ATRIA INSTITUTE OF TECHNOLOGYANAND NAGAR BANGALORE 24

(Prepared by T.G.Murali M.Sc. MPhil. B.Ed. (PhD) Retd.General

Manager reputed MNC pharma limited and Assistant professor
chemistry ATRIA)

MODULE 1

Module-1 (5 Hours) BIOMOLECULES AND THEIR APPLICATIONS

(QUALITATIVE):
Carbohydrates (cellulose-based water filters, PHA and PLA as bioplastics),
Nucleic acids (DNA Vaccine for Rabies and RNA vaccines for Covid19, Forensics
– DNA fingerprinting), Proteins (Proteins as food – whey protein and meat
analogs, Plant based proteins), lipids (biodiesel, cleaning agents/detergents),
Enzymes(glucose-oxidase in biosensors, lignolytic enzyme in bio-bleaching).

Carbohydrates (cellulose-based water filters, PHA and PLA as bioplastics

What is carbohydrates?
 Carbohydrates or carbs are sugar molecules.
 Carbohydrates are one main nutrients found in foods and drink.
 Break breaks down carbohydrates into glucose
Cellulose-based water filters
 Filtration is a common way to obtain pure drinking water by removing
particles and microorganisms based on size exclusion.
 Cellulose-based filters are affordable and biobased option for the
removal of particles but bacteria are usually too small to be removed by
size exclusion alone.
  Cellulose fibres in two types of commercial paper filters have been given
a positive net charge to trap bacteria through electrostatic interactions
without releasing any biocides.
 The fibres were modified with the cationic polyelectrolyte polyvinylamine
polymer in single layers (1 L) or in multilayers together with the anionic
polyelectrolyte polyacrylic acid (3 L or 5 L) using a water-based process at
room temperature.
 Filtration tests show that all filters, using both types of filter papers and a
number of layers, can physically remove more than 99.9% of E. coli from
water and that the 3 L modified filters can remove more than 97% of
cultivatable bacteria from natural water samples.
 The bacterial reduction increased with increasing number of filter sheets
used for the filtration and the majority of the bacteria were trapped in
the top sheets of the filter.
 The results show the potential for creating water purification filters from
bio-based everyday consumable products with a simple modification
process.
 The filters could be used in the future for point-of-use water purification
that may be able to save lives without releasing bactericides.
 Antibacterial metal nanoparticles into cellulose-based water filters; both
silver nanoparticles (AgNPs) and copper nanoparticles (CuNPs) are known
to have good antibacterial effects.
 Cellulose fibres treated with multilayers of cationic polyvinyl amine
(PVAm) and anionic polyacrylic acid (PAA) have previously been shown to
adsorb more than 99.9% of E. coli in a fibre suspension without leaching
any biocides.
PHA bioplastic
 Polyhydroxyalkanoates (PHAs) have a number of appealing properties
that make PHAs a feasible source material for bioplastics, either as a
direct replacement of petroleum-derived plastics or as a blend with
elements derived from natural origin, fabricated biodegradable polymers,
Polyhydroxyalkanoates or PHA bioplastic

 Polyhydroxyalkanoates or PHAs are linear polyesters produced in nature by

bacterial fermentation of sugar or lipids.
 They are produced by the bacteria to store carbon and energy.
  These plastics are biodegradable and are used in the production of
bioplastics. They can be either thermoplastic or elastomeric materials, with
melting points ranging from 40 to 180 °C.
 The mechanical and biocompatibility of PHA can also be changed by
blending, modifying the surface or combining PHA with other polymers,
enzymes and inorganic materials, making it possible for a wider range of
applications.

Production of PHA bioplastic

 PHA are produced by bacteria and micro-organisms. It’s more

appropriate to refer to it as biosynthesis than production.
 To make PHA, a culture of a micro-organism are fed with nutrients so that
they multiply rapidly.
 Once the population has reached a certain level, the nutrient
composition is changed to force the micro-organism to synthesize
PHA.PHA is be stored by the micro-organisms.
 The PHA can weight as much as 80 % of the organism’s dry weight.
 The biosynthesis of PHA is usually caused by certain deficient conditions
such as the lack of macro elements (phosphorus, nitrogen, trace
elements, oxygen) and the excess supply of carbon sources.

