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CHAPTER 1
INTRODUCTION
Biopolymers are of interest to both academicians and industrialists
involved in polymer matrix research. A polysaccharide derived from tamarind
seed (TSP), and its application as a matrix for embedding biological samples
were studied. A novel enzyme assay was further developed for lipases using
porphyrin as an indicator and final part of the work involved using biological
template (TSP) for synthesis of nano metal oxides.
1.1
ENZYME IMMOBILIZATION
1.1.1
Immobilization
Enzymes are protein molecules which serve to accelerate the
chemical reactions of living cells (often by several orders of magnitude).
Without enzymes, most biochemical reactions would be too slow to even
carry out life processes. Enzymes display great specificity and are not
permanently modified by their participation in reactions. There have been
numerous efforts devoted to the development of insoluble enzymes for
various applications. 1) The re usability of heterogeneous biocatalysts with
the aim of reducing the production cost by efficient recycling and control of
the process; 2) as stable and reusable analytic devices for analytic and medical
applications; 3) as selective adsorbents for purification of proteins and
enzymes; 4)as fundamental tools for solid-phase protein chemistry and 5) as
effective micro devices for controlled release of protein drugs are some of the
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benefits of using immobilized enzymes rather than their soluble counter parts
(Linqiu et al 2005).
However, regardless of its nature or preparation , an immobilized
enzyme by definition, has to perform two essential functions: namely, the
non-catalytic functions (NCFs) that are designed to aid separation and the
catalytic functions (Cfs) designed to convert the targeting compounds within a
desired time and space (Cao et al 2003). Generally, the peculiarities of these
two essential elements dictate the scope of application of the immobilized
enzymes. Further more, diversity of the process necessarily requires the
design of specific immobilized enzymes that can match the corresponding
requirements for the desired process.
Therefore, it is hardly surprising that there is no universally
applicable method of enzyme immobilization. The main task was to select a
suitable carrier (defined as the non-catalytic part of an immobilized enzyme,
on which the catalytic part was constructed), condition (pH, temperature, and
nature of the medium) and enzyme itself (source, nature and purity) to design
an immobilized biocatalyst. The selected method should meet both the
catalytic needs (expressed in productivity, space-time yield, stability and
selectivity) and the non-catalytic needs (separation, control, and down
streaming process) that are required by a given application. As a result, an
immobilized enzyme could be labeled robust, when both the catalytic and
non-catalytic functions
meet the requirements of a specific application
(Bornscheuer et al 2003).
1.1.2
Methods of Enzyme Immobilization
It is important to choose a method of attachment that would prevent
the loss of enzyme activity by not changing the chemical nature or reactive
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groups in the binding site of the enzyme while immobilizing an enzyme to a
substrate. In other words, attach the enzyme but do as little damage as
possible. Considerable knowledge of the active site of the enzyme prove
helpful in achieving this task. It is desired to avoid reaction with the essential
binding site of the enzyme. Alternatively, an active site can be protected
during attachment as long as the protective groups can be removed later on
without loss of enzyme activity. In some cases, this protective function can be
fulfilled by a substrate or a competitive inhibitor of the enzyme. The surface
on which the enzyme is immobilized is responsible for retaining the structure
in the enzyme through hydrogen bonding or the formation of electron
transition complexes. These links prevent vibration of the enzyme and thus
increase thermal stability. The micro environment of surface and enzyme has
a charged nature that can cause a shift in the optimum pH of the enzyme up to
2 pH units. This may be accompanied by a general broadening of the pH
region in which the enzyme can work effectively, allowing enzymes that
normally do not have similar pH regions to work together (Alexander 1979).
1.1.2.1
Carrier-binding
The carrier-binding method was the oldest immobilization
technique for enzymes. The amount of enzyme bound to the carrier and the
activity after immobilization depends on the nature of the carrier (Figure 1.1).
The selection of the carrier depends on the nature of the enzyme
itself, as well as on the particle size, surface area, molar ratio of hydrophilic to
hydrophobic groups and the chemical composition. In general, an increase in
the ratio of the hydrophilic groups and in the concentration of bound
enzymes, results in higher activity of the immobilized enzymes. Some of the
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Figure 1.1
Graphical representation of Carrier-binding technique of
immobilization
most commonly used carriers for enzyme immobilization were polysaccharide
derivatives such as cellulose (Kurokawa et al 2004), dextran (Manuel Fuentes
et al 2005) and agrasoe (Jaromir et al 2006). According to the binding mode
of the enzyme they were further classified as
a)
Physical adsorption : This method for the immobilization of
an enzyme is based on the physical adsorption of enzyme
protein on the surface of water-insoluble carriers. Hence, the
method causes little or no conformational change of the
enzyme or destruction of its active center. If a suitable carrier
is found, this method can be both simple and cheap. However,
the leakage of adsorbed enzyme from the carrier during use
due to a weak binding force between the enzyme and the
carrier is a disadvantage. The earliest example of enzyme
immobilization using this method was the adsorption of betaD-fructo-furanosidase
onto
aluminum
hydroxide.
