recent approaches for therapeutic enzymes - immobilization...

Vol - 4, Issue - 3, Apr-Jul 2013 ISSN: 0976-7908 Pawar et al

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PHARMA SCIENCE MONITOR

AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

RECENT APPROACHES FOR THERAPEUTIC ENZYMES -

IMMOBILIZATION AND APPLICATIONS: A REVIEW

Sonal A Pawar*1, Varsha B Pokharkar2, Nikunj R Solanki1 1Vidyabharti Trust College of Pharmacy, Umrakh, Bardoli-394345, Surat 2Bharati Vidyapeeth’s Deemed University, Poona College of Pharmacy, Erandwane, Pune-411038.

ABSTRACT Enzyme immobilization is undergoing an important transition since 1990s, hence immobilized proteins and enzymes have been widely used in processing of variety of products and increasingly used in the field of medicine. The review highlights on the evolution of the use of enzymes in therapy to the currently existing immobilized enzymes, along with the different matrixes used for the immobilization. It not only focuses on the therapeutic applications of the immobilized enzymes but also suggests a possible line of treatment for diseases like cancer, thromboembolic and certain genetic disorders. Keywords: enzyme immobilization, therapeutic applications, matrices. INTRODUCTION

Enzymes are biological catalysts consisting of proteins/ glycoproteins, which participate,

in numerous chemical reactions in living systems. They exhibit remarkable substrate

specificity and high efficiency due to which side reactions and by products are

eliminated. Also enzymes being biomolecules do not present disposable problems, as

they are biodegradable.

The stability and activity of enzymes can be improved when they are immobilized or

entrapped on water insoluble solid matrices and make them insoluble in aqueous media.

This process is referred to as “Enzyme Immobilization” which increases their efficacy as

well as their use in continuous processes where the product can be separated from the

reaction mixture and can be reused. Thus, this concept finds wide application in food

industry, brewing, pharmaceutical, textiles, medicines, detergents[1].

Enzymes as Therapeutic Agents:

The study of enzymes has immense practical importance. In some diseases, especially

inheritable genetic disorders, there may be a deficiency or a total absence of one or more

enzymes. Measurements of the activities of the enzymes in blood plasma, erythrocytes or



tissue samples are important in diagnosing certain illnesses (Fig 1). Many drugs exert

their biological effects through interactions with enzymes.[2]

Detailed understanding of enzymatic reactions, substrate specificity and their respective

role in integrated physiologic processes has allowed the development of therapeutic

applications that draw a unique, highly effective and specific catalytic function of the

enzyme. Traditional drug molecules which are designed as receptor agonist or antagonist

can provide some of the effects of enzyme therapy but lacking catalytic function they are

less efficient in mediating cascade events.

Figure 1: Therapeutic enzymes in different conditions

SCID

Gaucher’s

Thrombolytics

Infectious

Farby

Liver disorders

Digestive disorders

Phenyl ketonurea

Kidney

THERAPEUTIC

Cancer

Genetic Diseases

Pompe’s

Phenyl ketonurea



The novel facets of recombinant DNA technology and PEGylation technology are

proving useful to minimize the problems of enzyme based therapy and have boosted the

development of novel therapeutics worldwide. Enzymes as drugs have two important

features that distinguish them from all other types of drugs. Due to their specificity and

great substrate affinity they convert multiple target molecules to desired products. These

features make enzymes, as potent drugs that can accomplish therapeutic biochemistry in

the body resulting into the development of many enzyme drugs in a wide range of

disorders. Owing to this, the various therapeutic enzymes have been put into practice

(Table 1). However, the major hurdles of enzyme therapy are the development of

immune reaction by the host and reduction in efficacy of the enzyme due to repeated

administration but enzyme immobilization seems promising to minimize these problems[]

Table 1: Examples of some Therapeutic Enzymes and their Market Status.

Sr.No. Category Examples/Generic name

Trade name Potential Treatment

1. Pancreatic Enzymes

Lipases TheraCLEC-Total Steatorrhea

Proteases (Trypsin) TheraCLEC-Total Pancreatitis Amylases TheraCLEC-Total Lactose

intolerance 2. Thromobolyt

ic Enzymes Streptokinase, Myocardial

infarction, ischeamic necrosis

Urokinase, - (Alteplase)Tissue

plasminogen activator

Activase -

3. Oncolytic Enzymes

L-asparaginase Oncoaspar Acute lymphocytic leukaemia

Glutaminase, leukaemia Trans glutaminase leukaemia PEGylated arginine

deaminase Melanocid Invasive

malignant melanoma

PEGylated arginine deaminase

Hepacid Hepatocellular Carcinoma

4. Kidney disorders

Ureases



Uricases Rasburicase (Elitek) PEG-uricase (Puricase)

Gout

5. Antidiabetic Enzymes

Glucokinase Diabetes

Glucose-6-Phosphate, - Glycogen synthase - - 6. Liver

Enzymes Alcohol dehydrogenase

Liver cirrhosis

Alkaline phosphatases,

-

Catalases (superoxide dismutase)

Amytrophic lateral sclerosis

Serum transaminases - 7. DNAse

Enzyme therapy

Dornase- α Pulmozyme Cystic fibrosis

Adenosine deaminase Adagen Severe combined immuno deficiency syndrome

Ribonuclease RNA hydrolysis 8. Antibiotic Lysozyme Bacterial

infections Others Phenylalanine

Hydroxylase Phenylase Phenylketonuria

Hyaluronidase Heart attack Gluco

cerebrosidase(β - Glucosidase)

Ceredase Gaucher’s disease

α-Glucosidase - Pompe disease

PEG-glucocerebrosidase

Lysodase Gaucher’s disease

Agalsidase-β Fabrazyme Farby’s Disease α-Galactosidase A CC-galactosidase Farby’s Disease

Laronidase Aldurazyme MPS I Vianain Ananain, Cosmosain Enzymatic

debridement of severe burns.

Collagenase - Skin ulcers

β- Lactamase - Penicillin allergy



Enzyme applications include dissolving of blood clots (streptokinase), to promote

reperfusion and enhancement of cytotoxicity in cancer cells (L-asparaginase). Lysozyme

is an antibacterial agent which posseses activity against HIV. Similarly, cystic fibrosis is

a life threatening disease caused by a dysfunctional transmembrane regulator, CFTR

protein which modulates transport of salt and water leading to thick mucus secretions to

accumulate in the respiratory airways causing respiratory failure (Dornase- α). Thus

enzymes are important practical tools in medicine.

The manufacture or processing of enzymes for use as drugs is a minor but important

aspect of today's pharmaceutical industry. Attempts to capitalize on the advantages of

enzymes as drugs are now being made at virtually every pharmaceutical research center

in the world.

Rationale of Enzyme Immobilization:

Several techniques have been used to immobilize the enzymes like cross linking, physical

adsorption, ionic binding, metal binding, covalent binding and entrapment methods like

gel entrapment, fiber entrapment, microencapsulation for insoluble enzymes, while ultra

filtration membranes and hollow fiber devices are used for soluble enzymes. The choice

of method essentially depends on the nature of the enzyme and the kind of its application.

A matrix judiciously chosen can enhance the operational stability of the immobilized

enzyme system. Although it is recognized that there is no universal carrier, there are

number of characteristics which should be common to any material considered for

immobilization[1] (Table 2).

Table 2: Classification of Enzyme Matrixes

Organic Natural polymers Synthetic polymers Polysaccharides Proteins Carbon

materials Polystyrene Polyacrylate

Cellulose Collagen Polymethacrylates Starch Gelatin Polyacrylamide Dextran Albumin Hydroxy

alkylmethacrylates Agar/agarose Silk Maleic anhydride

polymers Chitin/chitosan



Vinyl and allyl polymers

Carrageenan Inorganic Minerals Fabricated materials Attapulgite clays Non porous glass Bentonite Controlled pore glass Keiselgur Controlled pore metal oxides Pumic stone Metals

SPECIALIZED CARRIER MATRIXES.

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.[4] 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).

1. Immobilized Cell Systems (Natural cells):

Immobilized cell (IC) technologies have developed since 1980s. Very briefly IC can be

divided into artificially and naturally occuring ones. In the former, microbial (eukaryotic)

cells are entrapped/ attached onto various matrices where they keep or not in a viable

state depending on the immobilization procedure. Polysacccharide matrices particularly

calcium alginate hydrogels used which are harmless for cell entrapment. The covalent

binding method of immobilization is generally incompatible with the cell viability. While

the spontaneous adsorption of the microbial cells to different types of carrier gives

natural IC systems in which the cells are attached to their carriers by weak, non-covalent

or electrostatic interactions. In suitable environmental conditions, the initial adsorption

may be followed by the colonization of the support leading to the formation of a biofilm

in which micro-organisms are entrapped within the matrix of extracellular polymer

secreted by themselves. Owing to the presence of this polymer paste, the biofilms are



more firmly attached to the substratum than merely adsorbed cells. The definite

importance of the biofilms in various areas of industrial and human health are recently

recognized. Main application fields of IC systems include; enzymes viz. lipases,

amylases, ribonucleases, L-glutaminases, inulases, penicillin V acylases, cellulolytic and

chitinolytic enzymes. Antibiotics viz. Penicillin G, ampicillin, candidicin, nikkomycin,

clavulanic acid , cephalosporin C5 etc.

