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What are the advantages of immobilized enzymes? 

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Enzyme and Cell Immobilization Techniques PPT by easybiologyclass

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Immobilization is defined as the imprisonment of cell or enzyme in a distinct support or matrix. The support or matrix on which the enzymes are immobilized allows the exchange of medium containing substrate or effector or inhibitor molecules. Advantages of immobilized enzymes are Increased functional efficiency of enzyme, Enhanced reproducibility of the process they are undertaking, Reuse of enzyme, Continuous use of enzyme, Less labour input in the processes, Saving in capital cost and investment of the process, Minimum reaction time, Less chance of contamination in products, More stability of products, Stable supply of products in the market, Improved process control and High enzyme substrate ratio. Methods of Immobilization: Based on support or matrix and the type of bonds involved, there are five different methods of immobilization of enzyme or whole cells, they are Adsorption, Covalent bonding, Entrapment, Copolymerization and Encapsulation
Immobilization of whole cells:
Immobilization of whole cells is an alternative to enzyme immobilization and it is a well-developed method for the utilization of enzymes from microbes. Immobilization of whole cells become particularly effective when the individual enzymes become inactive during direct immobilization, or the isolation and purification of enzyme is not cost effective. The greatest advantage of whole cell immobilization is that here the enzymes will be active and stable for long period of time since they are in their natural environment. Use of immobilized cells for fermentation is a very old practice. Bacteria or yeast cells are immobilized by adsorbing it on woodchips. This is practiced in many parts for different types of fermentations.
Advantages of whole cell immobilization:
 Multiple enzymes can be introduced to a single step
 Extraction and purification of enzymes are not required
 Enzymes are stable for long time
 Native conformation of enzyme is best maintained
 Cell organelles like mitochondria and chloroplasts can be immobilized
 Cost effective method
Disadvantages of whole cell immobilization:
 Concentration of enzymes will be less
 Production of unwanted enzymes and unwanted products
 Modification of end products by other enzymes produced by immobilized cells
Methods of immobilization of whole cells:
Methods of immobilization of whole cells are same as that described for enzyme immobilization and they include:
1. Adsorption
2. Covalent bonding
3. Cell to cell cross linking
4. Encapsulation

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Enzyme and Cell Immobilization Techniques PPT by easybiologyclass

  1. 1. l. ._——/ .~. _ <__, .r . ._-. _.: r- + -. _—: ,. . ._-_. .. _ _o. _ - .1 — I” ENZYME TMMQBEETZATTQIN I I Techniques of Enzyme & Whole Cell Immobilization I I I e-mail: easybioIogycIass@gmaiI. com maiI@easybioIogycIass. com www. easybiologyclass. com
  2. 2. Learning objectives: A I Understand immobilization of enzyme/ whole cells I Advantages and disadvantages of immobilization I Applications of enzyme immobilization I Supports/ matrix used in immobilization technique I Types/ methods of immobilization www. easybiologyclass. com > Adsorption > Covalent bonding > Entrapment > Co-polymerization/ cross linking > Encapsulation Immobilization of whole cells Advantages and disadvantages of whole cell immobilization Methods of whole cell immobilization Ix)
  3. 3. IMMOBILIZATION TECHNIQUES EASY BIOLOGY CAMS What is immobilization? I Definition: imprisonment of cell or enzyme in a distinct support/ matrix I The support/ matrix allows exchange of medium I The medium contains substrate or effecter or inhibitor molecules I First immobilization technology: amino acylases by Aspergillus oryzae for the production of L-amino acids in Japan I Two main advantages of enzyme immobilization: 1. Increased functional efficiency 2. Enhanced reproducibility
  4. 4. IMMOBILIZATION TECHNIQUES EASY BIOLOGY GA” What are the advantages of immobilized enzymes? . ° Reuse °. ° Continuous use V Less labour input V Saving in capital cost/ investment v Minimum reaction time °. ° Less chance of contamination in products *2’ More stability . ° Improved process control . * High enzyme substrate ratio
  5. 5. IlVllVIOETl_. EZATI~Oll TECHNIQUES What are the disadvantages of enzyme immobilization? 5 Uses in industrial applications are limited Loss of catalytic properties in some enzymes Some enzymes become unstable Enzymes are inactivated by heat generated in the system High cost for isolation, purification and recovery of active enzyme www. easybiologyclass. com on
  6. 6. ,. I ‘{ ~ ‘ . /‘usv I1‘/ lM*0BlIl_. lZATl'Oll TECHNIQUES What are the applications of enzyme immobilization? Industrial production: E g. Antibiotics, beverages, amino acids etc. i Biomedical applications: treatment, diagnosis and drug delivery Food industry: production of jams, jellies and syrups Research: HRP in blotting experiments, proteases for cell lysis Production of biodiesel: from vegetable oils Waste water management: treatment of sewage & industrial effluents Textile industry: scouring, bio-polishing and desizing of fabrics Detergent industry: immobilization of lipase for effective dirt removal www. easybiologyclass. com V, 77 i 1 . . I -1 r »-~ .
