Enzyme immobilization ,Methods ,advantages and disadvantages and applications

Introducing immobilized enzymes ,reasons and limitations and their application .

1. Immobilization of Enzyme 3/24/2020 1 Taufica Nusrat MS in Biotechnology Institute of biotechnology and genetic engineering

2. Learning Outcome What does Immobilized enzyme means? Important aspects of the immobilization procedure. Reasons and limitations for enzyme immobilization? Components of Enzyme Immobilization. What are the properties of support material? Classification of support. Techniques of Enzyme Immobilization? Kinetics of Immobilized Enzyme. Factors affecting immobilized enzyme production. What are the advantages and disadvantages of using immobilized enzyme are? What are the application of Immobilized enzyme? 3/24/2020 2

3. Enzyme immobilization Immobilization, is the term that expresses of making something immobile or fixed. The first immobilized enzyme was amino acylase of Aspergillus oryzae for the production of L-amino acid in japan. Immobilized enzymes are enzyme that are either covalently bound or absorb on the surface of an insoluble support or incorporate in a gel matrix. 3/24/2020 3

4. Enzyme immobilization 3/24/2020 4

5. PROPERTIES OF THE IDEAL CARRIER REQUIRED FOR ENZYME IMMOBILIZATION Regeneratiblity. Reduction in product inhibition. Enhancement of enzyme specificity. Ferromagnetism. cheap, inert, physically strong and stable. catalytic surface. 3/24/2020 5

6. Important aspects of the immobilization procedure The properties of the free enzyme. The type of support used. The methods of support activation and enzyme attachment . 3/24/2020 6

7. Reasons and limitations for enzyme immobilization Reasons Reuse enzymes . Protection from degradation and deactivation. Lowering the total production cost of enzyme mediated reactions. Ability to stop the reaction rapidly by removing the enzyme from the reaction solution. Enhanced stability. Easy separation of the enzyme from the product and recovery. Product is not contaminated with the enzyme. Limitations Cost of carriers and immobilization Problems with cofactors and regeneration Mass transfer limitations Problems with multi enzyme systems Changes in properties (selectivity) Activity loss during immobilization 3/24/2020 7

8. Components of Enzyme Immobilization Enzymes : Enzymes are both proteins and biological catalysts (biocatalysts) that accelerate chemical reactions. Example: Pyruvate kinase . Support matrix : The support or matrix on which the enzymes are immobilized allows the exchange of medium containing substrate or effector or inhibitor molecules. It is a material that uses to keep enzyme during catalytic reaction. Enzyme Immobilized Techniques: The most commonly used techniques for immobilization of enzymes on solid support include adsorption, covalent bonding, entrapment, cross linking, and encapsulation methods based on support or matrix and the type of bond. 3/24/2020 8

9. Properties of support materials 3/24/2020 9

10. Classification of support Supports can be categorized as organic and inorganic support based on their chemical composition. 3/24/2020 10

11. Classifications of Organic and Inorganic Supports: Organic support materials O rganic natural polymers used as support material for enzyme immobilization are water-insoluble polysaccharides. O rganic synthetic polymers have been employed as supports of immobilized enzymes, such as polystyrene, polyamides and vinyl . Advantages: Large surface areas, cost effectiveness, good mechanical rigidity, enhanced thermal and storage stability, reduced susceptibility to microbial attacks and allow reusability . Inorganic supports materials Inorganic supports materials have greater stabilities than organic supports due to their higher resistance to extreme operating conditions. Inorganic synthetic mineral, such as glass bead, metal and metal oxides, have been widely used as support to immobilize enzyme. Advantages: Large specific surface area, enhanced catalytic activity, and improved thermal and mechanical stabilities. 3/24/2020 11

12. Enzyme Immobilized Techniques Based on support or matrix and the type of bond; enzyme immobilization techniques are five types. 3/24/2020 12

13. Adsorption method The oldest method of enzyme immobilization . Nelson and Griffin used charcoal to absorb to external surface of the support. The enzyme is physically adsorbed or attached onto the carrier surface via weak forces, such as ionic interaction, hydrogen bonds and Van der Waals interaction. Advantages Simple and cheap High catalytic activity No conformational change of the biocatalyst No need to use reagents Reuse of expensive material Disadvantages Low stability Possible loss of biomolecules Weak bonds might cause desorption of biocatalyst 3/24/2020 13

