1

CHAPTER 1

INTRODUCTION

1.1 IMMOBILIZATION OF ENZYMES

Immobilization involves binding the enzyme with an insoluble

matrix, so that it can be retained in proper reactor geometry for its economic

reuse under stabilized conditions. The first published report of enzyme

immobilization was that of Nelson and Griffin in 1916, describing the

adsorption of invertase on charcoal and alumina. However, it was not until

after World War II that further studies on the binding of enzymes appeared

and it was shown that synthetic polymers could be used to bind

physiologically active proteins (Hartmeier 1986). In pioneering efforts

adsorption work was primarily performed upon inorganic carriers, while

covalent attachment was reserved for organic carriers. From the 1960's

onward, the explosive increase in publications reflected the worldwide

interest on immobilization and most of the development in this area of

biotechnology has occurred in this period.

Invertase, which hydrolyzes sucrose into glucose and fructose, was

the first enzyme to be used commercially in immobilized form during World

War II when sulfuric acid, used in the traditional process, was unavailable

(Cheetham et al 1995). Although enzyme immobilization has been performed

for over 50 years, its use in industrial processes has been limited due to

limited enzyme stability and specificity. Recent developments have made

the use of enzymes in industrial processes much more feasible

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(Ikehata and Buchanan 2004). Advancements in bioengineering have allowed

for the over-expression of selected enzymes by fermentation and coupled with

improved purification techniques lowered the cost of enzymes (Kim and Kang

2004 and Koizumi 2004). In addition, the developments in protein

engineering and advanced screening techniques have introduced a large

variety of designed enzymes: providing greater selection for the improvement

of particular reactions (Selber et al 2004). Most recently, the development of

non-aqueous enzymatic processes has drawn interest from many groups due

to improved product and enzyme recovery (Gupta 1992, Braco 1995 and

Krishna 2002).

1.2 ADVANTAGES OF IMMOBILIZED ENZYMES

The use of free enzymes is limited by several factors such as high

cost of enzyme, their instability and availability in small amounts. Also

enzymes are soluble in aqueous media and it is difficult and expensive to

recover them at the end of the catalytic process.

Immobilization of enzymes helps in their economic reuse and in the

development of continuous bioprocess. Immobilization often stabilizes the

structure of the enzymes, thereby allowing their applications even under harsh

environmental conditions of pH, temperature and organic solvents.

Immobilized enzyme has the ability to stop the reaction rapidly by removing

the enzyme from the reaction solution. In immobilized enzyme catalytic

reactions product is not contaminated with the enzyme. This is especially

useful in the food processing and pharmaceutical industries.

The advantages of immobilized enzymes may be summarized as

follows (Messing 1975, Hartmeier 1986 and Zaborsky 1973):

Low downstream processing cost

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Better stability, especially true for ADH dependent enzymes

towards heat (Liao et al 2001 and Julliard et al 1985).

Cofactor binding to enzyme (less waste).

Easy realization of continuous production of desired products.

Enzymes can be recovered from solution and reused.

Adaptability to a variety of configurations and to specific

processes.

Controlled product formation and possible greater efficiency in

consecutive multi step reactions.

Chemically based methods for immobilizing enzymes possess

the distinct possibility of changing the chemical or physical

properties of enzymes. For example, a change in pH optimum of

an enzyme may allow an enzyme which normally operates

around neutral pH to be used to process alkaline solutions. This

may remove a need to alter the pH of the substrate solution or

may be used as a device to help limit contaminating microbial

growth.

. The main disadvantages of enzyme immobilization are:

Possible loss of absolute enzyme activity due to the

immobilization process in certain cases

Additional cost of carrier or other reagents used for

immobilization process

Mass transfer limitations

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1.3 METHODS OF ENZYME IMMOBILIZATION

Based on the nature of interaction responsible for immobilization,

methods for immobilizing enzymes may be divided into two broad

classes- physical and chemical (Zaborsky 1973). Physical methods involve

non-covalent localization of an enzyme. In principle, these techniques are

reversible. Chemical methods of immobilization involve the formation of at

least one covalent bond between the enzyme and support. These techniques

are usually irreversible in that the original enzyme can not be regenerated or

recovered.

Enzyme immobilization can be accomplished in a variety of ways,

by entrapment, cross-linking, or physical attachment (Tischer and Kasche

1999). Enzyme entrapment is accomplished through the polymerization of a

matrix from a solution containing both the monomer and the enzyme. Enzyme

entrapment generally does not involve any modification of the enzyme and

therefore may not adversely affect activity. However, this method can be

susceptible to leaching of enzymes out of the matrix by diffusion when pore

diameters are larger than the enzyme.

1.3.1 Physical Methods of Immobilization

Physical Methods of Immobilization of includes

i) Physical adsorption

ii) Ionic bonding

iii) Gel entrapment

iv) Lattice entrapment

v) Encapsulation.

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1.3.1.1 Physical Adsorption

Physical adsorption is the earliest known method of enzyme

immobilization, based upon invertase adsorption onto aluminum hydroxide

(Nelson and Griffin 1916). This is the easiest method of preparing

immobilized enzymes, and includes physical adsorption of an enzyme onto an

inert support such as glass beads, charcoal or polysaccharides. The method

generally requires contacting an aqueous solution of the enzyme with the

carrier. A major disadvantage of this method is that binding forces between

protein and adsorbent are weak (hydrogen bonds, van der Waals’ forces and

hydrophobic interactions).

