The document discusses immobilization of enzymes. There are several reasons for immobilizing enzymes, including easy separation of the enzyme from reaction products and reuse of the enzyme. Common immobilization methods include covalent binding of the enzyme to a support, entrapment within a polymer matrix, and cross-linking. Supports can be inorganic materials like silica or organic polymers. Immobilization can impact properties of enzymes like activity, pH optimum, and stability. Reversible immobilization allows regeneration of supports and is important for labile enzymes.

1. Immobilization of Enzymes
Prepared by ABDUL QUDOOS
SARHAD UNIVERSITY PESHAWAR
PAKISTAN

2. immobilized enzymes
• The term “immobilized enzymes” refers to “enzymes physically
confined or localized in a certain defined region of space with
retention of their catalytic activities, and which can be used
repeatedly and continuously.
• Man-made usage of binding enzymes onto solid materials goes
back to the 1950s, when immobilized enzymes, that is enzymes
with restricted mobility, were first prepared.

3. Why Immobilize Enzymes?
• There are several reasons for the preparation and use of
immobilized enzymes. In addition to a more convenient handling
of enzyme preparations, the two main targeted benefits are
• Easy separation of the enzyme from the product, and
• Reuse of the enzyme.

4. • Easy separation of the enzyme from the product simplifies enzyme
applications and supports a reliable and efficient reaction
technology.
• On the other hand, reuse of enzymes provides cost advantages
which are often an essential prerequisite for establishing an
enzyme-catalyzed process in the first place

7. Immobilization Methods
• Immobilization of macromolecules can be generally defined as a
procedure leading to their restricted mobility.
• A suitable reaction to activate the enzyme for the immobilization
process is performed prior to binding. This approach often suffers from
significant loss of activity, because the protein is modified by highly
reactive chemical compounds that are often not strictly group specific
and may alter catalytically or structurally essential residues. Also intra-
and intermolecular cross-linking has to be considered.

8. • The support is modified and activated. The native enzyme is bound in
a subsequent step under well-defined conditions using the natural
reactivity of the molecule. This is the most prominent technique to
covalently bind enzymes to carrier surfaces.
• A bi- or multifunctional coupling agent is used to mediate between
carrier and enzyme functional groups. This can also lead to intra- and
intermolecular enzyme cross-linking.
• The enzyme is modified by recombinant DNA techniques to generate a
protein with “(bio)specific” groups, so that it can adsorb onto special
carriers using (bio)affinity binding.

10. Choice of Supports
• The characteristics of the matrix are of paramount importance in
determining the performance of the immobilized enzyme system.
Ideal support properties include physical resistance to
compression, hydrophilicity, inertness toward enzymes ease of
derivatization, biocompatibility, resistance to microbial attack, and
availability at low cost

11. • Supports can be classified as inorganic and
organic according to their chemical
composition The organic supports can be
subdivided into natural and synthetic
polymers

13. Inorganic Carriers
• Inorganic carriers (e.g. glass, silica gel, alumina, bentonite,
hydroxyapatite, nickel/nickel oxide, titania, zirconia) often show good
mechanical properties, thermal stability, and resistance against
microbial attack and organic solvents.
• On the other hand, non-porous materials like metal and metal oxides
only have small binding surfaces.
• Minerals usually display a broad distribution of pore size.

14. Inorganic Carriers
• Silica gels are available under the trade names Promaxon (Promat), Spherosil
(Rhone-Poulenc) or Aerosil (Degussa).
• Silica compounds can be prepared with defined pore sizes and binding surfaces
(controlled pore glass), but they suffer from high production costs and show limited
stability under alkaline conditions.
• Furthermore, silica carriers are chemically inert and need activation and modification.
• Usually, they are treated with aminoalkyl triethoxysilanes to introduce amino
groups, which can subsequently be activated for enzyme coupling reactions by a
variety of different methods.

