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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
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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
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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
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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
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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
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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
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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
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(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
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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]
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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-
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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
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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
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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
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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
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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,
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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.
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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
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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
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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
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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:
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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]
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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
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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
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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
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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,
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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
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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:
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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
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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]
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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]
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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
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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]
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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
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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 :
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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
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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
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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
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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]