Module – 1: Biomolecules and their application (quantitative)

Module-1
Biomolecules and their application (quantitative)
Biomolecules

Biomolecules also called biological molecule, any of the numerous substances that are produced by cells and
living organisms. Biomolecules have a wide range of sizes and structures and perform a vast array of functions.
The four major types of biomolecules are carbohydrates lipids nucleic acids and proteins.
The chemical composition of living tissue from abundance point of view is given below:
Compound % of the total cellular mass
Water 70-90
Carbohydrates 3
Lipids 2
Nucleic acids 5-7
Ions 1

Carbohydrates: They are basic component of food and principal source of energy which are composed of
carbon(C) hydrogen(H) and oxygen(O) in the approximate ratio of [Link]. Carbohydrates or carbs, are sugar
molecules. Body breaks down carbohydrates into glucose. Glucose, all blood sugar, is the main source of energy
for your body's cells, tissues, and organs. Glucose can be used immediately or stored in the liver and muscles for
the later use.
Types of carbohydrates: The carbohydrates can be divided into three major groups on the basis of hydrolysis
products as follows:
Monosaccharides: These are the simplest group of carbohydrates and cannot be hydrolyzed further to give
simpler units of poly-hydroxy-aldehyde or ketone. They are referred as simple sugar, sweet in taste, colorless,
having solubility in water but sparingly soluble in alcohol and insoluble in ether. These have at least one
asymmetric carbon atom hence they exist in different isomeric form.
Monosaccharides are reducing sugars. The term reducing reflects the fact that some sugars have carbonyl group
(C=O), which can be oxidized to carboxylic acid (-COOH) reducing other chemicals in the process.
Standard test for reducing sugar is benedict’s solution a blue solution that contain copper sulphate. If a reducing
sugar is present the copper ions (Cu I) result in an orange precipitate. Glucose, fructose, galactose, maltose and
lactose are all reducing sugars, but sucrose is a non-reducing sugar.
D-ribose a another monosaccharide sugar is an important sugar used in genetic material this sugar is not used in
energy source but is a part of the backbone of RNA. When OH group of C-2 position is removed (H in place of
OH) from ribose the sugar becomes deoxyribose, which is used in the backbone of DNA. Pentose sugar ribose is
found in every animal cell. It is the main constituent of ATP ADP riboflavin and RNA.

Oligosaccharides: The group of components, which on hydrolysis produce two or more molecules of same or
different monosaccharides until held together by a glycosidic bond are called Oligosaccharides. The carbon that
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 Module – 1: Biomolecules and their application (quantitative)
carries the aldehyde or the ketone can react with any hydroxyl group on a second sugar molecule to form a bond
called glycosidic bond. Based on position of the one OH, glycosidic bonds may be of two types.
α-glycosidic bond linkage between a C-1 α OH and a C-4 OH.
β-glycosidic bond linkage between a C-1 β OH and C-4 OH.
They are crystalline water soluble and sweet to taste. They can be di-saccharides, tri-saccharides, tetra-saccharides
and so on.
Examples of oligosaccharides:
Sucrose or table sugar is found in sugar cane and sugar beet up to 20% by mass. Maltose or malt sugar is not
common in nature except in germinating starchy seeds.
Maltose is produced commercially from starch by a starch hydrolyzing enzyme diastase.
Lactose or milk sugar is a dimer of β-D galactose and either the alpha or beta D glucose. Lactose does not occur
in nature except as a product of mammary gland. Compared to milk of cow Buffalo and goat, lactose quantity is
highest in human milk.

Structure of oligosaccharides. (Picture for education purpose adopted from [Link])

Polysaccharides: These are long chains of sugars stock. They are thread containing different monosaccharides
units as building blocks. For example, cellulose is a polymeric polysaccharide consisting of only one type of
monosaccharides i.e glucose. Polysaccharides glycogen, found in certain animal tissues and starch and cellulose
both of plant origin. Both these polysaccharides consist of only glucose units.

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Polysaccharides can be classified as follows:

Classification of polysaccharides

Some of the complex carbohydrate present in nature are as follows:

Glucans: are polymers of glucose monomer, for example starch, glycogen, cellulose, and chitin
Galactans: are polymers of galactose monomers, e.g. agar-agar, pectin, galacton from snails.
Fructans: are polymers of fructose monomers for example insulin.
Agar-Agar: is a galactan consisting of both D and L galactose. It is used as a microbial culture medium.
Glycogen (animal starch): is the commonly found in fungi and animals. It is water soluble, which gives a red
color with iodine. A starving man first consumes reserve glycogen.
Cellulose: Human cannot digest cellulose. It is digested by termites and ruminants by harboring bacteria and
protozoans that synthesize the necessary enzyme cellulase.
Insulin (Dahlia starch): is the polymer of fructose units linked by beta - 1, 2 glycosidic linkages.

Cellulose
Cellulose was discovered in 1838 by the French chemist Anselme Payen, who isolated it from plant matter and
determined its chemical formula. Cellulose was used to produce the first successful thermoplastic
polymer, celluloid, by Hyatt Manufacturing Company in 1870. Cellulose is the most abundant organic substance
in plant kingdom. Cellulose forms via β(1→4)-glycosidic bonds between D-glucose units.
The linkages in cellulose make it a straight chain polymer. The hydroxyl groups on the glucose molecules form
hydrogen bonds with oxygen atoms, holding the chains in place and conferring high tensile strength to the fibers.
Chemical formula for cellulose is (C6H10O5)n. It is complex carbohydrate, non-water soluble, biodegradable,
tasteless and colorless. Cotton contains 90% and dried hemp 57% of cellulose. In plant cell walls, multiple chains
bond together to form microfibrils. It is predominant constituent of plant cell wall and absent in animals.
In human nutrition, cellulose is a non-digestible constituent of insoluble dietary fiber, acting as
a hydrophilic bulking agent for feces and potentially aiding in defecation. Fibers may protect against the
development of colon cancer, relation and consuming high fiber diets have a low incidence of this disease.
Some animals, particularly ruminants and termites, can digest cellulose with the help of symbiotic micro-
organisms that live in their guts, such as Trichonympha.

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 Module – 1: Biomolecules and their application (quantitative)

Picture is adopted from [Link]

Uses of Cellulose
1. It is used to produce paperboard and paper products.
2. It helps as an additive in various food items.
3. It is used in the production of rayon.
4. It is used as a preservative in cheese as it plays the role of an anti-clumping agent.
5. It is used in making explosives.
6. It is used in the manufacturing of nitrocellulose.
7. It is used in the diet as a fiber supplement

Commercial applications of cellulose

1. Raw material in paper production.

2. Cellulose fiber is used in textile industry.
3. Cotton linen can directly be converted into rayon.
4. Microcrystalline and powdered cellulose are used as drug fillers as well as thickener, emulsifier, and stabilizer in
food.
5. Cellulose is used by scientist in liquid filtration and thin layer chromatography.
6. Cellulose is a building material, and electrical insulator.
7. Cellulose powder is used as inactive tablet fillers.
8. Cellulose powder is used as anticaking agent in food.
9. Cellulose occurs naturally in some foods and also as additive in food manufacturing.
10. Cellulose is used to make water soluble adhesives, for example methyl cellulose and carboxymethyl cellulose.
11. Cellulose is used in making hydrophilic and highly absorbent sponges.
12. Cellulose is converted to nitrocellulose which is used in smokeless gunpowder.

Cellulose based water filters

Creating efficient filters is critical to environmental and human health. While many conventional filters
already exist, they can be expensive, inefficient, or contribute to polluting the environment when disposed of
improperly or if they are not recyclable or biodegradable.
Biopolymers offer an attractive alternative for filter materials that can potentially address the above-
mentioned problems. Biopolymers are readily available and often cheap and easy to process. They mainly
encompass proteins and polysaccharides from both animal and plant sources, including silkworm silks, animal
wools, corn and soy proteins, and cotton cellulose. Biopolymer-based materials and their applications in
filtering have received increasing attention in the past decade.

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 Module – 1: Biomolecules and their application (quantitative)
For biopolymers to be useful in filter applications, they should exhibit certain desirable physical and chemical
properties that are important for absorption and the elimination of specific chemical contaminants or provide
bactericidal or viricidal functionality. The wide range of functional groups available in proteins and
polysaccharides allows for highly selective filtration for pollutants and other contaminants.

(Picture adopted for education purpose from Biopolymer-Based Filtration Materials Christopher R. Gough)

Cellulose based material such filters have the potential to be affordable, lightweight, and biodegradable.
Wood-based cellulose pulp fibers are today used in disposable everyday filters, such as coffee filters and air
filters, and some of the first membranes for microfiltration were produced from cellulose nitrate and
cellophane in the late 1920s. Microfiltration uses membranes with a pore size less than 0.5 μm to physically
remove bacteria from water, such as faecal bacteria e.g. Escherichia coli and Vibrio cholera, which are
approximately 1–2 μm in length. Lately, research has been focused on creating biobased membranes for
micro- and ultrafiltration from cellulose nanofibrils (CNFs).
Filters based on cellulose pulp filters do usually have large ports that facilitate water percolation, but they do
not sufficiently remove bacteria through size exclusion, other techniques are therefore needed to achieve a
bacterium reducing effect. Several groups have addressed this issue by incorporating antibacterial metal
nanoparticles into cellulose based water filters both silver nanoparticle (AgNPs)and copper nanoparticles
(CuNPs) are known to have a good antibacterial effect. These metal nanoparticles can however have a negative
impact on the environment if they end up in water sources as both AgNPs and CuNPs are acutely toxic towards
aqueous organisms. Additionally, the exposure to low concentrations of silver ions, e.g., released by the
AgNPs that end up in nature, could select for silver resistant bacteria.
An alternative method to physically remove bacteria from water, while keeping the filter pore size larger than
bacteria, is to use positively charged filters that adsorb negatively charged bacteria onto the surfaces of the
filters. This allows negatively charged particles much smaller than the filter pore size to be efficiently removed
from water and this is an interesting approach for removing bacteria from water without adding any toxic
chemicals or reducing the flow by reducing the pore size. Both Gram-positive and Gram-negative bacteria
have a negative net surface charge on the cell envelope, due to peptidoglycans, liposaccharides, and proteins
in the cell wall, and this makes their removal non-selective and efficient for most types of bacteria.
Surface grafting, by covalent linkage, is one of the more common ways to alter membrane surfaces. The
surfaces of natural cellulose pulp fibers have a negative net charge but can be modified to obtain a positive
surface charge. Recently, in an article Peña-Gómez et al. have showed that it is possible to remove E. coli
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 Module – 1: Biomolecules and their application (quantitative)
from water using amino-functionalized cellulose membranes, where a cationic polyamine has been covalently
linked to paper filters using dimethyl sulfoxide (DMSO).
Another way to change the surface charge of cellulose fibers, without using organic solvents, is to use
polyelectrolyte adsorption. In this simple and environmentally sustainable process, cationic polyelectrolytes
are physically adsorbed onto the fiber surface. To increase the surface coverage, the adsorption can be repeated
in steps using alternating charged polyelectrolytes using the layer-by-layer (LbL) method. LbL modification
amplifies the amount of cationic polymer adsorbed onto the substrate for each layer leading to a surface charge
overcompensation. Cellulose fibers 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 fiber suspension
without leaching any biocides.
Cellulose filter papers are versatile and diverse tool for microfiltration, that works by trapping particulates
within a random matrix of cellulose fibers full cellulose filter papers can be categorized as quantitative or
qualitative depending on their application.