1.3.1.2 Ionic Binding

Immobilization via ionic binding is based on the electrostatic

attraction between oppositely charged groups of the carrier and the enzyme

molecules. The matrices used for ionic bondings are ion-exchangers most

frequently prepared from organic supports. The organic polymers are

derivatives of polysaccharides, such as dextran and cellulose or synthetic

polymers mainly derived from polystyrene. The carriers are classified as

anion or cation exchangers depending on their ability to exchange anions

(chloride or hydroxyl) or cations (hydrogen and sodium ions) of the carrier

with anionic or cationic residues of the enzymes. Basically bound enzymes

may be prepared by stirring carrier particles in a solution of the enzyme or by

pumping an aqueous solution of the enzyme over the carrier particles.

1.3.1.6 Gel Entrapment

The gel entrapment method involves entrapping enzymes within the

interstitial spaces of cross linked polymer gels. A widely used system for

enzyme entrapment in a polymer lattice is polyacrylamide gel. The usual

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method of gel formation is to polymerize acrylamide in an aqueous solution

of the soluble enzyme and a cross linking agent such as N.N-

methylenebis(acry1amide). The immobilization process can be harsh leading

to inactivation of enzyme during the process. The broad distribution of pore

sizes in the gel inevitably results in leakage of the entrapped enzyme even

after prolonged washing (Kennedy and White 1985). The cross linked

polymer gels offer diffusion limitations and their application is restricted to

those reactions which involve relatively small substrate and product

molecules.

1.3.1.7 Lattice Entrapment

Entrapment involves entrapping enzyme within the interstitial

spaces of a carbon linked water-insoluble polymer. Some polymer systems

such as polyacrylamide, polyvinyl alcohol etc. and natural polymer (starch)

have been used to immobilize enzyme using this method (D’Souza 1999a).

1.3.1.8 Microencapsulation

Microencapsulated enzymes are formed by enclosing enzymes

within spherical semi permeable polymer membranes having diameters in the

1-100 µm range. Enzymes immobilized in this manner are physically

contained within the membrane. While substrate and product molecules are

free to diffuse across the membrane provided that their molecular sizes are

sufficiency small enough to allow this.

Microencapsulation methods have the potential to offer a very large

surface area. However the membrane is a significant mass-transfer barrier, so

that the effectiveness factor for the enzymes may be quite small (Bailey and

Ollis 1986). Disadvantages of this method include possible inactivation of the

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enzyme during microencapsulation and possible incorporation of the enzyme

into the membrane wall, which allows it to be released from the capsule.

1.3.2 Chemical Methods of Immobilization

1.3.2.1 Cross linking

Cross-linking, which involves the attachment of enzymes to each

other, is another method used to immobilize enzymes (Tyagi and Gupta

1998). This method is based on the formation of covalent bonds between

enzyme molecules by means of bi- or multi-functional reagents, leading to

three-dimensional cross linked aggregates which are insoluble in water. This

method involves the addition of the appropriate amount of cross linking agent

to an enzyme solution under conditions which give rise to the formation of

multiple covalent bonds. The most widely used method employs

glutaraldehyde which establishes intermolecular (and possible intramolecular)

cross-links with amino groups on the enzyme. Particles of cross-linked

enzyme alone are gelatinous and lack the mechanical properties required in

many applications.

1.3.2.2 Covalent Binding

The covalent binding method is based on the covalent attachment of

enzymes to water-insoluble matrices. Although the earliest immobilized

enzymes were produced by adsorption, the field of immobilized enzyme

technology was limited until methods of covalently coupling of enzymes were

introduced (Scouten 1987). Covalent binding is the most prevalent and most

investigated method for immobilizing enzymes.

The immobilization of an enzyme by covalent attachment to a

support matrix must not involve those functional groups of the enzyme that

are essential for catalytic action. The principal groups for coupling an enzyme

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are hydroxyl and amino groups and to a lesser extent sulphydryl groups

(Trevan et al 1987). One major problem with this technique is that the enzyme

may be inactivated owing to conformational change as a result of reaction or

reaction with a group at the enzyme's active site. To prevent inactivation

reactions with the essential amino acid residues of the active site, a number of

methods have been devised, such as conducting the covalent attachment of the

enzyme in the presence of a competitive inhibitor or substrate (Zaborsky

1973).

Covalent attachment of an enzyme to a water-insoluble support

offers numerous advantages as a method for immobilizing enzymes. Covalent

linkages provide strong, stable enzyme preparations which do not leach

enzyme into solution in the presence of substrate or ionic solutions. Chemical

alteration in the structure of a covalently-bound enzyme may also lead to

superior chemical or physical properties relative to its soluble counterpart,

such as reduced deactivation rates or improved enzyme specificity (Bailey

and Ollis 1986).

The functional groups that may take part in covalent binding

include, amino, carboxyl sulfhydryl group, hydroxyl group, imidazole group,

phenolic group, thiol group, threonine group and indole groups.

Covalent attachment to a support matrix must involve only

functional groups of the enzyme that are not essential for catalytic action.

