, (mL-1 s-1cm2)

1
2
3

Sample filtration: Table B P=……… psi Area: 50 ......... cm2

Vol. of permeate Time Flow rate, mL/s Flux, (mL-1 s-1cm2)
(mL) (s)

Resistance after filtration:

Table C Area: 50….cm2  = …0.01. g/cm/s

S.no P, psi Volume, mL Time, s Flow rate, mL/s Flux, mL/s/ cm2
1
2
3

Draw graphs:
(1) Time (s) in x-axis and Flux (mL-1 s-1cm2, using the table B data) {graph B}
(2) P, g-1cm-1s2 in x-axis and Flux (mL-1 s-1cm2) in y-axis using data- table A
{graph A}
(3) P, g-1cm-1s2 in x-axis and Flux (mL-1 s-1cm2) in y-axis using data table C {graph
C}

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The slope of the graph A will give the inverse value of (Rm.*) while the slope of the
graph
C will give the inverse value of (Rm+Rc+Rf) . The difference between these two
values will give the resistance due to fouling and resistance due to cake formation (Rf
+Rc). Also calculate the number of fold increase in the concentration of the component
/ protein//sugar collected in the permeate.

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 6. Free enzyme kinetics & effect of pH and temperature on enzyme activity

Aim
To determine the Michaelis Menten kinetic parameters of invertase enzyme and to
study the effect of temperature and pH on the enzyme activity.

Enzyme Activity
For the free enzyme, one unity of enzymatic activity (U) corresponds to the quantity of
enzyme that produces one micromole of glucose and fructose in the hydrolysis of a
5% (w/v) sucrose solution, at 55°C and pH 5.0.
The enzyme invertase catalyzes the cleavage of the disaccharide sucrose to the
monosaccharide glucose and fructose, as below

Sucrose ⎯⎯ ⎯⎯→ glucose + fructose

Invert ase

Enzyme Kinetics and estimation of kinetic constants

The enzyme (E) and substrate (S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of product (P).

Increase in the substrate concentration gradually increases the velocity of enzyme

reaction within the limited range of substrate levels. A rectangular hyperbola is
obtained when velocity is plotted against the substrate concentration.

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Estimation of Km and Vmax
Enzymes have varying affinities to bind their substrates. An enzyme's Km describes
thesubstrateconcentration at which half the enzyme's active sites are occupied by a
substrate. A high Km means more molecules of substrate must be present to saturate
the enzyme, meaning the enzyme has a low affinity for the substrate. Graphically, the
Km is the substrateconcentration that gives the enzyme one-half of its Vmax. The values
of kinetic constants Km and Vmax can be calculated from the Line-Weaver Burk plot,
from its slope and intercept

Effect of temperature on enzyme activity

Temperature affects the reaction rate of enzymes. At low temperatures, enzymes
have low activity. The activity peaks at a specific temperature unique to the enzyme.
This is known as the optimum temperature at which an enzyme is maximally active.
Beyond the optimum temperature, the activity of the enzyme decreases. At extreme
temperatures, the enzymes are denatured and activity ceases.

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Effect of pH on enzyme activity
Increase in the hydrogen ion concentration (pH) considerably influences the enzyme
activity and a bell-shaped curve is normally obtained. Each enzyme has an optimum
pH at which the velocity is maximum.

Reagents
1. Invertase stock solution 5 g/L
2. Sucrose solution 50 g/L
3. GOD-POD reagent
4. Different buffers with varying pH (Acetate Buffer & Phosphate Buffer, Tris Buffer)