Properties of PHA bioplastic

 PHA polymers are thermoplastic; they become liquid at their melting

point.
 They are UV stable and have a low permeation of water.
 Processability, impact strength and flexibility can be improved with a
higher percentage of valerate in the material.
 PHAs are soluble in halogenated solvents such chloroform,
dichloromethane or dichloromethane.

PLA bioplastic

 PLA is classified as a 100% biosourced plastic: it's made of renewable

resources such as corn or sugar cane.
 Lactic acid, obtained by fermenting sugar or starch, is then transformed
into a monomer called lactide.
 This lactide is then polymerised to produce PLA.
 PLA is also biodegradable since it can be composted.
Definition of Biodegradable polymer
The biodegradation of biodegradable polymers is defined as chemical
decomposition of substances, which is accomplished through the enzymatic
work of microorganisms that lead to a change in chemical composition,
mechanical and structural properties and forming metabolic products, which are
environmentally friendly materials such as methane, water and biomass and
carbon dioxide.
Synthesis and properties of Polylactic acid or Polylactide (PLA)
1. PLA is a biodegradable thermoplastic aliphatic polyester derived from
renewable resources such as corn or sugar canes.
2. Lactic acid is produced by bacterial fermentation of sugarcane or from the
conversion of starch from corn.
3. The lactic acid is oligomerized and then catalytically dimerized to make the
lactide monomer.
4. High molecular weight PLA is produced from the lactide monomer by ring
opening polymerization using a stannous octoate catalyst.
5. Polymerization of a racemic mixture of Land D Lactides leads to synthesis of
poly -DL-Lactide.
6. PDLLA which is not crystalline but amorphous.
7. Poly-L-Lactide PLLA is the product resulting from polymerization of L, L-
Lactide.
8. PLLA has a crystallinity of around 37%, a glass transition temperature 50-
800C and Tm 173-1780C.
9. PLLA can be processed like most thermoplastic into fiber (using conventional
melt spinning) and film.
10. Biodegradation of PDLA is slower than for PLLA due to the higher crystallinity
of PDLA.PLA is used in a number of biomedical applications, such as sutures,
stents, dialysis media and drug delivery devices.
11. Because of its biodegradability it can be used in preparation of bio plastics
useful for food packing and disposable table ware.
Properties (PLA)
 They are thermoplastic.
 Stable to UV radiation.
 The crystallinity found up to 70%
 Good resistance to moisture.
 Good elasticity properties.
 The glass transition temperature (600- 650C)
 PLA possess melting points 173-1780C.
PLA Application medical field
1. PLA used orthopaedic device.
2. PLA Widely used as a material for drug delivery system.
3. PLA used in tissue engineering or regenerative medicine
4. PLA used in cardio vascular implants and dental.
5. PLA used in cancer therapy.
6. PLA used in skin and tendon healing.
Nucleic acids (DNA Vaccine for Rabies and RNA vaccines for Covid19,
Forensics – DNA fingerprinting)
DNA Vaccine for Rabies

Several facts explain the interest in a DNA-based rabies vaccine approach:

 Protection from rabies is commensurate with the presence of adequate
amounts of virus neutralizing antibodies, principally targeted against the
rabies virus glycoprotein.
 The tools of recombinant DNA technology allow facile cloning of the
glycoprotein gene into suitable expression vectors which mediate efficient
in vivo expression of glycoprotein.
 Nanogram amounts of a plasmid-encoded protein in transfected cells can
generate high-affinity antibodies.
 In situ production of the protein within transfected cells facilitates native
post-translational modifications and obviates the issues associated with
protein purification in vitro.
 DNA vaccines provide unique approaches for expanding the spectrum of
immune responses, such as by employing multiple or modified antigens
and targeted delivery to specific cell types or locations.
 Remarkable thermal stability of plasmids (over 50˚C) may be of particular
advantage in tropical climates.
 Generic nature of the production and purification processes of plasmid
vaccines reduces cost and facilitates easier product development,
compared to conventional vaccines.
 A DNA rabies vaccine, in its simplest embodiment, is a eukaryotic
expression vector containing the rabies virus glycoprotein gene, under a
suitable promoter.
 The glycoprotein sequences of Pasteur Virus (PV), Challenge Virus
Standard (CVS), EvelynRokitnicki-Abelseth (ERA), or street virus isolates
have been used by different investigators who evaluated DNA rabies
vaccines.
RNA vaccines for Covid19
 Coronavirus (Covid-19) is an infection diseases by the SAR-CoV-2 Virus.
 Messenger RNA, or mRNA technology, instructs cells to make a protein
that generates an immune response in the body, thus producing the
antibodies against a disease.
 It is basis for Pfizer/BioNTech and Moderna COVID-19 vaccines being
used by governments worldwide, and the UN -supported COVAX global
vaccine solidarity initiative.
 Messenger ribonucleic acid (mRNA) is molecule that provides cells with
instructions for making Protein.
 mRNA vaccines contains the instructions for making the SARS-CoV-2
spike protein. This protein is found on the surface of the virus that cause
COVID-19.
 The mRNA molecule is essentially a recipe, telling the cells of the body
how to make the spike Protein.
 COVID -19 mRNA vaccines are given by injection, usually into muscle of
upper arm.After the protein piece is made, the cell breaks down the
instruction and gets rid of them.
 The mRNA never enters the central parts (nucleus) of the cells, which is
where our DNA (genetic material) is found. Your DNA cannot be altered
by mRNA vaccines.

 Forensics – DNA fingerprinting

Definition:
DNA fingerprinting is a laboratory technique used to determine the probable
identity of a person based on the nucleotide sequences of certain regions of
human DNA that are unique to individuals.
Introduction:
 DNA fingerprinting is used in a variety of situations, such as criminal
investigations, other forensic purposes and paternity testing.
 In these situations, one aims to “match” two DNA fingerprints with one
another, such as a DNA sample from a known person and one from an
unknown person.

Methodology:
  DNA fingerprinting.
 I think a lot of people are first introduced to DNA fingerprinting while
watching crime shows.
 An officer collects some samples from the crime scene.
 They put it in a tube. And then an hour later, they hold up a brightly
colored gel, squint at it, and say, aha, we have a match for the killer's
DNA.
 Then the show is over. Of course, that isn't exactly how things work in
real life. But DNA fingerprinting is an important part of forensic science.
 Although it can't really tell you exactly who committed a crime, it can be
used to help narrow down a list of suspects based on how well their DNA
matches the samples that were found at the crime scene.
 Investigators can also use the DNA results to search specific databases to
find other potential suspects.
 The DNA testing process is comprised of four main steps, including
extraction, quantitation, amplification and capillary electrophoresis.

Proteins (Proteins as food – whey protein and meat analogs, Plant based
proteins)

Proteins as food – whey protein and meat analogs

 The major market for protein application is as a replacement of soya
protein, due to concern for GMO (genetically modified soybean) soybean,
or for vegetarian products in which market animal-base protein such as
whey or casein are getting replaced with pulse protein.
 A drastic global population increase will further require the food industry
to develop nutritious protein source that can replace scarce animal
protein supply in underdeveloped countries.
 Two types of meat products utilizing pulses are meat enhancers and meat
analogues (replacer).
 Meat enhancers utilize protein or flour for gelation, emulsification and
water-binding functionalities. Examples of these processed meat are
salted meat, luncheon meat, boneless ham, sausage, and bologna.
 Meat analogues have appearance and texture resembling meat.
 The basic function of pulses in meat analogue is to form a complex,
three-dimensional gel network in which fine particles of emulsified meat
is trapped, using the starch and non-meat protein as a filler.
 Increased cooking yield for retaining water and fat reduces purge loss
during cooking yet the effect varies depending on different pulse flours.
 Meat replacers or meat analogue use protein for creating texture
resembles meat. Texturized vegetable protein (TVP) processed with
extrusion technology is commonly used for production of meat replacers
and analogues.
 Types of meat analogues vary on its processing and texture.
 Dry products such as structured meat analogues and fibrous protein
products resembles to whole-muscle meat and relatively high density
 High moisture meat analogues are sold refrigerated or frozen and contain
high moisture.
 TVP usually employs mechanical processing with an extruder to obtain
meat like chewy and stringy texture when hydrated. Either flour, protein
concentrate, isolate or a blend can be used as a raw material. Extrusion
process for TVP can denature protein, inactivate anti-nutrient and
provide better flavour.

