Immobilized Microbial cell

Immobilized Microbial Cell, Types of Immobilization technique , Immobilization yeast culture

1. IMMOBILIZED MICROBIAL CELL BY, U.AATHILAKSHMI(117011101273), G.ABIRAMI(117011101274), R.ARAVINDHAN (117012101275), 3 RD B.TECH BIOTECHNOLOGY . 4/2/2020 1 DEPARTMENT OF BIOTECHNOOGY

2. Objective Introduction. Need for immobilization. Support/matrix used. Types of immobilization. Carrier binding. Cross linking. Entrapping. Advantage & Disadvantage. Yeast cell immobilization. 4/2/2020 2 DEPARTMENT OF BIOTECHNOOGY

3. Introduction: Immobilized microbial cells can be defined by substituting the word " enzymes" for the microbial cells. The definition given for immobilized microbial enzymes is " microbial cells physically confined or localised in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously”. The immobilized microbial cells can be growing ,resting or dead but the enzyme activity remains in the active state. The immobilized microbial cells are otherwise called as "immobilized living cells". 4/2/2020 3 DEPARTMENT OF BIOTECHNOOGY

4. Need for immobilization: Protection from degradation and deactivation. Recycling, repetitive use. Cost efficiency. Enhanced stability. Use as controlled release agents. The ability to stop the reaction rapidly by removing the enzyme from the reaction Solution (or vice-versa) Allows development of multi-enzyme reaction system. 4/2/2020 4 DEPARTMENT OF BIOTECHNOOGY

5. Supports/ Matrix used in immobilization technology: The matrix / support hold the enzyme. The matrix used should be cheap and easily available. A wide range of matrix are used in immobilization of enzyme / whole cells. The matrix/ supports are grouped into three major categories ; 1. Natural polymers. 2. Synthetic polymers. 3. Inorganic materials. 4/2/2020 5 DEPARTMENT OF BIOTECHNOOGY

6. Natural polymer: Gelatin: partially hydrolyzed collagen, good water holding capacity. Cellulose: cheapest support available . Starch &Pectin : good water holding capacity . Synthetic polymer: They are ion exchange resins / polymers . They are insoluble supports with porous surface The porous surface trap and hold the enzymes / cells Example: DEAE cellulose , Polyvinyl chloride (P VC) , UV activated Polyethylene glycol (PEG) Inorganic material: Example : Zeolite ,Ceramics , Silica , Activated carbon ,charcoal. 4/2/2020 6 DEPARTMENT OF BIOTECHNOOGY

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8. Carrier binding Attachment of an enzyme to an insoluble carrier creates an active surface catalyst. Modes of surface attachment classify carrier methods into : 1.Physical adsorption. 2.Covalent binding. 4/2/2020 8 DEPARTMENT OF BIOTECHNOOGY

9. PHYSICAL ADSORPTION This technique is based on the physical interaction between the microorganism and the carrier surfaces In 1916, Nelson and Griffin showed the invertase could be adsorbed onto activated charcoal without any change in enzymatic activity, thus producing the first immobilized enzyme , although they made no subsequent use of it. 4/2/2020 9 DEPARTMENT OF BIOTECHNOOGY

10. The support materials may be inorganic (e.g. alumina, silica gel, calcium phosphate gel, glass) or organic (starch, carboxymethyl cellulose, DEAE- cellulose, DEAE-sephadex). Adsorption is based on weak forces such as van der Waals forces and hydrogen bonds . The immobilization of microorganisms on properly chosen adsorbents stimulates microbial metabolism, protects cells from unfavorable agents, and preserves their physiological activity. Porous solid support Enzyme + Absorbed enzyme on the support 4/2/2020 10 DEPARTMENT OF BIOTECHNOOGY

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12. Advantages of adsorption method : Easy to carry out. No reagents are required. Minimum activation steps involved. Comparatively cheap method. Less disruptive to protein than chemical methods. Disadvantages of adsorption method : Desorption of enzymes from the carrier. Efficiency is less. 4/2/2020 12 DEPARTMENT OF BIOTECHNOOGY

