Immobilized enzymes pharmaceutical technology

1. ENZYMES
10th SEMESTER
Pharm.D
RIPS. RIU
LAHORE

2. Enzymes:
• Enzymes are biological molecules (typically proteins tertiary and
quaternary structures) that significantly speed up the rate of
virtually all of the chemical reactions that take place within
cells.
• Not permanently changed in the process
• They are vital for life and serve a wide range of important
functions in the body, such as aiding in digestion
and metabolism.
• Some enzymes help break large molecules into smaller pieces
that are more easily absorbed by the body. Other enzymes help
bind two molecules together to produce a new molecule.
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3. •Enzymes are the biological substance or biological
macromolecules that are produced by a living organism
which acts as a catalyst to bring about a specific biochemical
reaction. These are like the chemical catalysts in a chemical
reaction which helps to accelerate the
biological/biochemical reactions inside as well as outside
the cell.
•These are generally known as “Biocatalyst.”
•Enzymes are highly efficient, which can increase reaction
rates by 100 million to 10 billion times faster than any
normal chemical reaction.

4. •Act as Catalyst to accelerates a reaction
•Are Reusable
•End in –ase
-Sucrase
-Lactase
-Maltase
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5. Enzyme-Substrate Complex
The substance (reactant) an enzyme acts on is the
substrate
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6. Active Site
A restricted region of an enzyme molecule
which binds to the substrate.
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7. Induced Fit
•A change in the shape of an enzyme’s active
site
•A change in the configuration of an enzyme’s
active site (H+ and ionic bonds are involved).
•Induced by the substrate
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When an enzyme binds to the appropriate substrate, subtle changes in the active
site occur. This alteration of the active site is known as an induced fit. This
alteration of the active site is known as an induced fit

10. What Affects Enzyme Activity?
• Concentration of Substrate
• Concentration of Enzyme
• Environmental Conditions
• Cofactors and Coenzymes
• Enzyme Inhibitors
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11. Immobilized Enzymes
• An immobilized enzyme is an enzyme attached to an inert, insoluble
material
• Immobilized enzymes are enzymes which are attached in or onto the
surface of an insoluble support
Fixation, the act of limiting moveme
nt or making incapable of movement

12. Definition
• An enzyme is said to be immobilized when it is attached to
(bound) or located within the network of a support material
(entrapped) , thereby separating it physically , but not
catalytically, from the bulk of the solution where the substrate
of the enzyme is usually found. Thus, the immobilization of an
enzyme (and cells) creates a heterogenous 2-phase system.
• Enzyme immobilization is a process whereby the enzymes are
entrapped on a carrier phase which allows them to take part
in reaction catalysis of bulk phase (reactants) without
affecting its own separate phase.

13. •Immobilized enzymes have several advantages over the soluble
enzyme:
–Convenience:
• Miniscule(extremely small or tiny) amounts of protein dissolve in the
reaction, so workup can be much easier.Upon completion, reaction
mixtures typically contain only solvent and reaction products.
–Economical:
• Easily removed from the reaction  reusage
–Stability:
• Immobilized enzymes typically have greater thermal and operational
stability than the soluble form of the enzyme

14. •Immobilizing enzymes is cheaper.
•Immobilized enzymes are usually used in
continuous flow-through re-actors, which have a
low volume.
•Isolated enzymes can be immobilized so that
they do not contaminate the end product and
can be used again and again.

15. Immobilization criteria
• There are a number of requirements to achieve a successful
immobilization:
•The biological component must retain substantial
biological activity after attachment.
•It must have a long-term stability.
•The sensitivity of the enzyme must be preserved after
attachment .
•Overloading can block or inactivate the active site of the
immobilized biomaterial, therefore, must be avoided

16. Immobilized Enzyme Systems
Matrix Materials:
Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and
polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagen
Inorganics: activated carbon, porous ceramic.
Immobilization procedures:
Enzyme + polymer solution → polymerization → extrusion/shape the
particles
Shapes: particle, membrane, fiber

17. Reasons for Immobilization
• As the enzymes are very expensive material so it is very crucial to
utilize them as efficiently as possible.The utility of enzymes is limited
in industrial processes because of their high cost isolation,
purification and recovery of active enzymes from the reaction mixture
after catalyzing the reaction process.
• Because of the ability of the enzymes to catalyze the reaction without
being utilize in the reaction process, they can be used again and
again.
• In the absence of any accidental damage to the enzymes they will
continue their work until converting all available substrate into
product.

