immobilization of beta galactosidase.pptx
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IMMOBILIZATION OF β-GALACTOSIDASE ON A MAGNETIC POLYSILOXANE-POLYANILINE PARTICLE AND MESOPOROUS
SUPPORT – A COMPARITIVE STUDY OF GLUCOSE PRODUCTION
A Project Work byVikram Ramakrishnan- 011255031
M.Tech Industrial Biotechnology (5-yr Integrated)SASTRA University
Under the guidance ofProf.A.Arumugam – Asst Prof II
SCBT , SASTRA University
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.
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.
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)
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 .
Materials and Methods – Synthesis and magnetization of Polysiloxane-Polyaniline particles
5ml of TEOS = Ethanol (1:1)
Temp raised to 70 ‘C under stirring +100µl of Conc.Hcl + incubated for 50 min .
Solidified for 3 days at 25’C . Smashed and powder weighed
2g of powder+100 ml de-ionized water +10 ml of 0.6M FeSO4 and 1.1M FeCl3 solution (1:1) added dropwise under magnetic stirring .
pH adjusted to 11 using 33% w/v liquor ammonia , incubated for 30 min at 100’C
Resulting Magnetic particles washed with de-ionized water , dried overnight at 105’C and sieved.
0.1M KMnO4 immersion at 50’C overnight + 0.5 M aniline +1.0 M HNO3 . Polymerization for 2h at 25’C . Activated with 2.5%w/v glutaraldehyde and 20mM , pH 4.5 stirred for 2h at 25 ‘C .
FeCl3+FeSO4 +POS under magnetic stirring at 700C
Brown Magnetic particles upon alkali addition
Filtered , washed mass
Immersion in KMnO4 mPOS-PANI upon polymerization
Synthesis and Functionalization of SBA-15 Mesoporous
4g of Pluronics P123 in 150 ml 1.6MHCl at 20 ‘C
2g TMB added and stirred for 5h
Solution aged at 40’C for 20h under magnetic stirring , further aged at 100’C for one day under static condition.
Product filtered and calcined at 550’C for 4h at the rate of 0.5’C/min.
Functionalized using ethylenediamine +acetone +30g dried MAC .
100ml glutaraldehyde(25%w/v) +100 ml acetone +amino functionalized MAC . Stirred for 30 min under magnetic stirring.
Dried under vaccum , heated at 600’C for 4h . Washed with water . Dried for 6h at 110’C .
Pluronics P123 + HClPluronics P123 + TMB + TEOS
Aging for 24 hrs under stirring
Calcination @ 5500C
Calcined mesoporous powder
Results and Discussions :
Substrate Conc (mM)
Rate of Rection
(µmol/min/ml)
1/S (1/mM)
1/V S/V Specific Activity ( U/mg)
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
-0.03 -0.02 -0.01 0.00 0.01 0.020
5
10
15
20
25
30
35
40
45
50
55
60
1/Vm
-1/Km
1/V
min
/µm
ol/m
l
1/S mM-1
B
-350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 2500
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
-Km
Km/Vm
S/V
ml/m
in
S mM
B
50 100 150 200 2504
6
8
10
12
14
16
18
20
Sp
ecif
ic A
ctiv
ity
(U/m
g s
olid
)
Substrate Concentration (mM)
Free
Lineweaver Burke Plot – Km= 303.33mM . Vm= 0.131 mmol/ml/min Hanes Woolf Plot – Km= 297.50 mM. Vm= 0.124 mmol/ml/min. Activity maximum at 250mM
Substrate Conc (mM)
Rate of Rection
(µmol/min/ml)
1/S (1/mM)
1/V S/V Specific Activity ( U/mg)
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
-0.03 -0.02 -0.01 0.00 0.01 0.020
2
4
6
8
10
12
14
16
18
20
22
24
26
28
1/Vm
-1/Km
1/V
min
/µm
ol/m
l
1/S mM-1
Act. Meso
-500 -450 -400 -350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 2500
500
1000
1500
2000
2500
3000
3500
4000
-Km
Km/Vm
S/V
S
B
50 100 150 200 25010
12
14
16
18
20
22
24
26
28
30
Sp
eci
fic A
ctiv
ity(U
/mg
so
lid)
Substrate Concentration (mM)
Activated Meso
Lineweaver Burke Plot – Km = 33.33mM . Vm= 0.06 mmol/ml/min. Hanes-Woolf Plot – Km= 137.50 mM . Vm= 0.1375 mmol/min/ml. Activity increase shown at 150 mM and 250 mM .
Substrate Conc (mM)
Rate of Rection
(µmol/min/ml)
1/S (1/mM)
1/V S/V Specific Activity ( U/mg)
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
-350 -300 -250 -200 -150 -100 -50 0 50 100 150 200 2500
1000
2000
3000
4000
5000
6000
Km/Vm-Km
S/V
ml/m
in
S mM
B
-0.03 -0.02 -0.01 0.00 0.01 0.020
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
-1/Km 1/Vm
1/V m
in/µ
mo
l/m
l
1/S mM-1
B
50 100 150 200 250
10
12
14
16
18
20
22
24
Spe
cific
Act
ivity
(U/m
g)
Substrate Concentration(50mM)
Activated Nano Lineweaver Burke Plot – Km= 144.90mM . Vm= 0.125 mmol/min/ml Hanes-Woolf Plot – Km= 38.60 mM . Vm= 0.054 mmol/ml/min . Activity Maximum at 175 mM .
pH Specific Activity FREE
(U/mg)
Specific Activity ACTI
MESO (U/mg)
Specific Activity ACTI
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
3.5 4.0 4.5 5.0 5.5 6.00
2
4
6
8
10
12
14
16
Sp
ecif
ic A
ctiv
ity
(U/m
g)
pH
FREE ACTI MESO ACTI mPOS-PANI
Time (min) Specific Activity
FREE(U/mg)
Specific Activity ACTI
MESO (U/mg)
Specific Activity ACTI
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
4 6 8 10 12 14 16 18 20 22
10
20
30
40
50
60
70
Sp
ecif
ic A
cti
vit
y (
U/m
g)
Time ( Min)
Free Meso mPOS-PANI
Substrate Conc (mM)
Glucose Production FREE (mMol/L)
Glucose Production NonActi Meso (mMol/L)
Glucose Production Acti Meso (mMol/L)
Glucose Production Non Acti mPOS-PANI (mMol/L)
Glucose Production Acti mPOS-PANI (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
50 100 150 200 250
20
22
24
26
28
30
32
34
36
38
40
Glu
co
se
Co
nce
ntr
atio
n (
mM
ol/L
)
Substrate Concentration (mM)
mPOS-PANI Activated mPOS-PANI Inactivated
50 100 150 200 250
20
22
24
26
28
30
32
34
36
38
40
42
44
Y A
xis
Titl
e
X Axis Title
Meso Activated Meso Inactivated
50 100 150 200 2500
5
10
15
20
25
30
35
40
45
50
Glu
cose
Co
nce
ntr
atio
n (
mM
ol/L
)
Substrate Concentration (mM)
Free
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
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.
SEM Images
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 .
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