Credit: [Link] Credit: [Link]

Bioplastics
The prefix ‘bio’ in bioplastics can mean monomers were derived from renewable resources (biomass) and
then polymerized through chemical mechanisms or the polymer was extracted from biomass; the polymer or
the plastic is biodegradable.
The use of ‘bioplastics’ for fossil-derived degradable plastics is discouraged. Using more descriptive
terminologies can be helpful: for example, bio-based durable polyethylene (bioPE) is made from biomass
derivatives but is not readily biodegradable, polybutylene succinate (PBS) is typically fossil-based yet
biodegradable (that is, easily hydrolysable), and poly-hydroxy-alkanoates (PHAs) are biodegradable and bio-
based, at least when the synthesizing microorganisms are grown on biomass.
History of bioplastics:
1862 – Alexander Parkes creates the first man-made polymer from an organic material derived from cellulose.
It was a bio-based plastic and was called Parkesine.
1926 – French scientist Maurice Lemoigne developed poly-hydroxy-butyrate (PHB) from bacterium Bacillus
megaterium. The first bioplastics made from bacteria.
1907 – Leo Baekeland invents Bakelite, and it will be described as a National Historic Chemical Landmark
due to its importance. Bakelite was a synthetic plastic that was revolutionary for its electrical nonconductivity
and heat-resistant properties in electrical insulators, radio and telephone casings and such diverse products as
kitchenware, jewelry, pipe stems, children’s toys, and firearms.

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1990 – Imperial Chemical Industries (UK) developed a bioplastic that was biodegradable. It was called named
Biopol.
1990 – Commercial demand for bioplastics starts to develop, driven by oil price volatility and environmental
concerns.
Bioplastics have two aspects: ‘‘green’’ educt and/or ‘‘green’’ product (where ‘‘green’’ stands for ‘‘sustainable’’).
Use of a ‘‘green’’ feedstock to produce conventional polymers renewability. Synthesis of ‘‘green’’ polymers,
biodegradability.
Biobased polymers are neither necessarily biocompatible nor biodegradable. According to industry association
European Bioplastics, bioplastics are ‘‘polymers that are biobased, biodegradable, or both’’. So, the industry has
adopted a rather large definition. An alternative expression could be ‘‘technical biopolymers.’’ 0.02 global
agriculture land producing precursor for bioplastics. It is produced from corn, cellulose, hemicellulose, sea weeds
and vegetable oils.
Below given table shows, a material that is either renewable or biodegradable qualifies as biopolymer. There are
also ‘‘partly biobased’’ biodegradable and nonbiodegradable biopolymers, if, for instance, only one blending
partner or only part of the feedstock is derived from renewable resources.

Feed stock crop for bioplastic production:

1. First generation biomass: corn, sugarcane, vegetable oils.
2. Second generation biomass: cooking food, foresting, municipal landfills, agriculture industry.
3. Third generation biomass: seaweeds, cyanobacteria and microalgae.

Types of bioplastics
Starch-Based
Simple bioplastic derived from corn starch. Often mixed with biodegradable polyesters
Example: Green Dot Bioplastics has successfully developed cell phone cases from compostable, starch-based
plastics used in food packaging.

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 Module – 1: Biomolecules and their application (quantitative)
Cellulose-Based
Produced using cellulose esters and cellulose derivatives. Example: Major applications for cellulose plastics
include thermoplastics, extruded films, eyeglass frames, electronics, sheets, rods, etc.
Protein-Based
Produced using protein sources such as wheat gluten, casein, and milk. Example: Biopolymers that are protein-
based have become a leading alternative for food packaging. There have been major advances in protein-based
films and coatings for food packaging made from plant and animal proteins.
Bio-derived Polyethylene
Polyethylene that has been produced from the fermentation of raw agricultural materials like sugarcane and corn
rather than fossil fuels. Example: High Density Polyethylene is used in several packaging applications including
crates, trays, bottles for milk and fruit juices, caps for food packaging, jerry cans, drums, industrial bulk containers
etc.
Aliphatic Polyesters
A collection of bio-based polyesters including PLA, PHB, PGA, among others.
Example: Some bio-based polyesters that have gained commercial use or that are currently being investigated are
polylactic acid (PLA), polyglycolic acid (PGA), poly-ε-caprolactone (PCL), poly-hydroxy-butyrate (PHB), and
poly(3-hydroxy valerate). However, only a small number of them are commercially available.

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 Module – 1: Biomolecules and their application (quantitative)

Poly lactic acid (PLA)

Polylactic acid or poly-lactate is obtained from the monomer lactic acid, which is produced from the
microorganism-catalyzed fermentation of sugar or starch. Common raw materials are corn starch, sugarcane, and
tapioca (starch extracted from cassava root). PLA is not a polyacid (polyelectrolyte), but rather a polyester.
PLA is not only biocompatible and biodegradable (N.B. under controlled composting conditions), but it is a
thermoplastic aliphatic polyester produced from non-fossil renewable natural resources by fermentation of
polysaccharides or sugar, e.g., extracted from corn, potato, cane molasses, sugar-beet, etc., therefore allowing the
biological cycle to come full circle with PLA biodegradation, as well as the photosynthesis process.

Poly lactic acid (PLA) production: Steps 1. Corn must first be converted to corn sugar (dextrose, the common
commercial name for D-glucose) through a process that begins with wet milling, a mechanical process in water
that separates starch, as well as other valuable components, from the corn kernel. The starch is then heated with
acid or enzymes, or both, to completely hydrolyze the starch to dextrose. The dextrose is isolated by crystallization
or used as a liquid concentrate.
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The process converts glucose into two molecules of pyruvate, along with two molecules of ATP and two of
NADH. Pyruvate can be metabolized further in several ways depending on the organism and environment. When
oxygen is available, animals, plants and some microorganisms oxidize pyruvate completely to CO2 through a
process known as respiration.
On the other hand, in an anaerobic (without oxygen) environment where oxygen is not available, pyruvate convert
into lactate (the conjugate base form of lactic acid) by reduction by hydrogen supplied by oxidation of NADH
back to NAD+. This route is favored by some microorganisms and occurs in animal muscle tissue during vigorous
exercise, when oxygen transport can’t keep up with demand.
Simple steps:
1. Food source (corn, starch, tapioca and sugarcane)
2. Milling of source release dextrose sugar
3. Bacterial treatment in fermentation for the production lactic acid in a controlled environment
4. further processing leads to production of oligomers of lactic acid called polylactic acid oligomers
5. Next component formation is lactide and then polymer of PLA

Poly-hydroxy-alkanoates (PHA):
PHA is homo-polymer of 3-hydroxybutyrate. It is 100% biobased and biodegradable, insoluble in water, non-
toxic linear polymer and produced from bacteria. PHA microbiologically produced polyester that have tunable
physical and mechanical properties. It is accompanied by low environmental impact due to their biodegradability
and non-toxic nature. Therefore, they are promising candidate for a sustainable future manufacturing. Ranging
from brittle thermoplastic 2 gummy elastomer, PHA properties can be altered by the selection of bacteria,
fermentation conditions and substrate. Due to their flexible properties, PHA eventually substitute polyethylene
(PE) and polystyrene (PS), which are main polymers of today's global polymer market.
Production of PHA:
Microorganism grown in an aqueous solution containing sustainable resources such as starch, glucose, sucrose,
and fatty acids are changed in fermenter to produce PHA.
Biosynthesis - Produced from microbial process (fermenters)-microorganism (alcaligenes eutophus) grown in
an aqueous solution of simple biomolecules such as starch, glucose, sucrose, and fatty acid. Another bacterium
Cupriavidus nector and recombinant Bacillus subtill. Nutrient required are carbon, nitrogen, sulfur and NaCl.
Polyester is deposited in the form of granules in the cell. Later recovered by disrupting the cells. Later conditions
are changes in fermenter and produce PHA.

