MODULE 5

1. Write about the industrial production or large scale production of enzymes? And write
its applications?
Industrial Production of Enzymes (With Applications)

Industrial production of amylases, glucose isomerase, pectinases, lipases, cellulases and

proteases are described.
1. Amylases:
Amylases are a complex group of enzymes that hydrolyse polysaccharides like starch and
glycogen to glucose. During the hydrolysis of 1, 4-glycoside, linkages present in above
polysaccharides are degraded which result first in the formation of short chain dextrins, then
to maltose and glucose. Different enzymes involved in the degradation of starch to glucose .

There are mainly two groups of amylases such as –

1. α -amylose and
2. β-amylose.
1. α -amylose – α -amylases are also called as 1,4 – α – glucan – glucano hydrolase.
Extracellular enzymes hydrolyze 1,4 – glycosidic bonds. These enzymes are also called as
endoenzymes as they split the substratum in the interior of the molecule in a random fashion.
On the other hand,
2. β-amylose – β-amylases split the substratum from one end of the molecule in a successive
fashion.
On every alternate 1, 4 glycosidic bond releasing maltose. These enzymes are also called exo
amylases. The other enzymes involved in the degradation of starch include amyloglycosidase
(Aspergilus niger and Rhizopus niveus), isoamylase and pullulanase (Klebsiella pneumoniae
and Bacillus acidopullulyticus). The molecular weight of various α -amyloses do not differ
considerably (table 8.3) and require calcium as a stabilizer.

α-amylases are secreted by many bacteria and fungi.

They are classified according to their:
1. Starch-liquefying or saccharogenic effect
2. pH optimum
3. Temperature range
4. Stability
Saccharogenic amylases produce free sugars (β-amylose and glucoamylase), whereas starch-
liquifying amyloses (α-amylose) break down the starch polymer but do not produce free
sugars.
Bacteria which produce α -amylase are Bacillus subtilis, B. cereus, B. amylo- liquefaciens, B.
coagulans, B. polymyxa, B. stearothermophilus, B. caldolyticus, B. acidocaldarius, B. subtilis
amylosaccharaticus, B. licheniformis, species of Lactobacillus, Micrococcus, Pseudomonas,
Arthrobacter, Escherichia, Proteus, Thermomonospora and Serratia. Some α-amylase
producing fungi are species of Aspergillus, Penicillium, Cephalosporium, Mucor, Candida,
Neurospora and Rhizopus.
Although all the above bacteria and fungi are capable of producing α-amylase, the most
important of them from which α -amylase are produced commercially through fermentation
process include Bacillus amyloliquefaciens, B. licheniformis and Aspergillus oryzae.

However, Bacillus are used much more extensively than those of Aspergillus.

Fungal α -Amylase:
Fungal α-amylase is produced commercially by employing either Aspergillus oryzae or
Aspergillus niger. Stationary culture method is used when A. oryzae is employed, while
submerged culture method is used when Aspergillus niger is employed.
The process of submerged culture method is only described here:
(a) Preparation of Inoculum:
Suitable and pure seed culture is generally selected as inoculum. In certain cases mutants
capable of giving higher yield are selected as inoculum.
(b) Preparation of Medium:
The following medium is generally employed for submerged fermentation:

Amylase biosynthesis is inhibited when there is glucose in the medium. The medium is steam
sterilized. The sterilized medium is passed into a production fermenter for α-amylase
production.
(c) Fermentation Process:
A cylindrical fermenter made up of stainless steel is generally used in the fermentation
process. It is equipped with an agitator, an aerating device, a cooling system and other
ancillary equipment like a device for foam control, monitoring of pH, temperature and
control of oxygen tension etc.
A sufficient quantity of pre-sterilized production medium is taken in the fermenter and is
inoculated with spores of the selected species of the fungus. The spores are allowed to
germinate and produce sufficient mycelium by controlling the fermentation conditions.
Control of fermentation conditions plays a vital role in the success of the process, which
include pH, temperature, aeration, agitation, oxygen supply etc.
The optimum pH for the fermentation is 7.0. Calcium carbonate is used as buffer to maintain
pH. The fermentation process is generally operated at a temperature of 30 to 40°C. Aeration
and agitation of the production medium is needed because of high viscosity of the medium
due to the presence of mycelial mat.
(d) Harvest and Recovery:
The following steps are followed during the recovery of the enzyme after the completion of
fermentation. In order to avoid denaturation of the enzyme, the fermentation broth is
subjected to rapid cooling at 5°C temperature immediately and the enzyme is extracted.
1. Separation of fungal mycelium is accomplished by filtration of the refrigerated broth.
2. The suspended particles present in the broth are removed with flocculating agents like
calcium phosphate.
3. The enzyme is precipitated, in order to get high degree of purity, by using acetone or
alcohol or even inorganic salts like ammonium sulphate or sodium sulphate.
4. Sometimes fractional precipitation of the enzyme is done to obtain it in purest form.
Bacterial α -Amylase:
Bacterial α -amylase is produced by Bacillus subtilis or B. amyloliquefaciens or B.
licheniformis. For industrial production of bacterial α -amylase, nowadays submerged culture
method is generally employed in many countries.
(a) Preparation of Inoculum:
Pure culture of any of the above-mentioned species of Bacillus is selected as inoculum.
Mutants which produce 250 times greater yields than the wild strain are preferred as
inoculum.
(b) Preparation of Medium:
The formulation of the production medium and control of fermentation conditions play a
major role in the success of enzyme fermentations. The production medium should basically
contain an energy source, a carbon source, a nitrogen source and growth requirements such as
essential amino acids or vitamins. For obtaining high yields of enzyme, the production
medium should also contain certain inducers like lactose.
Sometimes certain compounds like glucose present in the production medium act as repressor
for certain enzymes like α-amylase. In such conditions either the concentration of glucose
should be kept low or it should be fed intermittently.
The following production medium is generally employed in a submerged culture method