The main advantages of whole cells over enzymes include avoidance of the extraction

and purification steps and their effects on enzyme activity, stability, cost, easier

downstream processing, enhanced operational and storage stability and reusability. Since

viable IC are able to multiply during substrate metabolism, high cell densities may be

expected in the cultures giving high volumetric reaction rates.[5]

2. Red Blood Cells (RBCs)/ Erythrocytes:

Human and animal RBCs have been used as a carrier vehicle for a number of exogeneous

enzyme drugs intended as a therapeutic approach. RBCs being biocompatible in nature

elicit very little or virtually no immune response thereby protecting the activity of the

encapsulated enzymes from rapid clearance and thus eliminating the toxic effects.

Another advantage being that RBCs can be readily obtained and a large quantities of

enzymes can be entrapped into rather small volume of the cell using certain techniques

like endocytosis, passive diffusion, electroporation and hypotonic dialysis etc. Among

these, hypotonic dialysis is widely used currently since it is simple and efficient

encapsulation of large amount of protein and it provides a carrier cell possessing the same

in-vivo half life as the normal cell.[6]

Application of erythrocytes, the most abundant cells of the human body with desirable

physiologic and morphologic characteristics, in drug delivery has been exploited

extensively. These cellular carriers, having remarkable biocompatibility,

biodegradability, and life-span in circulation, can be loaded by a wide spectrum of

compounds of therapeutic value using different chemically, as well as physically, based

methods. Most of the characteristics of the erythrocytes, including shape, membrane

fragility, deformability, and hematologic indices undergo some degree of irreversible

changes during the loading procedure. The efflux pattern of the encapsulated compounds

from the carrier erythrocytes covers a wide range between a relatively rapid release



(complete release within a few hours) and no detectable release until the cell lysis. A

series of methods have been tested successfully for improvement of in vitro storability of

the carrier erythrocytes without any significant changes in cell biology as well as drug

delivery efficacy. Carrier erythrocytes have been exploited for several potential

applications, including intravenous slow release of therapeutic agents, enzyme therapy,

drug targeting to reticuloendothelial system (RES)[6], improvement of oxygen delivery to

tissues, and preparation of fused cells.[7] Red blood cell substitutes based on modified

hemoglobin are already in Phase 3 clinical trials in patients.[8]

Ingo Gottschalk et al (2002) studied the non-specific interactions of the solute with

immobilized biomembranes using chromatographic methods. Liposomes,

proteoliposomes, RBC membrane vesicles were immobilized by freeze-thawing

procedure, whereas whole RBCs were adsorbed in the gel beads using electrostatic

interactions, binding to wheat-germ agglutinin (WGA) or the streptavidin-biotin

interaction. Super porous agarose gel coupled with WGA was the most promising matrix

for RBC adsorption and allowed frontal chromatographic analyses of the cells for one

week. Dissociation constants for binding of cytochalasin B and glucose to glucose

transporter GLUT1 were determined under equilibrium conditions. The number of

cytochalasin B binding sites per glut 1monomer was calculated and compared to the

corresponding results measured on the free and immobilized vesicles and GLUT

1proteoliposomes allowing the proteins binding state invivo and invitro. They mainly

aimed at improving the cell adsorption stability and capacity.[9]

3. Artificial Cells (Microencapsulation):

Microencapsulation is a procedure by which enzymes, genes or even whole cells within

microscopic, semipermeable containers. The cell can be thought of as a naturally

occuring microcapsule in which enzymes and organells are contained within the plasma

membrane. Synthetic semipermeable microcapsules sometimes referred to as ‘artificial

cells’ are designed to retain artificial materials while allowing permeant molecules to

cross the membrane. The permeability of the membrane can be varied using different

types of synthetic or biological materials like ultrathin synthetic membranes. The large

surface area of the microcapsules allows the permeant substrates and products to diffuse

rapidly. The numerous variations in content, permeability and size of the microcapsules



make them a versatile tool adapted to treat wide range of diseases. A further advantage is

that the microcapsules are prevented from the immune rejection because the leukocytes

and antibodies cannot penetrate the capsule. This allows even the allogenic or the

xenogenic cells to be implanted into the organism. The encapsulated cells are supported

by external oxygen and nutrients and their secreted products can diffuse out of the

microcapsules to carry out their functions. However, the microcapsule causes

complement activation after the implantation the breakdown products may be small

enough to enter the microcapsules and damage the enclosed cells. Also recognition of the

microcapsules as foreign bodies leads to becoming coated with fibrous tissues resulting

into decreased mass transfer of oxygen and eventual death of encapsulated cells. [10]

The basic principles of artificial cells, encapsulation and immobilization form the basis of

a number of bioartificial organs. Hemoperfusion based on encapsulated adsorbent has

been in routine clinical uses for many years to remove toxins or drugs from the circulating

blood. Enzyme therapy using microencapsulated enzymes have been studied in animal

studies and in a preliminary human study. Encapsulation or other ways of immobilization

of cells are being developed extensively by many groups. This includes the encapsulation

or immobilization of islets, hepatocytes and genetically engineered cells. [11]

Artificial cells are prepared in the laboratory for medical and biotechnological

applications. Artificial cells containing enzymes are being developed for clinical trial in

hereditary enzyme deficiency disease and other diseases. They are also being investigated

for drug delivery and for use in other applications in biotechnology, chemical

engineering, and medicine.[8] Similarly, encapsulated cells are also being studied for the

treatment of diabetes, liver failure, and other conditions. More recently, there have been

extensive studies into the use of encapsulated genetically engineered cells for gene

therapy. It was recently found that daily orally administered artificial cells, each

containing a genetically engineered microorganism, can lower the elevated urea level in

uremic rats to normal levels. This may solve the final obstacle of the lack of an effective

oral urea removal system for the simple and inexpensive oral treatment of uremia. This is

important because 85% of the world's uremic population cannot afford standard dialysis.

Other areas of artificial cell application include use in hemoperfusion, blood substitutes,

cancer therapy, treatment of inborn errors of metabolism and endocrine disorders.[11]



4. Liposomes:

Liposomes can also act as enzyme carriers due to their ability to reduce toxicity and

enhance the efficacy of the encapsulated enzymes. But their therapeutic applicability has

been limited owing to their rapid clearance from the bloodstream and their uptake by the

macrophage cells in the liver and spleen. Recently the liposomes with the ability to evade

the rapid uptake by the reticuloendothelial system have been developed and the

significant improvement in the blood half-life has been achieved using liposomes whose

surfaces have been modified by the polyethylene glycol (PEG). Since the liposomes

exhibit dramatically different pharmacokinetics and biodistribution properties they

present a new avenue with regard to therapeutic applications.

Shaoqing et al (2003) prepared liposomes containing glucose oxidase by entrapping the

glucose oxidase in the liposomes (GOL) composed of phosphatidyl choline, dimyristoyl

L-α-phosphatidylethanolamine and cholesterol and then covalently immobilized in the

glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads were

characterized to obtain a highly stable biocatalyst applicable to bioreactor. Finally they

compared it to the conventionally immobilized glucose oxidase (IGO) , the higher

operational stability of the IGOL was verified by either using it repeatedly (4 times) or

for a long time (7days) to catalyze glucose oxidation in a small-scale airlift bioreactor. [12]

4. Cross Linked Enzyme Aggregates:

Though immobilization circumvents the problem of enzyme activity and stability and

improves the economy by the reuse of the biocatalyst, it is costly and requires the use of

an inert matrix for immobilization. Cross-linked enzyme crystal (CLEC) technology

provides a unique approach to ammeliorating the disadvantages of immobilization.

Cross-linking of enzymes results in both stabilization and immobilization of the enzyme

without dilution of enzyme activity since the interaction between the enzyme and matrix

leads to dilution incase of immobilization. The advantages of CLECs over

immobilization are that they have a higher activity per unit volume, they can withstand

high shear forces and high mixing speeds associated with stirred tanks, they can be

readily isolated, recycled and reused many times. The high operational stability allows

the reaction at high temperatures in aqueous organic mixtures thus increasing substrate

solubility. Similarly the intermolecular contacts between the enzymes in the crystal-



lattice of the CLEC stabilize the enzyme and prevent the denaturation. They can be easily

freeze-dried/ air-dried and stored indefinitely at room temperature. Long shelf life solves

storage problems and eases the handling of enzymes as ordinary chemicals. CLECs have

many synthetic, biomedical and biosensor applications. Enzyme therapy such as ‘lipase

therapy’ can be performed by administering cross-linked lipase crystals orally. Also

CLEC- glucose oxidase test strips can be used as a diagnostic reagent to detect the level

of glucose in blood.[13, 14, 15] (Table 3).

Table3: Comparison between free, immobilized and cross-linked enzyme.

