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1. IMMOBILIZATION OF β-GALACTOSIDASE ON A MAGNETIC
POLYSILOXANE-POLYANILINE PARTICLE AND MESOPOROUS SUPPORT
– A COMPARITIVE STUDY OF GLUCOSE PRODUCTION
A Project Work by
Vikram Ramakrishnan- 011255031
M.Tech Industrial Biotechnology (5-yr Integrated)
SASTRA University
Under the guidance of
Prof.A.Arumugam – Asst Prof II
SCBT , SASTRA University

2. Aims and Objectives
1. Aim : The primary aim of this project is to immobilize the enzyme β-galactosidase
on a magnetic polymeric particle and on a mesoporous support to compare the
immobilization efficiency and estimate glucose production from the same.
2. Objectives : The bigger picture ?? – the conversion of lactose to its
monosaccharides – Glucose and Galactose .
• Lactose hydrolysis by the enzyme β-galactosidase to alleviate lactose
malabsorption in a large human population.
• Treating whey permeate in dairy effluents to recover the excess lactose and reuse
it for producing digestible products.
3. Future Scope : To synthesize novel nanoparticle that serve as reactor systems and
enhance lactose hydrolysis.
• To develop reactor models for continuous conversion.

3. Introduction : The enzyme – β-galactosidase
• Sources – Plant , Microbial and Mammalian .
•E.C – 3.2.1.23
•Also known as Lactase
•Catalytic agents for transglycosylation and hydrolytic reactions.
• 2 types : Thermo stable and Cold-Active.
• Aspergillus Oryzae , Aspergillus niger & Pyrococcus sp; - Thermo stable 35-80’ C .
• Arthrobacter psychrolactophilus , Pseudomalteromonas haloplanktis – Cold-adapted
0-25 ‘C
Applications : 1. Used in medical and veterinary applications to treat digestive
disorders.
2. Used in studying the structure and function of blood groups containing glyco-
conjugates.

4.  The process : Immobilization – The enzyme is said to be immobilized when it is
physically confined or localised in a certain defined region or space with retention of its
catalytic activity which can be used repeatedly and continuously.
 Methods of Immobilization :
 Physical Adsorption
 Covalent Binding
 Entrapment
 Crosslinking
 Reasons to Immobilize : 1. To localise the enzyme and retain its activity.
2. Stabilize it to cater to industrial implementations.
3. Enhance its biocatalytic activity , to convert maximum substrate to product without
inhibitory effects.
 Immobilization matrices employed : 1. Polysiloxane-Polyaniline and 2. Ordered
Mesoporous Silica (SBA-15)

5.  Polysiloxane-Polyaniline – WHY ????
Properties – 1. Magnetic nature
2. Smaller Size (1 µm)
3. Polymeric composition
So ?? - The magnetic nature helps in separation in a magnetised fluidised bed
reactor .
Smaller Size helps in effective entrapment and binding of the enzyme-
substrate due to its large surface area .
Polymeric composition is necessary so that there are many functional groups
to attach itself covalently to the incoming enzyme/substrate .
 Mesoporous Silica : Properties – 1. 2 nm- 50 nm .
2. Ordered – rod shaped morphology
3. SBA-15 particles prepared from triblock non-
ionic copolymerization
So ?? - Mesoporous nature effective in binding of enzyme .
 Activation : Glutaraldehyde is used as an activating agent for the magnetic particle.
Ethylenediamine and Glutaraldehyde are used as activators for
Mesoporous particles .

6. Materials and Methods – Synthesis and
magnetization of Polysiloxane-Polyaniline particles
Temp raised to 70 ‘C Solidified for 3
5ml of TEOS =
under stirring days at 25’C .
Ethanol (1:1)
+100µl of Conc.Hcl + Smashed and
incubated for 50 min powder weighed
.
0.1M KMnO4 2g of powder+100 ml
immersion at Resulting pH adjusted to
Magnetic 11 using 33% de-ionized water +10
50’C overnight + ml of 0.6M FeSO4
0.5 M aniline particles washed w/v liquor
with de-ionized ammonia , and 1.1M FeCl3
+1.0 M HNO3 . solution (1:1) added
Polymerization water , dried incubated for
overnight at 30 min at 100’C dropwise under
for 2h at 25’C . magnetic stirring .
Activated with 105’C and
2.5%w/v sieved.
glutaraldehyde
and 20mM , pH
4.5 stirred for 2h
at 25 ‘C .