Higher activities result from prevention of inactivation reactions with amino

acid residues of the active sites. A number of protective methods have been

devised:

Covalent attachment of the enzyme in the presence of a

competitive inhibitor or substrate.

A reversible, covalently linked enzyme-inhibitor complex.

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A chemically modified soluble enzyme whose covalent linkage

to the matrix is achieved by newly incorporated residues.

A zymogen precursor.

Hence, covalent binding can be brought about by the following:

diazotization, amide bond formation, alkylation, arylation, Schiff's base

formation, amidation reaction, thiol-disulfide interchange, mercury-enzyme

interchange, gamma-irradiation induced coupling and carrier binding with

bifunctional reagents.

Various physical forms of the support material can be employed: the

enzyme- polymer conjugate can be in the form of a powder, fiber etc. Almost

any polymer may be used and the polymer can be constructed to carry any

charge. The variations in the chemical and physical nature of the support

render these immobilized enzymes adaptable to a variety of specific

engineering requirements, such that almost any enzyme is a candidate for

immobilization (Trevan et al 1987). In principle, there are two ways of

covalently binding an enzyme to a carrier. First, by direct linkage using a

reactive group to activate the carrier and second, by using a so-called spacer

to bridge between the enzyme and carrier. Examples of spacer molecules used

for the immobilization of glucose oxidase on nylon include:

polyethyleneimine (Thompson et al 1985, Garcia and Galindo 1990), amino

acids, such as lysine and arginine and phenylenediamine.

1.4 SUPPORTS USED FOR ENZYME IMMOBILIZATION

The appropriate immobilization matrix is chosen based on several

different properties which affect the production process (Tischer and Kasche

1999).

Insolubility in process solutions

High surface area

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Physical rigidity to withstand the operational stress

Presence of reactive functional groups to derivatize and bind

ligand molecules

Chemical stability towards harsh conditions during

derivatization or regeneration

Minimal non-specific adsorption characteristics

Should be reusable and preferably of low cost

Chemical reactivity, allowing spacers and enzymes to be

introduced and coupled.

Compatibility with the enzyme, the substrate, and the material

being processed.

Resistance to microbial degradation.

Size and shape: To simplify handling of the immobilized

enzyme (i.e. stirring, filtration) it is ideal to have particles of

uniform shape and size. For this reason, the use of a uniform

spherical matrix is preferred

Hydrophobic/hydrophilic nature: The compatibility of the

support with the liquid phase is important to insure the free

exchange of substrate and product between the matrix and bulk

phase.

Inorganic supports generally have fewer binding sites than organic

supports. Inorganic structures are generally rigid while many organic

polymers are flexible and elastic. The elastic support can be used in thin

membrane form and can be made to conform about another structure. A thin

membrane would imply a small diffusional path and an optimal available

surface for reaction. The classification of carriers into organics and inorganic

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is not wholly adequate for a full description of the carrier. Parameters such as

surface area and pore diameter will affect the loading of the enzyme, and

therefore, a further classification based on morphology can be considered,

into nonporous and porous carriers.

Examples of carriers used enzyme immobilization include :

Organic Carriers: natural polymer, activated carbon, agarose

(Sepharose), cellulose, collagen, dextran (Sephadex), gelatin and starch.

Synthetic polymer: acrylamide-based polymers, maleic anhydride

copolymers, nylon, styrene-based polymers.

Inorganic Carriers: glass beads (porous and nonporous), kaolinite,

metal oxides (e.g., ZrO2, TiO2, Al2O3 and NiO, sand, silica gel and porcelain

beads

1.4.1 Organic Carriers

Organic support matrices include both synthetic and natural

polymers. Nylon-6,6 tubing was used by Thompson et al (1985) to separately

immobilize glucose oxidase, catalase and urease by means of O-alkylation.

Serigraphy nylon nets (150-mesh) were used for the immobilization of

glucose oxidase (Garcia and Galindo 1990).

Generally, there is no universal carrier or immobilization technique:

the choice of carrier and coupling method are specific to the enzyme and its

ultimate applications. Beads of nylon-6,6 have been used in this work as the

carrier for a variety of reasons:

Nylon structures are mechanically strong and non-biodegradable,

which renders possible their prolonged exposure to biological

media without impairment of their structural integrity.

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Some of the nylons, such as nylon-6 and nylon-6,6 are relatively

hydrophilic; this means that they can support an environment for

enzyme immobilization which is conducive to the stability of the

protein (Hornby and Goldstein 1976).

Nylon is readily available, at relatively low cost, in a wide

variety of physical forms, such as films, membranes, powders,

hollow fibers and tubes. Thin membranes of nylon 66 have been

employed in this work to minimize diffusion limitations.

The variety of nylon forms makes it possible to consider the

preparation of various immobilized enzyme structures which

have both a common support matrix and a common method of

immobilization.

Finally, the chemistry of enzyme immobilization onto nylon

surface is well established.

1.4.2 Inorganic Carriers

1.4.2.1 Immobilization of enzymes on silica gel

A variety of gel matrixes have been used as carriers of immobilized

enzymes. Many of these carriers fall under the category of “soft gels” because

of their low mechanical strength. Silica gels offer a number of advantages

over these “soft gels” for use in industrial processes. Silica’s higher

mechanical strength allows a much wider range of operating pressures as

evidenced by their preferential use in High Performance Liquid

Chromatography (HPLC) (Majors 2003, Cabrera 2004, Xu and Feng 2004).