The
processes available for physical adsorption of enzymes were:
static procedure, electro deposition, reactor loading process,
and mixing or shaking bath loading. Of the four techniques,
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the most frequently used was mixing-bath loading and reactor
loading for commercial purposes.
A major advantage of adsorption as a general method of
immobilizing enzymes was that usually no reagents and only a
minimum of activation steps were required. Adsorption tends
to be less disruptive to the enzymatic protein than chemical
means of attachment because the binding is mainly by
hydrogen bonds, multiple salt linkages, and Van der Waal's
forces. In this respect, the method bears the greatest similarity
to the situation found in natural biological membranes and has
been used to model such systems. Because of the weak bonds
involved, desorption of the protein resulting from changes in
temperature, pH, ionic strength or even the mere presence of
substrate, was often observed. Further adsorption of other
proteins or other substances can take place when immobilized
enzyme is used. This may alter the properties of the
immobilized enzyme or, if the substance adsorbed is a
substrate for the enzyme, the rate will probably decrease
depending on the surface mobility of enzyme and substrate.
Adsorption of the enzyme may be necessary to facilitate the
covalent reactions. Stabilization of enzymes temporarily
adsorbed onto a matrix has been achieved by cross-linking the
protein in a chemical reaction subsequent to its physical
adsorption (Felipe et al 1996; Subramanian et al 1999).
b)
Ionic binding : The ionic binding method relies on the ionic
binding of the enzyme protein to water-insoluble carriers
containing
ion-exchange
residues.
Polysaccharides
and
synthetic polymers having ion-exchange centers usually used
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as carriers. The binding of an enzyme to the carrier is easily
carried out, and the conditions are much milder than those
needed for the covalent binding method. Hence, the ionic
binding method causes little changes in the conformation and
the active site of the enzyme. Therefore, this method yields
immobilized enzymes with high activity in most cases.
Leakage of enzymes from the carrier may occur in substrate
solutions of high ionic strength or upon variation of pH. This
is because the binding forces between enzyme proteins and
carriers are weaker than those in covalent binding. The main
difference between ionic binding and physical adsorption is
that the enzyme to carrier linkages are much stronger for ionic
binding although weaker than in covalent binding (Wilhelm
et al 1999).
c)
Covalent binding: The most intensely studied of the
immobilization techniques was the formation of covalent
bonds between the enzyme and the support matrix. While
trying to select the type of reaction by which a given protein
should be immobilized, the choice is limited by two
characteristics: (1) the binding reaction has to be performed
under conditions that do not cause loss of enzymatic activity,
and (2) the active site of the enzyme must be unaffected by the
reagents used (Karrasch et al 1993). The covalent binding
method is based on the binding of enzymes and waterinsoluble carriers by covalent bonds. The functional groups
that may take part in this binding are amino, carboxyl,
sulfhydryl, hydroxyl, thiol and phenolic groups (Jan et al
1975).
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This method can be further classified into diazo, peptide and
alkylation methods according to the mode of linkage. The
conditions for immobilization by covalent binding are much
more complicated and less mild than in the cases of physical
adsorption and ionic binding. Therefore, covalent binding may
alter the conformational structure and active center of the
enzyme, resulting in major loss of activity or changes in the
substrate. However, the binding force between enzyme and
carrier is so strong that no leakage of the enzymes occurs, even
in the presence of substrate or solution of high ionic strength.
Covalent attachment to a support matrix must involve only
functional groups of the enzyme that are not essential for
catalytic action. Higher activities result from prevention of
inactivation reactions with amino acid residues of the active
sites (Andrei et al 2006, Yong et al 2006, Ansil et al 2003).
1.1.2.2
Cross Linking
Immobilization of enzymes has been achieved by intermolecular
cross-linking of the protein, either to other protein molecules or to functional
groups on an insoluble support matrix (Figure 1.2). Cross-linking an enzyme
to itself is both expensive and insufficient, as some of the protein material will
inevitably be acting mainly as a support. This will result in relatively low
enzymatic activity. Generally, cross-linking is best used in conjunction with
one of the other methods. It is used mostly as a means of stabilizing adsorbed
enzymes and also for preventing leakage from polyacrylamide gels.
Since the enzyme is covalently linked to the support matrix, very
little desorption is likely using this method. For example, reported carbamyl
phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only
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16% of its activity after continuous use in a column at room temperature for
fourteen days. The most common reagent used for cross-linking is
glutaraldehyde. Cross-linking reactions are carried out under relatively severe
conditions. These harsh conditions can change the conformation of active
center of the enzyme; and so may lead to significant loss of activity (Walt
et al 1994).
Figure 1.2
Graphical representation of crosslinking technique of
immobilization
1.1.2.3
Entrapping Enzymes
The entrapment method of immobilization is based on the
localization of an enzyme within the lattice of a polymer matrix or membrane.