Sr. No. Character Soluble enzyme Immobilized Enzyme

Crosslinked enzyme

1. enzyme purity enzyme of any purity

even crude enzyme can be immobilized

only pure enzymes can be used

2. Stability can be stored in concentrated form at refrigerated temperature

store at refrigerated temperature

higher stability due to cross-linking; can be stored at room temperature

3. specific activity

high specific activity

dilutes the activity due to the interaction with the matrix

high specific activity due to high volumetric activity

4. reaction in aq/org

only in aqueous media

react in both aqueous and less in organic media

react in both aqueous and organic media

5. separation from the reaction mixture

difficult to separate from reaction mixture

can be separated by filtration or centrifugation

easily separated by filtration or centrifugation

6. pH and thermal stability

not stable over a range of pH and temperature

not stable over a range of pH and temperature

stable over a range of pH and temperature

7. Productivity Low productivity

High productivity

Very High productivity

5. Microcapsules/ Nanoparticles.

One of the approaches of enzyme immobilization is based on entrapment of enzymes in

microparticles or nanoparticles. Microcapsules of calcium alginate coated with a



polycation have been investigated for applications like immunoprotective containers in

cell transplantation, enzyme immobilization and drug release systems. [16] As Chang and

Prakash 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.[17]

Esqueisabel et al (1999) have prepared microcapsules using calcium alginate as core

material and three different polycations viz. Quitosan (QUI), poly-L-lysine(PLL),

polymethylene-co-guanidine. They assayed the ability of these microcapsules to remove

urea in vitro in freshly prepared microcapsules, in freeze-dried microcapsules and in

microcapsules exposed to pancreatic enzymes and can effectively remove urea within 60

mins.[18]

Silva et al and colleagues (2004) prepared chitosan- coated alginate microspheres by

emulsification/ internal gelation chosen as model carriers for model protein, haemoglobin

(Hb) due to non toxicity of the polymers and mild conditions of the method. The

influence of the process variables related to the emulsification step, microsphere

recovering and formulation variables such as alginate gelation and chitosan coating on

the size distribution and encapsulation efficiency were studied. The effect of

microspheres on the coating as well as its drying procedure on the release profile were

also evaluated. Chitosan coating process was achieved by a continuous

microencapsulation procedure or a two stage coating process. All microspheres showed

encapsulation efficiency above 90% and calcium alginate crosslinking was optimal by

using acid/ calcium carbonate molar ratio of 2.5 while microsphere recovery in acetate

buffer lead to higher encapsulation efficiency. Hb release in gastric fluid was minimal for

air-dried microspheres. Coating effect revealed total release of 27% for 2 sage coated

microspheres while other formulations showed Hb release above 50%. Lyophilized

microspheres behaved similar to wet spheres although higher total protein content was

obtained with 2 stage coating. At pH 6.8 uncoated microsphere dissolved in less than an

hour however Hb release from air dried spheres was incomplete. Chitosan coating



decreased the Hb release rate. 2 stage coating process showed no burst effect while one

stage process permitted higher release.[19]

6. Encapsulation of enzymes in sol-gel:

Sol-gel is a chemical synthesis technology utilized in preparing gels. Synthesis of

materials by sol-gel process generally involves the use of metal alkoxides which undergo

hydrolysis and condensation polymerization reactions to produce the gel. The beauty of

this system is that the activity and the function of the enzymes and proteins can be

retained when they are being confined within the pores of sol-gel matrix. Furthermore,

the porosity of the sol-gel allows small molecules to be diffused in whereas the large

enzyme macromolecule remains physically entrapped in the matrix. Other advantages

include: relatively uniform pore size and distribution, their thermal stability, controlled

surface area, ability to enhance the stability of the encapsulated enzymes and finally they

can prevent leaching of the entrapped enzyme.

Reetz et al (1995) have presented a novel method top achieve lipase immobilization by

entrapment in chemically inert silica gels which are prepared by the hydrolysis of alkyl

substituted silanes in the presence of the enzyme. They used aqueous lipase solution ,

sodium fluoride as a catalyst polyvinyl alcohol or proteins as additives and alkoxy silane

derivatives like RSi(OMe)3 with R= alkyl, aryl or alkoxy as gel precursors. They studied

the various immobilization parameters like stoichoimetric ratio of water , silane, type and

amount of additive, amount of catalyst etc. This new method is applicable to wide variety

of lipases yielding immobilized lipases with esterification activities enhanced by a factor

of up to 88. They showed that the sol-gel entrapped lipases are highly stable and can be

stored at room temperature for months without significant loss of activity.[20]

Hsu et al (2002) have immobilized Lipase PS (Pseudomonas cepacia) and Lipase

F(Rhizopus oryzae) within a phyllosilicate matrix, which catalyzed the esterification of

glycerol with short medium and long chain fatty acids to produce mono (MAG), di

(DAG), tri (TAG) acylglycerols. They compared the results from the above esterification

reactions to the reactions using commercially available immobilized lipase, Lipozyme

IM-60. Time course studies showed that free Lipase PS 30 or Lipase F enhanced

esterification reactions with the use of silica supported glycerol. In contrast the

immobilized Lipase PS-30 catalysed reactions occurred at the same conversion rate when



using either free or silica supported glycerol. For immobilized Lipase F and Lipozyme

IM-60 reactions the use of silica supported glycerol favored the production of DAG and

TAG over MAG. All three lipases could be reused for acylglycerol production.[21]

Categories of Therapeutic Enzymes:

1. Digestive Enzymes:

• Pancreatic Enzymes:

Pancreatic juice contains

1. triglyceride digesting enzymes: pancreatic lipases

2. Protein digesting enzymes: trypsin, chymotrypsin, carboxypeptidase, elastase

3. Carbohydrate digesting enzymes: pancreatic amylases.

Lipases:

Lipases (triacylglycerol ester hydrolase) are very relevant enzymes from both

physiological and biotechnological point of view. They are applied in organic solvents

and therefore various methods of their stabilization including immobilization have been

established. It is well known that lipases have been used in steatorrhea , pancreatic

insufficiency, chronic alcoholic pancreatitis, cystic fibrosis, coronary atherosclerosis,

hyperlipidemia, familial combined hyperlipidemia (FCHL) etc.[22] For these patients,

pancreatic enzyme preparations of porcine or bovine origin have been available in the

United States for the treatment of exocrine pancreatic insufficiency (EPI) in children and

adults with cystic fibrosis and chronic pancreatitis since before the enactment of the

Federal Food, Drug, and Cosmetic Act of 1938 (the Act). [5]

Most of the reports on lipase are focused on the use of the biocatalyst for industrial

applications.

Rosa.M.Blanco et al. (2004) have utilized lipase from C.antartica which was

immobilized on activated mesoporous silica in a monolayer avoiding the formation of

enzyme aggregates. The monolayer enzyme loading onto this support was as high as

200mg protein/gm. The strong interaction of the enzyme with the hydrophobic groups

from the support contributed to decrease the mobility of the immobilized protein and thus

to increase its thermal and operational stability.[23]

Pablo Dominguez et al.(2004) have explained the catalytic activity of crude lipases from

Candida rugosa, free or immobilized, in microemulsion based organogels gelled with



hydroxy propyl methyl cellulose (HPMC) as biopolymer and lecithin as surfactant.

Lipase factor obtained from initial reaction rate profile is a useful parameter to explain

the catalytic activity of this crude enzyme. Esterification of fatty acids in organic media

and LF values can be used to evaluate the amount of lipase in different crude enzymes.[24]

Desai et al.(2004) have demonstrated the immobilization of porcine pancreas lipase by

entrapment in the beads of κ-carrageenan beads which is found to be superior to the free

enzyme under all conditions tested for enzyme efficacy and stability. This results into

32mg/gm enzyme on dry weight of κ -carrageenan. The enzyme is found to retain 50% of

its activity after repeated use of 5 cycles. The Michaelis constant (Km) and maximum

reaction velocity (Vmax) of free and immobilized enzyme were almost same indicating

that there is no conformational change during immobilization.[25]

Palomo et al (2005) immobilized lipase from Pseudomonas cepacia on glyoxyl-agarose

and the active site was blocked after incubation with diethyl-p-nitrophenylphosphate

obtaining a new “glyoxyl-BCL” matrix to adsorb lipases .Then soluble lipase was

adsorbed selectively on this matrix at very low ionic strength. This lipase-lipase

interactions could be neglected with the use of Triton X-100 as detergent. Moreover the

close contact between the adsorbed lipase and immobilized lipase permitted to alter the

catalytic and functional properties of this lipase like the enantioselectivity of the BCL

adsorbed on the glyoxyl-BCL varied its E value from 10 at pH 7, 250C up to greater than

100 at pH 5, 250C in the hydrolytic resolution of (+_)-2-hydroxy-4-phenylbutyric acid

ethyl ester. They also reported that dimeric form of lipase is more stable than the

monomeric and the stabilized open conformation of lipase is more stable than even the

multipoint covalently attached lipase. The lipase adsorbed on this matrix showed 100%

activity even after 60h whereas 50% activity of the multipoint covalently attached

preparation was lost in around 40h.[26]

Dumitriu et al (2001) immobilized lipases noncovalently on Chitoxan, a polyionic

hydrogel obtained by complexation between chitosan and xanthan. They compared the

properties of free and immobilized lipases. In the aqueous medium the activity was twice

as high as for immobilized lipases as for free lipases. Immobilized lipases in chitoxan

were able to hydrolyze triacylglycerols in three distinct organic solvent media viz.

toulene, iso-octane and cyclohexane. Also, they showed that at microstructural level,



lipases were not distributed uniformly in the chitoxan beads. Higher concentration was

found in the outer membrane like layer of the beads.[27]

Fadnavis et al (1999) observed an unusual phenomenon for gelatin solutions (1.7-6.8%)

in the microemulsion system of 0.3M bis(2-ethylhexyl) sulfosuccinate sodium salt in

isooctane and 14.5% distilled water. Highly viscous gels obtained at temperatures

above300C become free-flowing liquids at low temperatures(5-10OC) .This reversible

temperature dependent sol-gel transition phenomenon is used to immobilize several

enzymes like lipase from Candida rugosa, alcohol dehydrogenase from baker’s yeast,

mandelonitrile lyase from Sorghum bicolor and horseradish peroxidase in gelatin matrix

by solubilizing the enzyme in the microemulsion based gelatin solution at low

temperature (<5oC) and then crosslinking with glutaraldehyde. The enzymes retain 70-

80% activity after immobilization and can be used in biotransformations in organic

solvents without any changes in enantioselectivity. This work provides a unique low

temperature technique for enzyme immobilization in a biocompatible gelatin matrix with

a great flexibility of size and shape.[28]

Similar kind of experiments were carried out using lipases obtained from different

sources like Candida rugosa, Pseudomonas cepacia, Rhizopus oryzae, and were

immobilized on different novel matrices like solid carriers (derivatives of cellulose,

diatomaceous earth , modified porous glass)[29],Accurel EP-100 (IM-PS) [30] or

microporous polypropylene supports [31] , Polyphenyl acetylene (PPA)[32], Poly

(acrylonitrile co-maleic acid)(PANCMA) [33], (ultrahollow fiber membranes)[34], silica

gel.[35, 36] or silanized[37]controlled pore silica previously activated with glutaraldehyde.