Additionally, silica also has relatively higher thermal and chemical stabilities,

except at extreme pH which is rarely experienced in enzymatic reactions, and

is resistant to microbial degradation (Nefedov 1992, Pang and Qiu 2002).

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Silica gels also provide high surface areas and high porosity which can be

used to increase enzyme loadings and accessibility.

According to the review, significant advantages of silica

encapsulation are: excellent optical and mechanical properties, high resistance

to (biological, chemical, and thermal) degradation, simple fabrication,

enhanced activities, long and stable shelf life, and application versatilities.

Silica gel was used for the covalent immobilization of urease.

Covalent immobilization of enzymes, onto silica surfaces, has been done for a

number of years (Weetall 1993). These methods commonly involve the use of

pre-fabricated silica gels that have chemically inactive surfaces (Hossain and

Do 1985). The gel surface must be activated, with strong acids, to produce the

reactive surface hydroxyl groups. The activated surface is then modified with

silane couple reagents, such as aminopropyltriethoxy-silane (APTES), which

link functional groups (e.g. amino) to the silica surface via siloxane bonds

(Moreno and Sinisterra 1994). The cross-linking agent (e.g. glutaraldehyde) is

then introduced to react with the modified surface and finally the enzyme is

added. In addition to being process intensive, these methods have been

limited to relatively low enzyme loadings.

Inorganic carriers are generally rigid and have fewer binding sites

per mass unit than do organic supports. In contrast, many organic polymers

are flexible and can be employed in a variety of physical forms; the enzyme-

polymer conjugate may be in the form of a thin membrane, powder, fiber, etc.

A thin membrane may help eliminate internal diffusion limitations which can

mask intrinsic enzyme kinetics.

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1.5 APPLICATIONS OF IMMOBILIZED ENZYMES

Applications of immobilized enzymes are well documented in at

least four areas. These include

i) Industrial applications

ii) Analytical applications

iii) Therapeutic uses

iv) Environmental applications

1.5.1 Industrial Applications of Immobilized Enzymes

Enzymes are extensively used in the manufacture of pharmaceutical

drugs and fine chemicals, among many other applications. The use of

enzymes in manufacturing processes does possess a few drawbacks; enzymes

are a relatively expensive reaction component and therefore increase

production costs. They are also extremely sensitive to environmental

conditions and can be easily denatured. Any manufacturing process must

consider the conservation of the enzymes within the reactor while also

maintaining their activity.

Often, the most expensive step in enzyme production is the

downstream separation and purification of the desired protein (Conder and

Hayek 2000). Therefore, use of these methods for enzyme recovery from a

homogeneous catalysis process can significantly increase production costs.

One method of lowering these processing requirements is to use a

heterogeneous catalyst – an immobilized enzyme (Beck and Suginome 1991,

Chitnis and Sharma 1997 and Laszlo 1998).

In 1978, Ichiro Chibata succeeded in the industrial application of an

immobilized enzyme, i.e., immobilized aminoacylase, for the continuous

production of L-amino acids from acetyl - DL-amino acids. This new

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procedure is the first industrial application of immobilized enzymes in the

world. Since then we also have carried out the industrial application of

immobilized microbial cells for the continuous production of L-aspartic acid

and L-malic acid have been reported.

1.5.1.1 The resolution of DL-amino acids

The chemically synthesized acylated DL-amino acid is selectively

hydrolysed by immobilized aminoacylase to the L-amino acid and the D-acyl

amino acid.

R-CH-COO- Aminoacylase (E.C.3.5.1.14) R-CH-COO- + R-CH-COO-

NH-COR NH 3 + NH-COR

DL-Amino acid L-Amino acid D-Acylamino acid

The L-amino acid and D-acylamino acid can be easily separated by

virtue of their differing solubilities. The D-acyl amino acid is then racemized

to the DL form and used again (Ichiro Chibata 1978).

1.5.1.2 High fructose syrups

The most important application was the conversion of glucose

syrups to high fructose syrups by employing soluble glucose isomerase. The

first commercial production of high fructose syrups using glucose isomerase

immobilized on to a cellulose ion- exchange polymer in a flat bed reactor was

initiated by Clinton corn processing. (Sharma and Messing 1980).

1.5.1.3 Production of 6-amino penicillanic acid

One of the major applications of immobilized enzymes in

pharmaceutical industry is the production of 6-aminopenicillanic acid

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(6-APA) by the deacylation of the side chain in either penicillin G or V, using

penicillin amidase (Pastore and Morisi 1976).

O

NHN

S

COOH NNH2

OCOOH

O

OHPencillin Amidase

Pencillin G

+

6 APA

More than 50% of 6-APA produced today is enzymatically using the

immobilized route. One of the major reasons for its success is in obtaining a

purer product, thereby minimizing the purification costs.