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 Module – 1: Biomolecules and their application (quantitative)
Simple steps:
1. Renewable feedstock (Hexoses, pentoses, starch, sucrose, lactose etc.)
2. Growing bacteria in fermenters with controlled environment
3. Creating unfavorable conditions for bacteria by reducing nutrients, oxygen, nitrogen, or phosphorus,
increase carbon supply
4. PHA will accumulate naturally at the periphery of the bacterial cell wall.
5. Extraction of PHA from bacteria by centrifugation or cell lysis

Figure adopted Bioplastics- Based plastics as renewable and /or biodegradable alternatives to petro-plastics author- Fachhochschule Technikum)

Nucleic acid
Nucleic acids are macromolecules, essentials to all known form of life. They are composed of nucleotide, which
are monomer made-up of three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. Nucleic
acids are of two types are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Nucleic acids are polymer of nucleotides and hence known as polynucleotides. It is represented by either ribose
sugar C5H10O5 OR C5H10O4. Both the ribose sugars have a furanose ring structure. A molecule of nucleotide is
composed of three smaller molecules phosphate sugar and a nitrogen base. Nitrogenous base purines which have
double ring structure. Purines are of two types, adenine (A) and guanine (G). Pyrimidine has a single ring structure.
Pyrimidines are of two type - cytosine (C) and thymine (T). Thymine is present in DNA, and it is substituted with
uracil (U) in RNA. The nitrogen-based molecule is attached to the sugar molecules by glycosidic bond. A
combination of nitrogen base with sugar is called nucleoside. When phosphate group attach to nucleoside is called
nucleotides.
DNA (deoxyribose nucleic acid) The structure is well explained by double Helix model proposed by Watson
and Crick in 1953. It is a genetic material in all living organism except viruses where genetic material may be
either DNA or RNA. A small amount of DNA is also found in the cytoplasm in cell organelles like mitochondria
and plastids known as extranuclear DNA. Uracil nucleotides are absent in DNA. A DNA molecule is composed
of two polynucleotide chains. The two polynucleotide chains are coiled around each other like a spiral staircase
double Helix. The cross rungs (steps) are formed by nitrogen bases, while phosphate and sugars form the uprights.
The two polynucleotide chains run in opposite direction antiparallel. One chain runs in the 3’-5’ directions, while
the other chain is in 5’-3’ directions. The average distance between the two chains in DNA is 20 Å. One full turn
off Helix called gyre, measures 34 Å in length. The distance between two successive sugar molecules 3.4 Å. Each
gyre accommodate 10 nucleotides.

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The nitrogen bases of two opposite chain exhibit highly specific base pairing. A purine one chain always pair with
pyrimidine in the opposite chain. Among purine adenine (A) pairs only with pyrimidines thymine (T)and vice
versa. Similarly, guanine(G) pairs only with cytosine(C) and vice versa.

Nucleotide structure
Picture adopted from [Link]

Functions of DNA:
It is a genetic material in all prokaryotes and eukaryotes.
It is capable of replication through which it can be faithfully passed on the successive generation.
It is involved in the synthesis of RNA.
It provides the code for protein biosynthesis.
It is involved in mutation and genetic recombination, which brings about variations.

DNA and RNA structure

Picture is adopted from [Link]

RNA (Ribonucleotide): It occurs mostly in the cytoplasm in the eukaryotic cells. Some viruses contain RNA as
genetic material. RNA virus has ribonucleic acid (RNA) as its genetic material. The nucleic acid is
usually single-stranded RNA (ssRNA) but it may be double-stranded (dsRNA). Notable human diseases caused
by RNA viruses include the common cold, influenza, SARS, MERS, Covid-19, Dengue Virus, hepatitis
C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, and measles.
RNA is a single polynucleotide chain composed of nucleotides of adenine guanine cytosine and uracil. Thymine
nucleotides are absent. There are three types of RNA namely ribosomal RNA (rRNA), messenger RNA (mRNA),
and transfer RNA (tRNA) participate in protein synthesis.

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Central Dogma of Life
Life of an organism required co-ordinate function of different organs or tissues mediated by different proteins.
The protein is made up of amino acids and every protein has unique amino acid arranged in a specific sequence.
The information to synthesize proteins with unique amino acid sequence is provided by the nucleic acid present
within the nucleus. DNA present in the nucleus give rise to the specific RNA sequence and that in turn guide the
cellular machinery to synthesize protein. This is considered as the fundamental event which run the life known as
"central dogma of life.
In another word “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of
sequential information. It states that such information cannot be transferred back from protein to either protein or
nucleic acid”

Picture credit [Link] for education purpose

Immunity: Immunity can be defined as the body's ability to guard itself against disease-causing organisms. In
other words, it is the ability to resist an infection by the action of particular antibodies.
Types of Immunity
The two major types of immunity are:

Innate Immunity or Non-specific Immunity: Innate immunity is referred to as the body’s defence system and
helps us by providing the natural resistance components that include natural killer cells, salivary enzymes, intact
skin and neutrophils, etc. This produces an initial response against the viruses at birth preceding the
vulnerability to a pathogen or antigen. Innate immunity can be referred to as a long-term immunity, wherein the
antibodies are produced on itself.

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Acquired Immunity or Adaptive Immunity: Acquired immunity or adaptive immunity is the one which our body
acquires over time. The main function of acquired immunity is to relieve the patient of the infectious disease
and also defend it from attacks in future.

The two types of acquired immunity include:

Active Immunity-Active immunity can be said to be present when an organism's immune system is involved
actively in the production of immune competent cells and antibodies.

Passive Immunity-In passive immunity, the immune competent cells as well as antibodies are transferred from
one organism to another in order to increase the resistance against a pathogen.

Vaccine: A vaccine is a preparation that improves immunity to a particular disease. It is a biologically

prepared product which contains typical agents resembling a microorganism that causes diseases, made from
weakened or dead forms of the microbes, one of its surface proteins or its toxins (antigens). It helps in the
stimulation of the immune system and to identify the invaded microbes as the foreign agent and destroy it so
that the immune system can be recognized and to destroy any microorganism encountered later.

Vaccination is the injection of a dead or weakened organism that forms immunity against that organism in the
body.

Different types of Vaccines

Inactivated Vaccine: Vaccines of this type are created by inactivating a pathogen, typically using heat or
chemicals such as formaldehyde or formalin. This destroys the pathogen’s ability to replicate but keeps it “intact”
so that the immune system can still recognize it.

Attenuated Vaccine: Attenuated vaccines can be made in several different ways. Some of the most common
methods involve passing the disease-causing virus through a series of cell cultures or animal embryos (typically
chick embryos). When the resulting vaccine virus is given to a human, it will be unable to replicate enough to
cause illness but will still provoke an immune response that can protect against future infection.

Toxoid Vaccine: Some bacterial diseases are not directly caused by a bacterium itself, but by a toxin produced
by the bacterium. Immunizations for this type of pathogen can be made by inactivating the toxin that causes
disease symptoms. As with organisms or viruses used in killed or inactivated vaccines, this can be done via
treatment with a chemical such as formalin, or by using heat or other methods.

Conjugate Vaccine: Conjugate vaccines are somewhat similar to recombinant vaccines: they’re made using a
combination of two different components. Conjugate vaccines, however, are made using pieces from the coats of
bacteria. These coats are chemically linked to a carrier protein, and the combination is used as a vaccine

mRNA Vaccine: An mRNA vaccine (or RNA vaccine) is a novel type of vaccine which is composed of the
nucleic acid RNA, packaged within a vector such as lipid nanoparticles.

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How vaccines work?

Vaccines contain weakened or inactive parts of a particular organism (antigen) that triggers an immune response
within the body. Newer vaccines contain the blueprint for producing antigens rather than the antigen itself.
Regardless of whether the vaccine is made up of the antigen itself or the blueprint so that the body will produce
the antigen, this weakened version will not cause the disease in the person receiving the vaccine, but it will prompt
their immune system to respond much as it would have on its first reaction to the actual pathogen.

Working of vaccine. Picture is adopted for educational purpose from [Link]

Some vaccines require multiple doses, given weeks or months apart. This is sometimes needed to allow for the
production of long-lived antibodies and development of memory cells. In this way, the body is trained to fight the
specific disease-causing organism, building up memory of the pathogen so as to rapidly fight it if and when
exposed in the future.

DNA vaccine for Rabies

Rabies:
The word rabies is derived from the Latin word rabere, which means to be mad, to rage, or to rave. The first
written description of rabies in the literature is cited in the Babylon Codex.
In 1885, Louis Pasteur obtained his first success against rabies through postexposure vaccination, but even more
than 125 years later, the disease continues to affect mankind, especially in developing countries in Africa, Asia,
and Latin America.
Rabies is regarded as one of the most important zoonotic diseases in the world. Commonly known as hydrophobia
in man, it is a viral disease that affects the central nervous system (CNS) of humans and warm-blooded animals.
Rabies is transmitted from animal to animal and from animal to man through saliva.
Animal bites introduce the virus into muscle and nerve ending-rich tissues from which it penetrates nerve cells
where it replicates and progressively travels through the spinal cord to the brain. This process usually requires
weeks or even months depending upon the distance from the site of the bite to the brain. The disease causes
hydrophobia in man, hallucinations, aggressive behavior, and paralysis, eventually leading to coma and death.
Once the symptoms appear, rabies is nearly always fatal. Rabies has been recognized for centuries.
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The transmission of Rabies virus could be with different ways, most commonly by bite of a infected animal. It
could be also spread when the virus comes in contact with mucous membrane or an open wound.
Kingdom-Orthornavirae, Phylum-Negarnaviricota, Class-Monjiviricetes, Order-Mononegavirales
Family-Rhabdoviridae, Genus-Lyssavirus, Species-Rabies lyssavirus
Symptoms of Rabies: The initial onset of rabies begins with flu-like symptoms, including fever, muscle
weakness, and tingling. You may also feel burning at the bite site. Other symptoms include insomnia, confusion,
agitation, hallucinations, excess salivation, problems swallowing, and fear of water.
Structure and Genome: Rabies virions are bullet-shaped with 10-nm spike-like glycoprotein peplomers covering
the surface. The virus is enveloped and has a single stranded, negative sense RNA genome. The RNA genome of
the virus encodes five genes whose order is highly conserved. These genes codes for: nucleoprotein (N),
phosphoprotein (P), matrix protein (M), glycoprotein (G), and a viral RNA polymerase (L). All rhabdoviruses
have two major structural components; helical ribonucleoprotein core (RNP) and surrounding envelops. The two
proteins, P and L are associated with RNP. The glycoprotein forms approximately 400 trimeric spikes, which are
tightly arranged on the surface of the virus. The virus nucleoprotein (N) plays critical role in replication and
transcription. Both viral transcription and replication are reduced, if the nucleoprotein is not phosphorylated.