Parameters like activity, hydrolytic activity, enzyme adsorption capacity, thermal stability

and kinetic constant like Km and Vmax were assayed for free and immobilized.

Thus, lipases can be used industrially as well as therapeutic enzymes if immobilized and

administered could be of significant importance to the above mentioned disorders.

Proteases:

Microbial proteases are among the most hydrolytic enzymes and have been studied

extensively in enzymology. The extracellular proteases are of commercial value, many

companies are constantly trying to use the proteases directly or to create modified

enzymes which have catalytic activity.



Hayashi et al (2006) prepared water-insoluble proteases by immobilizing papain, ficin,

and bromelain onto the surface of porous chitosan beads with any length of spacer by

covalently fixation. The activity of the immobilized proteases was found to be still high

toward small ester substrate, N-benzyl-L-arginine ethyl ester (BAEE), but rather low

toward casein, a high-molecular-weight substrate. The relative activity of the

immobilized proteases with spacer gave an almost constant value for the substrate

hydrolysis within the surface concentration region studied. The values of the Michaelis

constant Km and the maximum reaction velocity Vm for free and immobilized proteases on

the porous chitosan beads are estimated. The apparent Km values were larger for

immobilized proteases than for the free ones, while Vm values were smaller for the

immobilized proteases. The pH, thermal, and storage stability of the immobilized

proteases were higher than those of the free ones. The initial enzymatic activity of the

immobilized protease maintained almost unchanged without any elimination and

inactivation of proteases, when the batch enzyme reaction was performed repeatedly,

indicating the excellent durability.[38]

Elibol et al. (2002) did immobilization of cells of Teredinobactor turnirae in calcium

alginate beads used for alkaline protease production. Maximum proteolytic activity was

obtained at 3% sodium alginate and 3% CaCl2 concentration with a 1:2 cell:alginate

ratio.(approx. 2400 U/ml).Similarly a drastic fall in protease production was observed

when the cells were treated with gluteraldehyde. The beads were used for 8 successive

fermentation batches each lasting 72 hrs.[39]

Zaghloul et al.(2001) have worked on the expression and stability of the cloned Bacillus

subtilis alkaline protease (aprE) gene was monitored through the growth of free and

alginate immobilized β-subtilis cells. Time and level of expression of aprE gene in

alginate immobilized cells were found to be close to that of free cells. The multicopy

plasmid that carries the aprE gene was stably maintained in alginate immobilized cells.

Plasmid stability was greatly enhanced, this effect was observed with cell immobilization

matrices such as carrageenan, alginate, silicon polymer and gelatin beads.[40]

Tanksale et al. (2001) have demonstrated the immobilization of alkaline protease from

Conidiobolus macrosporous on polyamide using glutaraldehyde as a bifunctional agent.

The immobilized enzyme was optimally active at 50oC and free enzyme at 40oC and



showed a ten fold increased thermal stability at 60 oC. The efficiency of immobilization

was 58% under optimal conditions of temperature and pH. The immobilized enzyme was

fully active even after 22 cycles of repeated use and retained 80% activity at 50 oC in

presence of 8M urea.[41]

Johanna Mansfeld et al. (2001) have focused on different phenomena of immobilization

suggesting that immobilization of proteins usually leads to a random orientation of the

molecules from the surface of the carrier material. Recently, several mutant enzyme of

the thermo lysine like neutral protease from Bacillus stearothermophillus containing

cysteine residues in different positions on surface of the protein molecule. The basic

matrix was Sepharose 4 B in all the carriers. The enzyme bound site specifically to

activated thiol sepharose showed first order inactivation kinetics. Immobilization to a

highly functionalized carrier via amino groups increased stability suggesting that multiple

fixation outside of the unfolding region 56-65 is able to increase the stability of enzyme

molecules.[42,43]

Enterokinase:

Eun Kyu Lee et al.(2005) have covalently immobilized recombinant enterokinase as the

model proteolytic enzyme on glyoxyl agarose and evaluated in terms of immobilization

yield, activity and the cleavage performance of the immobilized enzyme.The specific

activity was only 20-30% of that of the soluble enzyme at the various pH conditions but

the cleavage rate by the covalenlty immobilized enterokinase was higher than that of the

soluble enzyme at various pH conditions and undesirable side reaction i.e. cryptic

cleavage was significantly reduced. In order to reuse the immobilized enterokinase

repeatedly solid phase refolding of immobilized enterokinase was attempted. The

covalently immobilized enterokinase showed almost 100% refolding yield whereas the

soluble enterokinase showed only 36% yield. It was confirmed then only covalent

conjugation maintained the rigid ‘reference structure’ during a denaturant - induced

unfolding step which could provide a more efficient route to refolding in the subsequent

renaturation step.[44]

Trypsin/ Chymotrypsin:

Trypsin has many industrial applications, and it is a very important enzyme in the food

industry. However, the cost of the enzyme is so high, in addition to its sensitivity to



reaction conditions. Immobilizing the enzyme can make the trypsin stable and reusable.

Among the various immobilization methods, covalent binding of enzymes to water-

insoluble carriers seems to be the most attractive method for enzyme stabilization,

recovery and reuse.

Kang et al. (2006) produced poly(methyl methacrylate-ethyl acrylate-acrylic acid) latex

particles with narrow size distribution and with surface carboxyl groups by soap-free

emulsion polymerization, and covalent immobilization of trypsin onto these particles was

carried out by using the water-soluble carbodiimide (EDC) as an activating agent under

various conditions. Different immobilization methods were employed and the factors

affecting the efficiency and activity of the immobilized enzyme, such as the amount of

trypsin and EDC, pH and temperature of the immobilization reaction were investigated.

Results showed that both relatively high immobilization efficiency and high enzyme

activity were achieved when pre-adsorption method was employed. The immobilization

efficiency decreased as the trypsin amount increased, and increased as pH and

temperature increased. When the EDC amount varied, the immobilization efficiency first

increased significantly and then decreased slowly. A maximum of enzyme activity can be

obtained at the optimum value of 958.0 mg trypsin/g dried particles and 372.5 mg EDC/g

dried particles at 25 °C and pH 5.0. The immobilized trypsin exhibited much higher

relative activity than its free

counterpart. [45]

Jang S et al.(2006) investigated the effects of pore size, structure, and surface

functionalization of mesoporous silica on the catalytic activity of the supported enzyme,

trypsin. For this purpose, SBA-15 with 1-dimensional pore arrangement and cubic Ia3d

mesoporous silica with 3-dimensional pores were prepared and tested as a support.

Materials with varying pore diameters in the range 5–10 nm were synthesized using a

non-ionic block copolymer by controlling the synthesis temperature. Thiol-group was

introduced to the porous materials via siloxypropane tethering either by post synthesis

grafting or by direct synthesis. These materials were characterized using XRD, SEM,

TEM, N2 adsorption, and elemental analysis. Trypsin-supported on the solids prepared

was active and stable for hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide

(BAPNA). Without applying thiol-functionalization, cubic Ia3d mesoporous silica with



ca. 5.4 nm average pore diameter was found to be superior to SBA-15 for trypsin

immobilization and showed a better catalytic performance. However, enzyme

immobilized on the 5% thiol-functionalized SBA-15 prepared by directly synthesis was

found to be the most promising and was also found recyclable.[46]

E.Magner et al.(2005) studied the immobilization of hydrolytic enzyme trypsin onto

various mesoporous silicates(MPS).They prepared MPS by using cationic surfactants

having average pore diameters in the range of 28-300 A0.They found that enzyme purity

strongly influenced loading trypsin adsorbed on MPS was found to be desorbed more

readily by polyethylene glycol than by ammonium sulphate suggesting that hydrophobic-

hydrophillic interactions were important. Immobilized trypsin showed 10-20 times more

activity and stability(for 4-6 weeks at 40 or 250 ) and was successfully used upto 6

cycles.[47]

Trypsin has been immobilized on supports like controlled pore glass beads (CPG) with

glutaraldehyde as activating reagent[48], microporous membranes[49], water-soluble acrylic

polymer[50] with spacer arms bearing benzamidine groups, and was synthesized as a

polymeric inhibitor to protect trypsin during immobilization of this enzyme on two

different water-insoluble carriers, i.e., VINAC-S and ACAPROSUC. This method of

enzyme protection should prove very useful in immobilized enzyme methodology and

technology. Similarly, Covalent attachment of enzymes and other proteins to the smart

polymer, poly(N-isopropylacrylamide) [poly (NIPAAm)],[51]has been widely used as a

method for the preparation of thermosensitive protein conjugates.