1.5.1.4 Production of L-aspartic acid

L-Aspartic acid is used for medicines and food additives, and it has

been industrially produced by enzymic methods from ammonium fumarate

using the action of aspartase:

HOOC-CH=CH-COOH + NH3HOOC - CH2 - CH - COOH

NH2Fumaric acid

Aspartase

L-Aspartic acid(EC 4.3.1.1)

1.5.1.5 Production of L-malic acid

L-Malic acid is an essential compound in cellular metabolism and is

mainly used in pharmaceutical field. L-Malic acid can be produced by

enzymatic methods from fumaric acid by the action of fumarase:

HOOC - CH =CH-COOH + H2O HOOC - CH2 - CH - COOH

OH

Fumaric acid

Fumarase(EC 4.2.1.2)

L - Malic acid

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1.5.1.6 Urocanic acid production

Urocanic acid is commercially used as an UV filter in sun tan

preparations. It is produced from L-histidine by the action of histidine

ammonia lyase (Yamarnoto et al 1974).

L- Histidine Urocanic acid + NH3

L -Histidine ammonia lyase(EC 4.3.1.3)

1.5.2 Analytical Applications

It may be classified into two areas (Michael Trevan 1980a), the

enzyme electrodes and automated analyzers. Enzyme electrodes are probes

capable of generating an electrical potential as a result of a reaction catalyzed

by an immobilized enzyme that is fixed on to (or) around the probe. The first

enzyme electrode to be prepared was glucose – sensitive electrode made by

immobilizing glucose oxidase around an oxygen electrode.

The use of immobilized enzymes in automated analysis implies that

the immobilized enzyme is used to replace soluble enzyme in an existing

automated analyzer system. There are two areas of automated analysis in

which immobilized enzymes may find general application, repeated

automated analysis of small samples (e.g., blood samples) and continuous

stream monitoring of large volumes.

The enzyme electrode (biosensors) has the following advantages,

long half-life, predictable decay rates, elimination of reagent precipitation etc.

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1.5.3 Therapeutic Applications

The study of immobilized enzymes for biomedical applications

started in the 1960s, aiming to solve some of the limitations to the use of

enzymes in clinics, to make them more stable, less immunogenic and

toxicologic and to present a longer in vivo circulation lifetime. Since then,

several approaches have been used in enzyme therapy either for the detection

of bioactive substances in the diagnosis of diseases or with the aim to treat a

disease condition, such as the correction of inborn metabolic defects,

cardiovascular diseases, cancer, intestinal diseases or for the treatment of

intoxication (Liang et al 2000). One of the approaches used for enzyme

immobilization is based on the entrapment of the enzyme in a matrix

(i.e. liposome, red blood cell, microparticle or nanoparticle). In fact,

microcapsules of calcium alginate coated with a polycation have been widely

investigated for applications like immunoprotective containers in cell

transplantation, enzyme immobilization and drug release systems (Gaserod

et al 1999).

As Chang and Prakash et al (1998) proposed, orally administered

microcapsules might be suitable for some applications, since the need for

implantation is avoided. During the passage of microcapsules through the

gastrointestinal tract, small molecules (urea, aminoacids) from the body enter

the microcapsules where they can be metabolized by the enzymes in the

microcapsules.

Replacement of enzyme: The replacement of enzyme in genetic

disorder due to the absence of particular enzyme either by genetic

malfunction/tissue malfunction. Examples of genetically derived enzyme

deficiency include the broad classification of lysosomal storage diseases,

where a lysosomal enzyme is absent and its substrate will accumulate within

the cell often leads to disastrous results and also to diseases such as favism

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where an enzyme involved in a metabolic pathway, in this case glucose- 6

phosphate dehydrogenase of the red blood cell, is absent. Potentially the most

useful form of immobilization is encapsulation within a non-antigenic

polymer material, example nylon or collodian whereas the majority of

enzyme replacement will involve simple hydrolytic or oxidase reaction

(example urease or catalase). Removal of toxic materials: Tissue malfunction,

particularly of the liver and kidney, may result in the accumulation of toxic

waste materials, for example urea, within the body. Such diseases can be

controlled, at least in theory, by injecting soluble enzymes but it may lead to

an allergic response that in itself may be fatal or it may cleared from the body

by the natural immune response system due to its instability. Immobilization

may overcome both these problems, by preventing the interaction between

enzyme and body’s immune response system, and stabilizing and protecting

the enzyme (D’Souza 1999c).

1.5.4 Environmental Applications

It includes the following (Michael Traven 1980b):

Waste water / effluent treatment: Immobilized enzymes have

found use in the treatment of effluents from industries. Especially urease

found its application in the hydrolysis of urea containing effluent from

fertilizer and from other industrial effluents.

Waste utilization: Effluents containing cellulose, hemicellulose,

lignin can be hydrolysed by enzymes and they can be used in some other

processes. For example the glucose syrup obtained from cellulose waste can

be used for the preparation of foodstuffs.

Energy production: Energy can be produced either from

electrobiochemical cell or combustible product, for instance methane or

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hydrogen. Biochemical fuel cell utilizing as the source of electrical current the

electrochemical oxidation of hydrogen at Pt black electrode (anode). The

hydrogen may be produced from a carbohydrate source by micro-organism,

for example E. coli that contain a hydrogen- producing system based on

hydrogenase.

