Introducing immobilized enzymes ,reasons and limitations and their application .

1. Immobilization of Enzyme
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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?
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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.
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4. Enzyme immobilization
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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.
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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.
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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
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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.
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9. Properties of support materials
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10. Classification of support
Supports can be categorized as organic and inorganic support based
on their chemical composition.
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11. Classifications of Organic and Inorganic
Supports:
Organic support materials
 Organic natural polymers used as
support material for enzyme
immobilization are water-insoluble
polysaccharides.
 Organic 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.
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12. Enzyme Immobilized Techniques
Based on support or matrix and the type of bond; enzyme immobilization techniques are five
types.
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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
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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.
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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
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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
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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
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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 :
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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.
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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
microenviron
ment of
carrierPhysical
post-
treatment
s
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21. Advantages and disadvantages of using Immobilized enzyme
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22. Application of Immobilized enzyme
Brewing
industry
Pharmaceut
ical
Biomedical
applicationWastewater
treatment
Detergent
industry
Biofuel
Pulp and
paper
industry
Textile
industry
Food
industry
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23. Application of Immobilized enzyme
Food and beverage
 Starch processing, cheese making, food preservation, lipid hydrolysis, lactose
hydrolysis, whey processing, skimmed milk production, amino acid production.
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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
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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.
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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.
 Diagnosis 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.
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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.
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28. Application of Immobilized enzyme
Detergent industry
 Biodegradable
 Protease 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.
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29. Application of Immobilized enzyme
Biodiesels production
 Biodiesel is monoalkyl esters of long chain fatty acids.
 Produced by renewable biological sources such as vegetable
oils and animal fats.
 Immobilized enzyme for biodiesel production is to make the
process more economical by reducing the cost of enzyme
production and reusability.
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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.
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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.
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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.
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34. Thank you!
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