It is done in such a way as to retain protein while allowing penetration of
substrate. It can be classified into lattice and micro capsule types (Figure 1.3).
This method differs from the covalent binding and cross linking in that the
enzyme itself does not bind to the gel matrix or membrane. The conditions
used in the chemical polymerization reaction are relatively severe and result
in the loss of enzyme activity. Therefore, careful selection of the most suitable
conditions for the immobilization of various enzymes are required.
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Figure 1.3
Graphical representation of entrapping technique of
immobilization
Lattice-Type entrapment involves entrapping enzymes within the
interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic
polymers such as polyarylamide, polyvinylalcohol, (Hidekatsu et al 2004 and
natural polymer (starch) (Muetgeert et al 1998) have been used to immobilize
enzymes using this technique.
Microcapsule-Type entrapping involves enclosing the enzymes
within semi permeable polymer membranes. The preparation of enzyme
micro capsules requires extremely well-controlled conditions and the
procedures for micro capsulation of enzymes can be classified as: a) Inter
facial polymerization method b) Liquid drying c) Phase separation.
Immobilized enzyme can be classified into four types: particles, membranes,
tubes, and filters. The solid supports used for enzyme immobilization can be
inorganic or organic. Some organic supports include: Polysaccharides,
Proteins, Carbon, Polystyrenes, Polyacrylates, Maleic Anhydride based
Copolymers, Polypeptides, Vinyl and Allyl Polymers, and Polyamides
(Bajpai et al 2003).
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1.1.3
Tamarind seed polysaccharide
1.1.3.1
Tamarind (General Introduction and uses)
Tamarindus indica is a tropical fruit growing tree which grows in
dry/monsoonal climates. It belongs to the family Leguminosae (Fabaceae).
The fruits are usually between 5 and 14 cm in length and approximately 2 cm
wide. The ripe fruit is filled with a sticky pulp which can be used both in
industry and for domestic purposes in different ways. The tree averages
20-25 m in height and 1 m in diameter, it has a wide spreading crown and a
short, stout trunk. It is slow growing, but long lived, with an average life span
of 80-200 years. Tamarindus is a monotypic genus (having only one species)
the closet relative is thought to be Heterostemon which is native to the upper
Amazon region. Tamarind is well adapted to semi-arid tropical conditions, it
also grows well in many humid tropical areas with seasonally high rainfall. It
grows in well drained, slightly acidic soils and although it cannot withstand
stagnant inundation, it can tolerate a wide range of physical site
characteristics.
There are 2 main varieties, sweet and sour, though the genetic
diversity in Asia and Africa is high with varying fruit and flower colors and
sugar/acid ratio in the fruits. The sweet tamarind is produced mainly in
Thailand where it is grown on a commercial scale and is exported both in the
fresh and processed form. Approximately 140,000 tons of tamarind is
produced annually in Thailand. India is also a major producer of tamarind,
where it is collected and marketed mainly by the rural communities.
The sticky pulp is often eaten fresh but has many other culinary
uses for example in pickles, jams, candy, juice and drinks. The pulp can also
be used, when mixed with salt, to polish brass, copper and silver, it can be
used as a fixative with turmeric and also serves to coagulate rubber. Extracts
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from the fruit pulp have shown some molluscicidal activity and has been
reported to have potent fungicidal and bactericidal properties. Extracts from
the plant also have an inhibitory effect on plant viruses. The leaves and
foliage of tamarind can be used as forage for cattle and the timber though very
hard, can be used for making furniture and tools. Tamarind fruits and leaves
are reputed to have medicinal properties and have been used in the past for
complaints such as intestinal ailments and skin infections. The American
pharmaceutical industry process 100 tons of tamarind pulp annually and it is a
common ingredient in cardiac and blood sugar reducing medicines.
Tamarind seed kernel powder (TKP) is a major industrial product,
which is used in the sizing of textile, paper and jute. A substance known as
"jellose" can be extracted from the seed which is a polysaccharide with gel
forming characteristics and has both food and industrial applications. The
seed and its extracts can be used in the food processing industry, as an
adhesive in the plywood industry and in the tanning industry due to the high
tannin content in the seed testa (Hughes et al 1999).
1.1.3.2
Tamarind seed polysaccharide
Tamarind seed polysaccharide (TSP) is extracted from the seed
kernels of the tamarind tree (Tamarindus indica). There have been numerous
publications in the past 30 years concerning the primary structure of TSP.
There is general agreement about the nature of the backbone and the side
chains. The polymer consists of a cellulose-type spine, which carries xylose
and galactoxylose substituents. About 80% of the glucose residues are
substituted by  1-6 linked xylose units, which themselves are partially
substituted by  1-2 galactose residues. These structural units are displayed in
Figure 1.4.
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Native TSP was shown to exhibit a strong tendency to
self-aggregation when dispersed in aqueous solvents. These aggregates
consist of lateral assemblies of single polysaccharide strands, showing a
behavior that could be well described by the worm like chain. Static light
scattering data on these particles shows that their stiffness is determined by
the number of aggregated strands.