Procedure
I) Determination of Michaelis Menten kinetic parameters
1. Pipette out 0, 0.1, 0.2, 0.3, 0.5, 0.6, 0.7, 0.8, and 0.9 mL of sucrose solution in a
series of test tubes and make up the volume to 0.95 mL with buffer pH 5.0.
2. Add 50 μl invertase enzyme solution (μg/L). Put parafilm or aluminium foil over the test
tube and immediately incubate the mixtures at 55 °C in a water bath for 10
minutes.
3. After incubation, estimate glucose by GOD-POD method (Appendix 2).
4. Measure A505 against blank. The blank should not have sucrose solution.
5. Calculate the enzyme activity (μM/min).
6. Draw the double reciprocal plot of substrate concentration vs enzyme activity and
calculate the kinetic parameters Vmax and Km
II) Effect of temperature on enzyme activity
1. To 0.5 mL of sucrose solution, add 0.9 mL of buffer along with 50 μl invertase
enzyme solution.
2. To study the effect of temperature on enzyme activity, perform the enzyme assay
at different temperatures (20℃, 30℃, 40℃, 60℃, 90℃) as instructed by the TA
incharge.
3. The control at respective temperature is to be set up and estimate glucose released
by GOD-POD method.
4. Cool the mixture and measure A505 against blank. The blank should not have
sucrose solution.
5. Calculate the enzyme activity (μM/min) and find the optimum temperature.

Page 23 of 42
III) Effect of pH on enzyme activity
1. To 0.5 mL of sucrose solution, add 0.9 mL of buffer at different pH (4, 5, 6, 7, 8, 10)
along with 50 μl invertase enzyme solution and do the assay at standard conditions.
2. The control at respective pH is to be set up and estimate glucose released by GOD-
POD method.
3. Cool the mixture and measure A505 against blank. The blank should not have
sucrose solution.
4. Calculate the enzyme activity (μM/min) and find the optimum pH.
Observation
Determination of Km and Vmax
Substrate 1/[S] A505 Enzyme 1/v
concentration activity (v)
([S]) (µmole/min)
(moles/L)

Effect of Temperature on enzyme activity

Temperature (℃) Enzyme Activity (v)

(μmoles/min)

Effect of pH on enzyme activity

pH Enzyme Activity (v)
(μmoles/min)

Results

Estimate Vmax, Km, optimum temperature and pH of invertase enzyme.

Page 24 of 42
 7. Immobilized enzyme kinetics
Aim

To study the immobilized enzyme kinetics using hydrolysis of sucrose catalyzed by

invertase enzyme immobilized by calcium alginate method

Introduction

The enzyme invertase catalyses the cleavage of the disaccharide sucrose to the
monosaccharide glucose and fructose, as below

Sucrose ⎯⎯ ⎯⎯→ glucose + fructose

Invert ase

Invertase immobilized calcium alginate beads will be prepared as per the standard
procedure. These alginate beads will be packed in a reactor to study the effect of flow
rate and the effect of substrate concentration on the hydrolysis rate. Reaction will be
monitored by measuring the reducing sugars (glucose and fructose) formed at various
time intervals.

Reagents

• 0.02 M sodium acetate buffer at pH 4.5

• 0.2 M calcium chloride solution
• Sucrose solution of various concentrations in 0.02 M sodium acetate buffer at
pH 4.5
• GOD-POD Reagent
• Immobilized invertase enzyme in the sodium alginate beads

Procedure

Preparation of calcium alginate beads

1. Dissolve 30 g of sodium alginate in 1 L to make 3% (w/v) solution

2. Mix 30 µL of 5 g/L enzyme solution in 10 mL of 3% (w/v) sodium alginate
solution (avoid clump formation)
3. Beads are formed by dripping the polymer solution from a height of
approximately 20 cm in to an excess (100 mL) of stirred 0.2 M calcium chloride
solution with a syringe and a needle at room temperature. Leave the beads in
the calcium solution to cure for 0.5 -3 h

Setting up the immobilized enzyme reactor

The given amount of enzyme beads (amount of enzyme will be informed by TA) are
added to different flasks containing various amounts of initial substrate concentrations.
The immobilized beads are allowed to react with the substrate solutions for 10 min

Page 25 of 42
and at the end of 10 min samples were collected and measure for amount of product
(glucose) released by enzymatic action using GOD-POD method (Appendix 2).