  7. 7. IMMOBILIZATION TECHNIQUES Supports/ Matrix used in immobilization technology: I The matrix/ supports hold the enzyme I The matrix used should be cheap and easily available I Their reaction with medium and enzyme should be minimums as possible I A wide range of matrix are used in immobilization of enzyme / whole cells I The matrix/ supports are grouped into three major categories 1. Natural polymers 2. Synthetic polymers 3. Inorganic materials Q3
  8. 8. IMMOBILIZATION TECHNIQUES EASY BOOLOGY CLASS Supports/ Matrix used in immobilization technology: (1). Natural polymers I Alginate: derived from algal cell wall (calcium or magnesium alginate) Chitosan and chitin: enzyme bins to the — OH groups Collagen: protenaceous support Carrageenan: a sulfated polysaccharide obtained from algae Gelatin: partially hydrolyzed collagen, good water holding capacity Cellulose: cheapest support available Starch: good water holding capacity Pectin: good water holding capacity
  9. 9. IMMOBILIZATION TECHNIQUES (MY BIOLOGY HMS Supports/ Matrix used in immobilization technology: (2). Synthetic polymers I They are ion exchange resins / polymers I They are insoluble supports with porous surface I The porous surface trap and hold the enzymes / cells I Example: > DEAE cellulose > Polyvinyl chloride (PVC) > UV activated Polyethylene glycol (PEG)
  10. 10. IMMOBILIZATION TECHNIQUES "5 :7.- EASV BIOLMY (M59 Supports/ Matrix used in immobilization technology: ( 3 ). Inorganic materials > Zeolites: > Ceramics: > Diatomaceous earth ( Trade name celite] > Silica: > Glass > Activated carbon > Charcoal
  11. 11. 4, I I1VlM'OETI_. fZATI0ll TECHNIQUES . ,=‘-in Types/ Meth ads of Immobilization Five different methods of immobilization of enzyme / cells 1. Adsorption 2. Covalent bonding 3. Entrapment 4. Copolymerization 5. Encapsulation www. ea. sybiologyclass. com . I , 1 . I .4: r V . 11
  12. 12. 4, I I1VlM'OETI_. fZATI0ll TECHNIQUES ll . /{Aw Cross Linking ": ;;x: ;:; ":. :;, ',: :,', " 5. Enzyme / Cell . Immobilization Adsorptmn Covalent Bonding . . . .. ... .f Encapsulation Entrapment Enzyme/ Cell Immobilization Methods . I , 1 _. I .4: r .
  13. 13. IlVl1VIOl. l.liZATI‘Oll TECHNIQUES (X ’ (1). Adsorption . Oldest method of enzyme immobilization Simplest method of enzyme immobilization Nelson & Griffin used charcoal to adsorb invertase Enzymes are adsorbed to external surface of support Support/ carrier may be : 1. Mineral support (aluminum oxide, clay) 2. Organic support (starch) 3. Modified sepharose and ion exchange resins www. easybiologyclass. com ' . J EASY Enzyme Enzyme Enzyme Support Enzyme Enzyme Adsorption 12
  14. 14. I . Y : K. -"usv " IMMoBiLrzA. r1oN TECHNIQUES I (1). Adsorption Weak bonds stabilize enzymes to the support/ carrier Bonds involved are low energy bonds such as: '2' Ionic interaction 6* Hydrogen bonds °2* Van der Waal forces Carrier particle size must be small (for appreciable surface bonding) Particle size used: 500 Ato 1 mm diameter N 0 pore diffusion limitations (since enzyme are immobilized externally) www. ea. sybiologyclass. com 1 3
  15. 15. IM1ViOEiLEZATI~Oll TECHNIQUES 1 Methods of adsorption: 1. Static process: Immobilization to carrier by allowing the solution containing enzyme to Contact the carrier (without stirring) 2. Dynamic batch process: Carrier is placed in the enzyme solution and mixed by stirring or agitation 3. Reactor loading process: Carrier is placed in the reactor, then enzyme solution is transferred to reactor 4. Electrode position process: Carrier is placed proximal to an electrode in an enzyme bath and the current is put on, the enzyme migrates to the carrier and deposited on the surface www. easybiologyclass. com 1 -'3-2
  16. 16. I1‘/ IMOBll. .liZATI: ON TECHNIQUES (1). Adsorption Advantages of adsorption method *4 Easy to carry out 5 No reagents are required “I Minimum activation steps involved 7 Comparatively cheap method ”-f Less disruptive to protein than chemical methods Disadvantages of adsorption method F2 Desorption of enzymes from the carrier r Efficiency is less www. easybiologyclass. com 0|
