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Immobilization of beta galactosidase.pptx

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  • 1. IMMOBILIZATION OF β-GALACTOSIDASE ON A MAGNETICPOLYSILOXANE-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 Objectives1. 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-adapted0-25 ‘CApplications : 1. Used in medical and veterinary applications to treat digestivedisorders.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 isphysically confined or localised in a certain defined region or space with retention of itscatalytic 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 withoutinhibitory effects. Immobilization matrices employed : 1. Polysiloxane-Polyaniline and 2. OrderedMesoporous 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 mlimmersion at Resulting pH adjusted to Magnetic 11 using 33% de-ionized water +1050’C overnight + ml of 0.6M FeSO40.5 M aniline particles washed w/v liquor with de-ionized ammonia , and 1.1M FeCl3+1.0 M HNO3 . solution (1:1) addedPolymerization water , dried incubated for overnight at 30 min at 100’C dropwise underfor 2h at 25’C . magnetic stirring .Activated with 105’C and2.5%w/v sieved.glutaraldehydeand 20mM , pH4.5 stirred for 2hat 25 ‘C .
  • 7. FeCl3+FeSO4 +POS Brown under magnetic Magnetic stirring at 700C particles upon alkali addition Filtered , washed massmPOS-PANI uponpolymerization 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 filteredDried under 100ml and calcined at Functionalized 550’C for 4h atvaccum , glutaraldehyde(25% using the rate ofheated at w/v) +100 ml ethylenediamine 0.5’C/min.600’C for 4h . acetone +amino +acetone +30gWashed with functionalized MAC dried MAC .water . Dried . Stirred for 30 minfor 6h at 110’C under magnetic. stirring.
  • 9. Pluronics P123 + Pluronics P123 + HCl TMB + TEOS Aging for 24 hrs underCalcined stirringmesoporous 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 5.93100 0.0226 0.01 44.248 4424.78 7.533150 0.02626 0.0066 38.08 5712.11 8.75200 0.0514 0.005 19.45 3891.05 17.13250 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 12.1100 0.0438 0.01 22.83 2283.1 14.6150 0.0536 0.0066 18.65 2798.5 17.86200 0.0524 0.005 19.08 3816.8 17.46250 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= 0.06Specific 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 10.5100 0.0357 0.01 28.01 2801.12 11.9150 0.064 0.0066 15.625 2343.75 21.33200 0.0703 0.005 14.224 2844.95 23.43250 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 94.0 4.3 3.3 12.674.4 5 4.2 13.334.8 7.52 4.2 14.675.2 15.43 4 15.735.6 14.33 3.67 12.67Variation of pH for Free and Activated mPOS-PANI , Mesoporous
  • 17. FREE ACTI MESO 16 ACTI mPOS-PANI 14Specific 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 60Specific 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 GlucoseConc (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 37.61100 39.55 42.39 25.61 33.27 30.4150 21.61 22.67 23.16 20.83 21.61200 42.83 38.55 23.4 37.72 38.89250 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 38Glucose 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 SupportFTIR 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 inthe immobilization efficacy.•In the estimation of Kinetic parameters , Activated Mesoporous support has a Km of 33.33 mM which isideal 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 beenachieved through the Lineweaver Burke Plot , so the particles have a greater binding strength and reactivesurface 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 rangewhich means it opens up to more substrate when the pH changes . While the Mesoporous supports showvery less activity variation within that range and hence the effect of pH has no profound significance on itssurface characteristics.•As Reaction time proceeds, both the particles show high activity at the start of time but as the reactionproceeds , the activity decreases . Compared to the Free and Mesoporous support activated enzyme , themPOS-PANI particles show less percentage reduction in activity which means the retention is not muchaffected 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-PANIsupports which shows that activation has no significant reduction in lactose hydrolysis though a littleincrease is shown in the activated particle .The activated mesoporous supports show reduced glucoseproduction than the Non-activated one which means there could be some hindrance in the conversionpreventing product formation. sOverall , the mPOS-PANI support proves conclusively to be the best support for immobilization owingto all the above results shown .
  • 25. THANK YOU