Rabies virus structure

DNA rabies vaccine:

DNA vaccine, using a pCl-neo plasmid encoding the glycoprotein (This is the surface glycoprotein spike and
exists as trimers. There are about 1200 G proteins (400 trimers) per virus particle) gene of a Mexican isolate of
rabies virus, was developed to induce long lasting protective immunity against rabbis virus in dogs. The
worldwide incident of rabbis and high rate of therapy failures, despite availability of effective vaccine indicate
the need for timely and improved prophylactic approaches. DNA vaccination based on optimized formulation of
lysosome-targeted glycoprotein of the rabies virus provides the ancient platform for preventing and controlling
rabies.
A range of parameters including physical, physiological, clinical, immunological, hematological along with the
histopathology profile of target organs was monitored to assess the impact of vaccination. There was no observed
adverse effect despite high dose administration of DNA vaccine formulation. Thus, this study indicates the safety
of next generation of vaccines as well as highlight their potential application.

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Recombinant technology for producing rabies DNA vaccine.

Covid-19
Coronaviruses are a family of viruses that can cause illnesses such as the common cold, severe acute respiratory
syndrome (SARS) and Middle East respiratory syndrome (MERS). In 2019, a new coronavirus was identified as
the cause of a disease outbreak that originated in China. The virus is known as severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). The disease it causes is called coronavirus disease 2019 (COVID-19). In March
2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic.

Symptoms of COVID-19: Signs and symptoms of coronavirus disease 2019 (COVID-19) may appear 2 to 14
days after exposure. This time after exposure and before having symptoms is called the incubation period. You
can still spread COVID-19 before you have symptoms (pre-symptomatic transmission). Common signs and
symptoms can include fever, breathing difficulty, sore throat, cough and tiredness and early symptoms of COVID-
19 may include a loss of taste or smell. People who are older have a higher risk of serious illness from COVID-
19, and the risk increases with age. People who have existing medical conditions also may have a higher risk of
serious illness.

Covid-19 RNA vaccine:

Coronavirus disease (COVID-19) is there an infectious disease caused by the SARS-CoV-2 virus. Messenger
RNA or mRNA, instructs cells to make a protein that generate an immune response in the body, thus producing
the antibodies that provide protection against disease. It is the basis for the Pfizer/ BioNtech and Moderna
COVID-19 vaccine being used by governments worldwide and in the UN supported COVAX global vaccine
solidarity initiative. Messenger RNA vaccine contains the instruction for making the SARS-CoV-2 spike protein.
This protein is found on the surface of the virus that causes COVID-19.
The mRNA molecule is essential recipe, telling the cells of the body how to make spike protein. COVID-19
mRNA vaccines are given by injections, usually into the muscles of upper arm. After the protein piece is made
the cell breaks down the instruction and get rid of them. The mRNA never entered the central part nucleus of the
cell, which is where our DNA genetic material is found. Your DNA can't be altered by mRNA vaccine. The cells
then display the protein piece of its surface. Our immune system recognizes this that the protein doesn't belong
there and being building an immune response and making antibodies for list.

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Covid vaccine. Picture credit [Link]

How does an mRNA vaccine work: mRNA acts as a cellular messenger. DNA, which is stored in a cell’s nucleus,
encodes the genetic information for making proteins. mRNA transfers a copy of this genetic information outside
of the nucleus, to a cell’s cytoplasm, where it is translated into amino acids by ribosomes and then folded into
complete proteins. mRNA is a short-lived molecule, meaning it degrades easily and does not last long inside
cells. By injecting cells with a synthetic mRNA that encodes a viral spike protein, an mRNA vaccine can direct
human cells to make a viral spike protein and evoke an immune response without a person ever having been
exposed to the viral material. These viral spike proteins, or antigens, normally coat the surface of the virus and
are recognized by antibodies and other immune cells that prepare and protect the body against the virus. If a
person is later exposed to the virus, antibodies and other parts of the immune system can recognize and attack the
virus before it can infect healthy cells or cause illness.

Mechanism of mRNA vaccine. Picture is adopted from national human genome research institute (NIH)

DNA fingerprinting
DNA fingerprinting also called DNA typing, DNA profiling, genetic fingerprinting, genotyping or identity testing,
in genetics, method of isolating and identifying variable elements within the base pair sequence of DNA
(deoxyribonucleic acid). DNA fingerprinting or DNA profiling is a process used to determine the nucleotide
sequence at a certain part of the DNA that is unique in all human beings. The process of DNA fingerprinting was
invented by Sir Alec Jeffrey at the University of Leicester in 1985.
The DNA of every human being on the planet is 99.9% same. However, about 0.1% or 3 x 106 base pairs (out of
3 x 109 bp) of DNA is unique in every individual. Human genome possesses numerous small non-coding but
inheritable sequences of bases which are repeated many times. They do not code for proteins. They can be
separated as satellite from the bulk DNA during density gradient centrifugation and hence called satellite DNA.
In satellite DNA, repetition of bases is in tandem for example AAATTTAAATTT or GCGCGCGC etc.
At specific loci on the chromosome the number of tandem repeats varies between individuals. There will be a
certain number of repeats for any specific loci on the chromosome. Depending on the size of the repeat, the repeat
regions are classified into two groups. Short tandem repeats (STRs) contain 2-5 base pair repeats and variable
number of tandem repeats (VNTRs) have repeats of 9-80 base pairs. Since a child receive 50% of the DNA from
its father and the other 50% from his mother, so the number VNTRs at a particular area of the DNA of the child
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will be different may be due to insertion, deletion, or mutation in the base pairs. As a result, every individual has
a distinct composition of VNTRs and this is the main principle of DNA fingerprinting.

Steps of DNA fingerprinting:

1. The first step in this process is to isolate the DNA from the sample material to be tested.
2. Once the required size of the sample is available, the DNA is isolated from the sample and is subjected
to restriction digestion using restriction endonuclease enzymes.
3. The digested DNA sample is then separated by agarose gel electrophoresis, in which the DNA is
separated based on the size.
4. The next step is transfer of separated DNA from gel slab onto the nitrocellulose membrane or nylon
membrane to hybridize with a labeled VNTR sequence probe that is specific for one VNTR region
(radio activity labeled complimentary sequence for VNTR region nucleotide sequence).
5. This technique of transferring and hybridizing DNA onto nitrocellulose membrane is known as
southern blotting, a most widely used DNA detection technique by molecular biologists.
6. After the hybridization with the radioactive probes, the X- ray film (autoradiography) is developed
form the southern blotting and only the areas where the radioactive probe binds will show up on the
film.
7. Now these bands when compared with the other known samples, will give the final result of the DNA
fingerprinting.

Steps of DNA fingerprinting. (adopted from [Link])

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Application of DNA fingerprinting

DNA Fingerprinting is used by scientists to distinguish between individuals of the same species using only
samples of their DNA. It is a primary method for identifying an individual.
Forensic Science: Biological materials used for DNA profiling are Blood, Hair, Saliva, Semen, Body tissue
cells etc. DNA isolated from the evidence sample can be compared through VNTR (Variable number of tandem
repeats) prototype. It is useful in solving crimes like murder and rape.
Paternity and Maternity Determination: A Person accedes to his or her VNTRs from his or her parents.
Parent-child VNTR prototype analysis has been used to solve disputed cases. This information can also be used
in inheritance cases, immigration cases.
Personal Identification: It utilizes the concept of using DNA fingerprints as a sort of genetic bar code to
pinpoint individuals.
Diagnosis of Inherited Disorders: It is also useful in diagnosing inherited disorders in both prenatal and
newborn babies. These disorders may include cystic fibrosis, hemophilia, Huntington’s disease, familial
Alzheimer’s, sickle cell anemia, thalassemia, and many others.
Development of Cures for Inherited Disorders: By studying the DNA fingerprints of relatives who have a
history of some disorder, DNA prototypes associated with the disease can be ascertained.
Detection of AIDS: By comparing the band of HIV “RNA” (converted to DNA using RTPCR) with the bands
form by the man’s blood, person suffering with AIDS can be identified.
Breeding Program: Breeders conventionally use the phenotype to evaluate the genotype of a plant or an
animal. As it is difficult to make out homozygous or heterozygous dominance from appearance, the DNA
fingerprinting allows a fastidious and precise determination of genotype. It is basically useful in breeding
racehorses and hunting dogs.

Proteins
The term protein was coined by Berzelius in 1837 and Mulder 1838. Do approximately 300 amino acids occur in
nature but only 20 make the composition of proteins. Proteins are polymers of amino acids. Protein is found
throughout the body in muscles, bones, skin, hairs and virtually every other body part or tissue full it makes up
the enzymes that power many chemical reactions and the hemoglobin that carries oxygen in your blood. Proteins
are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Protein
performs a vast array of functions within organisms, including catalyzing metabolic reactions, DNA replications,
responding to stimuli, providing structure to cells and organism, and transporting molecules from one location to
another. All amino acid has a common structure the only difference between the different amino acid lies with R
groups in general formula. The R group have quite diverse chemical properties.

Amino acid structure

All amino acid except glycine shows optical isomerism. This can result in two different arrangements beta
form and alpha form. Glycine is the simplest amino acid with lowest molecular weight. It is involved in-
formation of haeme found in hemoglobin. Tryptophane is the most complex amino acid containing indole
ring. Tryptophan amino acid forms the vitamin nicotinamide and a plant hormone indole acetic acid (IAA).

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Amino acid that cannot be synthesized in the body are called essential amino acids, while those which can be
synthesized in the body and need not to be supplied through diets are called non-essential amino acids. Infants
require arginine and histidine in addition. Methionine and cysteine are sulfur containing amino acids.