1.6 CLINICAL SIGNIFICANCE

1.6.1 Urea

Urea, the end product of nitrogen metabolism, has a considerable

significance in clinical chemistry, where blood urea nitrogen analysis gives an

important indication of possible kidney disease. Increased levels of blood urea

nitrogen occur in cases of renal failure (Abdel and Guilbault 1990). Urea is

formed in large quantities as a product of catabolism of nitrogen-containing

compounds, each human producing, for example, ca. 10 kg of urea per year.

Urea spontaneously decomposes with a half-life of ca. 3.6 years. A widely

accepted reference interval for serum urea is 2.3-8.3 mmol/L, derived from

young men on a normal diet, and the value between 24.9-41.5 mmol/L is

taken as a conclusive evidence of severe renal impairment (Anderson and

Cockayne 1993).

1.6.2 Cholesterol

Serum cholesterol serves as an indicator of propensity towards

coronary heart disease, liver function, biliary function, intestinal absorption,

thyroid function and adrenal disease. Primarily, two lipoprotein classes

transport the cholesterol. HDL transports cholesterol from tissues to the liver

for catabolism while LDL transports cholesterol from sites of origin to

deposition in tissues.

Cholesterol is a sterol that’s important as an essential structural

component of cell membrane, as a precursor to the sex and adrenal steroid

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hormones such as progesterone, estrogen and testosterone, as a precursor for

7-dehydrocholesterol (pro-vitamin D) and for the synthesis of cholic acid

which is then converted to bile salts. The bile acid aids the digestion and

absorption of fats in the diet. Cholesterol is also a part of lipoproteins, the

complexes that carry lipids (fats) in the bloodstream.

Cholesterol is a major component of mammalian cell membranes,

particularly of plasma membranes, and regulates membrane fluidity and

permeability. Cholesterol is found in the brain, nervous tissues, skin and

adrenal glands. Cholesterol is produced mainly in the liver. It is present in

saturated fats found in meat and dairy products and many processed foods.

Foods from animal sources that are high in saturated fats tend to be high in

cholesterol. In fact, cholesterol is only found in animal products. These

include dairy products, meat, fish, poultry, and fats.

Increased concentration: Increased cholesterol concentration is

found in idiopathic hyper cholesterolemia, hyperlipoproteinemia, nephritic

syndrome, hypothyroidism, nephrosis and diabetes mellitus.

Hypercholesterolemia is known to be associated with an increased risk of

Coronary Heart Diseases (CHD).

Decreased concentration: Decreased cholesterol concentration is

found in hepatocellular disease, hyperthyroidism, chronic anemia, starvation

and hypobetalipoprotenemia. Serum cholesterol concentration is very low in

rate genetic disease like abetalipoproteinemia.

1.7 UREASE

Urease is frequently used in medical field for the determination of

amount of urea present in blood or in urine. Urease serves as a virulence

factor in human and animal infections of the urinary and gastrointestinal

22

tracts, being involved in kidney stone formation, catheter encrustation,

pyelonephritis, ammonia encephalopathy, hepatic coma, and urinary tract

infections (Mobley and Hausinger 1989 and Collins and D’Orazio 1993). The

ureolytic activity of Helicobacter pylori is also the major cause of pathologies

(including cancer) induced by gastroduodenal infections by such

microorganisms.

Urease is found in a wide range of organisms, many have been

isolated from various bacteria, higher plants, fungi and some invertebrates

(Mobley et al 1995). The analysis of urea is also important in other fields,

such as agricultural chemistry, where urea is used in fertilizers, for

determination of water quality, and in seawater analysis (Abdel and Guilbault

1990). Urease is also used in agricultural field as urea is a major component

in nitrogenous fertilizers and has been recognized as a pollutant in agricultural

waste water.

Urease (urea amidohydrolase, EC 3.5.1.5) is a hexameric protein

(540 kDa) made up of six identical subunits (Hirai et al 1993) and a nickel-

dependent metalloenzyme, essential cysteine residue serves as a general acid

catalyst in the mechanism of action of urease, catalyzes the hydrolysis of urea

to ammonia and carbamate. The carbamate then spontaneously hydrolyzes to

form carbonic acid and a second molecule of ammonia has been used in an

immobilized form in kidney machines for blood detoxification (Thavarungkul

et al 1991 and Lee et al 1995). According to one report approximately half a

million patients worldwide are being supported by haemodialysis. Urease has

been used in an immobilized form in kidney machines for blood

detoxification. Immobilization of urease has been carried out on several

matrices for clinical analytical applications, and has also been used for the

treatment of urea-containing effluents. An acid urease from Arthtobacter

mobilis has been used for removal of urea from fermented beverages, such as

sake (Miyagawa et al 1999).

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1.8 IMMOBILIZED UREASE

Urease occurs in bacteria, molds and in a large number of higher

plants. It is found especially in beans. The jackbean contains as much as

0.15 % while the soybean contains 0.012 % by weight. Other important

sources of urease include Beach bean (C.oblusifolia), Squash (Cucurbita

maxima), Water melon seed (Citrullus vulgaris), Snake gourd (Trichosanthus

anguina), Horse gram (Dolichos biflorus), Bitter gourd (Monordica

chaorantia) and White gourd (Benicasa cerifera). Urease from jack bean

seed, soybean seeds, and bacteria have a lower optimum pH of 7-8 (Kerr et al

1983, Mobley and Hausinger 1989), whereas most fungal urease have an

optimum pH range of 8-8.5 (Mobley et al 1995 and Lubbers et al 1996).