The immobilization of α- chymotrypsin in κ-carageenan beads prepared with the static

mixer was demonstrated by Evgeniya Beryaeva et al.(2004) .The beads were obtained by

emulsification or thermal gelation with sunflower oil at ambient temperature as a

continuous phase using Sulzer SMX static mixer. The mean sauter bead diameter was

300 micrometer. α- Chymotrypsin encapsulation efficiency was increased two times by

preliminary enzyme cross linking by glutaraldehyde. The stability upon storage was

higher for beads containing cross linked α- chymotrypsin.[52]

Amylases:



Min-yun Chang et al. (2005) have narrated the comparison of thermal and pH stabilities

of free and immobilized α-amylase, β-amylase and glucoamylase in which

immobilization support was prepared by equal weights of chitosan and activated clay

which were cross linked with glutaraldehyde.The relative activities of the immobilized

enzyme are higher than free over a broad pH and temperature ranges. α-amylase and

gluco amylase immobilized on composite beads maintained 81% of the original activities

after 50 times of repeated use. It was seen that Km and Vmax of immobilized enzymes

are greater that those of free enzymes except for Vmax of glucoamylase.[53]

Ipsita Roy et al.(2004) have carried out hydrolysis of starch by a mixture of glucoamylase

and pullulanase and trapped individually in Ca-alginate beads in the ratio of 3:2. The

individually entrapped enzyme showed enhanced thermal stability at 55oC. Glucoamylase

hydrolysis α-1,4 links less rapidly and also reduces the extent of hydrolysis in the

formation of reversion products at high glucose concentration which are linked by

resistant α-1,6 linkages. Hence, a debranching enzyme, pullulanase was used.The

mixture of both enzymes can be used in both packed and fluidized bed formats as they

have pH optima in the same range.[54]

Work on the similar lines using supports like porous membranes (poly(HEMA-GMA-1-

3) membranes from the UV- initiated photopolymerisation of hydroxyethylmethacrylate

(HEMA) and glycidyl methacrylate (GMA) [55]and hydrogels (Poly(ethylene glycol

dimethacrylate-n-vinyl imidazole) [poly(EGDMA-VIM)]56has been reported in the

literature

• β- Galactosidase:

M.Portaccio et al.(1998) have studied the inhibition of β-galactosidase obtained from

Aspergillus oryzae immobilized on chitosan beads or a nylon membrane

(immunodyne).The enzyme has been immobilized on two different carriers,one natural

and other artificial to study the effect of nature of support on the catalytic activity. In both

cases inhibition was found. The Ki value for β-galactosidase/chitosan system was higher

than one for β-galactosidase/immunodyne showing that the former system is more

appreciate to perform lactose hydrolysis.A new technology based on the use if non

isothermal bioreactors is suggested to overcome the inhibition problems.[57]



Dong Ho Ahn et al.(1997) constructed a recombinant plasmid (pBCBD) for the

immobilization of Cellulomonas fimi β-glucosidase (CBD) of Bacillus subtilis BSU616 -

endo-1,4-glucanase(Beg). The Cbg-CBDBeg fusion protein which was 80 Kda was

expressed in E.coli and was immobilized to Avicel.Cellobiose was completely

hydrolysed with the immobilized fusion protein which was fully active during continuous

operation for 24 hour at 40 C.[58]

Paul.R.Oswald et al (1998) immobilized a very stable β-glucosidase to polyacrylamide

magnetite beads ,aminopropyl silica and chitosan using tris(hydroxymethyl)

phosphine(THP)or glutaraldehyde as a coupling agent.The use of THP on chitosan

resulted in greater than 90% yields with respect to free enzyme activity and 60% using

glutaraldehyde.Repetitive assays of THP and glutaraldehyde immobilized enzyme also

showed that THP was more able to retain active enzyme on silica based support.[59]

Giovanni Spagna et al. (2002) immobilized several glycosidases (β-d-glucopyranosidase,

α-l-arabinofuranosidase, α-L-rhamnopyranosidase) purified from Aspergillus niger by

inclusion on chitosan gels and subsequent crosslinking with glutaraldehyde followed by

addition of various agents inorder to improve the gels’ physical and mechanical

properties to reduce enzyme release phenomena and to increase immobilization yields

and operational stability.Gelatin and silica gels proved to be the best additives.[60]

2. Oncolytic Enzymes:

The oncolytic enzymes fall into two major classes: those that degrade small molecules for

which neoplastic tissues have a requirement, and those that degrade macromolecules such

as membrane polysaccharides, structural and functional protein, or nucleic acids. At

present, tumor-cell specificity observed only in the former category.

Following are the examples of most common oncolytic enzymes.

L-asparginase:

An important discovery showed that when cells become cancerous then their

biochemistry is changed. Certain tumor cells are deficient in their ability to synthesize the

nonessential amino acid L-asparagine, and are forced to extract it from body fluids; by

contrast, most normal cells can produce their own L-asparagine. Asparaginase given



parenterally acts in this way in many susceptible tumors. Unfortunately, only acute

lymphocytic leukaemia ordinarily responds to chemotherapy with the enzyme.

Nevertheless, the response of this one tumor type is promising (60% incidence of

complete remissions in 6000 cases), The search is being extended to other enzymes that

degrade small molecules. A bi-functional amidohydrolase, L-glutaminase-L-asparaginase

(L-glutamine and L-asparagine amidohydrolase), is undergoing clinical trials in the

United Kingdom and shows activity in other diseases.

Yu-Qing Zhang et al.(2004) have worked on the immobilization of L-Asparginase on the

microparticles of silk sericin protein.The natural silk sericin recovered from Bombyx mori

is a macromolecular protein with different molecular mass from 50-200Kda was poorly

soluble microparticles with an average size of 10 micrometer Anti-leukemic enzyme L-

Asparginase was covalently conjugated on the microparticles of silk sericin protein.The

immobilized L-Asparginase with glutaraldehyde as cross linking agent to maintain 62.5%

of original enzyme.The Km of sericin conjugates was times lower than that of native L-

Asparginase.The bioconjugation of L-Asparginase widened the optimum reactive

temperature range of the enzyme. The immobilized L-Asparginase showed higher

stability when the temperature was raised to 40-50oC.It also showed preferable resistance

to trypsin digestion as with native enzyme.[61]

Karsakevich et al (1992) have developed methods for obtaining soluble and insoluble

dextran carbonates by adding them to E.Coli L-asparginase which have resulted into

several forms viz. water-soluble, gel-like and water-insoluble of immobilized enzymes

which show a greater anti-leukemic action than the native enzyme[62]

Transglutaminase:

Noriho kamiya et al. (2004) have investigated a novel strategy for site specific

immobilization of recombinant proteins using microbial transglutaminase (MTG) and

alkaline protease(AP) as a model protein and tagged with a short peptide (MKHKGS) at

the end terminal to provide a reactive lysine residue for MTG. On the other hand, casein a

well known substrate for MTG was chemically attached onto a polyacrylic resin to

provide glutaminase residues for the enzymatic immobilization of recombinant AP. They

succeded in MTG mediated functional immobilization of the recombinant AP onto casein

coated polyacrylic resin.It was found that immobilized AP prepared by using MTG



exhibitedmuch higher specific activity than prepared by chemical modification.

Moreover,enzyme immobilization gave an immobilized formulation with higher stability

upon repeated use that obtained by physical adsorption.Use of this ability of MTG in post

translational modification will provide us with a benign sites for site specific

immobilization method for functional proteins.[63]

Glutamate Dehydrogenase:

Helen.H.Petach et al.(1994) have compared the activity of GDH on a variety of

transperant chitosan derivatives which provide different chemical environments for the

immobilized GDH. The amino group of the chitosan was modified to produce

succinyl,glutaryl and pthalyl derivatives.Thus,changing the close association of enzyme

with the carbohydrate based polymer to a chain(of varying composition) liking the

enzyme with carbohydrate backbone.The chain linkage may have some effect in the

enzyme flexibility as the enzyme is further removed from the polymer backbone and the

composition of the chain may alter the diffusivity of substrate and products within the

films. They observed that increased flexibility provided by succinyl, glutaryl, pthalyl side

chain over chitosan itself did not enhance GDH activity.Chitosan provides flexible

backbone foe enzyme attachment and the side chain increase the enzymes.[64]

Glycosyltransferases:

Paul L. DeAngelis et al. (2003) used Pasteurella multocida HA synthase, pmHAS, a

polymerizing enzyme that normally elongates HA chains rapidly (1-100 sugars/s), was

converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid

transferase and N-acetylglucosamine transferase). The two resulting enzymes were

purified and immobilized individually onto solid supports. The chemoenzymatic

synthesis of a variety of monodisperse hyaluronan (4-glucuronic acid-3-N-

acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA

oligosaccharides (10-20 sugars in length) include killing cancerous tumors and enhancing

wound vascularization is described. The two types of enzyme reactors were used in an

alternating fashion to produce extremely pure sugar polymers of a single length (up to

HA20) in a controlled, stepwise fashion without purification of the intermediates. These

molecules are the longest, non-block, monodisperse synthetic oligosaccharides. This

technology platform is also amenable to the synthesis of medicant-tagged or radioactive



oligosaccharides for biomedical testing. Furthermore, these experiments with

immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-

processive and that a monomer of enzyme is the functional catalytic unit.[65]

Serum amine oxidase:

Nicole Demers et al. (2001) have demonstrated the immobilization of native and poly

(ethylene glycol) treated (PEGylated) bovine serum amine oxidase(BSAO) in to a

biocompatible hydrgel.The hydrogel was obtained by cross linking of BSA with PEG

dinitrophenyl carbonates with a molecular mass of 10 Kda.Approximately 60% of the

amino groups at the surface of BSAO were modified by monoethoxy PEG with a

molecular mass of 5 Kda when reaction was carried out for 5 hrs in borate buffer pH9.