 While soybean has been a major ingredient for meat analogues and TVP
commercially available in the market, pea protein presents a high
potential partially due to its similar functionality with soy protein. For
example, gel forming capacity, solubility, and emulsifying capacity of pea
protein and soy protein are relatively similar compared to the other types
of legumes while pea protein exhibits better oil and water absorption
capacity than soya bean.

 Two popular types of meat analogue are produced with low moisture
extrusion (<35% moisture) and high moisture extrusion (>50% moisture)

 Low moisture extrusion has been used for many years with the product
made with soy ingredients; yet the sponge-like texture and appearance of
products from low moisture extrusion processing varies significantly from
meat, thus increased attention to using high moisture extrusion cooking
exists.

 Although there is little research published on the processing method,

extrusion processing parameters such as motor torque, die pressure and
specific mechanical energy (SEM) are critical.

 Texturization occurs in a cooling die; thus, a relatively long cooling die is

used for this process. Temperature has to be lower than 100 OC before
exiting the extruder to avoid the expansion, resulting in a high density
product .It showed that SEM (specific mechanical energy) controls
macromolecular transformation as a result of protein-water interaction
followed by cooking die which solidifies the structure to meal like texture.
  Additionally, functional and chemical properties of raw ingredients such
as water binding capacity, emulsion capacity and viscosity profile had
significant influence on texture properties of final products.

Plant based proteins

.

Definition of a plant-based protein. A plant-based diet consists of all minimally

processed fruits, vegetables, whole grains, legumes, nuts and seeds, herbs, and
spices and excludes all animal products, including red meat, poultry, fish, eggs,
and dairy products.
The following healthful, plant-based foods have a high-protein content per
serving:
1. Tofu, tempeh, and edamame
Soya products are among the richest sources of protein in a plant-based diet.
The protein content varies with how the soya is prepared:
Firm tofu (soybean curds) contains about 10 g of protein per ½ cup
Edamame beans (immature soybeans) contain 8.5 g of protein per ½ cup
Tempeh contains about 15 g of protein per ½ cupTofu takes on the flavour of
the dish it is prepared in so that it can be a versatile addition to a meal. These
soya products also contain good levels of calcium and iron, which makes them
healthful substitutes for dairy products.
2. Lentils
Red or green lentils contain plenty of protein, fiber, and key nutrients, including
iron and potassium. Cooked lentils contain 8.84 g of protein per ½ cup.Lentils
are a great source of protein to add to a lunch or dinner routine. They can be
added to stews, curries, salads, or rice to give an extra portion of protein.
3. Chickpeas
Cooked chickpeas are high in protein, containing around 7.25 g per ½ cup. They
can, for example, be added to stews and curries, or spiced with paprika and
roasted in the oven. A person can add hummus, which is made from chickpea
paste, to a sandwich for a healthful, protein-rich alternative to butter.
4. Peanuts
Peanuts are protein-rich, full of healthful fats, and may improve heart health.
They contain around 20.5 g of protein per ½ cup.
5. Almonds
Almonds offer 16.5 g of protein per ½ cup. They also provide a good amount of
vitamin E, which is great for the skin and eyes.
6. Spirulina
Spirulina is blue or green algae that contain around 8 g of protein per 2
tablespoons. It is also rich in nutrients, such as iron, B vitamins — although not
vitamin B-12 — and manganese. It can be added to water, smoothies, or fruit
juice.
7. Quinoa
Quinoa is a grain with a high-protein content, and is a complete protein. Cooked
quinoa contains 8 g of protein per cup. This grain is also rich in other nutrients,
including magnesium, iron, fibre, and manganese.
8. Mycoprotein
Mycoprotein is a fungus-based protein. Mycoprotein products contain around
13 g of protein per ½ cup serving.A very small number of people are allergic
to Fusarium venenatum, the fungus from which the mycoprotein brand known
as Quorn is made. People with a history of mushroom allergies or with
many food allergies may wish to consider another protein source.
9.Chia seeds
Seeds are low-calorie foods that are rich in fiber and heart-healthy Omega-
3 fatty acids. Chia seeds are a complete source of protein that contain 2 g of
protein per tablespoon.Try adding chia seeds to a smoothie, sprinkling them on
top of a plant-based yogurt, or soaking them in water or almond milk to make a
pudding.
10. Hemp seeds ;Hemp seeds offer 5 g of protein per tablespoon. They can be
used in a similar way to chia seeds.
11. Potatoes
A large baked potato offers 8 g of protein per serving. Potatoes are also high in
other nutrients, such as potassium and vitamin C.
12. Protein-rich vegetables
Many dark-colored, leafy greens and vegetables contain protein. Eaten alone,
these foods are not enough to meet daily protein requirements, but a few
vegetable snacks can increase protein intake, particularly when combined with
other protein-rich foods.
A single, medium stalk of broccoli contains about 4 g of protein. Kale offers 2 g
of protein per cup 5 medium mushrooms offer 3 g of protein
Lipids Definition
“Lipids are organic compounds that contain hydrogen, carbon, and oxygen
atoms, which form the framework for the structure and function of living
cells.”
What are Lipids?
These organic compounds are nonpolar molecules, which are soluble only in
nonpolar solvents and insoluble in water because water is a polar molecule. In
the human body, these molecules can be synthesized in the liver and are
found in oil, butter, whole milk, cheese, fried foods and also in some red
meats.