15. Organic Carriers
• Naturally Occurring Organic Carriers
• Synthetic Organic Carriers

16. Naturally Occurring Organic Carriers
• Natural organic polymers – such as structural proteins (ceratin, collagen),
globular proteins (albumin) or carbohydrates – are cheap starting materials for
the production of support materials and are available in large quantities.
• From this group, carbohydrates are of special interest, because they do not
suffer from biological safety aspects like protein matrices isolated from animal
sources and because they are highly hydrophilic which provides a desirable
microenvironment for many enzymes.
• Alginate , carrageenan , chitin or chitosan (prepared from chitin by
deacetylation) are particularly useful for encapsulating microorganisms by
ionotropic, respectively, acidic gelation.

17. Naturally Occurring Organic Carriers
• Enzymes have been linked to carbohydrates simply by adsorption followed by cross-linking.
• Chitosan is of importance because of its primary amino groups that are susceptible for coupling
reactions. Furthermore, porous spherical chitosan particles are commercially available
(Chitopearl, Fuji Spinning) allowing noncovalent or covalent attachment of enzymes. This
support matrix can be easily prepared and activation methods have been summarized.
• Treatment with polyethyleneimine or with hexamethylenediamine and glutardialdehyde can
improve the mechanical characteristics of the biocatalyst, which is poor otherwise.
• However, this is often accompanied by some activity loss or increase of diffusional limitations.

18. Naturally Occurring Organic Carriers
• Dextran and agarose have to be cross-linked,
for instance, with epichlorohydrin, to improve
their mechanical characteristics and
compressibility.
• Covalent immobilization to commercially
available beaded forms (Sephadex, Sepharose)

19. Naturally Occurring Organic Carriers
• Cellulose is also an acceptable support.
• The binding capacity for enzymes is generally lower as compared
to agarose but it is inexpensive and commercially available in
fibrous and granular forms.
• Some drawbacks are the low particle sizes, which impairs the use
in rapid highpressure applications, and its susceptibility to
microbial cellulases.

20. Synthetic Organic Carriers
• Synthetic organic polymers display the greatest variability with
regard to physical and chemical characteristics. In principle, they
can be adapted to the requirements of nearly any enzymatic
process. Furthermore, they are inert to microbial attack.
• They are commercially available as purely adsorptive resins, as ion
exchangers with a variety of different basic and acidic groups, or
as preactivated supports carrying for instance epoxide (Eupergit®,
Roehm Pharma) or azlactone groups (Emphaze™).

21. Synthetic Organic Carriers
• The main synthetic polymers are polystyrene, polyacrylate,
polyvinyls, polyamide, polypropylene and copolymers based on
maleic anhydride and ethylene or styrene, polyaldehyde, and
polypeptide structures.

22. Synthetic Organic Carriers
• Polystyrene was the first synthetic polymer used for enzyme
immobilization.

23. Immobilization
• Methods of Irreversible Enzyme Immobilization
• Methods of Reversible Immobilization

24. Methods of Irreversible Enzyme
Immobilization
• The concept of irreversible immobilization means that once the
biocatalyst is attached to the support it cannot be detached
without destroying either the biological activity of the enzyme or
the support.
• The most common procedures of irreversible enzyme
immobilization are covalent coupling, entrapment or micro-
encapsulation, and cross-linking.

25. Formation of Covalent Bonds
• Immobilization of proteins by methods based on the formation of
covalent bonds are among the most widely used.
• An advantage of these methods is that, because of the stable nature of
the bonds formed between enzyme and matrix, the enzyme is not
released into the solution upon use.
• However, in order to achieve high levels of bound activity, the amino
acid residues essential for catalytic activity must not be involved in the
covalent linkage to the support; this may prove a difficult requirement
to fulfill in some cases.

26. Formation of Covalent Bonds
• Covalent methods for immobilization are employed when there is a strict
requirement for the absence of the enzyme in the product.
• A wide variety of reactions have been developed depending on the functional
groups available on the matrix.
• Coupling methods in general can be divided in two main classes: (1) activation
of the matrix by addition of a reactive function to a polymer and (2)
modification of the polymer backbone to produce an activated group.