The apparent Km values of both forms of enzyme were decreased due to

preconcentration of benzyl amine substrate by the negatively charged hydrogel.Vmax

values were generally lower upon immobilization. Thus, hydrogel swelling has no

significant effect on enzyme structure. The operational stability increased upon

immobilization. The enzymic hydrogels were stable during storage in solution at

4mC,maintaining a high activity even after several weeks. The BSA-PEG hydrogel is a

good matrix for immobilization of enzymes with therapeutic potential such as BSAO.The

immobilization yield of about 40% of BSAO in to the hydrogel was markedly

compansated for the increase in operational half like which reach 3 days. High shelf

stability of more than 100 days is another important characteristic observed with the

immobilized BSAO.

Future experiments will be focused on synthesis of BSAO hydrogel microparticles for

further investigation of its therapeutic potential in mice bearing tumours of various

human origin. Thus, BSAO will have a great potential as enzymotherapeutic agent for

cancer.[66]

3. Thrombolytic enzymes:

Thrombolytic enzymes play an crucial role in management of patients with acute

myocardial infarction, pulmonary embolism, deep vein thrombosis, acute thrombosis of

retinal vessel, extensive coronary emboli, peripheral vascular thromboembolism.

Reconition of the importance of fibrinolytic system in thrombus resolution has resulted in

development of different fibrinolytic agents such as Streptokinase, Staphylokinase,



Urokinase, Prourokinase, Tissue plasminogen activators like:Alteplase, Reteplase,

Tenecteplase, Lanoteplase, Monteplase etc. Streptokinase and urokinase are widely used

for treatment of AMI due to lower cost than t-PA.[67]

Streptokinase:

Koneracka et al. and co-workers (2002) have been immobilized several clinically

important proteins and enzymes (bovine serum albumine, glucose oxidase, streptokinase,

chymotrypsine and dispase) to fine magnetic particles by means of 1-[3-(dimethylamino)

propyl]-3-ethylcarbodiimide hydrochloride (CDI) as a coupling agent. The coupling

reactions of these substances were carried out under different sets of conditions (change

the pH of the reaction mixture in areas from 4.5 to 6.5 and proportion of magnetic

particles to proteins and CDI) to determine the optimum conditions of the

immobilization. The usefulness of the presented method is discussed for biomedical and

biotechnological applications.[68]

US Patent: 4,305,926 by Everse et al. (1981) have reported the immobilization of

streptokinase or on diazotised copolymer of para-amino phenyl alanin and leusine and

urokinase on nylon. Further studies were carried out invivo,in rabbits by subcutaneous

implantation. The invention comprises the construction of an implantable device

consisting of a clot lysing initiator (made up of streptokinase and urokinase) immobilized

on to a biocompatible polymer viz. diazotized copolymer of para amino phenyl amine

and leucine and nylon 66 which is partially hydrolysed and then coupled with the

enzymes and finally implanted subcutaneousaly.They observed that stability of

streptokinase greatly increased invivo and may be useful in cases where prolong therapy

is required.They reported the blood cloting time increased from one minute from three

minutes and remained stable at three minutes for 150 days.Also that no antibodies were

formed throughout the tenure and thus no adverse reactions were reported.Thus,they

concluded that immobilized streptokinase could successfully be used in treatment of

thromboembolic disorders which require prolong fibrinolytic therapy and they could be

valuable therapy in cases of anticipated thromboembolic problems.[69]

Fernandes et al. (2006) immobilized streptokinase on dendrimers to obtain fibrinolytic

surfaces. Dendrimers are monodisperse, spherical and hyperbranched synthetic

macromolecules with a large number of surface groups that have the potential to act as



carriers for drug immobilization by covalent binding or charge transfer complexation.

Here, a biconjugate of streptokinase and polyglycerol of generation 5 (PGLD). The

dendrimer structure was confirmed by gel permeation chromatography and NMR. The

blood compatibility of the bioconjugate PGLD-Sk was evaluated by in vitro assays such

as platelet adhesion and thrombus formation. Uncoated polystyrene –microtitre plates

(ELISA) was used as reference.[70]

Urokinase:

Eun kyu Lee et al.(2003) have immobilized urokinase (UK) by covalent attachment to

sepharose6B-CL through multi point amine coupling and have evaluated its performance

in cleaving a fusion protein which consisted of recombinant human growth

hormone(hGH) and a fragment of glutathione - S - Transferase that was linked by a

tetrapeptide of a UK specific recognition sequence. Packing densities of aldehyde on the

activated agarose surface could be controlled in a gel range of 7-60 micromole/ml

aldehyde by the amount of glycidol used.The immobilization was nearly 100% at pH

10.5. The specific activity of the immobilized UK was equivalent to about 80% of the

soluble UK under assay condition. The cleavage rate by the immobilized UK was lower

than that of the soluble enzyme but the side reaction of the cryptic cleavage was

significantly decreased which might sugest that the enzyme specificity was altered by

immobilization. The immobilized UK showed an improvement in pH and thermal

stability. Cleavage yield in the column packed with immobilized UK was dependent on

the feed rate and the yield was approximately 80% of that of soluble UK.The monomeric

hGH could be obtained by selectivity, precipitating the uncleaved fusion protein and GST

fragment at an acidic pH.[71]

Urokinase has also been successfully immobilized on agarose gel[72] and sulphonated

polyvinylidene films[73] with substantial increase in activity. Fibrinolytic therapy is

widely available affordable and can be promptly administered. But the therapy causes

haemorrhagic complication and cannot be administered to a sizeable group due to

contraindications.

4. Enzymes for kidney disorders:

Uricase:



Nakamura et al (1986) found Uricase to be stabilized by protamine from salmon testis.

Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using

glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to

glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas

free uricase was inactivated at 45 degrees C and showed activity over a broader pH range

than free uricase. Automated analysis of uric acid was facilitated using the immobilized

uricase. The standard curve for uric acid was linear in the range of 2 to 10

micrograms/sample and passed through the origin. This automated procedure was also

applicable to the determination of uric acid in human serum. Protamine bound to glass

beads is expected to be useful for the simple immobilization and stabilization of

enzymes.[74]

Williams et al. (1999) have tentatively used uricase unmodified and modified with

poly(ethylene glycol) i.v. by humans. PEG conjugated uricase entrapped in liposome

formulation was orally administered to chicken surprisingly the enzyme activity appeared

in blood following the treatment indicating that the PEG-enzyme entrapped in liposomes

and reach the circulation via the gut. The therapeutic formulation of uricase should

exhibit low or nonimmunogenicity and the way to obtain this was to synthesize highly

PEG modified uricase which however lost more than 60% of its activity.It can be noted

that Williams et al. described PEG modification of uricase which has no immunogenicity

and with 75% of its activity retained.[75]

Mulhbacher et al. (2002) have demonstrated that the immobilization of uricase on

carboxy methyl high amylose starch cross-linked 35(CM-HASCL-35) as well as on

commercial supports, CNBr activity and diaminodipropyl amine agarose. The N- ethyl-5-

phenyl isoxazolium- 3’- sulphonate(Woodword reagent K)gave a high binding but totally

inhibited the enzyme activity best results were obtained with CM-HASCL-35 using 1

ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent.The immobilized

enzyme retain 88% of its initial limit rate [Vmax (approx.) = 16 EU/mg for immobilized

uricase versuses Vmax = 18 EU/mg for free enzyme with an apparent decreased affinity

for urate substrate Km (approx.) = 0.17 Mm vs. Km = 0.03 Mm for free enzyme.] The

coupling yield was 60 % and the modified uricase was found more resistant to proteolysis

than the free enzyme. The best immobilization yield was obtained with polymeric support



based on CM-HASCL-35 (35%) which gave better results than the commercial supports

based on agarose.[76]

Urease:

Elcin et al (2000) immobilized Urease (EC 3.5.1.5) within polyanionic

carboxymethylcellulose/alginate (CMC/Alg) microspheres coated with a cationic

polysaccharide, chitosan (C). Coating with chitosan improved the mechanically durability

of the polyanionic microspheres, as well as increased enzyme immobilization yield

[approximately 0.4 mg.mL-1 gel]. The effects of chitosan coating and CMC/Alg ratio on

the water uptake and spherical morphology of the microspheres were investigated. The

optimal pH of urease was not extensively affected by the immobilization procedure.

However, the optimal temperature of urease activity increased upto 60 and 65 0C within

CMC/Alg and C(CMC/Alg) microspheres, respectively, while the optimum for the free

enzyme was 500C. The half life (t1/2) and deactivation rate constant (kd) of free urease

were 79 min and 8.77 x 10(-3) min-1, respectively, whilst the t1/2 and kd values of

urease within polyanion and polycation-coated polyanion microspheres were 142 min and

4.88 x 10(-3).min-1, and 179 min and 3.87 x 10(-3).min-1, at 80 degrees C, respectively.