7. FeCl3+FeSO4 +POS Brown
under magnetic Magnetic
stirring at 700C particles upon
alkali addition
Filtered ,
washed mass
mPOS-PANI upon
polymerization Immersion in KMnO4

8. Synthesis and Functionalization of SBA-15
Mesoporous
4g of Pluronics 2g TMB added Solution aged at
P123 in 150 ml and stirred for 5h 40’C for 20h
1.6MHCl at 20 ‘C under magnetic
stirring , further
aged at 100’C for
one day under
static condition.
Product filtered
Dried under 100ml and calcined at
Functionalized 550’C for 4h at
vaccum , glutaraldehyde(25%
using the rate of
heated at w/v) +100 ml
ethylenediamine 0.5’C/min.
600’C for 4h . acetone +amino
+acetone +30g
Washed with functionalized MAC
dried MAC .
water . Dried . Stirred for 30 min
for 6h at 110’C under magnetic
. stirring.

9. Pluronics P123 +
Pluronics P123 + HCl TMB + TEOS
Aging for 24 hrs under
Calcined
stirring
mesoporous Calcination @
powder 5500C

10. Results and Discussions :
Substrate Rate of 1/S 1/V S/V Specific
Conc Rection (1/mM) Activity (
(mM) (µmol/mi U/mg)
n/ml)
50 0.0178 0.02 56.18 2808.98 5.93
100 0.0226 0.01 44.248 4424.78 7.533
150 0.02626 0.0066 38.08 5712.11 8.75
200 0.0514 0.005 19.45 3891.05 17.13
250 0.0567 0.004 17.636 4409.17 18.9
Variation of Kinetic Parameters for Free enzyme

11. B
60 B
6000
55
5500
S/V ml/min
50
5000
45
1/V min/µmol/ml
4500
40
4000
35
3500
30 3000
25 2500 Km/Vm
20 2000
-Km
-1/Km 15 1500
10 1000
1/Vm
5 500
0 0
-0.03 -0.02 -0.01 0.00 0.01 0.02 -350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 250
S mM
1/S mM-1
Free
20
18  Lineweaver Burke Plot – Km=
303.33mM . Vm= 0.131
Specific Activity (U/mg solid)
16
14 mmol/ml/min
12  Hanes Woolf Plot – Km=
10 297.50 mM. Vm= 0.124
8 mmol/ml/min.
6  Activity maximum at 250mM
4
50 100 150 200 250
Substrate Concentration (mM)

12. Substrate Rate of 1/S 1/V S/V Specific
Conc Rection (1/mM) Activity (
(mM) (µmol/mi U/mg)
n/ml)
50 0.0363 0.02 27.55 1377.41 12.1
100 0.0438 0.01 22.83 2283.1 14.6
150 0.0536 0.0066 18.65 2798.5 17.86
200 0.0524 0.005 19.08 3816.8 17.46
250 0.0868 0.004 11.52 2880.18 28.93
Variation of Kinetic Parameters for Activated Mesoporous
Support

13. Act. Meso
28
26 B
1/V min/µmol/ml
24
4000
22
20 3500
18
1/Vm 3000
16
14 2500
S/V
12
2000
10
8 1500
-1/Km 6 1000 Km/Vm
4 -Km
500
2
0 0
-0.03 -0.02 -0.01 0.00 0.01 0.02 -500 -450 -400 -350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 250
1/S mM-1 S
Activated Meso
30
28  Lineweaver Burke Plot – Km =
26
33.33mM . Vm= 0.06
Specific Activity(U/mg solid)
24
22
mmol/ml/min.
20  Hanes-Woolf Plot – Km= 137.50
18
mM . Vm= 0.1375 mmol/min/ml.
 Activity increase shown at 150
16
14
12 mM and 250 mM .
10
50 100 150 200 250
Substrate Concentration (mM)

14. Substrate Rate of 1/S 1/V S/V Specific
Conc Rection (1/mM) Activity (
(mM) (µmol/mi U/mg)
n/ml)
50 0.0315 0.02 31.746 1587.3 10.5
100 0.0357 0.01 28.01 2801.12 11.9
150 0.064 0.0066 15.625 2343.75 21.33
200 0.0703 0.005 14.224 2844.95 23.43
250 0.0446 0.004 22.42 5605.38 14.86
Variation of Kinetic Parameters for Activated mPOS-PANI
support

15. B
6000
B
S/V ml/min
34
32 5000
30
28
26 4000
1/V min/µmol/ml
24
22
20
3000
18
16
14
2000
12
10
-1/Km 8 1/Vm -Km
6 1000 Km/Vm
4
2
0 0
-0.03 -0.02 -0.01 0.00 0.01 0.02 -350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 250
1/S mM-1 S mM
Activated Nano
 Lineweaver Burke Plot –
24
Km= 144.90mM . Vm= 0.125
22
mmol/min/ml
Specific Activity(U/mg)
20
 Hanes-Woolf Plot – Km=
18
38.60 mM . Vm= 0.054
16
14
mmol/ml/min .
12
 Activity Maximum at 175
10
mM .
50 100 150 200 250
Substrate Concentration(50mM)

