SRI JAYACHAMARAJENDRA COLLEGE OF ENGINEERING,

MYSURU – 570 006

DEPARTMENT OF BIOTECHNOLOGY

BT 47L

Enzyme Technology and Biokinetics Lab Manual

Enzyme technology and Biokinetics lab

Prepared By LS and KRS
 Experiment - 1

Determination of alkalinity in the given water sample

AIM: To Determine the Alkalinity in a Given Water Sample

THEORY: Alkalinity is a measure of the capability of water to absorb H+ ions without

significant change of pH. In other words, alkalinity is a measure of the acid buffering capacity of
water. The determination of alkalinity of water is necessary for controlling the corrosion, to
calculate the amount of lime and soda needed for water softening; in conditioning the boiler feed
water, etc. Alkalinity of a sample of water is due to the presence of OH– (hydroxide ion), HCO3
– (bicarbonate ion) and CO3 2– (carbonate ion) or the mixture of two ions present in water. The
possibility of OH– and HCO3 – ions together is not possible since they combine together to form
CO3 2– ions. OH– + HCO3 – CO3 2– + H2O The alkalinity due to different ions can be
estimated separately by titration against standard acid solution, using selective indicators like
phenolphthalein and methyl orange. i) OH– + H+ H2O ii) CO3 2– + H+ HCO3 – iii) HCO3 – +
H+ H2O + CO2 The neutralization reaction upto phenolphthalein end point shows the
completion of reactions (i) and (ii) (OH– and CO3 2– ) and (CO3 2– and HCO3 – ) only. The
amount of acid used thus corresponds to complete neutralization of OH– plus half neutralization
of CO3 2– . The titration of water sample using methyl orange indicator marks the completion of
the reactions (i), (ii) and (iii). The amount of acid used after phenolphthalein end point
corresponds to one half of normal carbonate and all the bicarbonates. Total amount of acid used
represent the total alkalinity due to all ions present in water sample.

APPARATUS: Burette, pipette, conical flask, beakers, burette stand and clamp

CHEMICALS: Dry Na2CO3, concentrated 10(N) HCl, phenolphthalein and methyl orange
indicator

PROCEDURE:

1. Prepare Primary standard solution of Na2CO3 (0.1N).

2. Take Secondary standard solution of HCl and the water sample.

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3. Standardization of HCl solution by primary standard Na2CO3 solution– Pipette out 10 mL of
Na2CO3 solution in a conical flask, add 2 drops of methyl orange indicator, fill up the burette
with (10N) HCl solution and titrate till the color of the solution changes from yellow to red.

4. Analysis of water sample

i) Pipette 20 mL of the sample of water into a 100 mL conical flask and add2 drops of
phenolphthalein indicator and titrate against HCl till the color of the solution changes from pink
to colorless. Corresponding burette reading indicates the phenolphthalein end point (V1).

ii) Again pipette out 20 mL of the water sample in a conical flask, add 2 drops of methyl orange
indicator. Color of the solution becomes yellow. Continue the titration against the HCl solution
till the color changes to red. This burette reading corresponds to the methyl orange end point
(V2).

Analysis of water using phenolphthalein indicator

Strength of HCl solution = ____ (N)

Strength of water=(NHCl*VHCl)/20

Strength in terms of CaCO3 equivalent=Strength of water * equivalent weight of CaCO 3

Phenolphthalein alkalinity(P) =A g/L =A*1000mg/L

Slno Amount of water taken Volume of HCl rundown Mean volume of HCl
1
2

Enzyme technology and Biokinetics lab

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Analysis of water using Methyl orange indicator

Strength of HCl solution = ____ (N)

Strength of water=(NHCl*VHCl)/20

Strength in terms of CaCO3 equivalent=Strength of water * equivalent weight of CaCO 3

Methyl orange alkalinity(M) =B g/L =B*1000mg/L

Slno Amount of water taken Volume of HCl rundown Mean volume of HCl
1
2

CONCLUSION:

If P ˃ ½M Both OH– and CO3 2– ions are present in the water sample.

If P=0 Both OH– and CO3 2– ions are not present in the water sample

–
Alkalinity is present due to HCO3 ion only which can be determined using methyl orange
indicator and called methyl orange alkalinity (M).

If P = ½ M; indicates that only CO3 2– ions are present.

If P < ½ M; indicates that beside CO3 2– ions HCO3 – ions are also present.