14. Encapsulation Is the method is performed by enclosing the enzymes in selectively permeable membranes , such as nitrocellulose or nylon . Enhance the enzymatic activity as there is large contact surface between enzyme and substrate. Encapsulation method is widely used for application in biomedical, food, detergents, and wastewater treatment sectors. Advantages Large enzyme quantities may be immobilized. Easy preparation Low cost Disadvantages: Mass transfer limitation Leakage of enzyme High enzyme concentration required. 3/24/2020 14

15. Cross-linking method Cross-linking , which is also known as copolymerization, is the another irreversible enzyme immobilization method, whereby there is no matrix or support involved. Cross-linking is an immobilization technique that combines both covalent bonding and entrapment. Advantages Strong biocatalyst binding Prevents leakage Decreases desorption Increases the stability of biocatalyst Disadvantages Might cause alteration in active site Diffusion limitations Loss of enzyme activity 3/24/2020 15

16. Entrapment Enzymes are physically entrapped inside the porous matrix. Bonds involved in stabilizing the enzyme to the matrix may be covalent or non-covalent. The matrix is used will be a water soluble polymer. Advantages Easy to carry out Low cost Low enzyme quantity required High stability Disadvantages Continuous enzyme leakage Pore diffusion restraint Not suitable for large scale or industrial process 3/24/2020 16

17. Covalent binding Covalent binding is based on the formation of a covalent bond between the biomolecule and the support material. Covalent binding causes a tight binding so the biomolecule to be immobilized does not separate from supports during utilization. Because of this strong interaction between enzyme molecules and supports, there is high heat stability. Advantages Strong binding High heat stability Facilitates the enzyme contacts with its substrate Prevents elution of biocatalysts Flexibility in design of support material and method. Disadvantages Limited enzyme mobility causes decreased enzyme activity Less effective for immobilization of cells Support materials are not renewable 3/24/2020 17

18. Kinetics of Immobilized Enzyme The performance of both free and immobilized enzymes may be greatly influenced by higher temperatures or extremes of pH. As the temperature rises, the kinetic energy between the molecules also increases, causing the enzymatic activity to be increased. the use of multipoint interactions for the stabilization of enzymes : 3/24/2020 18

19. Kinetics of Immobilized Enzyme (chymotrypsin) (a) Activity of free Derivatization chymotrypsin. (b) Activity of chymotrypsin derivatised with acryloyl chloride. (c) Activity of acryloyl chymotrypsin co polymerized within a poly methacrylate gel. Up to 12 residues are covalently bound per enzyme molecule. Lower derivatization leads to lower stabilization. (d) Activity of chymotrypsin non-covalently entrapped within a poly methacrylate gel. The degree of stabilization is determined by strength of the gel, and hence the number of non-covalent interactions. 3/24/2020 19

20. Factors affecting immobilized enzyme production Effects of Immobilization on enzyme Hydrophobic partition Multipoint attachment of carrier Diffusion constraints Physical structure of the carrier such as pore size Presence of substrates Physical nature of the carrier Binding mode Effects of microenvironment of carrier Physical post-treatments 3/24/2020 20

21. Advantages and disadvantages of using Immobilized enzyme 3/24/2020 21

22. Application of Immobilized enzyme Brewing industry Pharmaceutical Biomedical application Wastewater treatment Detergent industry Biofuel Pulp and paper industry Textile industry Food industry 3/24/2020 22

23. Application of Immobilized enzyme Food and beverage S tarch processing, cheese making, food preservation, lipid hydrolysis, lactose hydrolysis, whey processing, skimmed milk production, amino acid production. 3/24/2020 23

24. Application of Immobilized enzyme Food and beverage High fructose syrup can be produced from glucose by using an immobilized enzyme glucose isomerase. The starch containing raw materials (wheat, potato, corn) are subjected to hydrolysis to produce glucose. Glucose isomerase then isomerizes glucose to fructose.  Starch(Wheat, potatoes, corn) Glucose High fructose syrup α -Amylase and glucoamylase Glucose isomerase 3/24/2020 24

25. Application of Immobilized enzyme Pharmaceutical Penicillin is used antibiotics, due to their antibacterial activity, low toxicity, and inhibitory effect on bacterial cell wall synthesis. L- Asparaginase is an important enzyme for pharmaceutical industry for acute lymphoblastic leukemia and lymph sarcoma treatment. Streptokinase is used in the treatment of deep vein thrombosis, pulmonary embolism, and acute myocardial infarction. 3/24/2020 25