Figure 1.4 The structure of Tamarind seed polysaccharide (TSP)
It exhibits properties like high viscosity, broad pH tolerance and
adhesivity. This led to its application as a stabilizer, thickener, gelling agent
and binder in food and pharmaceutical industries. In addition to these, other
important properties of TSP have been identified recently. They include noncarcinogenicity (Sano et al 1996), mucoadhesivity, biocompatibility, and high
thermal stability (Saettone et al 1997).
In recent years the polysaccharides have found
tremendous
application in the field of drug delivery. The tamarind seed polysaccharide
acts as a delivery system for the ocular administration of hydrophilic and
13
hydrophobic antibiotics and as an controlled delivery system for some drugs
such as Caffeine anhydrous, acetoaminophen etc. ( Sumathi et al 2002).
1.1.4
Lipase
The four main classes of biological substances are carbohydrates,
proteins, nucleic acids, and lipids. The first three of these substances have
been clearly defined on the basis of their structural features, whereas the
property that is common to all lipids is a physiochemical one. Lipids are a
group of structurally heterogeneous molecules, soluble in nonpolar and
slightly polar solvents such as benzene, ether, and chloroform, and insoluble
or partly soluble in water. Important lipids include fats and oils (triglycerides
or triacyglycerols), fatty acids, phospholipids, and cholesterol. Fats and oils, a
major form of metabolic energy in humans, are important sources of essential
fatty acids and fat-soluble vitamins. Metabolic turnover of these biomolecules
are achieved through hydrolytic enzymes.
Hydrolytic enzymes catalyzing the conversion of lipids include
phospholipases (EC 3.1.4.3), esterases (3.1.1.1), and lipases or, more
systematically, triacylglycerol hydrolases (3.1.1.3) (Figure 1.5).
Lipases are known for their excellent stereo specific nature in
various chemical reactions. They exhibit activity in organic solvents, which
makes them commercially important. They are used in various processes
ranging from pharmaceuticals to detergent
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Figure 1.5 Graphical representation of general reaction catalyzed by
lipase
1.2
ENZYME ASSAY
1.2.1
Various Assay System
Most lipases and esterases are water soluble enzymes that
hydrolyze ester bonds of substrates. The difference in the assay system
between these enzymes can be made out, by changing the substrate, i.e the
soluble substrates are exclusively for esterase and the in soluble substrates are
exclusively for lipase. There are various protocols developed to measure the
activity of lipase and esterase. These methods either depend on the
consumption of the substrate or release of a particular product over time.
1.2.1.1
Photometric methods
The chromogenic assay refers to the chrompophore which can be
monitored at a particular wavelength , either due to direct action of the
enzymes on the substrates or by indirect methods.
15
These are commonly used in the laboratory, and are replacing the
conventional pH-stat method due to their sensitive and user friendly
protocols.
A commonly used procedure to find out the esterase and lipase
activity is with 4-nitrophenyl esters of aliphatic acyl chains of varied lengths
(Huggins et al 1947). The release of the 4-nitrophenol is measured spectro
photometerically at 410 nm. A variety of 4-nitrophenyl esters with varying
acyl chain are commercially available. Short chain esters, like acetate or
butyrate, are used to measure esterase activity, while longer chains such as
sterate, palmitate or oleate are used to investigate lipase activity. Short acyl
chain esters are soluble in aqueous buffers, however, solubilization of
substrates like 4-nitrophenyl laurate, palmitate or sterate requires emulsifying
agents as additional reagents.
This method is convenient as it requires equipment, (ultravioletvisible spectrophotometer) that is normally found in research laboratory.
Further, many of these esters are commercially available and are relatively
inexpensive. The reactions are routinely scaled to a 96-well format (Jaeger
et al 2000) and measurements can be taken in a kinetic fashion.
These methods, however, have limitations. These esters particularly
with short acyl chains, can be hydrolyzed by non-specific esterases, non
specific proteins or proteases often found in biological samples. For example
serum albumin (Tildon et al 1972) as well as insulin (Hartley et al 1952) have
been shown to hydrolyze 4-nitrophenyl acetate. Therefore, these assays are
best suited for use with purified lipase, which exclude these interfering
catalyst. Measurements with these esters cannot be performed at acidic pH as
this dramatically affects the absorbance of 4-nitrophenol. Therefore, kinetic
assays can be performed at neutral or alkaline pH, which may not be suitable
16
for some lipases (Kademi et al 2000). Because 4-nitrophenol has different
absorption coefficient at different pH values, use of standards in different pH
environments is required. In addition, preparation of the samples containing
lipase is a consideration as spectrophotometeric analysis is confounded by
turbidity introduced in the reaction mixture , such as assaying cell lysates. The
chromophore being toxic in nature is again a disadvantage to the assay
system.
