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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