If P = M; indicates only OH– ions are present.

Enzyme technology and Biokinetics lab

Prepared By LS and KRS
 Experiment 2

Identification of enzymes in different sources

Aim: To identify the enzymes present in different solutions

Principle: Different enzymes will identify using following reactions

Starch Amylase Simple sugars

Sucrose Invertase Glucose +fructose

H2O2 Catalase water +oxygen

Sources: Dry leaves, fresh leaves, raw potatoes, Boiled potatoes, Sprouted seeds, apple, banana
and yeast.

Apparatus required: Glass wares, Pestle and Mortar, Water bath, Centrifuge, Testubes, Beaker.

Reagents Required: 2% solution of Glucose, Maltose , Starch and Sucrose, Benedicts reagent and
Iodine solution

Control preparation

 Take 3 test tubes

 Add 2.5ml of 2% solution of starch to 1 test tube maltose to the second and glucose to
third test tube

 Add 1 ml of 2% iodine solution to each test tube.

 Add 1 ml of Benedicts reagent to all test tubes and vertex

 Use this as reference for the color change in the sample.

Procedure

Sample preparation for Amylase and Catalase Assay

 Take 5 g of each sample and homogenize using distilled water in Pestle and
Mortar

 Transfer homogenate to centrifuge tubes and centrifuge for 3 min.

 Collect the supernatant in test tubes

Enzyme technology and Biokinetics lab

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  Mark the test tubes accordingly to the sources.

Amylase assay

 Take 2.5 ml of different samples in different test tubes.

 Add 2.5ml of 2% solution of starch,1 ml of 2% iodine solution and 1 ml of Benedicts

reagent to all test tubes and vertex

 Place the test tubes in boiling water bath for few minutes.

 Take out the test tubes from water bath and compare the color with control.

Invertase assay

 Take 2.5 ml of different samples in different test tubes.

 Add 2.5ml of 2% solution of sucrose and add 1 ml of Benedicts reagent to all test tubes
and vertex.

 Place the test tubes in boiling water bath for few minutes.

 Take out the test tubes from water bath and compare the color with control.

Catalase assay

 Take 5 ml of H2O2 in different test tubes

 Crush different samples and add it to the different test tubes

 Observe the test tubes for effervescence

Observation table

Source Amylase Invertase Catalase

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Experiment 3

Isolation of œ Amylase from different sources

Aim: To isolate œ Amylase from different sources

Sources: Sweet Potato and Potato

Reagents required: 10mM CaCl2 solution

Apparatus required: Beaker, Pestle & Mortar, Measuring cylinder, Centrifuge and its tubes

Procedure:

1. Take 2 g of given source and homogenize using 10ml 10mM CaCl 2 solution.

2. Incubate the homogenate for 24 h at 4o C

3. Centrifuge the homogenate at 10000 RPM for 20 min.

4. Collect the supernatant and discard the pellets.

5. Measure the volume of supernatant obtained.

Result: the volume of crude enzyme collected from the source is ……………

Enzyme technology and Biokinetics lab

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Experiment 4

Determination of œ Amylase Enzyme activity

Aim: To determine the enzyme activity of œ Amylase

Principle: The reducing sugars produced by the action of œ Amylase react with dinitrosalcylic
acid giving a brown colored product.

Materials and reagents:

1. 0.1 M Sodium acetate buffer: pH 4.7: Dissolve 3.4g of sodium acetate in 200ml of
distilled water and adjust the pH to 4.7 by adding glacial acetic acid and make the
volume up to 250ml.

2. 1% starch solution: Dissolve 1 g of starch in 100ml of distilled water and heat up to 50 o

C to get clear solution.

3. Dinitrosalicylic acid: Solution A: Dissolve 1 g of DNS in 20ml of 2N NaOH solution.

Solution B: Dissolve 30g of sodium Potassium tartarate in60mi of

distilled water.

Mix solutions A and B and heat up to 80-90o C.The resulting

solution is clear DNS

4. Standard glucose solution: Stock solution :1g/100ml, working solution0.4,0.8,1.2,1.6,2

mg/ml.

5. Enzyme dilution: Dilute crude enzyme in the ratio 1:25

Procedure:

1. Take 9 test tubes.

2. Pipette standard glucose solutions to test tubes and make the volume 2 ml by adding
distilled water and name the test tubes from 1-6.