Essetntial amino acids Non-essential amino acids

Arginine Glycine
Histidine Alanine
Isoleucine Serine
Leucine Aspartic acid
Methionine Asparagine
Phenylalanine Cysteine
Threonine Glutamic acid
Tryptophan Glutamine
Lysine Proline
Valine Tyrosine

Proteins are assembled from amino acid using information encoded in genes. Each protein has its own
sequence unique that is specified by nucleotide sequence of the gene encoding this protein. The genetic code
is a set of three nucleotide sets called codons and each three-nucleotide combination designated an amino
acid.
The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is coded
onto the ribosome and is read 3 nucleotide at a time by matching each codon to its pairing anticodon located
on a transfer RNA (tRNA) molecule which carries the amino acid.
The enzyme aminoacyl tRNA synthetase ‘’charges’’ the tRNA molecules with the correct amino acid. The
growing polypeptide is often termed the nascent chain. Proteins are always biosynthesized from C-terminal
to N-terminal. Proteins are chief actors within the cell, said to be carrying out the duties specified by the
information encoded in genes.
Depending on the number of peptide bonds present in the protein molecules:
1. Dipeptide when two amino acids are joined together via peptide bond a dipeptide is formed.
2. Oligopeptide it is a long unbranched chain of 2 to 9 amino acid residues, which are linked by peptide
bonds.
3. Polypeptide it is a long chain of many amino acids (more than 10 amino acids) linked each end to end by
peptide bond

Structural organization in protein:

1. Primary structure: It consists of linear sequence of amino acid residue in a polypeptide chain. The
enzyme ribonuclease and the protein myoglobin function only in their primary structure.
2. Secondary structure: It is a regular folding pattern of continuous portions of polypeptide chain for
example alpha Helix and beta sheets. Secondary structures are stabilized by hydrogen bonds.
Most globular proteins contain region of alpha Helix together with beta sheets.

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a.) Alpha Helix chains are coiled spirally in right-handed manner. At places the Helix is less regular
forming random coils. The Helix is stabilized by hydrogen bond between oxygen of one amino acid
residue and -NH group of next 4th amino acid residue. This secondary structure is found in several
proteins like keratin, myosin topomyosin, and fibrin.
b.) Beta pleated sheets: here alpha chains are bound by intermolecular hydrogen bonds.
3. Tertiary structure: They are formed by the folding of secondary structure into a complex shape. The
interactions involved in folding include weekend, agent bonds, hydrophobic interactions based on
disulfide bonds between neighboring cysteine amino acids. Enzymes are functional with the tertiary
structure only.
4. Quaternary structures: This consists of two or more polypeptide chains. Hemoglobin, a globular protein
composed of four polypeptide chains (two beta chains and two alpha chain) Each having a haeme group
at the center of chain.

Picture is adopted from Proteins: Structure and Function by Damodaran Vasudevan

Whey proteins: Whey protein is a mixture of proteins isolated from the liquid material created as a byproduct
of cheese production. The proteins consist of alpha lactalbumin, beta lactoglobulin, serum albumin and
immunoglobulins. Glycomacro peptide also makes up the third largest component but is not a protein. Whey
protein is commonly marketed as a protein supplement, and various health claim has been attributed to it.
There is leftover when milk is coagulated during the process of cheese production and contains everything that is
soluble from milk after the pH is dropped to 4.6 during the coagulation process. It is a 5% solution of lactose in
water and contains the water-soluble proteins of milk as well as some lipid contents. Processing can be done by
simple drying or relative protein content can be increased by improving the lactose, lipid and other known protein
materials. The primary uses of whey protein supplements is for muscle growth and development. Eating where
protein supplements before exercise will not assist athletic performance, but it will enhance the body's protein
recovery and synthesis after exercise because it increases the free amino acids in the body's free amino acid pool.

Steps for whey protein production:

Step 1. Whey protein is a dairy product and is obtained from cow’s milk.
Step 2. The milk obtained from the cows does have some harmful bacteria present, so it must go through
pasteurization. Some of the bacteria residing in milk die when cooled at around 4° C. Tons of liters of milk are
cooled under this process.

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Step 3. Cooled milk is then transported in big, refrigerated tankers to the nearest cheese processing facility. Whey
protein is the by-product of the cheese manufacturing process.
Step 4. In the cheese processing facility, the milk goes through a process called pasteurization. Pasteurization is
the process devised by Louis Pasteur. Pasteurization ensures that there are no bacteria breeding in the milk. Milk
is boiled until its boiling point (70-80 ° C) and then instantly cooled at 4° C. Scientists have proven that bacteria
don’t survive when subjected to extreme temperatures. Pasteurization ensures that the milk is free of harmful
bacteria. Post-pasteurization, the milk obtained has 20 % whey and 80 % casein.
Step 5. The pasteurized milk is subjected to enzymes to separate the whey from casein. Casein is the dominant
product in milk and is used to prepare cottage cheese or other variants of cheese.
On the application of enzymes, milk gets separated into two parts – liquid and solid particles. The solid particles
are collected and are processed as cheese. The liquid part is the one which contains whey as well as fats and carbs.
The liquid whey is sent for further purification to remove fats, carbohydrates, water, and minerals.
Step 6. The liquid input from Step 5 is treated to obtain Whey Protein Concentrate (WPC). The liquid whey is
sent to a protein manufacturing plant where it is instantly loaded into a massive web of stainless-steel turbines
that have special ceramic filters. The complete process is natural, and the environment is cold. The current process
separates fats and lactose from the liquid solution eventually producing Whey Protein Liquid.
Step 7. This is the last step in the processing of whey. The whey protein liquid is put into a dryer that has hot as
well as cold air to dry out the water and separate the solids from the liquid. The resultant powder might have
around 90% of Whey. This process also ensures that Whey doesn’t lose its nutritional value and the protein doesn’t
get denatured.
Step 8. The whey protein obtained here is packaged in containers and is tested for purity. The whey obtained is
unflavored and it is sent to Myoprotein facility for further processing.
Step 9. The protein is carefully examined and flavoring is added. The whey powder is put into high-speed blenders
along with flavors for the best taste and mixability. A portion of every batch is also tested for protein content by
a high-tech INR machine (infra-red). Post-mixing and evaluation, it is sealed and packed again and sent to the
warehouse.

Whey protein production. Picture adopted by publication DOI:10.1177/0884533617724759

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Whey Protein Benefits:
Body Composition: Whey protein is very beneficial for both men and women and may improve body
composition without leading to ‘bulking’.
Amino Acids: Amino acids are the chemical units which are not produced naturally in our body. Whey protein
is a good source of amino acids. Whey protein is high in Branched Chain Amino Acids (BCAA’s). They are
useful for muscle repair and preservation. Leucine, another amino acid, stimulates protein synthesis and sends
signals to the body to increase its storage of amino acid.
Aging: Whey protein contains glutathione. This is an antioxidant which scavenges free radicals and delays the
signs of aging. Glutathione is made from three main amino acids; cysteine, glutamic acid and glycine. Whey
protein also slows down the degeneration of muscles and keeps them strong during old age.
Weight loss: Whey protein is low in carbohydrates and high in protein. These speeds up the metabolism to burn
excess fat from the body. Researchers have concluded that whey protein can easily control appetite for up to
two hours. It contains very little fat, low cholesterol, and lactose. This makes it excellent for people on a low-
carbohydrate diet.

Cancer: Cancer, mainly breast and cervical cancer, is the second leading cause of death in women. Whey
concentrates and glutathione modulation is used in cancer treatment. Consumption of whey powder during
chemotherapy can help inhibit the growth of cancer cells.

Immunity: One of the best whey protein benefits is boosting immunity. Whey protein improves immune
response and helps fight infections in women. It boosts the immune system by increasing the production of
glutathione. This is a powerful antioxidant which helps in detoxification.

Meat analogue:

Meat analogues are plant-based and cultured products that are (or aim to be) equivalent substitutes for animal-
derived meat. They are produced from plant or animal cells cultured in a laboratory or bioreactor. What sets meat
analogues apart from well-known meat alternatives: for example, Quorn, or tofu and wheat-based processed
‘meat’ products is that they are aimed at meat-eaters rather than vegetarians or vegans.

Fake meat is raising interest in many consumers who are looking for indulgent, healthy, low environment impact,
ethical, cost effective, and new food products. High moisture extrusion cooking enables the production of fresh,
premium meat analogues that are texturally like cooked meat while high protein or animal proteins. The
appearance and eating sensation are like cooked meat, while high protein content offers a similar nutritional value.

Two broad categories of meat analogues – advanced plant-based ‘meat’ and cultured meat – mark a particularly
radical departure from the traditional meat and non-meat options seen till date. Advanced plant-based ‘meat’
products are those that use plant-derived ingredients to directly mimic animal-derived meat, and which are
designed to be indistinguishable from their animal-based equivalents.

‘Impossible Burger’ contains soy leghemoglobin (SLH), a plant protein. SLH is isolated from the root of the
soybean plant and, like haemoglobin in blood and myoglobin in muscles, it is a molecule that carries oxygen,
storing it in the roots of legumes. When the ‘Impossible Burger’ is cooked and eaten, SLH is exuded as a red
tinted liquid – comparable to myoglobin, the substance that ‘bleeds’ from minced beef – and gives a metallic iron-
like (and thus meat-like) flavor to this product.

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Picture credit [Link]

The factors that lead to this shift is due to low fat and calories food intakes, flexitarians, animal diseases, natural
resource depletion, and to reduce greenhouse gas emission. Currently, available marked meat analogue products
are plant-based meat which the quality (texture and taste) are like the conventional meat. The ingredients used
are mainly soy proteins with novel ingredients added, such as mycoprotein and leghemoglobin.