Another common colorimetric lipase assay is based on the
hydrolysis of naphtyl esters. Napthol produces a colored product after
complexing with a diazonium salt and generation of this product can be
measured at 560 nm (Lanz et al 1973). As with 4-nitrophenol, esters of
various chain lengths are available and the assay requires common laboratory
equipments and the reaction can be monitored in a kinetic fashion. The
problems faced in the 4-nitrophenol assay system prevails in this assay system
also. The auto hydrolyzing nature of the substrate of short acyl chain lengths
is a common problem. The change in the pH can also alter the reading value
of the assay system.
Resorufin incorporated into a triacylglycerol analogue can be used
as a chromogenic substrate for lipases. The ether bonds at one of the primary
and the sn-positions ensure these groups are not cleaved by lipases. The other
primary position on the glycerol backbone in the racemic substrate is an ester
linkage to a short acyl chain prior to the resorufin moiety that is also bound
via an additional ester. Mass spectrophotometry has shown that the major
product of hydrolysis by lipoprotein lipase is free resorufin (Bothner et al
2000). The release of free resorufin is monitored at 572 nm in a kinetic
fashion during incubations at 37 oC. The method is easily scalable to a 96 well
format. The substrate is suitable for measurements for plasma triacylglycerols
lipases and intracellular hepatic lipases, however, the resorufin moiety may
17
not be readily hyrdolyzed by some lipases as resorufin is polycyclic in nature,
not aliphatic like a fatty acid.
Spectroscopic assays have been employed to measure the increase
in turbidity generated when fatty acids liberated by lipase activity are
precipitated using calcium (Von et al 1989). The increase in turbidity is
measured at 500 nm. The turbidimetric method is described as being thirty
times more sensitive than the titrimetric assays and at least four times more
sensitive
than
a
spectrophotometeric
method
using
4-nitrophenyl palmitate (Winkler et al 1979). Clearly, this method is not
useful when activities in turbid solutions, such as cell extracts are to be
determined.
Recently various pH indicators are also used to find out the activity
of the enzymes. These are based on the drop in the pH of the solution that
leads to a change in absorbance value at a particular wavelength over a period
of time. The pKa value of a pH-color indicator should be located within or at
least close to the optimal pH range of the enzyme concerned so as to make the
change of color or absorbance of the indicator proportional to the changes of
hydrogen ion concentration in the solution (Yi et al 1998; Janes et al 1998).
1.2.1.2
Chromatographic methods
Chromatography is a common method for direct determination of
the released fatty acids
following lipolysis of a lipid substrate.
Chromatography allows use of the most physiologically relevant substrates,
which is critical when characterizing an enzyme. Although these methods of
analysis allow use of lipid substrates that occur naturally, they only allow end
point analysis and cannot be followed on a kinetic basis.
18
A simple method of detection of fatty acid released during the
reaction of the enzymes on the triacyl glycerols can be carried out using thin
layer chromatography. Lipids are visualized by exposure to iodine vapor, and
bands corresponding to the various lipid species are identified by comparison
to standards and scraped off the thin layer chromatography plate (Lehner et al
1999). The substrates can be radio actively labeled, if so the scrapped out
components can be quantified by scintillation counter (Lehner et al 1992).
This method can be used to screen enzymes in small scale, especially lipases,
as a true lipase substrate will not undergo auto hydrolysis.
Gas chromatography can be used to quantitatively determine
mono-, di-, and triacylglycerol as well as free glycerol and the methyl ester
derivatives of fatty acids (Christina et al 1995). Release of fatty acids by
lipase catalyzed cleavage of triacylgycerols and the generation of
diacylglycerol and monoacylglycerol intermediates can be monitored by GC
following the conversion of the reaction products of trimethylsilyl esters (fatty
acids) and ethers (partial acylglycerols) before performing GC. This method
is sensitive up to nanomole of products released and is suitable with both
purified lipase or with incubation mixture which does not contain other
glycerolipids. Although the method is highly sensitive, it requires specialized
expensive equipment (gas chromatograph) and is laborious. However, it is
very useful when fatty acyl chain length specificity of a given lipase is to be
determined since the various chain lengths and saturation of the released fatty
acid are easily detected.
High pressure liquid chromatography is another chromatography
technique that is widely used for the detection of the products released during
an lipolysis reaction. A detailed HPLC method for determining lipase activity
with 4-nitrophenyl palmitate as a substrate as been reported (Maurich et al
1991). HPLC can also be used to separate mixtures of free fatty acids,
19
mixtures of different triacyl glycerols, and mixtures of all fat classes
(monoacylglycerols, diacylglycerols, triacylglycerols, and free fatty acids). In
HPLC system the detection is accomplished with a refractive index detector.
Identification of fatty acid species is accomplished by comparison of retention
times with known standards. Quantization of fatty acid species can be
achieved using a known amount of fatty acid standard, which is often a
saturated fatty acid with an odd number of carbon atoms such as tridecanoate.