Results

Effect of substrate concentration

S.No Substrate A505 Glucose (g/l)

concentration

Substrate 1/[S] A505 Enzyme activity 1/v

concentration (v)
([S]) (µmole/min)
(moles/L)

Plot the graph of 1/v vs 1/S and calculate the values of vm and Km

References
1. Jose M. Guison, “Immobilization of enzymes and cells”, 2nd edition (2006), Human
Press, New Jersey.
2. Andres Illanes, “Enzyme biocatalysis-Principles and Applications”, (2008) Springer
Science

Page 26 of 42
 8. Cell disruption by sonication

Aim
To determine the protein release constant for ultrasonication of bacterial cells.

Principle
The treatment of microbial cells in suspension with inaudible ultrasound (greater than
about 18 kHz) results in their inactivation and disruption. The vibrating probe causes
cavities in the suspension. The collapse of these cavities results in pressure changes
and shear forces which cause cell disruption. Ultrasonication is a very vigorous
process, and complete solubilization of cells will occur if the process is carried out for
a sufficiently long period. However, the major problem with Ultrasonication is the heat
generated during the process.

The rate of protein released by cell disruption is usually found to be proportional to the
amount of releasable protein.
dp
= −kP
dt

Where P represents the protein content remaining associated with the cells, t is the
time and k is a release constant dependent on the system. Integrating from P = Pm
(maximum possible protein releasable) at time zero, to P = Pt at time t gives

pt dp t
 =  − kdt
pm p 0

 pm 
ln  = kt
 pt 

As the protein released from the cell, Pr is given by

Pr = Pm – Pt
The following equation for cell breakage is obtained
𝑃𝑚
𝑙𝑛 ( ) = 𝑘𝑡
𝑃𝑚 − 𝑃𝑟
Procedure

Page 27 of 42
 1. Collect overnight grown cells in tube and resuspend in the lysis buffer.
2. Take cell suspension in a micro centrifuge tube.
3. Sonicate the samples for different time lengths (on ice).Collect the samples at 1 min
interval till 10 mins.
4. Centrifuge the sample and estimate the total protein released in supernatant
by Bradford method (Appendix 1).
5. Prepare the standard curve using a standard protein (BSA).
6. Obtain the protein release constant using the equation.
7. Repeat the experiment for different cell concentrations.

References
1. Foster, D (1992) Cell Disruption: Breaking up is hard to do. Nature Biotechnology
10: 1539-1542.
2. Aidan Audouy, Constant Systems Ltd, Low March, Daventry, Northants, UK.
www.celldisrupter.co.uk
3. Dictionary of Microbiology and Molecular Biology 3rd Edition, 2001.

Page 28 of 42
 9. Protein precipitation

Aim
To concentrate/purify proteins from a solution by ammonium sulphate precipitation.

Introduction
The solubility of protein depends on the salt concentration in the solution. At low
concentrations, the presence of salt stabilizes the various charged groups on a protein
molecule, thus attracting protein into the solution and enhancing the solubility of
protein. This is commonly known as salting-in. However, as the salt concentration is
increased, a point of maximum protein solubility is usually reached. Further increase
in the salt concentration implies that there is less and less water available to solubilize
protein. Finally, protein starts to precipitate when there are not sufficient water
molecules to interact with protein molecules. This phenomenon of protein precipitation
in the presence of excess salt is known as salting-out. The salting out effect can be
explained empirically by Cohn Equation

ln S = β - KS′ I

where, S is the solubility of the protein,

I is the salt concentration,

KS′ is a salting-out constant characteristic of the specific protein and salt that
is independent of the temperature and pH above the isoelectric point

β is a constant for the hypothetical solubility of the protein at zero ionic strength
and it depends only on the temperature and pH for a given protein and
is a minimum at the isoelectric point