Urease was immobilized by adsorption onto inert solids, like flannel

cloth (Das et al 1998), ion-exchange resins, like DEAE-Cellulose (Reddy et al

2004), and physically entrapped/encapsulated in solids, such as cross-linked

gels like, polyacrylamide and calcium alginate (Das et al 1998). Urease was

immobilized by various derivates of chitosan supports, these are chitosan

beads (Kayastha and Srivastava 2001), chitosan-alginate (Kara et al 2006),

chitosan composite membranes (Gabrovska et al 2007) and chitosan

membrane (Ma and Yao 2005).

Urease immobilized by nano porous compounds and composites,

these are nanoporous alumina membranes (Yang et al 2007), reverse

micelles (Das et al 1997), hollow fibers polyacrylonitrile fibers (Lin and Yang

2003) and gelatin (Srivastava et al 2001). And urease was immobilized by

microbeads, these are microfluidic channel microbeads (Ayhan et al 2003),

silicon microchannels (Forrest et al 2005), deposition of bone-like

hydroxyapatite (Unuma et al 2007), magnetic polystyrene beads (Mustafa

et al 1992), non-porous poly methacrylate microbeads (Ayhan et al 2002) and

paramagnetic polyacrolein beads (Anca Roxana et al 1996).

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Urea sensors have been developed: thermal (Xie et al 1995; Xie and

Danielsson 1996), amperometric (Bertocchi et al 1996 and Adeloju et al

1996), conductimetric (Chen et al 1994 and Sheppard et al 1996), optical

(Stamm et al 1993, Li and Wolfbeis 1993), piezoelectric (Xu et al 1996) or

potentiometric biosensors (Mascini et al 1983, Gil 1992, Adeloju et al 1993,

Glab et al 1994 and Gracia et al 1996).

Synthesis and serological interactions of H. pylori urease fragment

321-339 n-terminally immobilized on the cellulose (Kolesinska et al 2006),

Recombinant H. pylori antigens, urease B subunit (UreB), vacuolating toxin

A (VacA) and cytotoxin associated gene A protein (CagA), were prepared and

immobilized in matrixes on nitrocellulose membrane to bind the specific

immunoglobulin G (IgG) antibodies in serum. (Han et al 2006).

1.9 CHOLESTEROL ESTERASE

Cholesterol esterase (CEase, 1 sterol ester hydrolase; EC 3.1.1.13) is

a lipolytic enzyme that is synthesized in the pancreas and secreted into the

duodenum in response to an oral fat load. The enzyme is also found in human,

feline, canine, and gorilla milk, wherein its digestive action allows alimentary

absorption of lipids in suckling infants. CEase has long been suspected of

playing a role in intestinal absorption of dietary lipids, including cholesterol.

Different laboratories have characterized the effect of removal of endogenous

CEase activity on lipid absorption in bolus-fed, duodenally rats. Contradictory

results have been reported:

Hui (1996) have addressed the role of CEase in the absorption of

dietary lipids by developing a CEase gene knockout mouse model, and report

that a loss of pancreatic CEase activity has no effect on cholesterol

absorption, but is a decisive element in cholesteryl ester absorption. Of

considerable interest are recent reports from the same group that plasma

25

cholesterol and plasma CEase levels are correlated, and that plasma CEase is

involved in LDL metabolism, may function as a lipid transfer protein, and is

involved in uptake of cholesteryl esters by liver cells in culture.

1.10 CHOLESTEROL OXIDASE

Cholesterol oxidase (CO, EC 1.1.3.6) is a flavoprotein that catalyses

the dehydrogenation of C(3)-OH of the cholestan system. It is therefore an

alcohol dehydrogenase/oxidase belonging to the Glucose-Methanol-Choline

(GMC) oxidoreductase family. Oxidized flavin is the primary acceptor of

hydride from the alcohol. The reduced flavin then transfers the redox

equivalents to dioxygen as final acceptor. CO has been isolated from a variety

of organisms, mainly fungi.

Cholesterol oxidase is a flavin-enzyme (with a FAD prosphetic

group) that produces hydrogen peroxide according to the reaction.

Cholesterol + O2 4 - Cholesten - 3 - one + H2O2

The structure of cholesterol oxidase reveals deeply buried active

sites occupied by water molecules in the absence of its substrate steroids.

Cholesterol oxidase is industrially and commercially important for application

in bioconversions for clinical determination of total or free serum cholesterol

and in agriculture.

1.11 HYDROGEN PEROXIDASE

Peroxidases (E.C. 1.11.1.7) are heme-containing enzymes that

catalyse the one-electron oxidation of several substrates at the expense of

H2O2. Catalysts cannot initiate reactions that would not happen in their

absence, but can, and do, radically affect reaction rates with the result that the

cell can carry out rapid and complex chemical activities at relatively low

26

temperatures. Most enzymes are highly specific. They tend to accelerate only

one or a group of related reactions. The result is that many different enzymes

may be present in a cell and may act simultaneously without mutual

interferences.