Cultured meat: Cultured meat is grown in vitro from animal-derived stem cells using a growth medium. The
cells used to initiate the cell culture can be sourced from primary animal tissue through a biopsy procedure;
alternatively, cell lines (stem cells) that can replicate indefinitely can be produced via genetic engineering, gene
editing or through induced or spontaneous mutations. Cells are cultured within specific liquid media, which
provide the conditions needed for tissue growth. The exact media used will depend on the cell species and tissue
type, but the process requires nutrients (supplied by foetal calf or horse serum, chicken embryo extract, collagen,
serum-free media, etc.). Other inorganic and organic components (antibiotic/antimitotics or carbohydrates, amino
acids and vitamins) can be added to the media to enable cell growth. A scaffold is required for cells to proliferate
and develop the structure required for producing a tissue (for example, a muscle) instead of an unorganized
collection of muscle cells. The components used in these processes are dependent on their stages of development,
and research in this area is still in its infancy. For example, even though a few companies, such as Higher Steaks
and Aleph Farms, already use only animal-free growing media, more research is needed for lowering the costs of
serum-free processes.

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Picture credit: [Link]

Plant based proteins: Plant protein is simply and meaningful food source of protein which is from plants. This
group can include pulses, tofu, soya, tempe, seitan, nuts seeds, certain grains and even peas. Pulses are a large
group of plants, which include chickpeas, lentils, beans and split peas.

Plant proteins are highly nutritious not only as good source of protein, but also because they provide other
nutrients such as fiber, vitamins, and minerals. Our intake of fiber tends to be too low, however by incorporating
certain plant proteins into your diet, such as pulses, peas and nuts, you can easily boost your fiber intake.

Consumer demand for plant protein waste product is high and expected to grow considerably in the next decade.
Factors contributing to the rise in popularity of plant proteins include: 1. Potential health benefits associated with
increased intake of plant-based diets 2. Consumer concerns regarding adverse health effect of consuming diets
high in animal protein (e.g increased saturated fat) 3. Increased recognition of the need to improve the
environmental sustainability of food production 4. Ethical issues regarding the Treatment of Animals and 5.
General consumer view of protein as a positive nutrient. While there are health and physical function benefits of
diets higher in plant-based protein, the nutritional quality of plant proteins may be inferior in some respect relative
to animal proteins.

Lipids:

The term ‘lipid’ was first used by Bloor in 1943. Lipids are water insoluble and consist of carbon hydrogen and
oxygen, but the ratio of H and O is more then 2:1. Lipids are a broad group of naturally occurring molecules
which include fats, waxes, sterols, fat soluble vitamins (vitamins A, D, E and K), monoglycerides, diglycerides,
phospholipids, and others. The functions of lipids include storing and signaling and acting as a structural
component of cell membranes. Lipids have application in cosmetic and food industries and in nanotechnology.
Lipids may be broadly defined as hydrophobic or amphiphilic small molecules the amphiphilic nature of some
lipids allow them to form structures such as multi lamellar or unilateral liposomes or membranes in an aqueous
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environment. Biological liquids originate entirely or in part from two distinct type of biochemical subunits or
building blocks ketoacyl and isoprene groups. Biological membrane is a form of lamellar phase lipid bilayer. The
formation of lipid bilayers is an energetically preferred process when the glycerophospholipids described above
are in an aqueous environment. This is known as hydrophobic effect. In an ecosystem the polar heads of lipids
aligned towards the polar, aqueous environment, while the hydrophobic tails minimize their contact with water
and tend to cluster together, forming A versicle, depending on the concentration of the lipid, this biophysical
interaction may result in the formation of micelles, liposomes, or lipid bilayers. Other aggregations are also
observed and form part of the polymorphism of amphiphilic behavior. Some of the applications are within the
body, lipid functions energy reserve, regulate hormones, transmit nerve impulses, cushion vital organs, and
transport fat soluble nutrients. Fat in food serves as an energy source with high caloric density, add texture and
taste.

Classification of lipids:

On the basis of their chemical structure, the lipids are classified into following classes:

1. Simple lipids: Simple lipids like triglycerides, fats, and waxes are formed from fatty acids and alcohol.
a. Triglycerides: Neutral fats such as butter and vegetable oils are mostly triglycerides. Each has three
fatty acids linked to a glycerol.
b. Waxes: These are long chain fatty acid linked to long chain of alcohol. In plants, they cover the surface
of leaf and other aerial surfaces to avoid excess transpiration. In animals, cutaneous glands secrete
wax (lanolin) for forming a protective water insoluble coating on animal fur.

2. Compound lipids
Complex or compound lipids contain an additional group in addition with alcohol and fatty acids.
a. Glycolipids: These contain sphingosine (alcohol) with the fatty acid and monosaccharide sugar i.e
cerebrosides, terpenes and gangliosides.
b. Phospholipids: These are triglyceride lipids with one fatty acid replaced by phosphoric acid which is
often linked to additional nitrogenous group like choline, ethanolamine, etc.
c. Lipoproteins: These are complex of lipids and proteins and are present in blood, milk, and egg yolk.

3. Derived lipids: These are derivative of lipids (steroid) or its chemicals (prostaglandins).
a. Steroids: The group of complex repairs that possess a rigid backbone of four fused together carbon
links. Sterols are the components of every eukaryotic cell membrane for example cholesterol.
b. Palmitic acid saturated fatty acid, found in coconut, etc.
c. Arachidonic acid an unsaturated fatty acid found in groundnut, etc.

4. Complex lipids:
a. very low-density lipoprotein (VDLD) which binds triglycerides in liver and carry them to fat tissue.
b. Low density lipoproteins (LDL) which carry cholesterol to peripheral tissues)
c. High density lipoproteins (HDL) which binds to plasma cholesterol and transport it to liver.

Fatty acids:

Most of the fatty acids found in nature has an even number of carbon atoms usually from 14 to 24. The
general formula for saturated fatty acid is CH3(CH2)nCOOH

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Fatty acids may be classified into following two types:

1. Saturated fatty acids have single bonds only, they are solid at room temperature and their melting point is
high. These are straight chain components, found more commonly in animal tissues for example palmitic acid,
lauric acid, stearic acid and arachidic acid.

2. Unsaturated fatty acids have one or more double bonds, they are liquid at room temperature and have a low
melting point. These components show more bending in their chains and are more common in plant tissues
for example oleic acid, linoleic acid, palmitoleic acid. Oils are rich in unsaturated fatty acids and have a low
melting point in hydrogenation the unsaturated fatty acids become saturated, and oil become solid fat e.g.,
Dalda ghee. Richest source of polyunsaturated fats in the diet is vegetable oils.

Functions of lipids:
a. Waxes and oils are secreted on surfaces to provide waterproofing in plants and animals.
b. Fats absorb shocks, organs that are prone to bumps and shocks are cushioned with a relatively thick layer
of fat.
c. Lipids are a source of metabolic water. During respiration stored lipid are metabolized for energy,
producing water and carbon dioxides.
d. Lipid constructed from 5 carbon compound isoprene are called terpenes. Isoprene and its derivatives are
joined in various combination to produce substances such as vitamin A and carotenoids. Natural rubber is
a poly-terpenes.

Biodiesel
Biodiesel is a mixture of fatty acid alkyl esters obtained from transesterification (ester exchange) of vegetable
oils and animal fats. It can be produced, from renewable fuel that can be manufactured from new and used
vegetable oils, animal fats, and recycled restaurant grease.

These lipid feedstocks for biodiesel production are composed of 90–98% (weight) of triglycerides and small
amounts of mono and diglycerides, free fatty acids (generally 1–5%), and residual amounts of phospholipids,
phosphatides, carotenes, tocopherols, sulfur compounds, and traces of water.

Biodiesel’s physical properties are similar to those of petroleum diesel, but it is a cleaner-fuel and a renewable
alternative. Using biodiesel in place of petroleum diesel significantly reduces lifecycle carbon emissions.
Rudolph Diesel used peanut oil as automobile fuel was a groundbreaking combustion engine presented in the
World Exhibition in Paris in 1900. Today biodiesel is the most important alternative diesel fuel in the EU
representing 82% of the total biofuel production (Bozbas, 2008) and has been reported to emit substantially
lower quantities of most of the regulated pollutants compared to mineral diesel.

Important features of biodiesel:

1. Biodiesel is non-aromatic, almost sulfur less, produced from renewable sources, presently vegetable oils
or animal fats. On another hand the lubricity property of biodiesel is much better than that of low-sulfur
diesel fuel.
2. On average, biodiesel emit less CO2 than conventional fossil fuels. The use of biomass energy has the
potential to greatly reduce greenhouse gas (GHG) emissions and thus biodiesel have a significant smaller
contribution to global warming when compared to fossil fuels.
3. Biodiesel tailpipe CO2 emissions per mass unit of biodiesel burnt are lower than for petroleum diesel. It
has reduced visible smoke, noxious fumes and odors, and emits 40–50% less particulate matter (PM), 30–

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70% less HC, 20–50% less CO, and less 50% in soot emissions, although the NOx may be about 10–15%
higher this problem can be overcome by retarding the injection timing.

Disadvantages of biodiesel:
1. They are not economically feasible yet, at least when competing with currently used fossil fuels. •
2. Feedstock costs account for a large percent of the direct biodiesel production costs, including capital cost
and returns.
3. The utilization of vegetable oils for biodiesel production, many of them used in human consumption, is
impacting the human food chain, and increasing the price of food.
4. Modifications in currently used engines may be necessary to better use biodiesel, namely changes in the
injection engine system and the utilization of heated fuel lines. As triglycerides have larger molecules, when
compared to normal diesel, its viscosity will be higher and this will impact especially the pump and injection
system, influencing the engine performance.

The Following are main steps involve in biodiesel production:

1. Feedstocks production
2. Feedstocks processing (extraction and pre-treatment)
3. Biodiesel production
4. Biodiesel post-treatment and blending and distribution.

(1) Feedstocks production, for example of vegetable oils, animal fats (e.g. Lard, tallow, poultry fat, fish oil)
microalgae, fat from slaughter house and waste oil.
(2) Feedstocks processing, including oil extraction and pre-treatment steps.
• Oil extraction: In mechanical press extraction the seeds are first heated to 40–50o C and then crushed in a
screw press. The mechanical extraction can be combined with solvent extraction to produce oil with a
higher degree of purity.
• Pre-treatment: The refined vegetable oils do not need a pre-treatment for biodiesel production. However,
waste oils and animal fats have a lot of impurities such as free fatty acids (FFA) and water that negatively
affect the reaction performance.