Procedure

1. Weigh 550 mg of protein and mix in 55 mL distilled water (final concentration: 10

mg/mL)
2. Take four centrifuge tubes, number them as 1 to 5. Take 10 mL of the protein
solution in each tube
3. Add ammonium sulphate corresponding to the saturation of 50%, 60%, 70%, 75%
and 80% (see table 1 for detail). One needs to be careful as the addition of the salt

Page 29 of 42
should be very slow. Add ammonium sulfate slowly and allow it to dissolve before
further addition

Test tube No. 1 2 3 4 5

Protein 10mg/mL 10 mL 10 mL 10 mL 10 mL 10

Ammonium sulphate (g) 2.95 3.66 4.42 4.83 5.23

Ammonium sulphate (%) 50 60 70 75 80

4. Mix them on magnetic stirrer for 30 minutes at 4 oC

5. Centrifuge the samples at 12000 rpm at 4 oC for 30 minutes
6. Collect the supernatant and the precipitate. Resuspend the pellet in 10 mL of
distilled water (buffer)
7. Measure protein concentration at 280 nm
8. The protein concentration can be taken from the standard graph for protein assay
Observation
Tube 1 Tube 2 Tube 3 Tube 4

Protein in precipitate (mg/mL)

Supernatant protein, S (mg/mL)

ln(S)

Ammonium sulphate added, I (in molarity)

Cohn equation: ln S = β - KS′ I

Plot the graph ln(S) on y-axis vs. I and calculate the slope (KS′) and the intercept(β)
From the values of the KS and β,calculate concentration of ammonium sulfate required
to get the desired recovery.

Result
The concentration of ammonium sulphate required to get the desired percentage
(concentration) of recovery is calculated using Cohn’s equation.

Page 30 of 42
 10. Batch and Fed-batch Reactor studies

Aim

To study the batch growth kinetics of bacterial culture in a bioreactor

Principle

Batch reactor

A batch reactor is a system with initial limited amount of nutrients run for a period time.
The microorganisms inoculated in a batch system exhibit lag, log and stationary growth
phases.

Fed-batch

A fed-batch reactor is a semi open system where limiting nutrients are added
intermittently. The advantages of fed-batch over batch fermentation include
overcoming catabolite repression, product inhibition and ability to maintain cells in
exponential phase and in high cell densities.

During exponential phase,

𝑑𝑋
= µ𝑋
𝑑𝑡

On integrating the above equation becomes,

𝑋
ln ( ) = µ(𝑡 − 𝑡0)
𝑋0

where X – Biomass concentration in reactor at time ‘t’

X0 - Biomass concentration in the reactor at time ‘t0’μ
– Specific growth rate

Biomass yield coefficient,

𝛥𝑋
𝑌𝑋 =
𝑆 𝛥𝑆

Page 31 of 42
 where YX/S – Biomass yield coefficient w.r.t. substrate concentration
(g of biomass/g of substrate)
∆X – Change in biomass concentration
∆S – Change in substrate concentration

Procedure

Batch Reactor

1. Prepare the medium for the pre-culture (100 mL) and the fermentation (1L) with
the following composition: Glucose 0.5 g/L, beef extract 1 g/L, Yeast extract 2
g/L, Peptone 5 g/L and NaCl 5 g/L.
2. Prepare inoculum in shaking flasks at a temperature of 37 °C by adding 2 mL
of the main bacterial suspension in 100 mL of culture medium. Incubate
overnight at 200 rpm in the shaker.
3. Autoclave separately the reactor tubings, Antifoam (polypropylene glycol, PPG)
10% (v/v) in a bottle fitted with a hypodermic needle and silicone tube by
covering the needle ends with cotton and aluminium foil.
4. Add 10% of grown seed inoculum to the growth medium in the reactor
aseptically with the help of a Bunsen burner.
5. Take samples at an interval of 20 minutes and analyze for biomass (A 600) and
glucose by GOD-POD assay (Appendix 2).
Fed-batch reactor