3. Add 1 ml of starch solutions to 7, 8 and 9 test tubes respectively.

Enzyme technology and Biokinetics lab

Prepared By LS and KRS
 4. Add 1, 0.5 and 0 ml Sodium acetate buffer to 7,8 and 9 test tubes respectively.

5. Add 0, 0.5 and 1ml of diluted enzyme to 7,8 and 9 test tubes respectively.

6. Vertex the test tubes and incubate at 37o for 15 min.

7. Arrest the enzyme substrate reaction by adding 1ml of DNS solutions to all test tubes.

8. Boil the test tubes in water bath for 5 min.

9. Add 5 ml of distilled water to all test tubes and vertex

10. Check the OD of solutions for all testubes.

Observation and Calculation

Standard Glucose curve

S Volu Concentratio Volu Volu Volume of distilled OD at

Incubation in hot water bath 5 min

l me of n(µg) me of me of water 540n

n gluco water DNS m
o se (ml)
(ml)

1 0 0 2

2 0.4 400 1.6

3 0.8 800 1.2 1ml 5ml

4 1.2 1200 0.8

5 1.6 1600 0.4

6 2 2000 0

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 Enzyme activity

Test Vol of Vol of Vol of Volume Vol of OD at

Incubation in hot water bath 5

Incubate for 15 min at room
tubes substrate buffer enzyme of DNS distilled 540nm
water
ml ml ml ml

7 1 1 0 5

1ml
temperature
8 1 0.5 0.5 5

9 1 0 1 5

min
Calculation:

Enzyme activity =……………..µg/15min/ml of diluted enzyme

=……………..µg/min/ml of diluted enzyme

Enzyme activity for undiluted enzyme=………………….*25 =µg/min/ml of undiluted

enzyme

Total volume of enzyme extract =……………………………..

Total enzyme activity for ………x……….ml of enzyme taken from …y…….g of

source= …………*x/y

Total œ Amylase Enzyme activity in y g of source= ………./1000 =

……………….mg/min/ml

Result : Total œ Amylase Enzyme activity=……………….mg/min/ml

Enzyme technology and Biokinetics lab

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Experiment 5

Specific activity of œ Amylase

Aim: To determine Specific activity of œ Amylase from different source

Principle: Specific activity is calculated by determining amount of proteins present in I mg in 1

ml of enzyme source and dividing it by the enzyme activity.

Materials and Reagents: Lowry’s reagent, Folin’s reagent, BSA standard solution

Procedure

1. Take 7 test tubes.

2. Pipette 0,0.2,0.4,0.6,0.8 and 1ml of working BSA solution to 6 test tubes and number it
from 1-6.

3. Make the volume as 1 ml in each test tube by adding water

4. Add 1ml of diluted enzyme to 7 th test tube.

5. Add 5 ml of Lowry’s reagent to all test tubes.

6. Incubate the test tubes at room temperature for 15 min.

7. After incubation add 0.5 ml of FC reagent to all test tubes

8. Keep the test tubes in dark at room temperature for 30 minutes.

9. Measure the OD and calculate the concentration of protein in 1ml of enzyme.

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 Tabular column

Sl Volu Volume Concentration Lowry’s FC OD at

no me of of water of protein in reagent reagent 660nm

Incubation at room temperature for 15min

Incubation at room temperature for 15min

BSA
ml µg
ml

0.5ml
5ml
4

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Calculations:

Enzyme activity of œ Amylase =………….mmole/min

1ml of 1:25ml diluted enzyme consists of…………… µg of protein

1ml of undiluted enzyme has ………………..mg of protein

Specific activity=enzyme activity/mg of proteins= ……………..µmol/min/mg

Nature of graph:

OD at 660nm

Con of protein in µg

Result: Specific activity of enzyme is ……………………………….

Enzyme technology and Biokinetics lab

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Experiment 6

Determination of Km and Vmax of œ Amylase

Aim: to determine Km and Vmax of œ Amylase

Reagents required :Citrate buffer (pH 5.3),Enzyme extract, Starch solution, DNS reagent.

Procedure:

1. Clean and dry 10 testubes

2. Mark test tubes as C1 T1 to C10 T10 depending on substrate concentration.(c- control

without enzyme).