27. Formation of Covalent Bonds
• The activation processes are generally designed to generate
electrophilic groups on the support which, in the coupling step,
react with the strong nucleophiles on the proteins.
• The most frequently used reactions involve the following side
chains of the amino acids: lysine (amino group), cysteine (thiol
group), and aspartic and glutamic acids (carboxylic group).

28. Formation of Covalent Bonds
• There are many commercially available supports for
immobilization, the best choice in each case requires the
consideration of some relevant properties of the catalyst and the
intended use.
• However, it is usually necessary to try more than one approach
and then adapt a method to the specific circumstances

29. Entrapment
• The entrapment method is based on the occlusion of an enzyme within a polymeric
network that allows the substrate and products to pass through but retains the
enzyme.
• This method differs from the coupling methods described above, in that the enzyme
is not bound to the matrix or membrane.
• There are different approaches to entrapping enzymes such as gel or fiber entrapping
and micro-encapsulation.
• The practical use of these methods is limited by mass transfer limitations through
membranes or gels.

30. Methods of Reversible Immobilization
• Because of the type of the enzyme-support binding, reversibly
immobilized enzymes can be detached from the support under
gentle conditions.
• The use of reversible methods for enzyme immobilization is
highly attractive, mostly for economic reasons because when the
enzymatic activity decays the support can be regenerated and re-
loaded with fresh enzyme.

31. Methods of Reversible Immobilization
• Indeed, the cost of the support is often a primary factor in the
overall cost of immobilized catalyst.
• The reversible immobilization of enzymes is particularly
important for immobilizing labile enzymes and for applications in
bioanalytical systems

32. Approaches to enzyme immobilization, reversible
method

34. Adsorption (Noncovalent
Interactions)
• Nonspecific Adsorption
• Ionic Binding
• Hydrophobic Adsorption
• Affinity Binding

35. Nonspecific Adsorption
• The simplest immobilization method is nonspecific adsorption, which
is mainly based on physical adsorption or ionic binding .
• In physical adsorption the enzymes are attached to the matrix through
hydrogen bonding, van der Waals forces, or hydrophobic interactions;
whereas in ionic bonding the enzymes are bound through salt linkages.
• The nature of the forces involved in noncovalent immobilization
results in a process can be reversed by changing the conditions that
influence the strength of the interaction (e.g., pH, ionic strength,
temperature, or polarity of the solvent).

36. Nonspecific Adsorption
• Immobilization by adsorption is a mild, easy to perform process,
and usually preserves the catalytic activity of the enzyme.
• Such methods are therefore economically attractive, but may suffer
from problems such as enzyme leakage from matrix when the
interactions are relatively weak.

37. Ionic Binding
• An obvious approach to the reversible immobilization of enzymes is to base the protein–ligand
interactions on principles used in chromatography.
• For example, one of the first applications of chromatographic principles in the reversible
immobilization of enzymes was the use of ion-exchangers.
• The method is simple and reversible but, in general, it is difficult to find conditions under which
the enzyme remains both strongly bound and fully active.
• More recently, the use of immobilized polymeric-ionic ligands has allowed for modulation of
protein– matrix interactions and has thus optimized the properties of the derivative.
• A number of patents have been filed on the use of polyethyleneimine to bind a rich variety of
enzymes and whole cells.

38. Ionic Binding
• However, problems may arise from the use of a highly charged support
when the substrates or products themselves are charged; the kinetics
are distorted as a result of partition or diffusion phenomena.
• Therefore, enzyme properties, such as pH optimum or pH stability,
may change.
• Although this could pose a problem it could also be useful to shift the
optimal conditions of a certain enzyme towards more alkaline or acidic
conditions, depending on the application.

39. Hydrophobic Adsorption
• Another approach is the use of hydrophobic interactions.
• Hydrophobic adsorption has been used as a chromatographic
principle for more than three decades. It relies on well-known
experimental variables such as pH, salt concentration, and
temperature.

40. Hydrophobic Adsorption
• The strength of interaction relies on both the hydrophobicity of the
adsorbent and the protein. The hydrophobicity of the adsorbent can be
regulated by the degree of substitution of the support and by the size
of the hydrophobic ligand molecule.
• The successful reversible immobilization of β-amylase and
amyloglucosidase to hexyl-agarose carriers has been reported.
• Several other examples of strong reversible binding to hydrophobic
adsorbents have also been reported.