To detect the acylglycerols present after the lipase reaction, the sample is
injected onto a reverse phase column and eluted. A flow gradient is required
to achieve appropriate separation of the lipid classes. Peak areas are
calculated and the concentrations of lipid species are quantified by
comparison to peaks arising from known standards using appropriate
software. An alternate method of detecting all the lipid species in a single
HPLC run combined with mass detection has been described (Christie et al
1985). These methods are found to be highly sensitive, and can be used to
find out the substrate specificity of the enzymes. However these methods are
expensive, and laborious similar to that of GC. The choice of the mobile
phase for the separation of the lipid classes is another major consideration
with this technique.
1.2.1.3
Fluorescent methods
These methods involve measurements of reaction products that
become fluorescent upon hydrolysis. The assay is usually very sensitive and
can be continuously monitored. The overall sensitivity of a fluorescent assay
using synthetic triacylglycerols or esters depends on the sensitivity of
detection and on the specific activity of the lipase for that substrate.
Fluorescence based assay are also much less confounded by turbidity in
samples that may arise when analyzing cell lysates (Gilham et al 2005).
20
It is possible to use triacylglycerols that have one of the alkyl
groups substituted with a fluorescent moiety such as pyrene (Thuren et al
1987). A quencher residue (trinitrophenylamine residue) has been introduced
to this type of substrate molecule as a means of decreasing the basal
fluorescence of the triacylglycerol analogue containing the pyrene group
resulting
in
1-O-hexadecyl-2-pyrene-decanoyl-3-trinitrophenylaminodo-
decanoyl-sn-glycerol (Negre et al 1985). Pyrene fluorescence of the intact
lipid molecule is very low as the pyrene emission spectrum overlaps
efficiently with absorption of trinitrophenylamine. The pyrene fluorescence is
hence quenched intra molecularly, and the increase in fluorescence during a
lipase assay can be measured in a continuous fashion using this substrate.
Another method of monitoring lipase activity on pyrene modified
triacylglycerol analogues uses 1,2-diol-eoyl-3-(1-pyren-1-yl)decanoyl-racglycerol, which includes a pyrene decanoic acid as one of the three fatty acyl
groups of a triacylglycerol at a primary position. Pyrene forms excimers in
close proximity, which have unique fluorescence properties. Upon liberation
of the pyrene group by lipase activity, decreased excimer fluorescence can be
observed. Alternatively, the liberated pyrene can be monitored via an increase
in fluorescence that would appear in the aqueous phase after extraction with
organic solvent. The draw back in this method is that the chemical
modification of a triacylglycerol with a pyrene group can result in poor
hydrolysis of some lipases, likely due to steric considerations. This potential
downside is overcompensated by the high sensitivity and reproducibility of
the assays.
Esters of 4-methyl umbelliferone is another commonly used
substrate in flurogenic study of lipase activity. This compound becomes
highly fluorescent after hydrolysis of the ester linkage. The solubility of some
the substrates are poor in aqueous phase and not easily hydrolyzed, so it
21
cannot be used for non lipolytic esterases. The possible draw backs to using
these substrates for measurements of lipolysis are that the substrates more
closely resemble mono acylglycerol rather than triacylglycerol and it has been
reported that these substrates can spontaneously hydrolyze with or without
albumin at pH 8.8 and higher (Nyfeler et al 2003). The octonate derivatives is
more stable in aqueous environments and can tolerate a greater range of pH,
however, this compound is not currently available commercially (Gilham
et al 2005).
1.2.1.4
Other methods
Apart from the above common methods there are other existing
new methods for determination of enzyme activity. IR thermographic analysis
is a method in which they are able to visualize the activity based on the
temperature difference arising solely from the catalytic activity of the catalyst
on the substrates( Manfred et al 2001). Ciruclar dichrosim is another method,
which is mainly to find out the stereo specific nature of the catalyst. New
methods have been extended were there is no need for an HPLC system
coupled to the circular dichrosim detector to detect the activity of the enzyme
(Manfred et al 2000). Mass spectrometry is another method that is highly
precise and accurate developed to estimate the activity of enzymes (Zhouxin
et al 2004). All these new technology are found to highly accurate but, they
are expensive and laborious
1.2.2
Porphyrins
Porphyrins are a ubiquitous class of naturally occurring compounds
with
many
important
biological
representatives
including
hemes,
chlorophylls, and several others. There are additionally a multitude of
synthetic porphyrinoid molecules that have been prepared for purposes
22
ranging from basic research to functional applications in society. All of these
molecules share in common the porphyrin macrocyclic substructure. They are
aromatic and they obey Huckel's rule for aromaticity in that they posses 4n+2
pi electrons which are delocalized over the macrocycle. Porphyrins and their
derivatives are dyes with particular photo physical and photochemical
properties that strongly depend on the substituents attached to the tetrapyrrolic
ring and on the surrounding medium. They can be modified by connecting
different peripheral substitutes, changing the central metal or expanding the
size of the macrocycle. Their absorption spectra are characterized by a band in
the ‘red’ region and therefore due to this fact and to the better penetration of
red light through biological tissue they possess wide potential for use in
clinical treatment as photosensitizers in photo dynamic therapy (PDT) and
tumour diagnosis. PDT is based on selective accumulation of photo
sensitizing agents in tumours and is a method showing significant promise in
tumour therapy. The concentration of the pharmaceuticals used usually in
PDT is between 10-3 and 10 -6 M (Hill et al 1995).