1. Add feed of glucose (in 10 mL H2O) to the reactor at fermentation time ‘t’ when
the substrate concentration of batch reactor approach to 0 g/L.
2. Take samples at an interval of 20 minutes and analyze for biomass (A600) and
glucose by GOD-POD assay (Appendix 2).
3. Add a second feed of glucose (in 10 mL H2O) to the reactor at fermentation time
‘t’ when the substrate concentration approach to 0 g/L.
4. Repeat step 2 till substrate concentration becomes 0 g/L
5. The feed concentration of glucose will be provided by the TA incharge while
performing the experiment.

Page 32 of 42
Observations
Time A600 Biomass concentration ln (X/X0) A505 Glucose concentration
(h) (g/L) (g/L)

Plot a graph between biomass & substrate concentration versus fermentation time and
calculate specific growth rate, doubling time, Yx/s for batch and fed-batch processes.
Results
Compare specific growth rate, biomass yield for batch and fed-batch reactor

References:
J.E. Bailey and D.F.Ollis, “Biochemical Engineering Fundamentals”, 2nd edition
(1986). McGraw Hill, New York

Page 33 of 42
 11. Recombinant protein expression and purification

Aim
Overexpression and purification of a recombinant protein by Ni2+ -NTA affinity
chromatography

Introduction
Since isolation of protein of interest from the crude extract requires complex
downstreamstrategies, various affinity tags have been developed over the years which
include 6 X His tag, MBPtag, GST tag etc. In this experiment, a recombinant protein
would be overexpressed in E. coli as a 6XHis tag fusion protein and would be purified
by Immobilized Metal Ion Affinity Chromatography
(IMAC).

Principle
• Ni2+-Affinity Chromatography uses the ability of histidine (His) to bind to nickel. Six
histidine amino acids (polyhistidine tag / 6X His Tag) at the end of a protein (either
N or C terminus) is known as a 6X His tag.

• Nickel is bound to an agarose bead by chelation using nitrilotriacetic acid (NTA),

NTA binds Ni2+ions by four coordination sites. The general procedure involves batch
binding of the protein, the protein mixture is mixed with the sample which allows the
His-tagged proteins to bind to nickel in the chromatographic matrix, and the slurry
is then poured into a chromatographic column.

• The lower concentration of imidazole is used to remove low affinity bound proteins,
increasing
the imidazole concentration results in the elution of all bound proteins from the
column
depending on their strength of binding.

Requirements
1. LB broth: (10 g – tryptone; 5g – yeast extract; 10 g- NaCl for 1 L)
2. IPTG: Prepare a 1M stock solution of IPTG and store at -20 °C

Page 34 of 42
3. Antibiotics: Kanamycin stock 50 mg/mL, Chloramphenicol stock 35 mg/mL (store at
-20 °C)
4. 1 M Imidazole (5 mL)

Procedure
A. Overexpression by IPTG induction
1. Inoculate a single transformant of E. coli Rosetta with Pet28a(+) plasmid containing
the gene of interest into LB broth containing 50 μg/mL kanamycin and 35 μg/mL
chloramphenicol under sterile conditions and incubate it for 12-16 h at 37 °C and 180
rpm (seed culture).
2. From seed, culture take 2% (v/v) of inoculum into LB broth containing antibiotics
(above mentioned) under sterile conditions and incubate at 37 °C and 180 rpm till the
A600 reaches 0.6-0.8
3. Once the desired density is reached, induce the cells with 0.3 mM IPTG and keep
for postinduction incubation for another 6 h at 30 °C and 180 rpm
4. Harvest the cells by centrifugation at 8000×g for 5 min at 4 °C and then discard
thesupernatant and store the pellet at -80 °C till future use

B. Cell disruption by sonication

1. Resuspend the pellet in lysis buffer (20 mM Tris/HCl (pH 7.5) 200 mM NaCl) and
add 1 mM EDTA, 1mM PMSF and 1 mM DTT.
2. Sonicate cells using a probe sonicator with following parameters, Set time: 5 min,
37%vibration amplitude, pulse 2s on/ 5s off, at 4 °C.
3. Centrifuge the lysate at 10000×g for 30 min at 4 °C and collect the supernatant.

