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
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.
Introduction : The enzyme – β-galactosidase• Sources – Plant , Microbial and Mammalian .•E.C – 18.104.22.168•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 ‘CApplications : 1. Used in medical and veterinary applications to treat digestivedisorders.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 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)
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 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 .
FeCl3+FeSO4 +POS Brown under magnetic Magnetic stirring at 700C particles upon alkali addition Filtered , washed massmPOS-PANI uponpolymerization Immersion in KMnO4
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.
Pluronics P123 + Pluronics P123 + HCl TMB + TEOS Aging for 24 hrs underCalcined stirringmesoporous Calcination @powder 5500C
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 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.
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. sOverall , the mPOS-PANI support proves conclusively to be the best support for immobilization owingto all the above results shown .