Lipids (biodiesel, cleaning agents/detergents),

Biodiesel:
 Biomass based biodiesel is a recent promoting approach to study
alternative fossil based fuel due to concerns of decreasing oil reservoirs
and less emission of greenhouse gasses (GHG).
 The current research is focused on bio transforming industrial waste like
crude glycerol, lignocellulose waste, and municipal secondary sludge to
renewable fuel (biodiesel) using heterotrophic oleaginous
microorganisms.
 The microbial production of lipid will occupy less arable land and will
not affect the food supply chain.
 Biodiesel production utilizing oleaginous yeast consists of three major
steps, microorganism cultivation (lipid accumulation), cell wall disruption
and lipid extraction from the biomass, and transesterification.
 Lipid is energy storage (lipid droplets) and structural components of the
cell membrane. The lipid droplets are enveloped by phospholipid
membrane and outer cell membrane has to be disrupted to free the
microbial lipid.
 There are two widely known methods, i.e. organic solvent extraction and
mechanical pressing that have been used to extract lipid from lipid
bearing substances.
 The main disadvantages of these methods are the low lipid yield and
long process time required for extraction.
 Therefore, method with high lipid yield and less process time is
required. Traditionally chloroform and methanol based lipid extraction is
effective, but it is time consuming (8–12 h), needs temperature up to
600C and solvents are toxic having safety concern.
 Therefore, decreasing solvent volume and time of extraction are main
factors for cost effective lipid extraction and safety reasons.
Consequently, lipid extraction from dried biomass employing mechanical
cell disruption processes such as bead milling, homogenization,
microwave, ultra-sonication is an energy intensive process
 The non-mechanical methods such as lytic enzyme treatment, alkali and
acid are cost prohibitive for large-scale microbial lipid extraction.
 The moisture content of the cell biomass (which is more than 80% on
weight basis) needs to be removed by oven drying or lyophilization
(dewatering) process before lipid extraction.
 Various researchers have investigated different methods of lipid
extraction and in-situ trans- esterification from wet biomass. Enzyme
assisted extraction.
 Ethanol Simultaneous distillation and extraction process (SDEP) acid
and base hydrolysis.
 Unfortunately, the most cases still require high energy input, time.
Therefore, lipid extraction technologies are up to now within the
laboratory scale.
Detergents
 N-lauroyl sarcosine (N-LS), an amino acid derived detergent (an anionic
detergent, made up of amino acid sarcosine and fatty acid), is non-toxic,
and biodegradable.
 It can disrupt the cell wall by the formation of micelle at certain specific
N-LS concentration, incubation time and temperature.
 It can be safely used for permeabilization of yeast cells to release
intracellular enzyme activities
 There are several chemical surfactants like Triton-100, sodium dodecyl
sulfate (SDS),toluene and cetyltrimethylammonium bromide (CTAB) that
have been used for yeast cells permeabilization.

