Introduction• Immobilization of enzymes can be defined as the confinement of an enzyme (bio-catalyst) in a distinct phase, separated from the bulk phase but allowing it to exchange with the latter.• Bulk Phase consists of a substrate, an effecter or inhibitor.• Immobilized enzyme is either physically entrapped or covalently bonded by chemical means to an inert insoluble matrix or carrier.• In other words, it involves the restrictive localization of enzymes.• Matrix is generally a high molecular weight polymer. Ex : cellulose, polyacrlamide, alginate, etc. S
Need for Immobilization• Accelerates the chemical reaction.• Specificity & un-modified enzyme.• Cost effective.• Not difficult to separate.• Attachment to polymers/matrix, causes re-use. S
Advantages of Immobilized Enzymes• Recovered at the end of the reaction thereby can be reused.• Economy of the reaction is improved.• Easy separation of enzyme from the products occurs.• Stability of immoblilised enzyme increases.• Enhanced enzyme properties.• Efficiency of the catalytic reaction is better in a few cases.• Better control of reaction can be achieved.• Catalytic process can be operated continuously.• Multi enzyme reaction possible. S• Potential in industrial & medicinal use.
Methods of Immobilization• Parameters for Method Selection :- Overall catalytic activity. Effectiveness of the catalytic utilization. Deactivation & Regeneration characteristics. Cost effective. Intended application of immobilized enzyme. Toxicity of immobilized enzyme. Waste disposal (of immobilization process). S
Methods of Immobilization• Physical Methods • Chemical Methods Adsorption Covalent Bonding Entrapping Cross Linking Membrane confinement Complexation & Chelation S
Carrier for Immobilized Enzymes• Ideal Characteristics of the Carrier:- Low Cost & of optimum quality Inertness Physical Strength Stability Regenerability Enhancement of enzyme specificity Reduction of product inhibition S
A • Non-specific binding like electrostatic orD A hydrophobic affinity binding to special ligand. • Mostly explained in following terms:S Static pores Dynamic poresO Reactor LoadingR Electro DepositionP • NOTE: Adsorbent (mostly polymeric matrix)T Ex: alumina, bentonite, CMC, Silica gel, Titania, etc.ION
A • Advantages:D Simple & EconomicalS Limited Loss of activity Can be Recycled, Regenerated & Reused (R3)OR • Disadvantages:P Relatively Low surface area for binding Exposure of enzyme to microbial attack.T Smaller particles cause high Pressure drop in continuous packed bed reactor.I Yield are often low due to inactivation &O desorption.N
• Enzymes are held or entrapped within theE suitable gels or fibres.N • In a gel it may causes: Matrix polymerization orT Precipitation orR Coagulation • Entrapment in calcium alginate is the mostA widely used for entrapment for :P Microbial Animal &P Plant enzymes/cellsI Ex: Glucose oxidase + Polyacrlamide (gel entrapment)N • NOTE: Adsorbent (mostly commonly used) Ex: polyacrylamides, collagen, silicaG gel, alginates, etc.
E • Advantages:N No chemical modification Relatively stable forms.T Easy handling & reusage.R • Disadvantages: May diffusion of substrate & product occurs.A Substrate accessibility may reduced due toP free radical polymerization of gel. Enzyme in-activation.P Loss of enzyme content.I • NOTE: Sometimes covalent bonding may formsN between the entrapped enzyme & the matrix.G
E Enzyme + Sod.alginateN Mixture is added dropwise CaCl2 SolutionTR Beads of Calcium alginatesAPPING
Membrane • Enzyme molecules (usually in aq. form) are C confined within semi-permeable : Reaction vessel O o Partitioning into two chambers by a semi N permeable membrane F o One chamber contains the enzyme while I the other have substrate & product. Hollow fiber membrane N o Entrapment in semi permeable fibres E (cellulose, triacetate) or spheres M (nylon, collodion). E o In which, the enzyme will be in the lumen/hollow space, while the N fibres/spheres will be submerged in the T substrate.
Membrane Micro capsules C o Enzymes are packed in microcapsules formed by polymerization (like phase O separation or chemical polymerization). N Liposomes F o Enzymes can be bounded in a concentric I spheres of lipoidal membrane formed by addition of phospholipid. N • Advantages: E No enzyme leakage M No change in enzyme activity E • Disadvantages: N Diffusional barrier to the substrate & T product. Not cost effective.
Chemical • Enzyme forms co-valent link with active group of B the matrix (like terminal -NH2, -COOH,etc,). • Support with groups like : O -OH : support activation covalently by CNBr. N -COOH : supports (like CMC) activation D covalently by azide derivatives. I -NH2 : support activation covalently by forming diazonium chlorides on treatment N with NaNO2 + HCl. NOTE: The functional group of enzyme which is involved in the linkage, should not affect the active G properties of the said enzyme.
Chemical • Advantages: B Not affected by pH O Ionic Strength N • Disadvantages: Active site may be modified D Not cost effective. I NOTE: Adsorbent Ex: N Agarose, Cellulose, sepharose, Polyacrlamide,etc. G
Cross L • It involves cross linking of enzyme to a multi functional reagent without use of any solid I support. N • Alternatively, chemical bridge of some other molecule between & with the chemical support K (i.e., reaction of enzyme with reagent bridge or chemical bridge). I • Activated carriers are used. N Ex : Sepharose by CNBr (most commonly used) Ex : Sepharose by ethyl chloroformate G
Cross • Advantages: L • Strong linkage leads to low enzyme leakage I while use. • Higher stability (i.e., pH, ionic & substrate concentration. N • Disadvantages: K • Partially or wholly inactivation by active site modification. I • Not cost effetcive. N G
Enzymes in Medicine Glucose oxidase Glucose Hydrogen peroxide peroxidase Dye: Blue---Green---Brown Dye changes according to amount of glucoseEnzyme-linked immunosorbent assays (ELISAs) Sdetect antibodies to infections.
Enzymes in biological washing Powders• Proteases break down the coloured, insoluble proteins that cause stains to smaller, colourless soluble polypeptides.• Can wash at lower temperatures S
Enzymes in Food Industry• Pectinase break down substances in apple cell walls and enable greater juice extraction. Lactase breaks down lactose in milk into glucose and galactose. This makes milk drinkable for lactose intolerant people. S
Enzymes in Seed Germination starch amylase secretedembryo plant maltose S
ReferencesPharmaceutical Biotechnology By Dr. S.P. Vyas & Dr. V.K. DixitEnymes & its Immobilization Presentation By Dr. S. KhanamInternet Resources : http://www.eplantscience.com http://www.clickbiology.com http://www.lsbu.ac.uk http://www.tech-ceramics.co.uk