  17. 17. IlVll/ I*OBILIZATION TECHNIQUES * . " . . 1- ‘ . A A. ‘. T I’ . , , ’ I* ‘ . .}": Asv (2). Covalent bonding: Involves the formation of covalent bonds between enzyme and support Widely used method of enzyme immobilization Chemical groups in enzymes that forms covalent bonds with support are: > Amino groups, Imino groups > > > > > www. easybiologyclass. com Covalent bond between enzyme and support / Enzyme Hydroxyl gro ups C b I ar OW groups ,5 ‘ Enzyme Thiol groups and Methylthiol groups g I U: ; Enzyme Guanidyl groups and Imidazole groups * Enzyme Phenol rings 16
  18. 18. IlVll/ I*OBILIZATION TECHNIQUES * . " . . 1- ‘ . A A. ‘. T I’ . , , ’ I* ‘ . .a": Asv (2). Covalent bonding: Important functional groups of enzyme that provide chemical groups to form covalent bonds with support/ carrier are: 1. 2. 3. . °°. l. °‘. °": F‘ 9. Alpha carboxyl group at ‘C’ terminal Alpha amino group at ‘N’ terminal Epsilon amino groups of Lysine and Arginine Beta and gamma carboxyl groups of Aspartate and Glutamate Phenol ring of Tyrosine Thiol group of Cysteine Hydroxyl groups of Serine and Threonine Imidazole group of Histidine Indole ring of Tryptophan www. easybiologyclass. com 1 7
  19. 19. INIMQEILIZAITIQN TECHNIQUES E . ‘(QM 1" (2). Covalent bonding: Carriers / supports used for covalent bonding: Carbohydrates: Eg. Cellulose, DEAE cellulose, Agarose Synthetic agents: Eg. Polyacrylamide Protein carriers Amino group bearing carriers: Eg. amino benzyl cellulose Inorganic carriers: Porous glass, silica Cyanogen bromide (CNBr)-agarose and CNBr Sepharose ‘ Hydroxyl and Amino groups form covalent bonds more easily www. easybiologyclass. com I 8
  20. 20. IMMOBILIZATION TECHNIQUES '. ' EASY BIOLOGY CLASS Methods of covalent bonding 1. Diazoation: Bonding between amino group of support and thyrosil or histidyl group of enzyme 2. Peptide bond: between amino / carboxyl groups of support and enzyme 3. Poly functional reagents: Use of a bi-functional or multifunctional reagent (glutaraldehyde) which forms bonding between the amino group of the support and amino group of the enzyme O¢/ /E0 Glutaraldehyde @
  21. 21. IMMOBILIZATION TECHNIQUES '. ' [MY BIOLOGY CLASS (2). Covalent bonding: Advantages > Strong linkage of enzyme to the support > No leakage or desorption problem > Comparatively simple method > A variety of support with different functional groups available > Wide applicability
  22. 22. IMMOBILIZATION TECHNIQUES ': [MY BIOLOGY CLASS (2). Covalent bonding: Disadvantages: ( major problem with covalent bonding ): I Chemical modification of enzyme leading to functional conformation loss I Enzyme inactivation by changes in the conformation when undergoes reactions at active sites I This can be overcome through immobilization in the presence of enzyme substrate or a competitive inhibitor
  23. 23. IIVIIVIIOBILIZATIION TECHNIQUES (3). Entrapment: Enzymes are physically entrapped inside a matrix Bonds involved may be covalent or non-covalent Matrix used will be water soluble polymer Examples of matrix: § I °. ° polyacrylamide gels Enzyme Enzyme °I° Cellulose triacetate 6 I’ Agar Enzyme Enzyme °I° Gelatin °2° Carrageenan Enzyme Enzyme °. ° Alginate www. easybiologyclass. com Enzyme Enzyme Enzyme / Matrix Enzyme Enzyme Enzyme I») Ix)
  24. 24. IlVIMOEI§I_. IZA. TI*OIl TECHNIQUES (3). Entrapment: Form and nature of matrix varies ‘ Pore size of matrix is adjusted to prevent loss of enzyme i Possibility of leakage of low molecular weight enzymes Agar and carrageenan have large pore sizes i Pore size can be adjusted with the concentration of the polymer ‘ Entrapment of enzyme can be used for sensing application Not much success in industrial process ‘ Easy to practice at small scale www. easybiologyclass. com
  25. 25. II». /III/ IoEII. I:zATI«oN TECHNIQUES Methods of en trapmen t: 1. Inclusion in the gels: enzymes trapped in gels 2. Inclusion in fibers: enzymes supported on fiber format 3. Inclusion in microcapsules: Enzymes entrapped in microcapsules formed by monomer mixtures such as polyamine, calcium alginate www. easybiologyclass. com 2-I