18. Reasons for Immobilization
• As the enzymes and products are mixed together in final reaction mixture, so it is
very costly and difficult to re isolate enzymes from this mixture in order to utilize
them in another fresh reaction batch. So use of soluble enzymes is uneconomical
in batch operation because of their loss after each reaction.
• This wastage of enzymes is now avoided by using the technique enzyme
immobilization.
• Enzymes in their immobilized state after performing their job could be easily
isolated leaving behind the final product with out itself wasting in the reaction
catalysis.

19. Properties of support material
 The form, shape, density, porosity, pore size distribution, operational
stability and particle size distribution of the supporting matrix will influence
the result.
 The ideal support is cheap, inert, physically strong and stable
 Ideally, it should:
 increase the enzyme specificity (kcat/Km)
 shift the pH optimum to the desired value for the process
 discourage microbial growth and non-specific adsorption
 Some matrices may possess other properties which are useful for particular purposes such as:
 ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst by means of magnetic
fields)
 a catalytic surface (e.g. manganese dioxide, which catalytically removes the inactivating hydrogen
peroxide produced by most oxidases)

20. Methods of Immobilization Entrapment
The different methods are:
• Carrier-Binding: the binding of enzymes to water-
insoluble carriers
• Cross-linking: intermolecular cross-linking of enzymes by
bi-functional or multi-functional reagents.
• Entrapping: incorporating enzymes into the lattices of a
semi-permeable gel or enclosing the enzymes in a semi-
permeable polymer membrane

21. Immobilization methods
a) adsorption
b) covalent binding
c) entrapment
d) encapsulation

24. 1: Carrier-Binding
• The oldest immobilization technique for enzymes
• Some of the most commonly used carriers for enzyme
immobilization are polysaccharide derivatives such as:
cellulose, dextran, agarose, and polyacrylamide
gel.
• The selection of the carrier depends on the nature of
the enzyme itself, as well as the:
• Particle size
• Surface area
• Molar ratio of hydrophilic to hydrophobic groups
• Chemical composition

25. •The carrier-binding method can be
further sub-classified into:
•Physical Adsorption
•Covalent Binding
•Ionic Binding

26. Physical Adsorption
• The enzyme molecules are attached by weak physical forces to a support
matrix, such as glass beads or carbon particles.
• Physical Adsorption of enzyme protein on the surface of water-insoluble
carriers
• This does not chemically modify the enzyme molecules, but the adsorption
process may cause the enzymes to loose their shape and therefore their
activity.
• The molecules may also become detached during the bioconversion reaction.
• Simplest immobilization method; Mix the enzyme and support in suitable
conditions
• Forces are weak so leakage is generally a problem.
• Supports such as alluminium hydroxide are often utilized

27. Advantages :
• No reagents and only a minimum of activation steps are required
• Ease of immobilization process
• Readily available polymer matrix for enzyme adsorption.
• Great access to the substrate by bound enzymes
Disadvantages :
• the adsorbed enzyme may leak from the carrier during use due to a weak binding force
between the enzyme and the carrier. Moreover, the adsorption is non-specific, further
adsorption of other proteins or other substances
• pH changes causes the immobilized enzyme to leach into reaction mixture.
• In some cases ionic portioning between substrate and polymer matrix owning to have
same charge.