In nature, many porphyrin systems are already known – such as
hemoglobin, myoglobin (storage of oxygen), chlorophyll (solar energy
transfer) or cytochrome c (electron transfer) which play a significant role in
living organisms because of their properties. This has promoted the research
of these functions in non-living systems. Porphyrins are versatile molecules,
whose physiochemical properties are very sensitive to the modification of
their electronic distribution on the aromatic ring (Figure 1.6). This makes
them excellent building blocks, which can create supra molecular
architectures with very good spectroscopic properties. They can potentially be
used as sensors, opto-electronic devices, antenna systems, etc.
23
Figure 1.6 Structure of various Porphyrin molecule
Porphyrins are soluble in various organic solvents such as
dichloromethane, toluene, etc. For the past few years research is focused on
the synthesis of water soluble porphyrins and their application. A sufficient
solubility is achieved by the introduction of water-solubilizing groups on the
porphyrin periphery. Most of described derivatives possess positively charged
groups. On the other hand, there are only a few examples of negatively
charged porphyrins that are prepared mainly by the introduction of
carboxylate, sulfonate groups into the porphyrin periphery and recently
phosphonium based cathionic porphyrins.
Water-soluble porphyrins have attracted considerable attention due
to the binding affinity to synthetic or natural nucleic acids (Fiel et al 1979)
24
and the ability to selectively cleave DNA (Armitage et al 1998). Interaction
of proteins with water soluble proteins have also been invesigated in recent
years (Suzana et al 2002). They are used as receptors in saccharide
recognition (Oleksandr Rusin et al 2001) and as sensors to detect the level of
toxins in water Mufeed et al 2005).
1.3
METAL OXIDE
1.3.1
Nano size
A nanometer is about the width of six bonded carbon atoms, and
approximately 40,000 are needed to equal the width of an average human
hair. At the nanoscale, the physical, chemical, and biological properties of
materials differ in fundamental and valuable ways from the properties of
individual atoms and molecules or bulk matter. Nanotechnology is the
understanding and control of matter at dimensions of roughly 1 to
100 nanometers, where unique phenomena enable novel applications
(Figure 1.7).
Figure 1.7 Graphical representation of various size systems
25
1.3.2
Synthesis of Nano Sized Materials
The process conditions required for the synthesis of monodisperse
particles of micrometer size are relatively well established, and a similar
principle could be applied to the synthesis of uniform-sized nanocrystals. The
inhibition of additional nucleation during growth, in other words, the
complete separation of nucleation and growth, is critical for the successful
synthesis of monodisperse nanocrystals.
Thus, different techniques for the production of various materials
have been developed under different conditions. Generally, porous materials
can be prepared following three procedures. The first is the dealloying
process, which involves the selective dissolution of a specific metal from a
metal alloy. For example, porous gold can be prepared by dealloying a
silver–gold alloy—the silver phase is dissolved using nitric acid, leaving the
gold phase intact. The second is an electrochemical process in the presence of
templates (soft or hard). Ordered macroporous gold and platinum films, for
example, have been produced by electrochemical reduction of gold or
platinum complexes dissolved in aqueous solution within the interstitial
spaces of a polystyrene colloidal array. The third approach for preparing
porous materials is by a (thermal reduction of metal-ion-impregnated porous
supports and simultaneous) template-sacrifice route. This route involves first
soaking a porous template in a colloidal metal sol or metal salt solution to
load the template with the metal or its soluble precursor, then obtaining
porous metal structures by calcining the organic phase or, particularly in the
case of inorganic templates, by dissolving away the original porous materials.
For example, echinoid (sea urchin) skeletal plates were immersed in gold
paint and a continuous coating of gold was deposited over the whole surface
area. Dissolution of the original calcium carbonate support in acid solution
produced a porous structure with 15 mm channels. Dominic et al (2003) have
26
demonstrated the fabrication of macroporous frameworks of silver, gold, and
copper oxide, as well as composites of silver/copper oxide or silver/titania by
heating metal salt-containing pastes of the polysaccharide dextran to
temperatures between 500oC and 900 oC. Recently, Zhang and Cooper (2005)
modified this approach, by using emulsion-templated polymers as scaffolds,
for the production of macroporous materials from nano particulate building
blocks. In addition, Yamada et al ( 2004) recently prepared nanoporous films
with different particle sizes and agglomerated states by a two-step strategy,
i.e. preparing colloidal solutions and subsequent salting-out of the colloidal
solutions with salts.
Although
these
various
synthesis
approaches
have
been
successfully used to fabricate many porous metals, the preparations of
precursors for the dealloying process and the prerequisite interstitial spaces
for the electrochemical process make these two methods restricted.