  26. 26. I1‘/ EM-OBIMZATI-OlI TECHNIQUES (3). Entrapment: Advantages: ‘ Fast Cheap (low cost matrix available] ‘ Mild conditions are required Less chance of conformational changes in enzyme Disadvantages: Leakage of enzyme ‘ Pore diffusion limitation Chance of microbial contamination www. easybiologyclass. com lo '11
  27. 27. I1‘! /IMOEII. IZA. 'I'I'OI! TECHNIQUES (4). Cross linking (Copolymerization): F Cross linking: covalent bonding between various groups of enzymes via polyfunctional reagents If No matrix or support are involved Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme A Enzyme Cross links www. easybiologyclass. com
  28. 28. Ill/ IMOETMZATI-Oll TECHNIQUES (4). Cross linking (Copolymerization): F Commonly used polyfunctional reagents: Glutaraldehyde, Diazonium salt r Technique is cheap and simple but not often used with pure proteins rt It is widely used in commercial preparations Demerit: r Polyfunctional reagents can denature the enzyme j N= N O%/ /§O Glutaraldehyde Diazonium www. easybiologyclass. com 2 7
  29. 29. IMM-oBiLrzA: r1»oix1 TECHNIQUES I 3 ‘ l ‘l l’. ‘ . /tAsv ' (5). Encapsulation: Enclosing enzymes in a semi permeable membrane capsule Capsule is made up of nitro cellulose or nylon Effectiveness depends upon the stability of enzymes Enzyme Enzyme Enzyme A: Enzyme I i . - Enzyme Enzyme Enzyme I. .’ V , /i www. easybiologyclass. com I Semi-permeable Membrane 2 8
  30. 30. IlVllVI'OElLTZATI*ON TECHNIQUES (5). Encapsulation: Advantages: Cheap and simple method Large quantity of enzymes can be immobilized by encapsulation Disadvantages: ‘ Pore size limitation Only small substrate molecule is able to cross the membrane www. easybiologyclass. com
  31. 31. I1‘/ IM'0BILIZA. TI'0ll TECHNIQUES Immobilization of cells: An alternative to enzyme immobilization Well developed method for the utilization of enzymes from microbes Effective method when: > Individual enzymes become inactive during immobilization > Isolation and purification of enzyme is not cost effective Here enzymes will be active and stable for a long period of time Method of cell immobilization are same as described for enzyme immobilization Adsorption method is the oldest method [use of woodchips as a carrier] www. easybioIogycIass. com 30
  32. 32. I1V1'M*0ElLlIZA. TION TECHNIQUES Advantages of whole cell immobilization: Multiple enzymes can be introduced to a single step Extraction and purification of enzymes are not required Enzymes are stable for long time Native conformation of enzyme is best maintained Cell organelles like mitochondria and chloroplasts can be immobilized Disadvantages of whole cell immobilization: Concentration of enzymes will be less Production of unwanted enzymes and unwanted products Modification of end products by other enzymes www. easybiologyclass. com 31
  33. 33. IMMOBILIZATION TECHNIQUES Immobilization of cells: I Methods of whole cell immobilization are similar to enzyme immobilization I Methods of whole cell immobilization: CI Adsorption Cl Covalentbonding Cl Cell to cell cross linking Cl Encapsulation CI Entrapment
  34. 34. IlVIlVIOEll_. IZATIOlI TECHNIQUES Immobilization of cells: Methods, Support materials, Cells and Reaction Method Support Material i Reaction g Gelatin Lactobacilli Lactose = > lactic acid Porous glass Saccharomyces Glucose = > ethanol Adsorption Cotton fibers Zymomonas Glucose = > ethanol F DEAE Cellulose Nocardia Steroid conversion Cellulose + cyanuric . . Covalent chloride .5‘. cerevzsiae Glucose = > ethanol bonding _ _ _ _ Titanium oxide Acetobacter Vinegar J : - Cross linking Glutaraldehyde E. coli Fumaric acid Aluminium alginate Candida tropicalis calciumalginate Polyester Streptomycessps. Alginate polylysine Hybridoma cells Phenol degradation Entrapment Glucose = > ethanol Glucose : fructose Encapsulation Monoclonal antibodies www. easybiologyclass. com 33
  35. 35. i/1312; www. easybiologyclass. com You Tube‘ casybiologyclass (. l/Tltllili/ §’(l[éO>l, /£0 0 E-mail us @: easybiologyclass@gmail. com mail@easybiologyclass. com .5 visit: www. easybiologyclass. com 74° ‘-05"’ W“

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