Hydrogen peroxide (H2O2) is a common end product of oxidative

metabolism and, being a strong oxidizing agent, would be toxic if allowed to

accumulate. To prevent this, eukaryotic cells have enclosed the enzymes

producing peroxides within a membrane-bound organelle, the peroxisome,

which is similar in size and appearance to a lysosome. Peroxisomes also

contain high concentrations of peroxidase – the enzyme that functions to

reduce the peroxide to water, rendering it harmless. A variety of electron

donors can be used, including aromatic amines, phenols, and enediols like

ascorbic acid.

peroxidaseH2O2+ Colorless Dye (reduced) H2O + Colored Dye (oxidized)

A dye like o-dianisidine or 4-amino antipyrine can be used as the

electron donor (colorless) to easily detect peroxidase in vitro because its

oxidized product is highly colored (Extinction coefficient is 11.3 mM-1cm-1).

The rate of appearance of this colored pigment can be measured

calorimetrically and is equivalent to the rate of reaction.

1.12 CO-IMMOBILIZED CHOLESTEROL ESTERASE,

CHOLESTEROL OXIDASE AND PEROXIDASE

Among the various methods available for total cholesterol

determination, an enzymic colorimetric method employing cholesterol

esterase, cholesterol oxidase and peroxidase their first successful

immobilization on alkylamine glass by Huang et al. (Huang et al 1977),

27

cholesterol esterase and cholesterol oxidase have been immobilized on to

octyl-agarose gel (Karube et al 1982) and silica gel (Yao et al 1985).

Various biosensor methods are employed by mixture of enzymes,

these are sol–gel film through physical adsorption, Au nanowires based

micro-fluidic platform (Shyam Aravamudhan et al 2007), sol–gel films

(Suman et al 2007), polyaniline films (Suman et al 2006), carbon nanotube

(Li et al 2005), clay-modified electrodes (Christine Mousty 2004), alkylamine

glass beads (Suman and Pundir 2003), polypyrrole films (Yoshio Kajiya et al

1991), poly-N-methylpyrrole-p-toluene sulphonate films (Pratima et al 2007),

and thermistor-based assay (Venkatesh Raghavan et al 1999).

Further, glutaraldehyde used as a coupling agent for immobilization

of an enzyme on alkylamine glass has the disadvantage of self-

polymerization, protein cross-linking and reversibility of reaction at low pH,

due to Schiff base formation. Immobilization of an enzyme on to arylamine

glass through diazotization has no such problems (Foster et al 1980). The

level of total cholesterol in human blood serum samples is determined by

flow-injection analysis (Achim Krug et al 1994). Flow-injection system for

simultaneous assay of free and total cholesterol in blood serum by use of

immobilized enzymes (Toshio Yao and Tamotsu Wasa 1988).

Temperature enhanced chemiluminescence for determination of

cholesterol (Mike et al 1992), colorimetric determination of free and total

cholesterol by flow injection analysis with a fiber optic detector (Krug et al

1992) and controlled pore glass through glutaraldehyde (Masoom et al 1985)

are used in automated and flow-injection systems for measurement of total

cholesterol in serum.

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As awareness of the importance of total cholesterol levels has

increased, numerous methods for human blood cholesterol assays have been

developed (Crumbliss et al 1993, Martin et al 2003 and Shumyantseva et al

2004), including colorimetric spectrometric and electrochemical methods.

Mainly enzymatic procedures are employed in clinical diagnosis due to their

rapid, selective, sensitive nature and the great accuracy.

1.13 SCOPE AND OBJECTIVES

Urease and cholesterol esterase have significant applications in

clinical chemistry. Urea constitutes the largest fraction of the non protein

nitrogen component of the blood. Impaired renal function is associated with

elevated levels of urea. Determination of blood urea is important for

hydrolysis of urea in kidney. Urease also finds use in environmental

applications for the destruction of urea in effluents from fertilizer industries.

The determination of serum cholesterol is one of the impartment

tools in the diagnosis and classification of lipemia. High blood cholesterol is

one of the major risk factors for heart disease. Commercial use of enzymes is

limited by their relatively high cost. The major goal of this research is to

develop methods for the efficient immobilization of enzymes on organic and

inorganic supports.

The current work aims at immobilizing urease and co-immobilizing

cholesterol esterase, cholesterol oxidase and peroxidase such that the

immobilized enzymes exhibited desirable activity to be made useful for

clinical applications. The specific objectives of the present work are:

i) Immobilise the clinically important enzyme, urease extracted

from jack bean onto different matrices such as, nylon beads,

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gelatin supported on photographic film, sepharose gel and

tosylated silica gel using different coupling agents.

ii) Effect co-immobilization of cholesterol esterase, cholesterol

oxidase and peroxidase on to nylon beads and gelatin film

through glutaraldyde and ascorbic acid coupling and also on

to porcelain beads using physical adsorption.

iii) Compare the kinetic properties and activity of immobilized

enzymes with those of the free enzyme.

iv) Compare pH, thermal and storage stability of immobilized

urease and co-immobilized cholesterol esterase.

v) Evaluate the performance of the immobilized urease and

co-immobilized cholesterol esterase in the estimation of

blood urea and total cholesterol, respectively, and compare

the efficiency of the same with that of the commercially

available urea and cholesterol test kits.