28. Covalent immobilization
• Based on the binding of enzymes and water-insoluble carriers by covalent bonds
• The most widely used method for enzyme immobilization
• It is technically more complex
• It requires a variety of often expensive chemicals
• It is time-consuming
• But immobilized enzyme preparations are stable and leaching is minimal
• Enzymes are immobilized by a suitable group in the surface:
• Hydroxyl groups in supports (e.g cellulose, dextran, agarose)
• Amino, carboxyl and sulfhydryl groups in amino acids
(The functional groups that may take part in this binding are Amino group, Carboxyl group,
Sulfhydryl group, Hydroxyl group, Imidazole group, Phenolic group, Thiol group, Threonine
group,Indole group)

29. • Less mild than in the cases of physical adsorption and ionic binding.
• Covalent binding may alter the conformational structure and active center of the
enzyme, resulting in major loss of activity and/or changes of the substrate.
• This technique is extensively utilized for the immobilization of enzymes although it is not
equally good for immobilizing cells. In organic supports are widely investigated for
covalent binding of enzymes. Inorganic bounded enzymes are mostly used for industrial
purposes.
• Enzymes immobilized by this technique are glucose oxidase, peroxidase and invertase etc.

30. Disadvantages :
• Covalent binding may alter the conformational structure and active center of the
enzyme, resulting in major loss of activity and/or changes of the substrate
• Inactivation of enzymes during immobilization
• Complicated immobilization process
Advantages :
• The binding force between enzyme and carrier is so strong that no leakage of the
enzymes occurs, even in the presence of substrate or solution of high ionic
strength.
• Leaching of enzyme is protected by covalent immobilization.
• Almost any polymer material can be used for covalent binding.
• Variety of supporting matrix ultimately allows almost any enzyme to covalently
immobilize

31. Ionic binding Of the enzyme protein to water-insoluble carriers containing ion-
exchange residues.
Polysaccharides and synthetic polymers having ion exchange centers are
usually used as carriers
Advantages : the enzyme to carrier linkages is much stronger for ionic binding
Disadvantages : the binding forces between enzyme proteins and carriers are
weaker than those in covalent binding
Ionic Binding

32. 2: Cross-Linking
• Either to other protein molecules or to functional
groups on an insoluble support matrix
• It is used mostly as a means of stabilizing adsorbed
enzymes and also for preventing leakage from
polyacrylamide gels
• Enzyme molecules are chemically cross-linked by
covalent bonds using glutaraldehyde.
• This only works for some enzymes, but is very
successful.
• Used in Biosensors

33. • The most common reagent used for cross-linking is glutaraldehyde
E=CH–CH2–CH2–CH2– CH=E
• Disadvantages : Cross-linking reactions are carried out under
relatively severe conditions. These harsh conditions can change the
conformation of active center of the enzyme; and so may lead to
significant loss of activity.

34. 3: Entrapping Enzymes

35. Entrapment Method
• Entrapment method does not need polymer matrix as attachment surface rather enzymes are simply
entrapped inside the polymer matrix.
• This procedure is largely used for entrapping cells rather than isolated enzymes because they are more
easily retained inside the support.
• This method involves mixing the biocatalyst into the polymer monomers solution followed by initiating
polymerization through changes in temperature or chemical reactions.
• Most commonly used polymer matrix used for entrapment are collagen, cellulose acetate, calcium
alginate and polyacrylamide.
• A novel variation in this method involved preparation of enzyme entrapped fibers of polymer matrix
which relatively increase the surface area of catalyst.
challenges:
• enzyme leakage into solution
• diffusional limitation
• reduced enzyme activity and stability
• lack of control micro-environmental conditions.
• Advantages:
• Simple method for immobilization.
• Requires mild conditions during procedure.
• Nonliving cells could be immobilized well through this technique.
• Disadvantages:
• Free radicals produce during polymerization are toxic for sensitive
enzymes.
• Live, dividing entrapped bio catalytic cells could burst out while
immobilized by this method.

36. Encapsulation Method
• Encapsulation involves enclosing a droplet of biocatalyst solution inside a semi permeable
membrane capsule.
• Encapsulation involves entrapping the enzymes within a semipermeable membrane such as
cellulose nitrate and nylon-based membranes
• Immobilized enzymes through encapsulation depend for their utility applications, on the nature
of membrane.
• Membrane must confine(limits) the enzyme along with allowing the passage of reactants and the
product.
• The application of encapsulation is largely being restricted to the medical field.
• It is very cheap and simple method of immobilization if the enzyme to be immobilized show
stability is solution form.
• Very little diffusion problems are encountered and capsules of any required size can be prepared
by alteration of reaction conditions.