41. Affinity Binding
• The principle of affinity between complementary biomolecules has
been applied to enzyme immobilization.
• The remarkable selectivity of the interaction is a major benefit of
the method.
• However, the procedure often requires the covalent binding of a
costly affinity ligand (e.s., antibody,= or lectin) to the matrix

42. Chelation or Metal Binding
• Transition metal salts or hydroxides deposited on the surface of
organic carriers become bound by coordination with nucleophilic
groups on the matrix.
• Mainly titanium and zirconium salts have been used and the
method is known as “metal link immobilization”.

43. Chelation or Metal Binding
• The metal salt or hydroxide is precipitated onto the support (e.g.,
cellulose, chitin, alginic acid, and silica-based carriers) by heating
or neutralization.
• Because of steric factors, it is impossible for the matrix to occupy
all coordination positions of the metal; therefore some of the
positions remain free to coordinate with groups from the enzymes.

44. Chelation or Metal Binding
• The method is quite simple and the immobilized specific activities
obtained with enzymes in this way have been relatively high (30–
80%).
• However, the operational stabilities achieved are highly variable
and the results are not easily reproducible

45. Chelation or Metal Binding
• The reason for this lack of reproducibility is probably related to the
existence of nonuniform adsorption sites and to a significant metal ion
leakage from the support.
• In order to improve the control of the formation of the adsorption
sites, chelator ligands can be immobilized on the solid supports by
means of stable covalent bonds.
• The metal ions are then bound by coordination and the stable
complexes formed can be used for the retention of proteins.

46. Chelation or Metal Binding
• Elution of the bound proteins can be easily achieved by competition
with soluble ligands or by decreasing pH.
• The support is subsequently regenerated by washing with a strong
chelator such as ethylene diamine tetraacetic acid (EDTA) when
desired.
• These metal chelated supports were named IMA Immobilized Metal-
Ion Affinity (IMA) adsorbents and have been used extensively in
protein chromatography.

47. Chelation or Metal Binding
• The approach of using different IMA-gels as supports for enzyme
immobilization has been studied using Eschericia coli β-
galactosidase as a model

48. Formation of Disulfide Bonds
• These methods are unique because, even though a stable covalent bond
is formed between matrix and enzyme, it can be broken by reaction
with a suitable agent such as dithiothreitol (DTT) under mild
conditions.
• Additionally, because the reactivity of the thiol groups can be
modulated via pH alteration, the activity yield of the methods involving
disulfide bond formation is usually high—provided that an appropriate
thiol-reactive adsorbent with high specificity is used.

49. Properties of Immobilized Enzymes
• The behavior of immobilized enzymes differs from that of
dissolved enzymes because of the effects of the support material,
or matrix, as well as conformational changes in the enzyme that
result from interactions with the support and covalent
modification of amino acid residues.
• Properties observed to change significantly upon immobilization
include specific activity, pH optimum, Km, selectivity, and
stability.

50. Properties of Immobilized Enzymes
• Physical immobilization methods, especially entrapment and
encapsulation, yield less dramatic changes in an enzyme's catalytic
behavior than chemical immobilization methods or adsorption.
• The reason is that entrapment and encapsulation result in the
enzyme remaining essentially in its native conformation, in a
hydrophilic environment, with no covalent modification.

51. Properties of Immobilized Enzymes
• The stability of the immobilized enzymes is based on the temperature and time.
• The activity of the enzyme is retained throughout series of cycles.
• Due to immobilization, the properties of enzymes will be altered such as catalytic activity with
respect to the support matrix.
• The change in the enzyme properties in the immobilized enzyme is due to the enzyme and the
substrate reacts in the microenvironment which is different from the enzyme substrate reaction in
the bulk solution environment.
• The change is also due to the change in the three dimensional conformation of the protein when
linked with the support matrix. These conformational changes are to a lesser extent and these
changes occur in the limited enzyme systems

