Lipase-Catalyzed Synthesis and Applications of Bio-conjugates Report

This report provides a comprehensive overview of lipase-catalyzed synthesis and the applications of bioconjugates. It begins with an introduction to lipases and bioconjugates, detailing their properties and various types, including polymer, nanoparticle, fullerene, and dendrimer bioconjugates. The report then delves into the lipase-catalyzed synthesis using substrates such as polyphenols, ascorbic acids (Vitamin C), tocopherols (Vitamin E), and retinols (Vitamin A). Furthermore, it explores the food and health applications of these bioconjugates, including their roles as antioxidants, surfactants, antimicrobial agents, and their potential in anti-cancer and anti-tumour treatments. The report also discusses potential areas for future studies and concludes with a summary of the key findings, providing a detailed analysis of the subject matter and its implications.

Running head: LIPASE-CATALYSED SYNTHESIS AND BIO-CONJUGATES
LIPASE-CATALYSED SYNTHESIS AND APPLICATIONS OF BIO-
CONJUGATES
Name of the Student:
Name of the University:
Author note:

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