13. Covalent binding The mechanism involved in this method is based on covalent bond formation between activated inorganic support and cell in the presence of a binding agent. This technique is widely used. However, covalent binding is often associated with loss of some enzyme activity. 4/2/2020 13 DEPARTMENT OF BIOTECHNOOGY

14. T he following are the common methods of covalent binding. 1. Cyanogen bromide activation : The inert support materials (cellulose, sepharose, sephadex) containing glycol groups are activated by CNBr , which then bind to enzymes and immobilize them. 4/2/2020 14 DEPARTMENT OF BIOTECHNOOGY

15. 2.Peptide bond formation: Enzyme immobi­lization can also be achieved by the formation of peptide bonds between the amino (or carboxyl) groups of the support and the carboxyl (or amino) groups of enzymes. The support material is first chemically treated to form active functional groups. 4/2/2020 15 DEPARTMENT OF BIOTECHNOOGY

16. Showing the various enzyme immobilized by covalent bonding 4/2/2020 16 DEPARTMENT OF BIOTECHNOOGY

17. Cross linking The enzyme molecules are immobilized by creating cross-links between them, through the involvement of poly-functional reagents . These reagents in fact react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules . There are several reagents in use for cross-linking. These include glutaraldehyde , diazobenzidine , hexamethylene diisocyanate and toluene di- isothiocyanate . 4/2/2020 17 DEPARTMENT OF BIOTECHNOOGY

18. Glutaraldehyde is used to cross-link enzymes or link them to supports. It usually consists of an equilibrium mixture of monomer and oligomers. The product of the condensation of enzyme and glutaraldehyde may be stabilised against dissociation by reduction with sodium borohydride . 4/2/2020 18 DEPARTMENT OF BIOTECHNOOGY

19. Advantage and disadvantage of cross linking: 4/2/2020 19 DEPARTMENT OF BIOTECHNOOGY

20. Entrapment Entrapment is an irreversible method, where immobilized cells are entrapped in a support or inside fibers . This technique creates a protective barrier around the immobilized microbes , ensuring their prolonged viability during not only processing but also storage in polymers The matrices used are agar, alginate, carrageenan, cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-linkable resins, polyacrylamide, polyester, polystyrene and polyurethane . 4/2/2020 20 DEPARTMENT OF BIOTECHNOOGY

21. Method of entrapment (A) Enzyme inclusion in gels: This is an entrapment of enzymes inside the gels (B) Enzyme inclusion in fibers: The enzymes are trapped in a fiber format of the matrix. (C) Enzyme inclusion in microcapsules: In this case, the enzymes are trapped inside a microcapsule matrix. Microcapsule formed formed by monomer mixture such as polyamide or calcium alginate 4/2/2020 21 DEPARTMENT OF BIOTECHNOOGY

22. Lattice entrapment Entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide,polyvinylalcohol , etc... and natural polymer (starch) have been used to immobilize enzymes using this technique. 4/2/2020 22 DEPARTMENT OF BIOTECHNOOGY

23. Microencapsulation It refers to the process of spherical particle formation where in a liquid or suspension is enclosed in a semipermeable membrane . The membrane may be polymeric, lipoidal , lipoprotein-based or non-ionic in nature. There are three distinct ways of microencapsulation. 1. Building of special membrane reactors. 2. Formation of emulsions. 3. Stabilization of emulsions to form microcapsules. 4/2/2020 23 DEPARTMENT OF BIOTECHNOOGY

24. Uses: Microencapsulation is recently being used for immobilization of enzymes and mammalian cells. For instance, pancreatic cells grown in cultures can be immobilized by microencapsulation. Hybridoma cells have also been immobilized successfully by this technique. 4/2/2020 24 DEPARTMENT OF BIOTECHNOOGY