[77]

Neufeld et al.(2001) encapsulated urease in alginate beads coated with chitosan, poly-L-

lysine or poly (methylene-co-gaunidine) membrane to exclude alpha chymotrypsin and

other proteases. Urease in uncoated alginate was highly susceptible to 21.6 Kda. Alpha

chymotrypsin with 98% of the activity lost within 10 min. exposure chitosan and poly -L-

lysine in or poly(methylene-co-gaunidine) membrane protein provided 50 and 12 %

activity retension respectively after exposure while poly (methylene-co-gaunidine)

membrane both inactivated urease fail to provide protection from protease hydrolysis.

Lyophilization of beads caused shrinkage and rehydrated beads did not swell to their

original diameter effectively redusing permeability.This was evident through a large

improvement in protease exclusion for both chitosan coated (89% activity retension) and

uncoated (71 %) beads. Results with trypsin were similar to that observed with alpha

chymotrypsin likely due to similarity in molecular weight. A high level of exclusion and

urease production within chitosan protected alginate beads was observed with high

weight protease K.[78]



US Patent no.6534296 by Alther et al prepared immobilized enzymes in one step

operation by simultaneously adding urease (aqueous enzyme) to a quaternary ionic

compound and mineral (bentonite and a quarternary amine) in a mixer. This one step

operation results in an enzyme clad organoclay. A paste may be formed in the mixer

which can be extruded to form noodles that are air dried. Immobilized enzymes may

alternately be prepared by adding aqueous enzyme to an already formed organoclay to

confer stability through hydrophobic bonding. They showed that they could add urease at

a level as high as 40% by weight of the organoclay, with resulting immobilization.[79]

5. Enzymes for liver disorders:

Alcohol Dehydrogenase:

Fiona C. Cochrane et al. (1996) have used tris (hydroxy methyl)phosphene as a coupling

reagent for the immobilization of alcohol dehydrogenase onto chitosan films and for the

attachment of chitosan fim onto a glass support resulted in enzyme activity far above

those obtained by adsorption of enzyme and greater that those observed when using the

more conventional glutaralhyde coupling protocol. The stability of chitosan films was

dramatically increased by the co valent attachment to the glass using tris (hydroxy

methyl) phosphine and glutaraldehyde on amino propyl silica and amino propyl glass.

The tris (hydroxy methyl) phosphine coupling extended the longevity of alcohol

dehydrogenase activity but did not ulter the pH optima or Km of the enzyme.[80]

Ming-Hung Liao et al. (2001) studied the covalent immobilization of yeast alcohol

dehydrogenase (YADH) Fe3O4 magnetic nanoparticles (10.6nm) via carbodiimide

activation. The immobilization process did not alter the affect the size and structure of

magnetic nanoparticles were superparamagnetic with a saturation paramagnetism of

61emu/g only slightly lower than that of naked ones (63emu/g). Compared to the free

enzyme the immobilized YADH retained 62% activity and showed a 10-fold increased

stability and a 2.7 fold increased activity a at Ph 5. For reduction of 2-butanone by

immobilized YADH the activation energies within 25-45oC was27J/mol, the maximum

specific was 0.23mol/min and Michaelis constant for NADH and 2-butanone was

0.62Mm and 0.43M respectively. These results indicated structural change of YADH

with a decrease in affinity for NADH and 2-butanone after immobilization compared

with free enzyme.[81]



Catalase:

Senay akkus Centinus et al (2000) demonstrated the immobilization of catalase on the

chitosan film, which is a natural polymer. Experiments were performed on the free

catalase and immobilized catalase on chitosan film determining the optimum pH, thermal

stability, storage stability, operational stability and kinetic parameters: Km = 25.16mM

and Vmax = 24042 micromole/min.mg protein for free catalase and Km = 27.67mM and

Vmax = 1022 micromole/min.mg protein for immobilized catalase.It was found that the

storage stability at 5oC for immobilized catalase stored wet is greater than free catalase

and immobilized catalase stored dry. Also immobilized catalase showed operation

stability.This study also shows that catalase can be immobilized on glutaraldehyde

pretreated chitosan films and can be used for practical applications.[82]

The same authors in 2003 immobilized the bovine liver catalase in to chitosan beads

prepared in cross linking solution.Various characteristics of immobilized catalase were

evaluated.They reported that the pH optimum and temperature optimum of the free and

immobilized catalase were found to be pH 7.0 and 350C.The Km of immobilized catalase

(77.5mM) was higher than that of free enzyme (35mM). Immobilization decreased in the

Vmax value from 32000 to 122micromole/min.mg protein. It was observed that

operational, thermal and storage stabilities of enzyme were increased with

immobilization.[83]

Stefano Giovagnoli et al (2004) have encapsulated superoxide dismutase (SOD) and

catalase (CAT) in biodegradable microspheres (MS) to obtain suitable sustained protein

delivery. A modified water/oil/water double emulsion method was used for poly(D,L-

lactide-co-glycolide) (PLGA) and poly(D,L-lactide) PLA MS preparation co-

encapsulating mannitol, trehalose, and PEG400 for protein stabilization. In vitro activity

retention within MS was evaluated by nicotinammide adenine dinucleotide oxidation and

H2O2 consumption assays. SOD encapsulation efficiency resulted in 30% to 34% for

PLA MS and up to 51% for PLGA MS, whereas CAT encapsulation was 34% and 45%

for PLGA and PLA MS, respectively. All MS were spherical with a smooth surface and

low porosity with particle mean diameters 10 to17 µm. CAT release was prolonged, but

the results were incomplete for both PLA and PLGA MS, whereas SOD was completely

released from PLGA MS in a sustained manner after 2 months. CAT results were less



stable and showed a stronger interaction than SOD with the polymers. Mass loss and

mass balance correlated well with the release profiles. SOD and CAT in vitro activity

was preserved in all the preparations, and SOD was better stabilized in PLGA MS. PLGA

MS can be useful for SOD delivery in its native form and is promising as a new depot

system. Moreover, the 2-month SOD release from PLGA MS may be potentially useful

for long-term sustained release of the enzyme for the treatment of inflammatory

manifestations, such as rheumatoid arthritis or other intra-articular and joint diseases.[84]

6. Antidiabetics:

Glucose oxidase:

Shin-Ichiro et al.(1998) have described the immobilized glucose oxidase on a

polycarbonate membrane modified by a urethane coupling with a poly-(L-lysine)

activated with glutaraldehyde. The enzymic properties of immobilized enzyme were

investigated and compared with those of native glucose oxidase. The thermal stability

and pH stability of the immobilized glucose oxidase were greater than native enzyme.

The molecular mass of poly- (L-lysine) was investigated as a possible influencing agent

on immobilization of glucose oxidaes on porous polycarbonate membrane. They used

standard immobilization procedure except that the molecular mass of poly-( L-lysine)

was varied in the range 5-300 Kda.The effect of molecular mass on the immobilized

glucose oxidase activity showed that 50 Kda or above was required for optimum

immobilization of glucose oxidase. The comparison of enzyme activity with the method

of immobilization showed a quantity of glucose oxidase adsorbed on ordinary poly

carbonate membrane was negligible,while covalent binding with aldehyde groups in the

derivatized membrane was string and no leakage was observed.The membrane was

applied as glucose sensor.[85]

Blandino et al (2001) encapsulated glucose oxidase(GOD) within calcium alginate

capsules. The effects of gelation conditions on capsule characteristics such as thickness,

percentage of enzyme leakage and encapsulation efficiency were studied and the optimal

conditions for GOD encapsulation were obtained. Oxidation of glucose to gluconic acid

followed Michaelis –Menten Kinetics. The optimum conditions selected for the effective

encapsulation of glucose oxidase were 1%w/v sodium alginate, 5.5% w/v CaCl2 and 1

hour gelation with stirring rates 400rpm. [86]



Benoit Van Aken et al (2000) have co-immobilized manganese peroxidase (MnP) from

Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads.

Immobilization of both enzymes on same carrier provided an integrated system in which

H2O2 required MnP was produced by glucose oxidase. The immobilization process

resulted in a decrease of both enzymatic activities. However immobilization improved the

stability of MnP against H2O2 or high pH as well as the storage stability of the enzyme.

Surprisingly the immobilized system showed a lower thermal stability than the free

enzyme.[87]

US Patent no.4539294 by Metcalfe et al (1985) have immobilized glucose-oxidase and/

catalase on on a porous polymeric support by a first soaking in a dilute long-chain

cationic solution and a second soaking in a dilute aqueous protein solution. The long

chain cationic is preferably a nitrogen compound such as a diamine having at least one

alkyl or alkenyl group containing at least eight carbon atoms. A preferred diamine is N-

coco-1,3-diamino-propane and the cationic surfactant is 1%w/v acetone. They

demonstrated different immobilization processes for hormone (human chorionic

gonadotrophin) and enzymes (glucose oxidase and catalase),and evaluated enzyme

activities under different conditions, so that the proteins can be reused.[88]

7. Enzyme replacement therapy:

The treatment of the enzyme deficiency state represents an obvious use of

enzymes.More intriguing is the treatment of inborn errors of metabolism in which

deficiency of single enzyme leads to accumulation of abnormal amounts of substrate

.With the recognition that many errors are owing to the inadequacies of lysosomal

enzymatic catabolism ,it was reasoned that exogenously administered enzyme might react

with and dispose of these accumulations.