(3) Biodiesel production:

The biodiesel production process is based on the transesterification reaction between triglycerides and
alcohols. The transesterification reaction can be performed in various ways. Currently most processes involve
homogeneous catalysis normally using alkali as catalysts and based in stirred reactors operating in batch
models. Process is based on the transesterification reaction between triglycerides and alcohols.

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 Module – 1: Biomolecules and their application (quantitative)

Transesterification reaction (Picture credit In book: Biofuel Production-Recent Developments and Prospects)

(4) Biodiesel post-treatment and blending and distribution.

After the transesterification reaction, the mixture is allowed to separate into an upper layer of methyl esters
and a lower layer of glycerin diluted with methanol. The unreacted methanol is then air stripped, or vacuum
distilled away and recycled back to the process. Depending on the process, water can be used to wash catalyst
residues and sodium soaps from the methyl esters. Also, small amounts of concentrated phosphoric acid
(H3PO4) can be added to the raw methyl esters to break catalyst residues and sodium soaps.
Biodiesel can be blended with petroleum diesel or used as 100% biodiesel (called B100) in vehicles.
Predominantly biodiesel is available in the market as mixtures formulated with petroleum diesel on a volume
basis (v/v) to yield blends from B2 (2% biodiesel mixed with 98% petroleum diesel) to B99.9. However,
biodiesel blends higher than B5 can also be commercialized with variations depending on the country. For
example, in France it is B30.
In Germany it is common to use neat biodiesel (B100) instead of petroleum diesel. In Portugal it is possible
to commercialize B100 336 Recent Progress in Chemical Engineering and blends up to B15 according to
national legislation. It is expected in the future that both standards and blends used will change, because of
the evolution in the diesel engines and changes in regulations.

PICTURE CREDIT: [Link]

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 Module – 1: Biomolecules and their application (quantitative)

Cleaning agents/Detergents:
The hydrophobic end of the phospholipid bilayer stays away from the water. This avoids the dissolution
of cell membrane in water. But the detergent can bind to the hydrophobic end of the cell membrane and
form a solution with water, thus breaking the cell membrane barrier.
Detergent monomers solubilize membrane proteins by portioning into the membrane bilayer. With
increasing amounts of detergents, membrane undergo various stages of solubilization. The initial stage is
lysis or rupture of the membrane.
While lipids also have the same general structure as detergents - a polar hydrophilic head group and non-
polar hydrophobic tail - lipids differ from detergents in the shape of the monomers, in the type of
aggregates formed in solution, in the concentration, and range required for aggregation.

Soaps
A soap is a water-soluble compound which is made via a process called saponification by the reaction
between sodium hydroxide or potassium hydroxide with vegetable or animal oil (fats). They are
represented by general formula RCOOM, where RCOC is the acetyl ion of higher fatty acid and M+ is
alkali metal ion (Na+ or K+ ). Soap is produced by reacting fatty acid with alkali or hydrolyzing the
glycerides with alkali.

Saponification reaction (credit: [Link])

Raw Materials 1. Fats, Oils 2. Fatty acids 3. Alkaline Materials [Link] Salt 5. Other additives.

Working of soap:
The hydrophilic (carboxylate) end of the soap molecules attached to water. The hydrophobic (hydrocarbon
chain) end attached to oil and grease. The more and more soap molecules embedded into a grease to form
micelles around oil droplets. The hydrophobic ends of the soap molecules attach towards the grease while
the hydrophilic ends of the soap face outward into the water, resulting is an emulsion of soapy grease
particles suspended in the water. Then the oily particles are easily washed away. The water agitation can
help to clean greasy and oil. The micelles are dispersed into the water. Soap do not work well in hard
water. Hard water contains calcium and magnesium ions, these ions react with soap to form an insoluble
precipitate known as soap film. This precipitate is often seen as a gray line on a bathtub or sink and is
often called scum.

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 Module – 1: Biomolecules and their application (quantitative)

Working of soap (picture credit: [Link])

Detergents:

Detergents are the potassium or sodium salts of a long alkyl chain ending with a sulfonate group. They are surface
active agent nicknamed as surfactant. They clean the surface by reducing surface tension at the boundary between
two phases. So, soap, detergents, emulsifiers, wetting, and penetrants are all surfactants, because they modify the
properties. The chemical formula for detergent is: C18H29NaO3S. They are soluble in hard water. This solubility
is attributed to the fact that the sulfonate group does not attach itself to the ions present in hard water.

Non-polar detergents do not react with Ca2+ or Mg2+ions, while the polar detergents react to form soluble
salts. Therefore, they are most suited in textile processing and as cleansing agents in hard water. Compared to
soap, detergents are more potent surfactants even at much lower concentration. Detergents are excellent foaming
agents. They have bactericidal and germicidal properties. Commonly, anionic detergents such as alkyl benzene
sulfonates are used for domestic purposes. Because of their higher solubility in organic solvents, they are most
suited for dry cleaning of wool, and silk fabrics.

• 4.

Formula for soap and detergents

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 Module – 1: Biomolecules and their application (quantitative)
Uses of Detergents
Powder and liquid detergent can be used for other purposes besides cleaning clothes or dishes.

1. These are mostly used as cleaning agents in the laundry and dishwashing.
2. Tiles, floors, worktops, tubs, and toilets may all be cleaned with either type of detergent.
3. Oil spilled on a garage floor, or the street can be absorbed by powdered Detergent.
4. The detergents increase the softness of the water.
5. On moss growing in the cracks of your steps, sidewalk, or driveway, sprinkle powdered Detergent.
Allow it to be brown for a few days before brushing it out of the gaps with a broom.
6. These are also used for solubilizing and crystallizing membrane proteins, as well as preventing non-
specific binding.
7. Detergents are used for cell permeabilization, cell lysis, gel electrophoresis, and other procedures in
laboratories.

Enzymes
The term enzyme was coined by the Kuhne in 1878. There are approximately 3000 enzymes present in a cell. The
molecular weight of enzymes ranges from 10,000 to more than 100,000 Daltons. Enzyme “zymase” was
discovered by Buchner, he was awarded Nobel Prize for his studies based on yeast extract.
Enzymes are biocatalysts having prominent active sites. These are very efficient. A very small amount of catalyst
brings about the change of a large amount of substance.

They are highly specific, i.e an enzyme will generally catalyze only a single reaction. Metabolic reactions are
catalyzed reaction. There is no uncatalyzed, metabolic conversion in living systems. The constant making and
breaking of biomolecules in the living cell through chemical reactions is called metabolism. Each of the metabolic
reactions results in transformation of biomolecules.
.
Enzymes are also used for therapeutic means to treat disease e.g., streptokinase is used in cleaning blood clots
inside blood vessels. Peroxidase is the smallest enzyme. Diastase is the earliest known enzyme.

Cofactors
Enzymes are composed of one of several polypeptide chains. The cofactors are bound to the enzyme to make the
enzyme catalytically active.
Three kinds of cofactors may be identified as prosthetic group, co-enzymes and metal ions. A complete enzyme
is called a holoenzyme, it consist of an apoenzyme and a prosthetic group. Enzymes are Thermolabile, amphoteric,
colloidal and substrate specific. Working inside the cell, in which they are produced, they are called endo
enzymes. Enzyme secreted outside the cell and act on external medium are called exoenzymes.
Most human enzymes function best within a relatively narrow temperature range between 35 and 40o Celsius.

Naming and classification of enzyme

EC number is called enzyme commission number. It gives a code number to an enzyme, which is in 4 digits.
Enzyme are named by adding a suffix-ase do the root word of the substrate, on which that enzymes acts, e.g.
Lipase (fat hydrolyzing enzyme), Sucrase (breakdown sucrose).
Sometimes the enzymes are named on the basis of the reaction that they catalyze e.g. polymerase (aids in
polymerization), dehydrogenase (removal of H-atom ).

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 Module – 1: Biomolecules and their application (quantitative)
Mainly enzymes are classified into six classes:

Classification of enzyme. Tables is adopted from [Link]

Mechanism of enzyme action:

Enzymes perform the critical task of lowering a reaction’s activation energy that is the amount of energy that
must be put in for the reaction to begin. Enzymes works by binding to reactant molecules and holding them in
such a way that the chemical bond breaking and bond forming possession takes place more readily.
Enzymes possess active sites, where the reaction takes place. These have specific shapes. Enzymes remain
unaltered up to the end of chemical reactions therefore, it can be used again and again. Only a small portion (4-
12 amino acid) of the large enzyme molecule comes in direct contact with the substrate, this portion is called
active site.
An enzyme combines with its substrate(S) to form a short-lived enzyme substrate (ES) complex, which breaks up
into products and enzyme. Various models for enzyme mechanism are proposed over the time. Fisher (1980)
suggested the lock and key hypothesis (template theory) for enzyme action based on specificity.

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 Module – 1: Biomolecules and their application (quantitative)
Lock and key model by Emil Fisher:

Picture is adopted from the [Link]

This model was prepared in 1890 by Emil Fisher. In this model, the enzyme is pre-shaped, and the active site has
rigid structure which is complementary to that of the substrate. This is called lock and key model because the
substrate fits on the active site of the enzyme in the same way as the key fits in the lock. This model describes
that how enzyme binds only to particular specific substrate and will not bind to any other substrate with almost
identical structure.

Induced fit model or hand-in glove model of Daniel E koshland

This model explains both the aspects:

1. Specificity of enzyme substrate interactions

2. Dynamic changes that take place during catalysis

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 Module – 1: Biomolecules and their application (quantitative)
According to the model, the interactions between the substrates and binding sites are relatively weak at first. The
initial binding of the substrate causes change in the conformation of the binding site, causing it to become the
correct shape to bind with the rest of the substrate. This is also sometimes called the hand-in-glove model, due to
the analogy of a hand changing the shape of a glove as it is put on, progressively making it easier for the hand to
fit inside.