25. Advantages of entrapment method : Fast Cheap (low cost matrix available). Mild conditions are required. Less chance of conformational changes in enzyme. Disadvantages of entrapment method : Leakage of enzyme. Pore diffusion limitation. Chance of microbial contamination. 4/2/2020 25 DEPARTMENT OF BIOTECHNOOGY

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28. Advantage of 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. 4/2/2020 28 DEPARTMENT OF BIOTECHNOOGY

29. Disadvantage of immobilization: Concentration of enzymes will be less. Production of unwanted enzymes and unwanted products. Modification of end products by other enzymes. 4/2/2020 29 DEPARTMENT OF BIOTECHNOOGY

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31. Applications of immobilization : 1.  Industrial production: Industrial production of antibiotics, beverages, amino acids etc. uses immobilized enzymes. 2. Biomedical applications: Immobilized enzymes are widely used for diagnosis and treatment of many diseases such as inborn disorder. 3 . Food industry : Enzymes like pectinases and cellulases immobilized on suitable carriers are successfully used in the production of jams, jellys and syrups from fruits and vegetables. 4. Research : The use of immobilized enzyme allow researcher to increase the efficiency of different enzymes such as different proteases for cell and organelle lysis. 5. Biodiesel production from vegetable oils. 6. Textile industry : Scouring, bio polishing and desizing of fabrics. 7. Waste water management: Treatment of sewage and industrial effluents. 8. Detergent industry: Immobilization of lipase enzyme for effective dirt removal from cloths. Applications of immobilization 4/2/2020 31 DEPARTMENT OF BIOTECHNOOGY

32. Immobilization of Yeast Cells by Calcium- alginate Entrapment Method Requirement: Culture : Saccharomyces cerevisiae Yeast potato Dextrose (YPD)/ Potato Dextrose Agar (PDA) medium (for cultivation of Yeast cells) Sodium alginate solution (2.5% w/v in 0.1% NaCl ) Calcium Chloride (CaCl2 ) solution (0.05 N) Gluteraldehyde (2.5% v/v) Sucrose solution (1% w/v) 4/2/2020 DEPARTMENT OF BIOTECHNOOGY 32

33. Procedure for immobilization of Yeast Cells: Grow the yeast cells in YPD/PDA medium Keep it in shaker for 24hours at 100 rpm Filter out the yeast cells with the help of Whattman filter paper Take 20 ml - sodium alginate solution and add 3 ml-yeast cell in it(mix & kept at room temp for 30 min ) Then ,Add 3 ml -gluteraldehyde solution (incubate at room temperature for 90 minutes ) With the help of 10 ml pipette, drop wise add this mixture into the beaker containing 100 ml CaCl2 solution . 4/2/2020 DEPARTMENT OF BIOTECHNOOGY 33

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35. Filter out the beads with the help of normal filter paper. Wash the beads 2-3 times with sterile Distilled water Load these beads into thoroughly washed glass column/ beaker. Add 50ml of sterile 1% sucrose solution. After every 30 minutes interval collect the 1 ml of sample and estimate the amount of glucose by using Dinitrosalilcylic acid method (DNSA) method. 4/2/2020 DEPARTMENT OF BIOTECHNOOGY 35

36. Reference: https://www.slideshare.net/EasyBiologyClassEBC/enzyme-cellimmobilizationtechniquesppteasybiologyclass http://www.biologydiscussion.com/enzymes/immobilization/immobilization-of-enzymes-and-cells-methods-effects-and-applications/10208 http://www1.lsbu.ac.uk/water/enztech/immethod.html http://content.inflibnet.ac.in/data-server/eacharya-documents/53e0c6cbe413016f234436f5_INFIEP_17/49/ET/17-49-ET-V1-S1__pc-9_immobilization.pdf YouTube link- https://youtu.be/gK-_FhGAhUg 4/2/2020 36 DEPARTMENT OF BIOTECHNOOGY

37. THANK YOU 4/2/2020 37 DEPARTMENT OF BIOTECHNOOGY