37. Recycle packed column reactor:
- allow the reactor to operate at high fluid velocities.
Immobilized Enzyme Reactors

38. Fluidized Bed Reactor:
- a high viscosity substrate solution
- a gaseous substrate or product in a continuous reaction system
- care must be taken to avoid the destruction and
decomposition of immobilized enzymes

39. - An immobilized enzyme tends to decompose
upon physical stirring.
- The batch system is generally suitable for the production
of rather small amounts of chemicals.

40. Application of immobilized enzymes
Bioreactors
Large scale production or conversion of various compounds

41. Application of immobilized enzymes
Biosensors
An analytical device which can detect and quantify specific analytes in complex samples

42. Applications of Biosensors
• Health care and life sciences research applications
• Glucose and urea sensors
• Proteomics
• Genomics
• Toxicology
• Oncology
• Drug discovery
• Process industries
• Monitoring of active component or pollutants
• Food and drink
• Contaminant/Pathogen Detection
• Process/Quality Control
• Detection of Genetically Modified Organisms in Food
• Environmental monitoring
• Pesticide
• Defence and security
• Military; Nerve gases and explosives
• Forensics; DNA identification

43. Application of immobilized enzymes
Bioremediation:-
• For the removal/detoxification of contaminants
• E.g. Polyphenol oxidase immobilized on chitosan coated membranes

44. Applications
• Act as a tool in biochemical studies
• Medical/cosmetic application (eg immobilized heparinase, use of liposomes in cosmetics)
• Industrial applications
• Eg: immobilized glucose isomerase, lipase, L-amino acid acylase, penicillin acylase to produce 6-amino penicillinic
acid from penicillin, E.coli-bound aspartase for the production of aspartic acid from fumaric acid in the presence of
ammonia, B. ammoniagenes-bound fumarase to produce malic acid from fumaric acid.

45. Selective Adsorbent
•Applications in food industry:
•Lactose removal in milk
• Milk contains 5% lactose which must be removed before feeding babies deficient in
β-galactosidase (lactose hydrolyzing enzyme). A column packed with β-
galactosidase immobilized in polyacrylamide gel allows continuous lactose removal.
•Hydrogen peroxide removal in milk after sterilization
• Immobilized catalase by covalent binding with carboxychloride resin
•Bitterness removal in orange juice
• Naringinase immobilized on copolymer, used in both batch and column systems

46. In Food Industry
• Food additives: encapsulated β-galactosidase added to milk.
The enzyme is only active during digestion process, when the
enzyme is released from the lipid vesicles
• Acceleration of cheese ripening: hard cheeses take up to 2 yrs
to reach optimum flavor and texture; need to reduce ripening
costs. Immobilize ripening enzymes into liposomes.
• Method of release: many parameters in cheese can induce a
destabilization of lipid bilayers. Sensitivity to pH, temperature,
and interactions with other lipids could induce release.

47. •Applications in waste treatment:
• Reduction of phenol content in wastewater using immobilized polyphenol
oxidase
• Removal of cyanide:
• Improve use of microorganisms to decompose cyanide by entrapping enzyme in
polyacrylamide gel and packed into column
• Does not allow simultaneous removal of several different substances but
allows removal of specific substances

48. In medicine
• Blood detoxification: immobilization of phospholipase A2 and heparinase in
bioreactors
• Urease-containing microcapsules injected intraveneously decrease urea content in
blood
• Wound dressings: Immobilization of proteolytic enzymes (trypsin) onto
textiles used as wound dressings to accelerate cleaning and healing of
infected wounds and reduces amount of enzyme used as compared with
use of enzyme solutions
• Low Molecular Weight Protamine (LMWP)
Reverse haparin after surgery
Some patients are sensitive to protamine

49. •Controlled release
•Two major applications:
• Pharmaceutical (enzymes as drugs)
• Prolonged circulation in blood
• Local deposition in particular tissue, organ
• Food (release of enzymes from liposomes)