3. Add o.5 ml of diluted (1:5) enzyme to test tubes marked as T

4. Add substrate (in the range of 0.1-1) to different test tubes

5. Add buffer to make the volume as 2ml

6. Vertex the test tubes and incubate at room temperature for 15 min

7. Add 1ml of DNS and keep it in boiling water bath for 5 min.

8. Cool all test tubes and add 4 ml of distilled water to all test tubes.

9. Vertex the contents in test tube and read the absorbance at 540nm.

10. Calculate the activity for each test tube.

11. Plot a graph and determine the constants by using Michalis-Menton plot and Line-weaver
Burk plot.

Tabular column:

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Sl.no Test Vol of Vol of Vol of Vol of OD at Activity 1/ [S] 1/[S]

Incubate at room temperature for 15 min

Keep it in boiling water bath for 5 min .Add 4 ml of distilled water after 5 min
tube enzyme substrate buffer( DNS 540nm V
(ml) (ml) ml)
1 C1 - 0.1 1.9
T1 0.5 0.1 1.4

2 C1 - 0.2 1.8
T1 0.5 0.2 1.3

3 C1 - 0.3 1.7
T1 0.5 0.3 1.2

4 C1 - 0.4 1.6
T1 0.5 0.4 1.1

5 C1 - 0.5 1.5
T1 0.5 0.5 1.0

6 C1 - 0.6 1.4
T1 0.5 0.6 0.9

7 C1 - 0.7 1.3
T1 0.5 0.7 0.8

8 C1 - 0.8 1.2
T1 0.5 0.8 0.7

9 C1 - 0.9 1.1
T1 0.5 0.9 0.6

10 C1 - 1 1
T1 0.5 1 0.5

Enzyme technology and Biokinetics lab

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 Experiment 7

Mixed Flow Reactor

Aim: To find out order and rate constant of the reaction between no-equimolar quantities of
NaOH and ethyl acetate in a mixed flow reactor.

Principle:
Continuous stirred tank reactors (CSTR) or mixed flow reactor (MFR) is configured much like a
batch reactor except reactants and products continuously from in and out of the reactor. Steady
state flow reactors are ideal for industrial purpose when large quantities of material are to be
processed and when the rate of reaction is fairly high to extremely high. Flow reactors need
more supporting equipments like pump. But good product quality can be obtained space item (τ)
is the time required to process the reactor volume of feed at specified column.

τ= =

Theory:
The SCTR also known as backmix reactor is a common ideal reactor type in chemical
engineering. Perfect mixing is assumed in care of ideal CSTR. In a perfectly mixed reactors, the
outfeed composition material inside the reactor which is a function is residence time and rate of
reaction. The ideal CSTR model is used to simplify engineering claculations and can be used to
describe research reactors. In practise, it only to be approached in particular in in industrial size
reactors. Design equation for mixed flow reactor in steady state is
τ= =

Plot, ln v/s τ

If the points fits well into a straight line then the assumption of second order, kinetics is true.
The rate constant may be calculated from the slope of the line.

Apparatus:
Setup the vessels as shown in the figure with necessary connections, conical flask, burette,
measuring cylinder.

Reagents required:
0.04N NaOH, 0.04N ethyl acetate, 0.1N HCl, 0.04N oxalic acid.

Procedure:
 Volume of the MFR was noted down.
 The volumetric flow rate of NaOH and ethyl acetate was adjusted.
 The space time was calculated using the formula τ = VR – volume reactor; VT –volumetric
flow.

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 The reactants were allowed to flow through the reactor for τ minutes.
 The rection was arrested by adding 0.1N HCl.
 It was titrated against NaOH by adding 2-3 drops of phenolphthalein indicator till the color
changes from colourless to pale pink.
 The above procedure was repeated for different reactant flow rates.
 The order and rate constant was determined by plotting a graph of ln v/s τ.

Result:
Rate constant for the given reaction is k = ________ (mol/L)-1 min -1

Enzyme technology and Biokinetics lab

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 Experiment 8

Determination of Rate Constant and Order for Heating of Water

Aim: To find order and rate constant for heating of water.

Principle:
If the ration follows
 Zero order:
Then T-T0 = kt
The graph of T-T0 v/s time should pass through the origin with slope k or if it is
 First order:
Then ln T/T0 = kt
The graph of ln(T/T0) v/s time should pass through the origin with slope k, and if it is
 Second order:
- = kt

The graph of - v/s time pass through origin with slope.