52. Properties of Immobilized Enzymes
• Enzyme immobilization improves the operational stability.
• The stabilization as a result of, number of bonds formed between
the enzyme and the support matrix.
• When the immobilized enzyme acts on the macromolecular
substrates, active site of the enzymes does not able to access with
the substrates, hence the enzyme loses its activity

53. Application of the Immobilized Enzymes:

54. Biomedical Application:
• Immobilized enzymes are used in medicine from 1990.
• immobilized enzymes are used for diagnosis and treatment of
diseases in the medical field.
• The inborn metabolic deficiency can be overcome by replacing the
encapsulated enzymes (i.e, enzymes encapsulated by erythrocytes)
instead of waste metabolites, the RBC acts as a carrier for the
exogenous enzyme drugs and the enzymes are biocompatable in
nature, hence there is no immune response.

55. Biomedical Application:
• The enzyme encapsulation through the electroporation is a easiest
way of immobilization in the biomedical field and it is a reversible
process for which enzyme can be regenerated.
• The enzymes when combined with the biomaterials it provides
biological and functional systems. The biomaterials are used in
tissue engineering application for repair of the defect.

56. Biomedical Application:
• The advantage of the enzyme immobilization in biomedical is that
the free enzymes are consumed by the cells and not active for
prolonged use, hence the immobilized enzymes remains stable, to
stimulate the growth and to repair the defect.
• The cancer therapy is delivery of enzymes to the oncogenic sites
have been improved with new methods. The nanoparticles and
nanospheres are often used as enzyme carriers for the delivery of
therapeutic agents.

57. Food industry Application:
• In food industry, the purified enzymes are used but during the purification the
enzymes will denature. Hence the immobilization technique makes the enzymes
stable.
• The immobilized enzymes are used for the production of syrups.
• Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production
of bakers yeast. The enzyme is linked to porous silica matrix through covalent
linkage.
• This method is not preferably used due to its cost and the other technique developed
by Valio in 1980, the enzyme galactosidase was linked to resin (food grade) through
cross linking. This method was used for the various purposes such as confectionaries
and icecreams.

58. Biodiesel Production:
• Biodiesel is monoalkyl esters of long chain fatty acids.
• Biodiesel is produced through triglycerides (vegetable oil, animal
fat) with esterification of alcohol (methanol, ethanol) in the
presence of the catalyst.
• The production of catalyst is a drawback of high energy
requirements, recovery of glycerol and side reaction which may
affect the pollution.
• Hence the biological production of liquid fuel with lipases
nowadays has a great consideration with a rapid improvement

59. Biodiesel Production:
• Lipase catalyses the reaction with less energy requirements and mild conditions
required.
• But the production of lipase is of high cost, hence the immobilization of lipase
which results in repeated use and stability.
• The immobilization of lipase includes several methods entrapment,
encapsulation, cross linking, adsorption and covalent bonding.
• Adsorption method of immobilization is widely used in recent years when
compared to covalent bond, entrapment and cross linking.

60. Biodiesel Production:
• In the biological production of biodiesel the methanol inactivates the lipase,
hence the immobilization method is an advantage for the biodiesel production
.
• The nanostructured carriers are with high porosity, natural material activated
carbon, celite, zeolite.
• The carriers for lipase immobilization by covalent attachment of olive pomace
, resins , Polyurethane foam, chitosan , silica and magnetic nanostructures,
When compared to the natural support material chitosan is used for enzyme
binding, the immobilized lipase retains its stability for 10 cycles of pomace oil,
esterification, while maintaining 80% residual activity

61. Wastewater Treatment:
• The increasing consumption of fresh water and water bodies are
mixed up with polluted industrial waste water and the waste water
treatments are necessary at present. The sources of dye effluents
are textile industry, paper industry, leather industry and the
effluents are rich in dye colourants.
• These effluents are threat to the environment and even in low
concentration it is carcinogenic. Nowadays enzymes are used to
degrade the dye stuffs.