C. Purification by Ni2+ NTA affinity chromatography

1. Either at room temperature or at 4°C, load 5 mL of supernatant (with 10 mM
imidazole) onto Ni2+ affinity column (1 mL Ni-NTA agarose), which is previously
equilibrated with Tris-buffer saline (TBS) (20 mM Tris-HCl (pH 7.5), 200 mM NaCl)
and allow to pass through the column.
2. Later, wash the column with 30-bed volumes of TBS containing 20 mM imidazole.
3. The bound protein is then eluted by a gradient of 50, 100, and 200 mM imidazole
in TBS buffer (each 5 mL).

Page 35 of 42
4. All of the fractions (Flow through, 20, 50, 100, 200 mM imidazole elutions) flowing
through the column can be analyzed by SDS-PAGE (by loading same volumes of
each fraction).
5. The fractions containing pure protein (100, 200 mM imidazole elutions) will be
collected and subjected to dialysis. Estimation is done by Bradford method
(Appendix 1).
6. The recombinant protein is Xylose reductase from yeast. Estimate the activity of
the purified protein is to be done.

D. Xylose reductase assay

1. Pipette out 350 μl of 50 mM sodium phosphate buffer (pH 7.0), 75 μl of 2 M xylose and
75 μlof 2mM NADPH into a microcentrifuge tube and measure the decrease in
absorbance at 340nm. Note down the dA/dt as autohydrolysis.
2. Pipette out 340 μl of 50 mM sodium phosphate buffer (pH 7.0), 75 μl of 2 M xylose, 75
μl of 2 mM NADPH, 10 μl of the enzyme into a microcentrifuge tube and measure
the decrease in absorbance at 340 nm. Note down dA/dt as the test for each step.
Dilute the sampleswherever necessary.

Sample graph for autohydrolysis and test are shown above. dA/dt is the slope of the
graph.
3. Calculate activity, total activity and specific activity at each step.
Activity calculation
Activity is defined in units (U) where one unit is defined as amount of enzyme required
to convert one µmoles of NADPH per minute. Molar Extinction coefficient of NADPH
at 340nm is 6220 M-1cm-1. Use the following Beer-Lambertz law to determine the
activity of enzyme.
𝐴 = 𝜀𝑐𝑙

Page 36 of 42
 Where, A = Absorbance @ 340 nm
c = concentration of NADPH
l = Path length
𝜀 = Molar extinction coefficient

4. Calculate purification fold from specific activity and % yield from the total activity.

Step Protein Enzyme Total Total Specific Yield Purification

Concentration Activity Protein Activity Activity (%) fold
(mg.ml) (U/ml) (mg) (U) (U/mg)

Whole cell
lysate
Pellet
Supernatant

Wash
100 mM
fraction
200 mM
fraction

Results
• SDS-PAGE gel picture showing all different fractions flowed through the column
• Find out the molecular weight of purified protein
• Estimation of protein concentration
References
1. Foster, D (1992) Cell Disruption: Breaking up is hard to do. Nature Biotechnology
10: 1539- 1542.
2. Aidan Audouy, Constant Systems Ltd, Low March, Daventry, Northants, UK.
www.celldisrupter.co.uk.
3. Dictionary of Microbiology and Molecular Biology 3rd Edition, 2001.