Diabetes
Diabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the
body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood glucose. Symptoms
of diabetes include feeling very thirsty, needing to urinate more often than usual, blurred vision, feeling tired,
losing weight unintentionally. Over time, diabetes can damage blood vessels in the heart, eyes, kidneys and
nerves. People with diabetes have a higher risk of health problems including heart attack, stroke and kidney
failure. Diabetes can cause permanent vision loss by damaging blood vessels in the eyes. Many people with
diabetes develop problems with their feet from nerve damage and poor blood flow. This can cause foot ulcers and
may lead to amputation.

Types of diabetes:

Type1 diabetes: Type 1 diabetes develops due to an autoimmune reaction. This causes the immune system, or
T cells, to attack and destroy insulin-producing cells in the pancreas. This means the pancreas is unable to
produce insulin.

Type 2 diabetes: Insulin resistance causes type 2 diabetes. This means that the body does not respond to the
insulin that the pancreas creates. Typically, the pancreas increases the supply of insulin initially, but over time, it
reduces the amount of insulin it produces.

Prediabetes: Prediabetes means you have a higher-than-normal blood sugar level. It's not high enough to be
considered type 2 diabetes yet. But without lifestyle changes, adults and children with prediabetes are at high
risk to develop type 2 diabetes.

Gestational diabetes: Pregnancy causes some insulin resistance. The state that hormones and weight gain
during pregnancy cause the body to use insulin less effectively. This, in turn, causes insulin resistance, which
increases the body’s need for insulin. If the body is unable to produce enough insulin to compensate for the
insulin resistance that pregnancy causes, a person may develop gestational diabetes.

Blood glucose chart:

Picture credit: [Link]

Biology for Engineers
 Module – 1: Biomolecules and their application (quantitative)
Glucose oxidase in biosensors
Multiple laboratory tests are used for the diagnosis and management of patients with diabetes. The blood glucose
concentration is the major diagnostic criterion for diabetes with HbA1c level and is a useful tool for patient
monitoring. Self-monitoring of blood glucose (SMBG) has been established as a valuable tool for the management
of diabetes. The goal of SMBG is to help the patient achieve and maintain normal blood glucose concentrations
to delay or even prevent the progression of microvascular (retinopathy, nephropathy and neuropathy) and
macrovascular complications (stroke and coronary artery disease).

Basic Principles of self-monitoring glucose biosensors:

A biosensor can be defined as a “compact analytical device or unit incorporating a biological or biologically
derived sensitive recognition element integrated or associated with a physio-chemical transducer”.

There are three main parts of a biosensor:

1. The biological recognition elements that differentiate the target molecules in the presence of various
chemicals,
2. A transducer that converts the Sensors biorecognition event into a measurable signal, and
3. A signal processing system that converts the signal into a readable form.

The molecular recognition elements include receptors, enzymes, antibodies, nucleic acids, microorganisms, and
lectins. The five principal transducer classes are electrochemical, optical, thermometric, piezoelectric, and
magnetic. The majority of the current glucose biosensors are of the electrochemical type, because of their better
sensitivity, reproducibility, and easy maintenance as well as their low cost. Electrochemical sensors may be
subdivided into potentiometric, amperometric, or conductometric types. Enzymatic amperometric (detection of
ions in a solution based on electric current or changes in electric current.) glucose biosensors are the most common
devices commercially available and have been widely studied over the last few decades. Amperometric sensors
monitor currents generated when electrons are exchanged either directly or indirectly between a biological system
and an electrode.

Generally, glucose measurements are based on interactions with one of three enzymes: hexokinase, glucose
oxidase (GOx) or glucose-1-dehydrogenase (GDH). The hexokinase assay is the reference method for measuring
glucose using spectrophotometry in many clinical laboratories. Glucose biosensors for SMBG are usually based
on the two enzyme families, Gox (Gulcose oxidase and) and GDH (glucose-1-dehydrogenase). These enzymes
differ in redox potentials, cofactors, turnover rate, and selectivity for glucose. GOx is the standard enzyme for
biosensors; it has a relatively higher selectivity for glucose. GOx is easy to obtain, cheap, and can withstand
greater extremes of pH, ionic strength, and temperature than many other enzymes, thus allowing less stringent
conditions during the manufacturing process and relatively relaxed storage norms for use by lay biosensor users.
The basic concept of the glucose biosensor is based on the fact that the immobilized Gox catalyzes the
oxidation of β-D-glucose by molecular oxygen producing gluconic acid, gluco-lactone and hydrogen peroxide.
To work as a catalyst, GOx requires a redox cofactor—flavin adenine dinucleotide (FAD). FAD works as the
initial electron acceptor and is reduced to FADH2.

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 Module – 1: Biomolecules and their application (quantitative)

Oxidase is widely used enzyme in glucose biosensor due to its better stability and relatability inexpensive.
Glucose oxidase catalyzes the redox reaction and transfer electrons from enzyme active sites to electrode for
glucose level analysis in blood samples.
Amperometric Glucose biosensor was fabricated by immobilizing glucose oxidase. Glucose oxidase, the most
popular enzyme used for glucose detection, can reduce oxygen to oxygen per oxide while at the same time
transferring glucose to d-glucono- 1,5- lactone. Quantification of glucose can be achieved based on either the
detection of hydrogen peroxide produced, or the oxygen consumed.

Lignolytic enzyme in bio-bleaching:

Bleaching is the process of making pulp white to improve printing properties and its ability to absorb liquids.
Bleaching also attacks some contaminants to reduce stray dark colored particles in the final sheet of paper.
Bleaching processes add significantly to the total cost of making pulp. Also, the environmental impact of
bleaching has the close attention of the government.
Bleaching is one of the important process in paper production primarily for two purposes: first, to increase
brightness; second, to remove residual lignin. Lignin can be thought of as the ‘glue’ holding the cellulose fibers
of wood together; it accounts for up to 50 per cent of the weight of pulpwood, the basic feedstock for pulp
manufacture.
The removal of lignin in a single step is not feasible; therefore, to remove lignin at the desired level,
bleaching chemicals are used consecutively with intermediate washing and extraction stages. Several oxidants
such as chlorine, ClO2, sodium hypochlorite, H2O2, O2, and O3 are used for bleaching different types of pulps.
Chlorine and hypochlorite are used for chemical pulp bleaching for many years because they are less expensive
and produce high bright pulp. Bleaching with chlorine and chlorine derivatives undergoes many chemical
reactions resulting in highly toxic and non-biodegradable pollutants
All most all important chemical pulp bleaching agents are oxidants, e.g., chlorine, Cl2; chlorine dioxide, ClO2;
calcium hypochlorite, Ca(OCl)2; hydrogen peroxide, H2O2.

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 Module – 1: Biomolecules and their application (quantitative)
Biobleaching:
The environmental issues and growing market demand for superior pulp and paper products force the
development of green processing for a better quality of products along with the reduced generation of pollutants
during bleaching. The pretreatment of pulp with enzymes such as xylanase, manganese peroxidase, versatile
peroxidase or laccase is known as biobleaching or pre-bleaching. It has emerged as a viable option to minimize
the consumption of chlorine compound.

Ligninolytic enzymes biobleaching:

Ligninolytic enzymes are also found effective for bio-bleaching of different kinds of pulp. The ligninolytic
enzymes studied till now are identified as extracellular and nonspecific enzymes that are involved in oxidative
reactions, whenever bonds between the basic units and the aromatic structure of lignin are broken.
Bacteria and fungi produce extracellular oxidative enzymes that degrade the lignin. A small group of
basidiomycetes termed as white-rot fungi (WRF) has especially developed the ability to breakdown and
mineralize lignin by producing oxidative enzymes. WRF efficiently degrade the lignin within the plant cells using
extracellular ligninolytic enzymes.
These fungi secrete a number of oxidative enzymes and same hitherto unknown substances (mediators)
into their environment together effecting a slow but continuous degradation. The most important lignin oxidizing
and themes are lignin peroxidase, manganese peroxidase and laccases. Lignin peroxidase and manganese
peroxidase appear to constitute a major component of the lignol lytic system.
Ligninases directly and specifically attack lignin to oxidize it and make it water-soluble. Oxidoreductases involve
in lignin degradation are peroxidases and laccases. Peroxidases perform the oxidation of different types of
molecules utilizing H2O2 as the oxidant. Laccases, LiP, and MnP are the key enzymes in the oxidative enzyme
system of WRF that have been used for bio-bleaching of pulp.

Important enzymes for bio-bleaching:

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 Module – 1: Biomolecules and their application (quantitative)
Ignore lytic enzymes play a key role in degradation and detoxification of lignocellulosic waste in environment.
The major ligno lytic enzymes are lessees, lignin peroxidase, manganese peroxidase and versatile peroxidase.
Legno lytic fungi and enzymes (i.e laccase, manganese peroxidase and lignin peroxidase) Have been applied
recently in reduction of second generation refuels.

Sources and references:

Research papers:
PLA composites: From production to properties PLA bioplastic production: From monomer to the polymer
Jiaming Yu a b, Shengchao Xu a b, Biao Liu a b, Hailan Wang a b, Fengmin Qiao b, Xiulian Ren a b, Qifeng Wei

Knowledge of Constituent Ingredients in Enteral Nutrition Formulas Can Make a Difference in Patient Response to
Enteral Feeding. August 2017 Nutrition in Clinical Practice Authors: Patricia savino

Chlorine in the bleaching of pulp and paper Keith R. Solomon Centre for Toxicology, University of Guelph, Guelph ON
NlG 2 Wl, Canada

Biobleaching - An ecofriendly and environmental benign pulp bleaching technique: A review Shardesh Kumar
Chaurasia & Nishi K. Bhardwaj

Websites:
[Link]
Bioplastics and Biopolymers ([Link])
[Link]
[Link]
[Link]
[Link]
[Link]

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[Link]
[Link]
[Link]
[Link]
[Link]
[Link]
[Link]
[Link]

Biology for Engineers