Comparatively, the third route is more advisable and practical. The
hydrothermal method makes many starting materials undergo quite
unexpected reactions and serves as a useful tool for preparing fine inorganic
particles.
The pores material are the common type of nano materials
synthesized, apart from them in recent years there are vast number of
publications, reporting synthesis of materials in various forms.
The shapes and sizes of nanoparticles were controlled by changes
in the ratio of the concentration of the capping polymer material to the
concentration of the metal cations used in the reductive synthesis of colloidal
particles in solution at room temperature. Tetrahedral, cubic, irregularprismatic, icosahedral, and cubo-octahedral particle shapes were observed,
27
whose distribution was dependent on the concentration ratio of the capping
polymer material to the platinum cation (Temer et al 1996).
Ultra long belt (ribbon) like were successfully synthesized for
semiconducting oxides of zinc, tin, indium, cadmium, and gallium by simply
evaporating the desired commercial metal oxide powders at high
temperatures. They have a rectangle like cross section with typical widths of
30 to 300 nanometers, width-to-thickness ratios of 5 to 10, and lengths of up
to a few millimeters. The belt like morphology was found to be a distinctive
and common structural characteristic for the family of semi conducting oxides
with cations of different valence states and materials of distinct
crystallographic structures. The synthesized nanobelts could be an ideal
system for fully understanding dimensionally confined transport phenomena
in functional oxides and building functional devices (Zheng et al 2001).
Nano wires and oriented nanorod arrays of zinc oxide particles
were synthesized. The synthesis involved a template-less and surfactant-free
aqueous method, which enables the generation at large-scale, low-cost, and
moderate temperatures, advanced metal oxide thin films with controlled
complexity. The strategy consists of monitoring of the nucleation, growth,
and aging processes by means of chemical and electrostatic control of the
interfacial free energy. The methods enables to control the size of nano-,
meso-, and microcrystallites, their surface morphology, orientations onto
various substrates, and crystal structure (Vayssieres 2003).
1.3.3
Iron Oxide
The synthesis of magnetic nanoparticles has received increased
attention as the possibility of creating functional materials became more
apparent, generating interest as isolable sequestering agents for removal of
28
solution-phase contaminants (magnetically assisted chemical separation), heat
transfer reagents, and medical imaging enhancers (Jennifer et al 2007).
Typically, colloidal magnetite is synthesized through the reaction of
a solution of combined Fe(II) and Fe(III) salts with an alkali. Micellar
surfactants have been used as microreactors in the synthesis of maghemite
(Fe2O3). Ordered arrays of magnetic nanoparticles can also be synthesised
through templating methods. Maghemite structures were obtained by
depositing Fe(NO3)3
in between the pores formed by a network of
polystyrene beads (Yue et al 2004).
As with other nanomaterials,
functionalization chemistry provides an opportunity to alter solubility and
impart stability to as-synthesized materials. Surfactants have been used to
impart temporary stability to magnetic particles, allowing for subsequent
functionalization. Aqueous maghemite (Fe2O3) particles with average
diameters of 8 nm were synthesized by reacting a mixture of Fe(II)/Fe(III)
ions with NaOH in the presence of sodium dodecylsulfate (SDS) (Shi et al
2004).
Since some of the most promising applications of magnetic
nanomaterials lie within the medical imaging field, functionalization designed
with biological environments in mind has been an area of increasing focus.
The use of a cubic silsesquioxane ligand to functionalize magnetic materials,
resulting in excellent stability in a variety of aqueous solutions, resisting
aggregation upon encountering environmental variations such as changes in
pH and salt concentration (Benjamin et al 2006).
1.3.4
Copper Oxide
Copper oxide has been extensively studied because of its close
connection to high-Tc superconductors. The valence of Cu and its fluctuation
29
are believed to play important roles in determining the superconductivity of
various types of cupric compounds. Cupric oxide has also been known as a
p-type semiconductor that exhibits a narrow band gap (1.2 eV) and a number
of other interesting properties. For example, monoclinic CuO solid belongs to
a particular class of materials known as Mott insulators, whose electronic
structures cannot be simply described using conventional band theory
(Xuchuan et al 2002).
It is well known that copper oxides can be conventionally obtained
by the thermal decomposition of copper salts in solid state, for instance, the
nitrates, hydroxides or sometimes the hydroxysalts obtained from the direct
deposition method (Carel et al 1999). This simple method allows the
preparation of the tenorite copper oxide in large amounts. However, it is too
difficult to control the grain size of the resulting copper oxide particles
through this method (Carel et al 1999). Several new synthetic approaches
have been developed in the aim to achieve the preparation of nano-sized CuO
particles. Recent methods of preparation of nano sized copper oxide includes,
stable colloidal solution of copper oxide by inter phase synthesis (Vorobyova
et al 1999), sonochemical method in various organic solvents (Vijaya et al
2000), alcohol thermal deposition of copper acetate (Zhong et al 2002).