26. Application of Immobilized enzyme Biomedical Application Immobilized enzymes are used in medicine from 1990. The nanoparticles and nano spheres are often used as enzyme carriers for the delivery of therapeutic agents. D iagnosis and treatment of many diseases eg . inborn disorder. Role of Immobilization technique in biomedical industry as biosensors, controlled release, drug delivery, scaffolding materials has many promising advantages in biomedical application. Egg-white lysozyme was immobilized onto electro spin chitosan nanofiber via cross-linked enzyme aggregates to use as for effective antibacterial applications. Immobilized lysozyme showed high antibacterial activity against four pathogenic bacteria. 3/24/2020 26

27. Application of Immobilized enzyme Wastewater treatment The enzymes used in the wastewater treatments are preoxidases , laccase, azo reductases. Lipase has the ability to hydrolysis oil and fats to long chain fatty acid and glycerol. The immobilized lipase is of high interest for the hydrolysis of oils and fats for treating the waste water from the food industry. 3/24/2020 27

28. Application of Immobilized enzyme Detergent industry Biodegradable P rotease which is used to remove the stains of blood, egg, grass and human sweat .   Amylase used to remove the starch based stains like potatoes , chocolate.  Lipase used to remove the stains of oil and fats and also used to remove the stains in cuffs and collars. Research The use of immobilized enzyme allow researcher to increase the efficiency of different enzymes such as different proteases for cell and organelle lysis. 3/24/2020 28

29. Application of Immobilized enzyme Biodiesels production Biodiesel is monoalkyl esters of long chain fatty acids. P roduced by renewable biological sources such as vegetable oils and animal fats. I mmobilized enzyme for biodiesel production is to make the process more economical by reducing the cost of enzyme production and reusability. 3/24/2020 29

30. Application of Immobilized enzyme Pulp and paper industry immobilized Peroxidases from horseradish and soybean were is used to remove chlorophenol as important pollutants in industrial wastewater treatment especially from the pulp and paper industry. from aqueous solutions . The immobilized lipase is used to reduce pitch particles present in whitewater during papermaking because high operational stability . 3/24/2020 30

31. Application of Immobilized enzyme Textile industry Celluloses is used in textile for denim finishing and cotton softening. Amylase for de-sizing, pectate lyase for scouring, catalase for bleach termination, laccase for bleaching, and peroxidase for excess dye removal. Lipase from porcine-pancreas is an industrially important enzyme, which is commercially available, for its applications in textile industry . Peroxidase is used for treatment of industrial effluent. The reusability of the magnetic beads as support of Laccase can provide economic advantages for large scale applications. 3/24/2020 31

32. References Zdarta, Jakub, Anne Meyer, Teofil Jesionowski, and Manuel Pinelo . "A general overview of support materials for enzyme immobilization: characteristics, properties, practical utility."  Catalysts  8, no. 2 (2018): 92 . Datta , Sumitra, L. Rene Christena , and Yamuna Rani Sriramulu Rajaram . "Enzyme immobilization: an overview on techniques and support materials."  3 Biotech  3, no. 1 (2013): 1-9 . Eş , Ismail, José Daniel Gonçalves Vieira, and André Corrêa Amaral . "Principles, techniques, and applications of biocatalyst immobilization for industrial application."  Applied microbiology and biotechnology  99, no. 5 (2015): 2065-2082 . Nisha , S., S. Arun Karthick , and N. Gobi. "A review on methods, application and properties of immobilized enzyme."  Chemical Science Review and Letters  1, no. 3 (2012): 148-155 . Jun, Lau Yien , Lau Sie Yon, N. M. Mubarak, Chua Han Bing, Sharadwata Pan, Michael K. Danquah , E. C. Abdullah, and Mohammad Khalid. "An Overview of Immobilized Enzyme Technologies for Dye, Phaenolic Removal from Wastewater."  Journal of Environmental Chemical Engineering  (2019): 102961 . Basso, Alessandra, and Simona Serban . "Industrial applications of immobilized enzymes—A review."  Molecular Catalysis  479 (2019): 110607 . Chaplin, Martin F., and Christopher Bucke .  Enzyme technology . CUP Archive, 1990 . Buchholz, Klaus, Volker Kasche , and Uwe Theo Bornscheuer .  Biocatalysts and enzyme technology . John Wiley & Sons, 2012. 3/24/2020 32

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34. Thank you! 3/24/2020 34