Lipase-Catalysed Synthesis and Applications of Bioconjugates

This article discusses the industrial uses of lipases and the synthesis of bioconjugates using lipase-catalysis. It explores the applications of bioconjugates in health and food industries, such as surfactants, antioxidants, anti-tumour, anti-cancer, anti-inflammatory, and anti-microbial agents. The article also covers different types of bioconjugates, including polymer bioconjugates, nanoparticle bioconjugates, fullerene and carbon nanotube bioconjugates, and dendrimer bioconjugates.

Running head: LIPASE-CATALYSED SYNTHESIS AND BIO-CONJUGATES
LIPASE-CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-
CONJUGATES
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1LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
Table of Contents
Introduction....................................................................................................................2
Bio-conjugates................................................................................................................4
Polymer bioconjugates...............................................................................................5
Nanoparticle Bioconjugates.......................................................................................5
Fullerene and Carbon Nanotube Bioconjugates.........................................................6
Dendrimer Bioconjugates...........................................................................................6
Lipases............................................................................................................................7
Lipase-catalysed synthesis...........................................................................................10
Polyphenols..............................................................................................................10
Ascorbic Acids (Vitamin C).....................................................................................12
Tocopherols (Vitamin E)..........................................................................................13
Retinols (Vitamin A)................................................................................................15
Food and Health applications of Bio-conjugates.........................................................17
Antioxidant and Anti-Inflammatory Applications...................................................17
As Surfactant, Anti-Microbial and Anti-Allergen...................................................19
Anti-Cancer & Anti-Tumour Applications..............................................................23
Future Studies...............................................................................................................25
Conclusion....................................................................................................................26
References....................................................................................................................28

2LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES

3LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
Introduction
Lipases are enzymes which aid in the catalysis of lipid hydrolysis and belong to the
subclass of enzymes known as esterases. Lipases exert essential intrinsic functioning by
contributing extensively to the digestion, metabolism, transport and circulation of lipids
acquired from foods, such as oils, fats and triglycerides. In addition to humans, lipases are
also present in several microorganisms, for example certain virus also possess genes specific
to the coding of lipases (Hasan, Shah and Hameed 2006). For the purpose of hydrolysis of
lipids, lipases exert their function specifically upon a part of the backbone of a glycerol,
present in the lipid substrate. Such characteristic functioning can be explained best by taking
examples from lipase secreted by the pancreas in humans, known as human pancreatic lipase,
which metabolises triglycerides obtained from intake of fats and oils into two fatty acids and
monoglycerides – and is the primary driver in the breakdown and metabolism of lipids in the
human body (Verma, Thakur and Bhatt 2012).
Lipases are also employed extensively for the purpose of industrial functioning. The
following report will focus extensively on the various industrial uses of lipases. Since
traditional times lipases have been employed by humans for the purpose of dairy product
formulation, such as the fermentation of yogurt and cheese. Lipases area also considered and
utilised by industrial as an inexpensive catalyst with the wide range of uses and versatility
(Gandhi 1997). In biotechnology, enzymes which are recombinant lipases are being utilised
as biocatalysts as well as for the purpose of additional applications such as manufacturing of
laundry products, ingredients for bakery and detergent uses. Lipases are also being
considered for the purpose of producing alternative sources of energy, such as in the
conversion to fuel from vegetable oils (Schmid and Verger 1998) For the purpose of
processing of biodiesels, lipases are being utilised as an inexpensive and environmentally

4LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
feasible alternative, due to their ability to exert catalytic functioning similar to traditional
catalysts which consume high rates of energy. For the purpose of industrial applications,
lipases are generally extracted from animals, despite microbial sources are not new as
alternative methods of extraction. For adequate industrial usage and application of lipases,
these enzymes undergo process intensification with the aid of small scale continuous
processing and flow bioreactors (Pandey et al. 1999). The following paragraphs of this
review will aim to shed extensive light on the benefits associated with the industrial
application of lipases as well the characteristic functions of catalysis of product synthesis in
the presence of various substrates such as polyphenols, ascorbic acids, tocopherols and
retinols.
The process of bioconjugation involves chemical strategies for the purpose of
establishment of a link between two molecules, of which one molecule must be a
biomolecule (Medintz et al. 2005). Due to advancements in technology and science, and
growing field of knowledge on the various benefits and scope of functioning of biomolecules,
have resulted in the applicantion of synthetically formulated biomolecules in a wide variety
of applications and industry. Biomolecules which have been modified synthetically are now
being considered for applications such as determination of protein distribution, identification
of functioning of enzymes, monitoring of events and processes at the cellular level,
deliverance of drugs to targeted cells and for the purposes of imaging specific biomarkers
(Gil and Hudson 2004). With the aid of the bioconjugation process, it is possible to establish
associations between biomolecules and various substrates. The manufacturing and synthesis
of bioconjugates poses several challenges and may include simple, crude procedures such as
a fluorescent dye marker used non-specifically, to complex processes such as conjugates in
drug antibodies (Jaiswal et al. 2003). Hence, to enhance manufacturing and application of a
wide variety of bioconjugates with various functions, a number of chemical reactions are

5LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
considered and developed for the purpose of establishing links between tow molecules and
for the chemical modification of proteins. Some examples of bioconugation reactions include:
coupling of cysteine residues, coupling of resides of the amino acid lysine, coupling of the
residues of tyrosine, modification of residues obtained from the amino acid tryptophan and
modification of the C- and N- terminus (Meng, Hennink and Zhong 2009). However, due to
the reliance on residues of amino acids and presence of a large amount of residue disrupting
selectivity, such bioconjugation reactions are devoid of efficacy and chemoselectivity –
which is why, the need of the hour is for the development of chemical processes which can
establish specific links between proteins and synthetic molecules (Liao and Hafner 2005). In
addition to lipases, the following review will also focus extensively on the application of
bioconjugates in health as well as food based industries such as surfactants, antioxidants and
as various anti-tumour, anti-cancer, anti-inflammatory, antioxidant and anti-microbial agents.
Bio-conjugates
As researched by Kalia and Raines (2010), the process of bioconjugation involves the
procedure of linking two biomaterials or a biomaterial with a synthetic material, with the aid
of noncovalent intermolecular interactions or covalent bonds for the purpose of designing a
new product with features, properties and varied applications unique in comparison to the
original products. Taking insights from Veronese and Morpurgo (1999), examples of
bioconjugates include modified products being made biomaterial such as sugars, amino acids,
nucleotides or cell, enzyme or protein based complex moieties, or additional organic products
such as nanoparticles, polymers, dendrimers, fullerenes, microgels and liposomes. The
following paragraphs will highlight on the various examples and properties of biconjugae
based systems.

6LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
Polymer bioconjugates
As researched by Faghihnejad, Huang and Zeng (2014), an example of a polymer
bioconjugate involves obtaining a modified synthetic structure which is highly ordered and
can be developed after coupling a building block with a low molecular weight such as
nucleotides, amino acids or oligopeptides wiuth a synthetic polymer, which has the ability of
possessing structures which are self-organized. A key property of synthetic polymer
bioconjugates includes their ability to peform functions which are highly advanced such as
selective catalytic activity and recognition activities which are highly specific. An additional
property polymer conjugates is their high rates of solubility (Lutz and Börner 2008). An
example of this property can be observed into the coupling of a single amino acid into a
repeating unit of ethylene oxide, or polybutadiene-block poly, resulting in the formulation of
a polymer which was soluble in various mixtures alcohol and water and the ability of the
polymer to possess an altered hydrophobicity. Such polymer bioconjugates find wide
applicable in pharmaceuticals and research in pharmacy, and are considered for the
development of electronic nanodevices, biometrics, biosensors and artificially prepared
enzymes (Heredia and Maynard 2006).
Nanoparticle Bioconjugates
A key property of nanoparticle biocojugates is their possession of a diameter which is
less than 100 nm and generally can be characterised as being in the form of spheres, rods and
tubes. Examples of nanoparticle bioconjugates include their development of from various
materials such as polymers like polystyrene, polymethacrylate, inorganic products like silica,
semiconductors and composites with superparamagnetic properties (Wang et al. 2002).
Nanoparticle bioconjugates can be prepared from various processes such as ultrasonic, hot-
soap and sol-gel procedures. Nanoparticle bioconjugates with magnetic properties, due to
their conjugation with a biomolecular shell find applications in terms of imaging in

7LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
therapeutic healthcare and purification of cell components like DNA and RNA. Additional
applications for nanoparticle bioconjugates involve the generation of nanoparticle networks
and structures which find assembly in the nanowires and surfaces on nanoelecltronic devices
(Sperling and Parak 2010).
Fullerene and Carbon Nanotube Bioconjugates
Fullerenes, involving complex arrangement and bonding of carbon atoms, when
conjugated wit biomolecules, find their application in biological processes such as in
diagnostic processes and for the deliverance of genes and drugs. An example of fullerene
bioconjugate function which can be considered is the ability of C60 to perform a cleavage of
DNA strands in surroundings of visible light, after being conjugated with carboxylic acid
(Biju 2014). Additional examples and applications of fullerene bioconjugates in the field of
pharmaceuticals is their ability to exert antiviral and antioxidant properties after being
conjugated with ammonium grousps. Despite such benefits, additional procedures may have
to be considered for the production of fullerene bioconjugates since complex carbon
structures like fullerenes are often insoluble and hydrophobic and find difficulty in water
based solution usage. However, conjugation or covalent binding with hydrophilic groups can
be considerd to mitigate this shortcoming of fullerene bioconjugates (Djordjević, Bogdanović
and Dobrić 2006).
Dendrimer Bioconjugates
Dendrimer Bioconjugates are characterised by their structural properties of branching
and possession of a complex architecture which is multi-layered, with each layer being
referred to as a ‘generation’. Dendrimers bioconjugates can be manufactured using
conjugation with trifunctional aromatic units such as polyhydroxyls and polyethers.
Dendrimers which are smaller in size have structural properties characterising them to be flat
with accessible internal areas (Wu et al. 2006). With their enlargement in size, dendrimers

8LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
undergo alteration in properties resulting in obtaining a shape which is spherical with internal
void areas which can be accessed and hence find application in the pharmaceutical industry in
terms of encapsulating guest molecules such as drugs (Ong et al. 2001). Dendrimers which
are larger in size are completely spherical with no internal areas available for access. With the
increase in layers or generations in dendrimer bioconjugates, their properties alter further in
terms of gaining qualities of multivalency. Such multivalent properties dendrimers allow
expansion of pharmaceutical applications such as the enhancement of cell-ligand interactions
in interventions underlying HIV and cancer treatments (Wängler et al. 2008).
In addition to the above major biconjugate examples, examples such as liposome
based bioconjugates, microgel and hydrogel bioconjugates and cell based biconjugates are
also finding recent application in fields such as deliverance of drugs, engineering of tissues,
cell-based therapy, encapsulation of drugs, fluorescent based detection reagents and
transportation of petides, nucleic acids and proteins in to in vivo sites of cells (Canalle, Löwik
and van Hest 2010).
Lipases
The class of enzymes which are characterised by their ability to catalyse the
hydrolysis of long chain fatty acids such as triglycerides in organisms are known as lipases.
Within monogastric species such as humans, enzymes like lipases are present in the stomach
and in additional organs such as the pancreas, where they are involved in the digestion of
lipids and fats consumed from dietary sources (Ansorge-Schumacher and Thum 2013). In
addition to intrinsic functioning, the lipases to be used for industrial purposes, are obtained
generally derives from animal based sources, especially for pharmaceutical purposes. For
example, lipases derived from pigs’ pancreas are used for the supplementation of the enzyme
in patients who are deficient in the same. In addition to animal sources, lipase may also be

9LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
obtained from various fungal and bacterial strains. Lipases find extensive use in the industry
in applications and fields such as processing of foods, manufacturing of detergents, in
pharmaceuticals, in production of oleo chemicals, in waste as well as in the cosmetic industry
(Jooyandeh, Amarjeet and Minhas 2009).
As researched by Guerrand (2017), lipases present a wide range of functions in
various industrial applications. Lipases find prevalent use in the food industry in the
alteration of fatty acid and glycerol locations or in the replacement of fatty acids with
alternative ones resulting in production of fats and oils with unique processing and
palatability qualities. Lipases obtained from microbial strains can also be used extensively in
the nutritional alteration and modification of edible oils, using similar mechanisms mentioned
above to produce oils enriched with beneficial oleic acid and low triacylglycerol (Ray 2012).
Additionally, lipases can be also be used for the production lean meats due to their ability to
cleave fats from meat products. In the cleaning and detergent industry, the incorporation of
hydrolytic lipases lends household soaps superior dirt removal properties. In the paper
industry lipases enhance pulp and paper production via the removal of sticky deposits known
as ‘pitch’ from paper producing mills since these are generally acquired from wood
components which are hydrophobic in nature (Casas-Godoy et al. 2012). In the leather and
textile industries, lipases aid in the removal of fat and protein deposits from animal hides and
improve rates of fabric absorbency via removal of lubricants. In the cosmetic industry, lipases
are used widely in the production of emollients, creams and oils containing, ethylhexyl
palmitate, isopropyl myristate and isopropyl palmitate (Kademi, Lee and Houde 2003).
As researched by Andualema and Gessesse (2012), the application of lipases in the
industry are currently being valued and preferred in comparison to chemicals due to variety
of benefits and advantages. In comparison to chemical based catalysts, lipases do not require
adverse or specialised environmental modifications and can exert their function efficiently in

10LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
mild temperatures and pH conditions. In organic solvents, lipases also demonstrate greater
levels of stability in comparison to chemicals and also display a large range of specificity and
versatility in a number of substrates (Hill 2003). Additionally, lipases derived from bacteria
are easy to produce in mass quantities using relatively inexpensive techniques due to their
easy availability in microbial and animal based sources, in comparison to the high processing
costs involved in the manufacturing of chemical based biocatalysts (Rasor and Voss 2001).
As researched by Houde, Kademi and Leblanc (2004), a major property of lipases which
grants them advantage and greater industrial preference in comparison to chemicals is due to
their ability to exert optimum and active levels of functioning at relatively ambient
temperatures and do not require high energy expenditures, temperatures and pressures. Hence
the avoidance of such adverse environments for functioning, which is a common feature in
chemical oriented reactions, reduces the possibilities of products which are labile towards
pressure and temperature from encountering destruction. Such characteristics prevalent in
industrial usage of lipases also result in avoidance of incurring high cost during pre-
processing associated with achievement high temperature and pressure conditions
(Fernandez-Lafuente 2010). Additionally, lipases can also be suited to the performance of
industrial applications and manufacturing of products which require high temperatures by
obtaining the same from microbial strains which are thermophilic in nature, that is, are
characterised by their functioning in elevated temperatures. Enzymes like lipases also display
large rates of specificity in terms of substrates in comparison to chemicals, which results in
the prevention of the production of wastes in the processing steam, hence resulting in an
added advantage (Mayordomo, Randez-Gil and Prieto 2000). Additionally, the usage of
enzymes in replacement of chemicals also reduces the possibilities of producing by-products
and problems in the downstream process. An additional novel capability of lipases, which
makes their usage advantageous in comparison to chemicals, is their property to remain stable

11LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
in organic solvents. This allows immobilized lipases to maintain stable functioning even in
industrial reactor temperatures as high as 70C, as compared to the possibilities of
encountering degradation in the usage of chemicals (O’donnell et al. 2010).
Lipase-catalysed synthesis
Polyphenols
With growing technological improvement in terms of nutritional knowledge and novel
food products, current scientific support surrounding present day food systems are exploring
the benefits of nutraceuticals - bioactive compounds with diverse health benefits. Of the
various products available, polyunsaturated fatty acids are being regarded as a beneficial
source of omega 3 fatty acids. Such components remain beneficial, not only in terms of
possessing antioxidant properties but also exert benefits in terms of inflammation and
harmful platelet aggregation along with additional neurological protective consequences
(Sabally et al. 2006). Despite the benefits, it is worthwhile to remember that such nutritious
oil systems are prone to hydrolytic rancidity and oxidation due to environmental exposure
towards temperatyres and humidity not ambient to lipids. Hence, to retain nutritional qualities
of such unsaturated oils, administration of anti-oxidants is of utmost importance. However,
considering the possibilities of the carcinogenic nature of artificial antioxidants, the
incorporation of the natural antioxidant properties of nutraceuticals are being recommended
as a novel alternative – hence resulting in the entry of polyphenolic compounds (Sabally et al.
2006). . Administration of polyphenolic compounds not only lends natural, antioxidant
properties free of complications, but also exerts the associated biological benefits in oil and
lipid systems. However it must be noted that polyphenols remain in possession of a
characteristic low rates of solubility in non-polar systems prevalent in oils and lipids, hence
resulting in their inability to exert full antioxidant potential (Villeneuve 2007). Hence, as
researched by Stamatis, Sereti and Kolisis (2001), this is where lipase catalysed synthesis

12LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
comes in, considering the possible benefits of coupling structures of triacylglyceros with
polyphenols hence leading to the manufacturing of phenolic lipids with unique structures. As
researched by Lue et al. (2005), the structure of phenolic lipids are characterised by the
presence of polyunsaturated or saturated hydrocarbon chain and an aromatic ring containing
one or more substitutes of hydroxyl or methoxyl groups, linked with the aid of ester bonds.
Hence, the application of lipase catalysed synthesis can be considered to play a key
contributory role due to their ability to aid in the enzymatic synthesis of phenolic lipids in a
solvent system which is organic. As researched by Sorour et al. (2012), in the production of
novel compounds like phenolic lipids, lipases find essential application by administering
esterification of phenolic acids and fatty alcohols and in the transesterification of acylglycerol
models with phenolic acids. Such lipase catalysed synthesis of phenolic lipids involving
phenolic and lipid substrates are being considered in the nutritional alteration of edible oils
such as those obtained from flaxseeds and fishes.
Hence, taking insights from Ferreira-Dias et al. (2013), the novel role of lipase
catalyses synthesis in terms of polyphenolic substrates are currently finding application in the
pharmaceutical industry as well as in the growing field underlying manufacturing of
nutritional supplements to cater to the needs of a growing population exhibiting greater levels
of awareness in terms of importance of quality dietary and nutritional consumption. However,
the production of lipase catalysed phenolic lipids encounter major drawbacks in terms of a
low volumetric productivity. Additionally, as researched by Wang and Dordick (1998), the
amalgamation of organic solvents with functional components like polyphenols may hinder
organoleptic properties and disrupt rates of consumer acceptability in terms of consumption
of nutritional supplements. However, to combat these limitations, recent advancements are
incorporating the usage of a system free of solvents which require usage of increased

13LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
substrate concentrations and reduced reaction volumes hence resulting in avoidance in the
need to recover solvents (de Pinedo et al. 2005).
Ascorbic Acids (Vitamin C)
As discussed previously, lipids and unsaturated oils, which are rich in essential
bioactive components beneficial health, pose the highest risk of acquisition of spoilage due to
hydrolytic rancidity. To combat the same, synthetic antioxidants, such as butylated
hydroxyanisole, butyrated hydroxytoluene and butylhydroquinine are used extensively to halt
the detrimental effects of the same. Despite their widespread usage, such synthetic
antioxidants have been deemed to be carcinogenic in nature along with the risk of suffering
from fatal health symptoms associated with their toxic consumption in individuals (Humeau
et al. 1998). Hence, as researched by Stojanović et al. (2013), to combat such processes, the
need of the hour is to explore alternative antioxidant mechanisms, mainly those derived from
natural sources are being considered. Ascorbic acid or Vitamin C has been is one of the most
traditional dietary sources of natural antioxidants and are now finding their way as a novel
application in ensuring the shelf life stability of edible oils with nutritional properties. Hence,
taking insights from the research by Stojanović et al. (2015), lipase catalysed synthesis of
fatty acid ascorbyl esters are being considered as a novel product with extensive antioxidant
capabilities and hence, administer wide range of uses in the food, dosmestic, as well as the
pharmaceutical industry. Fatty acid ascorbyl esters, such as ascorbyl palmitate are
manufactured using immobilized lipase enzyme systems obtained from Candida sp., for the
esterification or transesterification of ascorbic acid in palmitic fatty acid oil systems. Due to
the incorporation of ascorbic acid – a potentially powerful antioxidant, such novel lipase
catalysed ester compounds possess high activity in terms of scavenging free radicals (Karmee
2009). Additionally, such lipase catalysed synthesis ascorbyl ester compounds also possess
an amphiphilic structure which allows them biosurfactant based properties and hence, can

14LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
also be used in detergent manufacturing as well as drug production containing nanoparticle
formulations (Viklund, Alander and Hult 2003). However, a key shortcoming underlying the
process of ascorbyl palmitate is their low rates of solubility in oils. However, the same may
be mitigated through the usage of fatty acid esters which are unsaturated or of shorter chain
length in the esterification of ascorbic acid substrates catalysed by lipase enzymes. Such
methods also aid in the production of ascorbyl esters in higher yields as well as with higher
rates of free radical scavenging activities. It has been estimated that incorporation of oleic
acid in the ascorbic acid and lipid system to be catalysed by lipases, results in the highest
yields of ascorbyl esters (Gulati et al. 1999). Key limitation in the production of vitamin C
based fatty acid esters with the aid of chemical processes are relatively low yields and a
reduced property of regioselectivity. In comparison, lipase catalysed synthesis of ascorbyl
esters, namely those based on palmitate or acyl associated fatty acids pose key advantages as
compared to chemical based ones, due to their association with high regioselectivity and
ability to exert optimum functioning even in moderate industrial pre-processing conditions
(Chang et al. 2009). Hence, as researched by Burham et al. (2009), the lack of a need to
administer high temperature and pressure conditions, ensures high substrate specificity in
lipase catalysed systems and hence prevention of heat labile product degradation and
secretion of unnecessary by-products characteristic in chemically produced ascorbyl ester
systems.
Tocopherols (Vitamin E)
In addition to previously discussed bioactive compounds and ascorbic acids, an
additional compound with powerful antioxidant properties includes alpha tocopherol,
otherwise known as vitamin E. Vitamin E finds itself in a wide variety of usage in terms of
nutritional supplementation or as in cosmetic ointments for topical application (Shimada et
al. 2009). However, despite its benefits, Vitamin E continues to remain a compound which is

15LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
highly labile to heat, oxidizing agents or oxygen hence rendering it low and unpredictable
levels of solubility (Chu, Baharin and Quek 2002). Hence, as researched by Jensen, Engberg
and Hedemann (1999), to mitigate the same, the administration of Vitamin E in the form of
its derivatives is considered as the most preferred industrial alternative. Recent research
implicates that Vitamin E succinate is the most efficient derivative of the same and exerts
excellent in industries associated with food, pharmaceuticals and cosmetics, especially in the
inhibition of cancer. Using the chemical means, as researched by Jiang et al. (2013), vitamin
E generally undergoes esterification by acylation of Vitamin E using chemical means with
carboxylic acids or additional acyl donors such as acid anhydrides, in which the catalysts
utilised include pyridine or tertiary amine. Due to the potential toxic nature of such chemical
based catalysts, the administration of lipase catalysed synthesis of vitamin E derivatives is
being considered as a potentially beneficial alterative. The usage of lipases in the production
of such derivates like Vitamin E succinate, can be advantageous in comparison to chemically
produced alternatives due to their ability to exhibit adequate performance in moderate
environmental processing conditions, their high rates of efficiency in terms of catalytic
functioning and their inherent ability towards selectivity (Hu et al. 2013).
As researched by Aouf et al. (2014), despite the potential advantages of such a lipase
catalysed system, such methods can pose several limitations. There is still a need to further
optimise the rates of lipase synthesised reaction considering the highly slow rates of kinetics.
Difficulties still remain on the need to obtain a suitable lipase and an efficient enzyme-
tocopherol based substrate which can ensure high rates of enzyme activity after dissolving
both the hydrophilic and hydrophobic substrates in the medium of organic solvents.
Additional research is required on improving conditions of enzyme operation to ensure
sufficient enzyme yields. Hence, the need of the hour is to evaluate the function of lipases

16LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
obtained from various organic sources and explore their effects on yields in various operating
conditions and organic solvents (Liu et al. 2014).
As researched by Wei et al. (2014), similar to Vitamin C, tocopherols like Vitamin E
can also undergo lipase catalysed modifications for the purpose of usage as a semisynthetic
antioxidants for the purpose of a wide range of nutrition and food applications, especially for
the purpose of ensuring natural antioxidant properties and stable shelf life in foods, such as
lipid based products susceptible to high rates of rancidity. Hence, as researched by Torres et
al. (2008) lipases play a key role in the production of alpha tocopheryl succinate or acetate
which finds exploratory use in the food and nutrition industry. When administered in
unsaturated oils, such as nutritiously beneficial polyunsaturated fatty acids, Vitamin E acetate
or succinate produced via the action of lipase aids in efficient scavenging of free radicals and
enhances storage and shelf life of the oil by providing protection against lipid peroxidation
and rancidity.
Retinols (Vitamin A)
Micronutrients like minerals and vitamins, are essential for the functioning of various
immunological, enzymatic and metabolic processes in our body, essential for ensuring
sustenance of daily life activities. Micronutrients like Vitamin A or retinols are essential for
the regulation of visual functioning, for administering proliferation and differentiation at the
cellular level and for the purpose of stabilization of free radicals responsible for the
production of mutations in the DNA (Maugard, Tudella and Legoy 2000). Hence,
considering the same, Vitamin A and its derivatives find usage in a wide range of
applications in the cosmetic industry for manufacturing of skin care products and also in the
pharmaceutical industry for production of drugs and nutritional supplements. A prevalently
used vitamin A derivative is Vitamin A alcohol, due to its highest rates of activity among
retinoids (Ansorge-Schumacher and Thum 2013). However, usage of the same has been

17LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
associated with a number of shortcomings. The derivative product is highly prone to
oxidation in the presence of air, light, heat, oxygen or oxidising agents and displays high rates
of instability. Excessive usage of such Vitamin A products also exerts symptoms or
irritability to the skin. Hence, to mitigate the same, there is a need to use esters of Vitamin A
(Orsat, Wirz and Bischof 1999).
As researched by Chunhua, Tao and Tianwei (2006), vitamin A esters like retinyl
esters are manufactured with the aid of chemical methods using catalysts such as inorganic
alkaline products which required high temperatures for optimum functioning. Such chemical
processes not only increased possibilities of degradation of retinols but also result in the
production of interfering by-products. Hence, for this reason, the alternative usage of lipase
catalysed synthesis can be considered as a beneficial means for safe production of vitamin A
esters with optimum anti-oxidant functioning. In comparison to chemically produced
methods, usage of lipase for the production of vitamin A based esters, is beneficial and
industrially economical due to their ability to operate in mild conditions of temperature and
pressure. Additionally, lipases also demonstrate highly efficient rates of catalysis and
selectivity. Esters of Vitamin A, such as retinyl palmitate exhibit impressive rates of stability
and find extensive application in manufacturing of commercial products (Turner King and
Mathiasson 2001).
Hence, from the extensive review highlighted above, it can be implied that lipases are
now emerging as a promising option in the production of a range of products of commercial
importance. As observed, lipases acquired from microbial sources are currently being
attached greater importance with the onset of advancements in science and enzyme
technology. As researched by Rajendran, Palanisamy and Thangavelu (2009), lipases now
comprise of the mostvast and essential group of biologically derived catalysts due to their
inexpensive origins and commendable rates of efficiency even in moderate to high

18LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
environmental processing conditions. However, there is a further need to develop improved
operating industrial processing systems considering the relatively low rates of kinetics and
difficulties in obtaining adequate yields. Lipases are at present, used widely in a variety of
industries and commercial fields such as nutritional supplements, food flavourings,
manufacturing of fine chemicals, agrochemicals, derivation of micronutrient and antioxidant
esters, production of perfumes and cosmetics and biocatalytic resolution of pharmaceuticals
(Gao et al. 2005).
Food and Health applications of Bio-conjugates
Antioxidant and Anti-Inflammatory Applications
As researched by Debets et al. (2011), bioconjugation involves the linking of
biomolecule with other synthetic molecules with the aid of covalent bonds to produce a
product with varied characteristics unique to those present in the previous original product.
Hence, bioconjugates or covalently modified products, due to their unique properties are now
used in wide range of applications especially in the food, nutrition and pharmaceutical
industry.
Bioconjugates are at present used widely in the food industry, especially in the
modification of bioactive compounds like antioxidants to result in improved properties. A
key application of bioconjugates can be observed in an alteration of the properties of
curcumim - a compound widely prevalent in Curcuma Longa, otherwise known turmeric.
Turmeric witnesses extensive application in cooking as a spice, for cosmetic application and
also as a traditional medicine in ancient subject of Ayurveda (Mishra et al. 2009). The unique
and vast nutritional properties of turmeric can be attributed to the compound curcumin – a
bioactive compound with potential anti-carcinogenic, anti-oxidant and anti-inflammatory
characteristics. Additionally, the key function of curcumin lies in its potential to prevent and

19LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
mitigate detrimental effects in Alzheimer’s due to its evidenced ability to dissolve fibrils and
beta – amyloid deposits in the cerebral region of the concerned patient (Singh et al. 2010).
However, despite the wide ranging abilities, it is worthwhile to mention the rates of solubility
in curcumin is grossly low, hence hindering its incorporation in manufacturing of medicinal
products. Hence, to mitigate this condition, bioconjugates play a major role (Kumar et al.
2001).
As researched by Ahmed and Narain (2013) bioconjugation of curcumin to form
dendrimer curcumin conjugates can be performed by performed by click chemistry and has
been implicated to be beneficial in the improvement of solubility of curcumin. Hence, the
current investigations now aim to explore the effects of antioxidant functioning exerted by
curcumin bioconjugates as compared to original curcumin formulations. It has been
evidenced that in comparison to the observed proliferation of amyloid fibrils in the absence of
curcumin, the growth and development of the same have been found to be inhibited in the
presence of curcimum bioconjugates. Hence, this confirms the ability of bioconjugates to
deliver efficient antioxidant properties (Harish et al. 2010).
Additional applications underlying exploration of the antioxidant and anti-
inflammatory properties of bioconjugates, includes the development of antidiabetic
biconjugates. Diabetes is known as a harmful metabolic with chronic endocrinal
complications. At present, with expansion and exploration using emerging clinical models,
treatment underlying diabetes can be implicated to no longer limit its procedures merely to
the alleviation of symptoms of hyperglycemia (Storr et al. 2003). Diabetes at present, due to
detrimental lifestyle behaviours, poses high susceptibility to the acquisition of long term
metabolic complications characterised by oxidative stress, high rates of inflammation and the
resultant fatal disease aggravations in the form of carcinogenic or cardiovascular diseases and
malfunctioning. Hence, medications classified as antidiabetic are current being manufactured

20LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
to possess multiple functions pertaining to inhibition of alpha-glucosidase as well as
administration of antioxidant properties (Zhang et al. 2006). Such novel drugs are considered
to yield beneficial effects since in comparison to general antidiabetic medications, their
functioning is not only limited to mitigation of hyperglycemia but are also aimed at
prevention of the aggravation of harmful metabolic consequences. Hence, taking insights
from the same, the development and manufacturing of antidiabetic bioconjugates is being
considered (Kong et al. 2010). In the research performed by Rattanangkool et al. (2013), the
study witnessed the development of fifteen quercitylcinnamates with the aid of coupling
directly using ester bonds in the DMAP (dimethylaminopyridene) and DCC
(dicyclohexylcarbodiimide). It was found that particularly quercityl esters 8a, 7a and 6a were
able to inhibit selectively maltase of rat intestinal origins as well as sucrose with a potency of
4 to 6 times greater than their parent compounds 8, 7 and 6. Out of the total manufactured
antidiabetic bioconjugates, 6a was discovered to inhibit sucrose and maltase with a 6 times
high potency of inhibiting maltase in comparison to its parent compound caffeic acid. The
radical scavenging property of 6a was found to be similar to that of butylated hydroxyanisole
(BHA). Extensive investigation of the underlying mechanisms of the inhibitory activity of 6a
revealed that it was able to exert its functions of maltase and sucrose inhibition with the aid
of non-competitive and competitive processes. Hence, such research paves the way for
exploration of the potential antioxidant, anti-inflammatory and metabolic altering properties
of bioconjugates hence proving to be a novel breakthrough in the pharmaceutical industry.
As Surfactant, Anti-Microbial and Anti-Allergen
The application of bioconjugates find further usage in the food industry especially in
ensuring the safety of food and commercial products. For the purpose of ensuring the
production, consumption and manufacturing of food which is safe and free from microbes,
the formulation of food preservatives with excellent anti-microbial activities and tolerable

21LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
levels of toxicities is of utmost importance (Ji, Liu and Zhao 2017). The major ingredient in
the production of items with antimicrobial properties include lysozymes obtained from hen
eggs, considering their high rate of antibacterial properties. Despite the widespread usage and
proven levels of effectiveness, the antimicrobial potential of such lysozymes are limited due
to their lack of ability to exert inhibition of gram negative bacterial considering difficulties
associated with the penetration of the lipopolysaccharide membrane. Usage of detergents and
increased temperatures are procedures considered beneficial in the prevention of the
proliferation of gram negative bacteria (Tao et al. 2010). Hence, as researched by Zhang et
al. (2013), the beneficial antimicrobial properties of lysozymes and the surfactant properties
of polysaccharides are coupled with the aid of manufacturing of chitosan-lysozyme
bioconjugates. Such bioconjugates have properties of high molecular weight and display
excellent antimicrobial potency against the growth and development of gram negative
bacteria at room temperature. Further such antibacterial action combined with the excellent
properties of emulsification in chitosan-lysozyme bioconjugates paves the way for the
development of an efficient bioconjugate with the ability to exert applicability in various
fields in the industry.
As researched by de la Cruz et al. (2017), an additional key application of
bioconjugates in the food industry lies in the need to counter the harmful effects of allergens.
The allergens present in proteins can be hidden with the aid of conjugation with moieties
which are biocompatible. The most prevalent allergen in soy is a 34 kDa protein and is the
causative factor for administering allergic symptoms in individuals. Using Maillard reactions,
this protein with allergenic properties can undergo bioconjugation with polysaccharides. Such
conjugation has been proven to significantly reduce the rates of allergic responses and
potential allergenicity in such protein foods hence resulting in increased food acceptability
among individuals susceptible to such responses. The beneficial effects of such conjugates

22LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
have been demonstrated in animal studies where significant levels of IgE were found in mice
after the administration of lysozyme. Whereas, upon administration of polysaccharide
lysozyme conjugates, such mice demonstrated reduced IgE levels (Yang et al. 2011).
Additional innovations in the application of bioconjugates as anti-microbial agents
have been underway. At present, the issues of multidrug resistance has compelled the global
pharmaceutical industry to explore novel approaches and alternative breakthroughs.
Considering their prevalent antimicrobial capabilities, the coupling of nanoparticles with
antimicrobial peptides seems to be an existing innovation in the eradication of microbial
strains which are drug resistant (van t Hof et al. 2001). Taking insights from the research by
Rajchakit and Sarojini (2017), antimicrobial peptides conjugated with god nanoparticles can
be considered as a remarkable breakthrough due to the potent antimicrobial capabilities of
such peptides, the finely tuned conjugation between such peptides and nanoparticles, and the
non-toxic and non-harmful, no-reactive properties of gold. Similarly, as evidenced in the
research by Kumari et al. (2018), bioconjugates prepared by coupling N-acetyl-D-
glucosamine with benothiazole derivatives using linkages of esters. These bioconjugates were
studied extensively to observe their antimicrobial properties against pathogenic microbial
strains such as Candida albicans, Staphylococcus aureus and Escherichia coli. The
bioconjugates were also evaluated to explore their antioxidant properties and anticancer
properties by monitoring against caco and Hep-2 cell lines. The results reported that such
bioconjugates were successfully able to inhibit the growth of the pathogenic microbial strains
in comparison to equal doses of benzothiazol which were not conjugated, along with
demonstrating commendable and significant levels of anticancer properties against cell lines
of caco and Hep-2.
Biconjugates have also been explored extensively in their utility as surfactants. As
researched by Mukhopadhyay et al. (2018), solvents deemed to possess low volatility are

23LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
subjects of eagerness in terms of biocatalytic synthesis of utility fuels and chemicals. In the
following study, the authors demonstrated the novel properties which were similar to a
solvent in neat and water-less polymer surfactant bovine serum albumin (BSA) conjugated
material (Wl-PspBSA). This bionconjugate demonstrated properties of a non-volatile,
increasingly viscous liquid above the its temperature of solid-liquid transition (25 to 27C) and
can be utilised as an effective solvent for the dispersal of a wide range of solutes with
absolute dryness and varied levels of surface chemistries and sizes. The research by Benkő et
al. (2015), aimed to explore the precipitation by anionic surfactants of bovine serum albumin
(BSA), with alkyl chains of increasing lengths, at pH 3.0, at room temperature and in isotonic
sodium chloride solution. The zeta potential, particle size of albumin, the surface charge and
the fluorescent properties of composites of BSA and surfactants were explored in terms of
incorporation of greater amounts of surfactant. Calorimetric analysis as used to evaluate the
thermal stability of the bioconjugate surfactant system while XRD measurement was utilised
to explore the formation of an adequately ordered structure during the process of self-
assembly. Small angle X ray scattering was utilised to characterise the structure of the
precipitated BSA-surfactant nanocomposites. The release properties of bioconjugated drug
was evaluated by enclosing ibuprofen molecules in the BSA-surfactant bioconjugate system.
The authors reported that as a result of increments in the number of atoms of carbon in the
surfactant’s alkyl chains, the fluorescent properties and the structure of the formulated
aggregates can undergo control owing to increments in the hydrophobic characteristics of the
BSA-surfactant conjugate composites. Hence, such bioconjugate surfactant systems display
commendable applicability as carriers and controlled drug delivery systems for the purpose of
regulated release of drug molecules.

24LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
Anti-Cancer & Anti-Tumour Applications
Taking insights from Al-Wabli, AboulWafa and Youssef (2012), it is worthwhile to
note that curcumin obtained from turmeric has been implicated to be in possession of anti-
tumour properties hence proving to beneficial in the development of chronic condition of
cancer. It has been found that dendrimer bioconjugates of curcumins demonstrate anticancer
activities due to their demonstration of apoptosis in metastatic cells. Such functions can be
measured by recording the rate of activation of caspase 3 in neurotumour cells undergoing
metastasis and have been found to demonstrate functions similar in potency to the ones
compared in the original curcumin pharmaceutical product.
A key step underlying the progression of tumours in cancer is the process of
angiogenesis - which is characterised by the growth, development and proliferation of new
blood vessels from the tumour or the surrounding existing vascular tissues. Hence, one of the
key strategies of current cancer therapy is the administration of drugs known as angiotensin
inhibitors (Vicent 2007). A key compound used as an inhibitor of angiogenesis is TNP-470,
which is a synthetic derivative of a fungal product names as fumagillin - and has been found
to be secreted by the fungal strain Aspeegillus fumigates fresenius. Despite its widespread
usage and efficacy, long term complications of nephrotoxicity has been associated which
results in limitations in the complete usage and utilization of TNP-470 in the treatment of
cancer (Ahmed and Narain 2014). Hence, for the purpose of enhancing the safety, efficacy,
accumulation and biological distribution of TNP-470, its bioconjugation in the form of
HPMA polymer conjugated TNP-470 analogs have been implicated to yield beneficial effects
in the prevention of cancer, proliferation of angiogenesis and the avoidance of symptoms of
nephrotoxicity (Ljubimova et al. 2008). The process of bioconjugation between HPMA
polymers and TNP-470 is performed using tetrapeptide linker of biodegradable nature. This
linker (Gly-Phe-Leu-Gly) will be able to encounter cleavage by cathepsin B – a key

25LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
compound which is expressed excessively in malignant cancer cells. Hence, the novel
bioconjugated drug undergoes rapid assimilation and has been found to display improved
anti-tumour activity, increased time of circulation and solubility in the blood, while the
conjugation restricts its possibility to overcome the blood brain barrier - key symptom
underlying nephrotoxic symptoms (Pasut et al. 2009).
Additional bioconjugation procedures which have proven to be beneficial in the
administration of anti-cancer and anti-tumour properties is the conjugation of gold
nanonparticles with biomolecules, as observed in the research by Mahendran and
Ponnuchamy (2018), the authors utilised methodologies which required the coupling of
coumarin on the surface of gold nano particles capped with citrate using simple in situ
procedures. With the aid of processes such as dynamic light scattering, zeta potential
measurements, transmission electron microscopy and ultraviolet spectroscopy, the authors
were able to observe the presence of an in-depth surface chemistry of the biconjugate
particles which were found to possess near spherical shape, improvements in surface plasmon
resonance, hydrodynamic diameter and charges on the surface. Additionally, the authors
reported a dose dependent response from the bioconjugates in the scavenging of free radicals
and were also demonstrated to exhibit cytotoxic functioning against cell lines of MCF-7
breast cancer. Additionally, analysis using a phase contrast microscope demonstrated that the
prepared bioconjugates exhibited apoptosis effects in a time dependent procedure on the
concerned cell lines of the cancer cells. Hence, such a research paves the way for
development and usage of inorganic molecules as bioconjugates and hence presents
possibilities of novel cancer treatment and drug formulation.

26LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
Future Studies
While bioconjugates have demonstrated significant application in a wide variety of
fields, their applications in the field of bioconjugate therapeutics are proving to be an exciting
opportunity in the field of nutrition and pharmaceuticals. The field of bioconjugate
therapeutics refer to the preparation of macromolecule drugs via formation of linkages with
therapeutic molecules to lipid or covalent chemical linking with polymer carrier molecules
(Hermanson 2013). For the deliverance of drugs, the alteration of therapeutic substances via
linking with carrier molecules introduced a wide range of advantages in terms of:
achievement of optimum chemical and physical properties, enhancement of targeting specific
to disease, reduction of toxicity and control of profile underlying drug release (West and
Halas 2003). Taking insights from the research by Li and Mahato (2017), an innovation in
this field is the PEGylation which is characterised by the conjugation of therapeutic proteins
with poly (ethylene glycol) in order to achieve improvements in stability and achieve
increments in half life. Currently, a number of such PEGylated products of therapeutic
proteins are undergoing development or have obtained approval for usage. Additional
innovative bioconjugates undergoing examination in their potency associated with targeting
and drug deliverance include bioxonjugates formulated with the coupling of polymers such as
N-(2-hydrooxypropyl) methacrylamide (HMPA), in which the primary backbone for linking
of functional nutrients and drug groups, comprise of the hydrophilic polymers (Shabangi,
Kuhlman and Flowers 1999).
With the development of such novel bioconjugate systems, the world is rapidly
witnessing a host of innovative developments and alterations in such therapeutic, nutritional
and pharmaceutical systems. With the advancements in biotechnology and material sciences,
a wide range of carrier molecules are now being considered for their incorporation in various
coupling and conjugation systems, such as natural as well as synthetic polymers, proteins like

27LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
antibodies and peptides (Lutz, Börner and Weichenhan 2007). Innovative methods in terms of
polymerization are currently witnessing emergence, such as reversible addition-fragmentation
chain transfer polymerization (RAFT) which provide for opportunities to grant polymers with
specifically controlled properties and less wide distribution in terms of molecular weight
(Bulmus 2008). To further enhance safety and efficacy, innovations including the design of
novel environment and biodegradable stimuli is underway. The development and
advancement in technologies of protein engineering and recombinant DNA have resulted in
the formulation of peptide or protein conjugated therapeutic substances. Lastly, the relatively
novel field of antibody-drug conjugate development holds a promising option for combatting
the menace of several global diseases (Chari, Miller and Widdison 2014).
Conclusion
Hence, to conclude, it can be observed that lipases have found use in a wide range of
industrial applications due to their potential advantage in terms of increased specificity
towards substrates, their ability to function adequately without the need to administer
specialised condition and their ability to not encounter unnecessary production of by-products
during the downstream process. Alternatively, the need of the hour is to explore alternatives
and improvements in processing considering the difficulties in yields and kinetics of lipase
usage in the industry. Nevertheless, lipases have displayed impressive qualities in the
catalysis of substances resultant in innovative production of a wide range of antioxidant
based esterified compounds. Likewise, there bioconjugation and the production of
bioconjugates have proved to be an essential innovation in the drug, pharmaceutical and food
industry, in terms of improve drug delivery, surfactant production and improvements of
existing bioactive compounds in terms of their existing anti-inflammatory, anti-cancer, anti-
tumour and anti-microbial properties. To conclude, the emergence of bioconjugate

28LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
therapeutics has proved to be an exciting opportunity paving the way for improvements,
achievements and innovations in pharmaceuticals and disease management.

29LIPASE CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-CONJUGATES
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