62. Wastewater Treatment:
• The enzymes used in the wastewater treatments are preoxidases, laccase, azo
reductases.
• These enzymes due to harsh conditions like extreme temperature, low or high
pH and high ionic strength may lose its activity; to overcome this problem
immobilized enzymes are used.
• The Horse radish Peroxidases are entrapped in calcium alginate beads, this
method is still in lab scale research.
• The immobilized laccase enzyme has the ability to degrade dyes anthracinoid
dye, Lancet blue and Ponceau red

63. Wastewater Treatment:
• Adsorption method is widely used because of its easy regeneration.
• During the covalent method of immobilization the conformational
change in the enzyme occurs which will affect the activity of the
enzyme.
• In Single Enzyme Nanoparticle, the enzyme is protected by a
nanometer thick substance as it provides the large surface area.
• SEN has the ability to retain its activity during the extreme
conditions. SEN is also used in the removal of heavy metals from
the waste water.

64. Wastewater Treatment:
• Lipase has the ability to hydrolyse 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.
• The drawback of the conventional treatment methods is slow biodegradability, oil
and fats are absorbed on the surface of sludge.
• Researchers are now focusing on the treatment with immobilized lipase. Lipase
immobilized on the sol gel / calcium alginate with the size of 82µm, immobilized
lipase. Immobilized lipase operated for 100 days in continuous sludge without any
problem, does not produced foam in the reactor .

65. Textile Industry:
• The enzymes derived from microbial origin are of great interest in
textile industry.
• The enzymes such as cellulase, amylase, liccase, pectinase, cutinase etc
and these are used for various textile applications such as scouring,
biopolishing, desizing, denim finishing, treating wools etc. Among
these enzymes cellulase has been widely used from the older period to
till now.
• The textile industries now turned to enzyme process instead of using
harsh chemical which affects the pollution and cause damage to the
fabrics

66. Textile Industry:
• The processing of fabrics with enzymes requires high temperatures and
increased pH, the free enzymes does not able to withstand the extreme
conditions.
• Hence, enzyme immobilization for this process able to withstand at
extreme and able to maintains its activity for more than 5-6 cycles.
• PolyMethyl Methacrylateis linked with cellulase covalently. In this
method the nanoparticle is synthesized with cellulase as core particle

67. Textile Industry:
• Endoglucanase is a component of Cellulase enzyme,
Endoglucanase is microencapsulated with Arabic Gum is a natural
polymer with the biodegradable property is used as a matrix for
encapsulation of endoglucanase.
• Encapsulation of endoglucanase prevented it to retain its activity
in the presence of detergents

68. Detergent Industry:
• The detergent industry also employs enzymes for removal of
stains. The enzymes used in detergent industry are 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.

69. Detergent Industry:
• improve softening, colour brightening and to remove soil stains.
• Nowadays Biotech cleaning agents are widely used in the
detergent industries. When compared to synthetic detergents the
biobased detergents have good cleaning property

70. Detergent Industry:
• The enzymes based detergents can be used in low quantity when
compared to the synthetic detergent, it has increased biodegradability,
does not affect the environment works well in low temperature, and
these are the advantages of enzymes in detergent industry.
• Proteases hydrolyse the proteins, and protease cannot be used for
keratin based fabric wool and silk which cause adverse damage to the
garment. So protease directly cannot be used for wool and silk
garments, protease loses its stability in the presence of surfactants and
oxidizing agents, hence protease is immobilized by covalently linking
with Eudragit S-100 using carbodiimide coupling.

71. Detergent Industry:
• The immobilized protease treated with wool for 72hrs with 100U
at 40oC the free enzyme was degraded the wool but the
immobilized enzyme retained 76% of the tensile strength of wool.
• Protease also immobilized by entrapment method with
polyacryamide gel, the enzyme retained its activity for about 6
cycles with incubation time of 20 min at 55 oC for each cycles, by
this immobilization method, protease activity is retained for about
83%of the initial activity after six cycles.

72. Detergent Industry:
• The lipase immobilization is carried in order to prevent lipase
from protease action and surfactant inhibition lipase is
immobilized on acrylamine glass beads coated with zirconia with
the size of 55nm.
• The Maximum activity was found for immobilized lipase at the
pH of 6.5 and for free enzyme it is 7.5.