Enzymes (glucose-oxidase in biosensors) lignolytic enzyme in bio-bleaching

  Electrochemical biosensor based on glucose oxidase
encapsulated within enzymatically synthesized poly(1,10-
phenanthroline-5,6-dione)
 Immobilization of GOx on the GR electrode surface In order to prepare
GOx-modified (GOx/GR) electrode, 3.0 L solution of GOx (1 mg/mL) was
dropped on the electrode surface three times.
 At the end of this treatment GOx/GR electrode was stored for 20 h over
a 5% solution of glutaraldehyde at +4 ◦C in a closed vessel.
 After cross-linking the GOx/GR electrode was thoroughly washed with
distilled water in order to remove noncross-linked enzyme.
 GOx/GR electrode surface was used as the substrate suitable for the
enzymatic polymerization of 1,10-phenanthroline-5,6- dione.
 For this aim, GOx/GR electrode was immersed into A-PBS, pH 6.0,
containing 25 mM of glucose and 5 mM of PD and it was stored at +4 ◦C,
for 24 h.
 By this treatment, enzymatically poly (1,10-phenanthroline-5,6-dione)
modified electrode (pPD/GOx/GR) was prepared.
 Prior to electrochemical measurements the pPD/GOx/GR electrode was
thoroughly washed with distilled water It was thought that the hydrogen
peroxide formed by the interaction between GOx and glucose could be
used as initiator of PD polymerization
Glucose + O2 GOx −→Gluconolactone + H2O2 (1)
Gluconolactone + H2O → Gluconic acid (2)
nPD → (PD)n

Enzymes lignolytic enzyme in bio-bleaching

 Biobleaching of newspaper pulp is a critical factor in the process

of paper recycling. There are many chemically based
technologies for deinking of paper pulp to remove the ink
entrapped in it.
 Enzymatic deinking has to overcome these disadvantages of
chemical treatment through its high efficiency and low
environmental impact.
 Bio-bleaching using enzymes could replace the use of chlorine
and chloride compounds in the bleaching process.
 The biological deinking method using microbial enzymes is quite
promising and eco-friendly, but highly challenging when
compared to chemical deinking
 Researchers have identified the bio-bleaching potential and pulp
modification properties of few microbial enzymes, such as
cellulase, xylanase, pectinase, mannase, laccase, and lipase.
 Their mode of action in bio-bleaching is to facilitate the residual
ink removal by fiber breakage.
 Laccases are an interesting group of multi copper enzymes,
which are widely distributed in bacteria, fungi and even in
higher plants.
 Ascomycetes and basidiomycetes were the major sources for
laccase.
 The ability of laccase to act on a wide range of substrates has
made them very important enzyme in many of the
biotechnological applications.
 They have the ability to withstand a wide range of temperature
and pH.
 They act upon both phenolic and non-phenolic lignin related
compounds as well as highly recalcitrant environmental
pollutants that makes it suitable for its application in
detoxification, de-colorization of industrial effluents and waste
water treatment.
 They can also be used effectively in paper and pulp industries as
well as textile industries.
 Newspaper is composed of ligno-cellulosic material and the
lignin content tends to decrease the paper brightness upon
aging process.
 Larger lignin residues will lower brightness of paper and hence
the paper brightness can be achieved by delignification, a
process of lignin removal.
 Reported the application of white-rot fungi to degrade residual
lignin in the pulp via lignolytic enzymes, such as manganese
 peroxidase and laccase, or by hemi-cellulolytic enzymes such as
xylanase.
 The major problem of paper bleaching is the removal of lignin
and its derivatives, which are linked to xylan and cellulose.
Hence, the lignolytic systems are essential in the bio-bleaching
process to achieve the paper brightness.
 Though many laccase producing strains have been reported
from multiple microbial sources, certain enzymes were unstable
and had poor efficiency in the deinking of paper waste.
 There is a need for prospecting better enzymes for application
in the paper and pulp industry, as paper pulp contains many
compounds and metal ions which might inhibit the enzyme
activity and thereby resulting in a poor deinking efficiency.
 A potential fungal isolate was obtained from mangrove
sediments and optimized for laccase production using solid
state fermentation in this study.