Dornase-α (Pulmozyme) :

Cystic Fibrosis (CF) (Mucoviscidosis)is one of the most common genetic diseases

affecting 1 in 2500 babies. It is estimated that 20% people carry abnormal recessive gene

which must be suppressed in both parents causing the disease.It is a life threatening

disease caused by a dysfunctional cystic fibrosis transmembrane regulator, CFTR protein

which modulates salt and water transport in and out of cells .This ion channel defect leads

to poorly hydrated, thick mucous secretions in the airways and severely impaired



mucociliary function leading to progressive pulmonary dysfunction and thus respiratory

failure.Retention of viscous purulent secretions which contain high concentrations of

DNA released by degenerating leukocytes that accumulate in response to infection in

airways cause reduced pulmonary function. Digestion of DNA polymers in purulent

secretions with DNAseI (dornase-α) has shown to reduce sputum viscosity in cystic

fibrosis patients. Genentech produces recombinant human Dnase I under the tradename

Pulmozyme. The availability of recombinant DNAse has allowed its use in an aerosol

formulations to deliver the enzyme into the alveoli of CF patients. Mucus also contains

the polysaccharide alginate, which is produced by the seaweeds in soil and marine

bacteria. Pseudomonas aeruginosa is one of the main infectious agent among

them.Alginate lyase is combination with Dnase is used to degrade alginate as well DNA.

Alginate lyase gene was isolated from the soil bacterium Flavobacterium and the alginate

degradation domain was amplified ,this was then cloned into the expression vector.89

β-Glucoronidase (Glucocerebrosidase) :

Gaucher’s Disease is agenetic defect in glucocerebrosidase enzyme which leads to

accumulation of glucocerebroside (a glycolipid) in lysosomes which is potentially

fatal.As β -glucoronidase is localized in lysosomes and exogeneously administered

enzyme is capable of accumulating in liposomes of most blood cells including leukocytes

enzyme replacement therapy is a logical strategy . β –glucoronidase extracted from

human placental tissues (Ceredase) was used initially and later substituted with human

recombinant form (Cerezyme) for treatment of Gaucher’s disease .Both placental and

human recombinant β –glucoronidase were modified to expose terminal mannose

residues on the glycosylated enzyme to enhance locolization of enzymes to lysosomes in

leukocytes which express a high density of mannose receptors .ERT is a chronic therapy

which typically requires administration every week or two.15% patients develop IGg

antibodies to Cerezyme and half of them develop hypersensitivity.Recombinant β –

glucoronidase have been engaged in suitable vectors for somatic gene therapy.We expect

that improvement in gene delivery systems along with enzyme immobilization will

provide a higher degree and more prolonged expression of the enzyme.[89]

Adenosine deaminase :



Severe Immunodeficiency Syndrome (SCID) is an autosomal recessive syndrome

characterized by the T and B cell function from birth due to the inhereht deficiency of

adenosine deaminase.Symptoms include frequent episodes of diarrhoea, pneumonia,

otitis,sepsis, and cutaneous infections. ADA catalyzes the irreversible deamination of

adenosine and 2’-deoxyadenosine to iosine as a part of purine nucleoside metabolism.

Adenosine and deoxyadenosine are suicide inactivators of S-adenosyl-homocyestine

(SAH) hydrolase and indirectly to intracellular accumulation of SAH which is a potent

inhibitor of methylation reactions. Cellular methylation function is essential for

detoxification of adenosine and deoxyadenosine. As a result ADA deficiency leads to

accumulation toxic levels of intracellular purine metabolism and impairment of T-and B-

cell functions.[89]

8. Enzymes for infectious diseases :

Lysozyme:

Near et al (1992) developed an immunoassay for lysozyme to see whether serum

lysozyme levels could be used to identify individuals with clinical leprosy or TB. Since,

active tuberculosis (TB) and leprosy are difficult to diagnose early because there are few

organisms to detect and the specific immune response does not distinguish between active

and inactive disease.The immunoassay for lysozyme proved superior to standard enzyme

assays that were less sensitive and reliable. The lysozyme assay was compared with

assays for antibodies to Mycobacterium tuberculosis lipoarabinomannan (LAM) and M.

leprae phenolic glycolipid-1. The sera tested were from Ethiopian leprosy (paucibacillary

and multibacillary) and TB patients and from healthy Ethiopian and U.S. controls. The

lysozyme assay was able to detect more of the individuals with TB (sensitivity, 100% for

19 patients) or leprosy (sensitivity, 86% for 36 patients) than either antibody assay. In

particular, lysozyme levels were raised in a higher proportion of the paucibacillary

leprosy patients (83% of 17), for whom the antibody assays were less sensitive; the LAM

IgG and the phenolic glycolipid-1 IgM levels were raised in only 62 and 44% of 16

patients, respectively. The data suggest that lysozyme measurements may be useful in the

diagnosis of mycobacterial infections and other chronic infectious granulomatoses.[90]

Odilio B.G.Assis et al (2003) deposited the protein lysozyme onto a permaeble a support

comprising chemically functionalized glass fibre.The main objective was to set a stable



organic weight with no effect on the medium bed permeability and a preliminary test of

this enzyme under immobilized conditions. The film formation is followed by atomic

force microscopy(AFM) surface imaging. The effect on Escherischia.coli was tested

using simple microfiltration column .The filtration results pointed uot around 75%

removal of bacteria in the effluent when compared to the influent concentration. The

removal mechanism is assumed as being essentially due to biointeractions. The surface

polarity characteristics of the fcrmed film were also considered as playing an important

role suggesting an electrostatic interaction mechanism in the micro-organism removal.[91]

M. Tortajada et al (2005) have shown the ability of hierarchial porous silica based

network with pore systems of different length scales for enzyme immobilization using

lysozyme- a relatively small globular enzyme and α-L-arabinofuranosidase –a large

enzyme. Lysozyme immobilization on several silica-gel supports have been studied(on

bimodal porous silicas denoted UVM7 and conventional silica xerogels ). They studied

the ability of the UVM-7 bimodal porous silica and HPNO an organosilica related

material for immobilizing the 2 enzymes. Lysozyme whose main function is degradation

of bacterial cell wallswas selected as a model enzyme for the immobilization .They have

compared the results of electrostatic and covalent immobilization concluding that the

covalent immobilization allows to shift the optimum enzymatic working conditions

towards lower pH values and higher temperature than free enzymes.[92]

Other gel matrices exclusively used for enzyme immobilization include polyacrylamide,

agar, alginate, κ-carragenan or synthetic such as gels derived from acrylamide [93],

modified silica composites [94], crosslinked agarose support(Sepharose-4B).[95]

Immobilization versus Genetic Engineering.

Enzymes belong to the category natural catalysts which includes DNA, RNA and

catalytic antibodies. Since enzymes already play a major role in synthetic chemistry,

pharmaceuticals as well as have industrial applications, they must be modified by genetic

engineering or by chemical modification with the objective of improving their selectivity,

activity and durability to be used furthermore in downstream processing in the

immobilized forms to reduce the production costs. In the last decade although it has been

increasingly appreciated that enzyme immobilization is a powerful tool for improvement



of enzyme performance, the rational combination of immobilization and genetic

engineering might an alternative technique for protein engineering.[96]

Prospective Of Therapeutic Enzymes:

Of late, it has been proved that therapeutic enzymes have a tremendous potential to

develop novel therapeutics. Similarly with the emergence of r-DNA technology,

PEGylation technology along with different immobilization techniques seem to have

made this a possibility for the treatment of both rare and common diseases . In addition

the changes in orphan drug laws and new initiatives by the FDA have been effective in

facilitating efforts to develop enzyme drugs. The major achievements include MPS VI,

genetic diseases, burn debridement, infectious diseases and cancer. For genetic disorders

gene therapy seems to be the first line of treatment but enzyme therapy will continue to

serve the purpose. Currently efforts are being channelised on the delivery of insulin,

glucose oxidase for the treatment of diabetes as well as on chronic liver failure,

phenylketonuria, removal of glutamine or aspargine in cancer. [97]

CONCLUSION

This paper presents a brief review of the recent (mainly during past decade)

developments and medical applications of immobilized enzymes particularly in therapy.

Faced with that situation, the emergence of enzyme therapy as a powerful tool to

compare the properties of free and immobilized therapeutic enzymes opens promising

avenues for future research and therapy .The alliance of the therapeutic approach with

classical tools of enzyme immobilization will in near future probably allow us to rectify

all enzyme based disorders. Will enzymes as therapeutic agents in the immobilized state

be useful to strengthen the therapeutic potentialities? An exhaustive answer to the

question is not easy at present times but studies will help to balance the two sides:

enzyme immobilization and therapeutic enzymes, as it seems much easier to hit the

target of therapy ,with the advent of biosensors, new techniques and novel matrices for

immobilization to fight against the untreated enzyme imbalances .Although the progress

in therapeutic use of immobilized enzymes is slow and somewhat staggered, because of

the complexity of the human body system to be applied; the future prospect for

application of immobilized enzymes in therapy seems to promising .Thus, the



improvement of the weapons of the immobilized systems will be an ambitious goal

offered to pharmacists in coming decade.

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For Correspondence: Sonal Pawar Email: [email protected]

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