Page 37 of 42
 Appendix

I. Estimation of Proteins by Bradford’s Method

Aim

To estimate the concentration of the protein in the given sample solution

Principle

The Bradford reagent can be used to determine the concentration of proteins in

solution. The procedure is based on the formation of a complex between the dye,
Brilliant Blue G, and proteins in solution. The protein-dye complex causes a shift in the
absorption maximum of the dye from 465 to 595 nm. The amount of absorption is
proportional to the protein present.

Materials

Prepare the Bradford reagent (0.5 mg/mL CB-G, 25% methanol and 42.5% H3PO4).
100 mg of Coomassie Blue G is dissolved in 50 mL of methanol and added to 100 mL
of 85% H3PO4 and diluted to 200 mL with distilled water.

Assay reagent: Dilute 1 volume of dye stock with 4 volumes of distilled water, solution
will appear brown. The reagent is stable for weeks in a dark bottle at 4 °C.

Buffer: 20 mM Tris-HCl, 200 mM NaCl (pH 7.4)

Procedure

Stock: 1 mg/mL BSA

1. Pipette 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 l of stock solution (1 mg/mL BSA)
into clean test tubes and make up the volume to 100 l using buffer. Blank will
have 100 l of buffer. Similarly take 100 l of test sample.
2. Add 3 mL of Bradford reagent to each test tube and mix by vortexing. Allow the
mixture to stand at room temperature for 5 min.
3. Read the absorbance at 595 nm within 5-60 min. The protein-dye complex is
stable for 60 min.

Page 38 of 42
 Standard table

S. No. Volume of Volume of Volume of Amount Bradford

stock BSA test buffer of BSA reagent
sample (mL) A595
(l) (g)
(l) (l)

Blank 0.0 - 100 0 3

1 10 - 90 10 3

2 20 - 80 20 3

3 30 - 70 30 3

4 40 - 60 40 3

5 50 - 50 50 3

6 60 - 40 60 3

7 70 - 30 70 3

8 80 - 20 80 3

9 90 - 10 90 3

10 100 - 0 100 3

Test - 100 0 - 3

Plot the absorbance versus concentration of the standard protein solutions.

Determine the concentration of the unknown samples from the standard curve.
Report on the sensitivity of this assay based on the results obtained for 1:10, 1:100,
1:1000 samples.

Page 39 of 42
 II. Estimation of glucose by GOD-POD method

Aim

Estimation of the glucose concentration by GOD-POD assay

Principle

Glucose is oxidized to gluconic acid and hydrogen peroxide by the enzyme glucose
oxidase (GOD). The hydrogen peroxide produced further reacts with phenol and 4-
aminoantipyrine by the catalytic action of peroxidase (POD) to form a red
coloredquinoneimine dye complex. The intensity of color formed is directly proportional
to the amount of glucose present in the sample.

Materials: GOD-POD reagent, samples

Procedure
1. Prepare standard glucose solution from 0.1 to 1 mg/mL by pipetting 0- 100 μLof
stock glucose solution (10 mg/mL) and make up to 0.1 mL using distilled water
as shown in the standard table.
2. Add 1 mL of GOD-POD reagent into test tubes with proper marking.
3. Incubate at 37 °C for 10 min.
4. Take water instead of glucose sample in the blank solution and add the reagent
and against this measure absorbance at 505 nm.
5. The amount of the unknown sample can be known by reacting 0.1 mL of the
sample with 1 mL of the reagent.
Plot a graph of OD vs glucose concentration, and from this graph calculate the
concentration of the unknown sample.

S. No Volume of Volume of Amount of Volume of A505

glucose (μL) water (μL) glucose (mg) GOD-POD
reagent (mL)
Blank 0 100 0 1

1 10 90 0.1 1

2 20 80 0.2 1

3 30 70 0.3 1

4 40 60 0.4 1

4 50 50 0.5 1

5 60 40 0.6 1

6 70 30 0.7 1

Page 40 of 42
 7 80 20 0.8 1

8 100 0 1.0 1

Unknown 100 0 - 1

Page 41 of 42