Theory:
Changing the concentration of substances taking part in a reaction usually changes the rate of the
reaction.
Order: It the power to which concentration is raised.
Zero order reaction:
A zero order reaction has a constant rate that is independent of the reactants concentration. The
rate law is as follows
rate = k
Where k has unit of ms-1. In other words, a zero order reaction has a rate law in which the sum
of the exponents is equal to zero. A reaction is zero order if concentration data are plotted versus
time and the result is a straight line.

First order reaction:

A first order reaction has a rate proportional to the concentration of one reactant.
rate = kCa1
First order rate constants have units of sec-1. In other words, a first order reaction has a rate law
in which the sum of the exponent is equal to 1.

Second order reaction:

A second order reaction has a rate proportional to the product of the concentration of a reactants,
or to the square of the concentration of a single reactant.
rate = kca2

Apparatus:
Heating mantle, stop watch, beaker.

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Procedure:
 1000 mL of water was collected in a beker.
 The initial temperature of water was noted.
 The beaker was then placed on a heating mantle (Bunsen burner).
 The temperature raise was noted for every 1 minute.
 The order of reaction and rate constant was checked for zero order, first order and second
order.
 Based on the correct and straight line fir from the graph order was found.

Result:
From the graph it is clear that the reaction is zero order and rate constant is __________
mol/L/sec.

Enzyme technology and Biokinetics lab

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 Experiment 9

Determination of Kinetics for Esterification

Aim: To find the order and rate constant for non-equimolar concentration reaction.

Principle: A batch reactor has neither inflow nor outflow of reactants or products while the reaction is
being carried out. This is an unsteady state operation where composition changes with time; however, at
any instant the composition throughout the reactor is uniform. As the concentration of the reactant
species in the reactor decreases, conversion increases.

Theory: The reaction of ester hydrolysis is chosen for this experiment for particular reasons. First, it
takes place with rather low rates so that changes can be observed on time scale of minutes. Second, it
requires an acid as catalyst. The amount of products can thus be followed as a function of time by
extracting small amounts from the reacting solution at certain time intervals. Third, the product itself is
an acid. So that its concentration can be easily and precisely determined by titration.

The batch reactor is the generic term for a type of vessel widely used in the process industries. In
a batch reactor, the reactants and the catalyst are placed in the reactor and the reaction is allowed to
proceed for a given time where upon the mixture of unreacted material together with the product is
withdrawn. In an ideal batch reactor, the concentration and temperature are assumed to be spatially
uniform. In practise, the condition can be approximately reduced by vigorous agitation or stiring.

Order and the rate constant of the reaction can be obtained by experiments. Mainly two types of
analysis may be used for rate law determination. a) Integral Method of Analysis. b) Differential Method
of Analysis.

Plot:
= Cao(µ-1)kt

Where t = time
µ=

xa is conversion of a at given time. If the data points fit well into a straight line then the assumption of
second order kinetics is true. The rate constant can be calculated from the slope of the line kC ao(µ-1).

Apparatus:
Conical flask, beakers, burette, burette stand, measuring cylinder, reactor, magnetic pellet and magnetic
stirrer.

Reagents required:
 0.08N 1 L sodium hydroxide solution.
 0.05N 1 L ethyl acetate solution.
 0.1N 250 mL hydrochloric acid.
 0.1n 100 mL oxalic acid.

Procedure:

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 Batch reactor of capacity 1 L was taken and 400 mL of prepared NaOH solution was added.
 The stirrer was switched on and 400 mL of ethyl acetate was added immediately after addition, the
stop watch was started.
 10 mL of the reaction mixture was withdrawn from the reactor after 5 mins. This was done after
every 5 mins intervals simultaneously, the reaction was arrested by adding 10 mL of 0.1N HCl each
time.
 The excess HCl in this mixture was titrated against standardized NaOH solution using
phenolphthalein as indicator and the volume of NaOH comnsumed was noted.
 The above procedure was repeated for all the reaction mixtures withdrawn at different time intervals
from the reactors.
 From the experimental data, Ca value was found and hence Xa value was calculated.
 A graph of v/s time was plotted and checked for a straight lune fit.
 The rate constant was determined from the slope of the graph.

Result:
Rate constant for given reaction k = _________ (mol/L)-1 min -1

Observation:
Time Volume of NaOH

Time ln

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