16. pH Specific Specific Specific
Activity FREE Activity ACTI Activity ACTI
(U/mg) MESO (U/mg) mPOS-
PANI(U/mg)
3.6 1.73 2.27 9
4.0 4.3 3.3 12.67
4.4 5 4.2 13.33
4.8 7.52 4.2 14.67
5.2 15.43 4 15.73
5.6 14.33 3.67 12.67
Variation of pH for Free and Activated mPOS-PANI ,
Mesoporous

17. FREE
ACTI MESO
16
ACTI mPOS-PANI
14
Specific Activity (U/mg)
12
10
8
6
4
2
0
3.5 4.0 4.5 5.0 5.5 6.0
pH

18. Time (min) Specific Specific Specific
Activity Activity ACTI Activity ACTI
FREE(U/mg) MESO (U/mg) mPOS-
PANI(U/mg)
5 56.6 67.33 47.33
10 36.6 42.66 20
15 25 27.66 13.6
20 12.66 14.66 11
Variation of Time
Free
Meso
70
mPOS-PANI
60
Specific Activity (U/mg)
50
40
30
20
10
4 6 8 10 12 14 16 18 20 22
Time ( Min)

19. Substrate Glucose Glucose Glucose Glucose Glucose
Conc (mM) Production Production Production Production Production
FREE NonActi Acti Meso Non Acti Acti
(mMol/L) Meso (mMol/L) mPOS- mPOS-
(mMol/L) PANI PANI
(mMol/L) (mMol/L)
50 24.27 30.94 24.66 35.83 37.61
100 39.55 42.39 25.61 33.27 30.4
150 21.61 22.67 23.16 20.83 21.61
200 42.83 38.55 23.4 37.72 38.89
250 39.22 20.78 34.89 22.89 33.55
Estimation of Glucose Production

20. Meso Activated
Meso Inactivated
mPOS-PANI Activated 44
mPOS-PANI Inactivated
42
40
40
38
38
Glucose Concentration (mMol/L)
36
36
34
34
Y Axis Title
32
32
30
30
28
28
26
26
24
24
22
22
20
20
50 100 150 200 250
Substrate Concentration (mM) 50 100 150 200 250
X Axis Title
Free
50
45
Glucose Concentration (mMol/L)
40
35
30
25
20
15
10
5
0
50 100 150 200 250
Substrate Concentration (mM)

21. Particle Characterization Studies - mPOS-PANI
Particle Size Analysis
– 1 µm
SEM images for(a) mPOS and
(b)mPOS-PANI
XRD for mPOS
and mPOS-PANI

22. SBA-15 Mesoporous Support
FTIR Analysis
Bare Group : Characteristic
peak observed at
1082.78cm-1 confirms the
presence of hydroxyl group.
This shows the presence of
hydroxyl group (Bare group)
in the sample
Amine Group :
Characteristic peak
observed at 104.70cm-1
confirms the presence of
Si-O-Si. The primary amine
bend was observed at
1569.11 cm-1 and the peak
at 3392.14cm-1 confirms
the presence of amine
group.

23. SEM Images

24. CONCLUSIONS
•Both prove to be equally competent but the magnetic POS-PANI particles seem to take an upper hand in
the immobilization efficacy.
•In the estimation of Kinetic parameters , Activated Mesoporous support has a Km of 33.33 mM which is
ideal for binding but has very less Vm value = 0.06mmol/ml/min through the Lineweaver Plot .
•For the Activated mPOS-PANI supports , a Km = 144.9 mM and a Vm = 0.125 mmol/ml/min has been
achieved through the Lineweaver Burke Plot , so the particles have a greater binding strength and reactive
surface area for it to covalently accommodate the enzyme and substrate .
• Variation of pH for the mPOS-PANI particles elucidates its increase in activity within this short range
which means it opens up to more substrate when the pH changes . While the Mesoporous supports show
very less activity variation within that range and hence the effect of pH has no profound significance on its
surface characteristics.
•As Reaction time proceeds, both the particles show high activity at the start of time but as the reaction
proceeds , the activity decreases . Compared to the Free and Mesoporous support activated enzyme , the
mPOS-PANI particles show less percentage reduction in activity which means the retention is not much
affected and can be proved to be stable over a larger amount of time .
•Glucose production is maximum in 200 mM for both the activated and non-activated mPOS-PANI
supports which shows that activation has no significant reduction in lactose hydrolysis though a little
increase is shown in the activated particle .The activated mesoporous supports show reduced glucose
production than the Non-activated one which means there could be some hindrance in the conversion
preventing product formation. s
Overall , the mPOS-PANI support proves conclusively to be the best support for immobilization owing
to all the above results shown .

25. THANK
YOU