(c) Fermentation Process:

The type of fermenter that is used for fungal α-amylase is also used for bacterial α-amylase
production. About 1000 to 30,000 gallons of production medium is taken in the fermenter and
inoculated. The fermentation is continued upto 4-6 days. The pH of the medium is maintained
at 7.0.
Calcium carbonate is used as a buffer for maintaining neutral pH. The temperature is
maintained at 30-40°C. The production of α-amylase starts when the bacterial density reaches
109-1010 cells ml-1. However, the enzyme production increases just before the growth rate of
the microorganism decreases and spore formation begins.
(d) Harvest and Recovery:
Bacterial α-amylase is harvested and recovered by the same method that is used for the
recovery of fungal α-amylase. The most active liquid enzyme preparation contains 2%
amylase protein and solid preparation contains 5% amylase proteins.
2. Proteases:
Proteases, second most important industrial enzymes, are produced about 500 tons per year.
Proteases are primarily used in detergent, dairy, leather firms, pharmaceutical industries, the
manufactured protein hydrolysates, food industry and waste processing.
Organisms:
Proteases are commercially produced both by fungi and bacteria based on optimum pH for
their activity. They can be grouped into alkaline, neutral and acid proteases and other groups
(i) Alkaline Proteases:
Most bacteria and some fungi excrete alkaline proteases. The most important producers of
alkaline protease producers are Bacillus licheniformis, B. amyloliquefaciens, B. firmus, B.
megaterium and B. pumilus species of Streptomyces such as S. fradiae, S. griseus and S.
rectus. Some fungi like Aspergillus niger, A. sojae, A. oryzae, and A. flavus. Proteases of this
type have many features of value for use as detergent enzymes. These enzymes are stable at
high temperature, at alkaline range of pH (9.0 – 11.0), in association with chelating agents
and perborates.
However, their stability is low in the presence of surface active agents resulting in the limited
self-life. Subtilisin Carlsberg from B. licheniformis, subtilisin BPN and subtilisin NOVO
from B. amyloliqui-faciens are some of the examples of this type of enzymes. These enzymes
contain serine at the active site of the molecules and are not inhibited by EDTA (Ethylene
diamine tetracetic acid) but are inhibited by DFP (diisopropyl fluorophosphate).
Screening for isolation of microorganisms with better production is done by using strongly
basic protein media at pH 10. As most of the wild strains are with low yield and insufficient
for industrial utilization, improvement of strain is being done through genetic engineering.
Protein engineering has been used to develop modified Bacillus subtilopeptidase with altered
amino acid sequences, corresponding changes in enzymatic properties such as substrate
specificity, pH optimum and stability to bleaching agents.
Protease production occurs in shaken flasks and small fermenters or in a production
fermenter of 40-100 ml capacity at 30-37°C. Production of extracellular proteases is chiefly
regulated by the medium composition. The fed-batch process is generally used in order to
keep down the concentration of ammonia ions and amino acids which otherwise may repress
protease production. High O2 partial pressure is generally necessary for proteases titres,
aeration rates are 1 V m-1 and incubation time of 48-72 hrs depending on the organism.
Proteases must be converted into particulate form before they are added to detergents since if
dry enzyme powder is inhaled by production workers or users, allergic reaction may result.
Enzyme is marketed in a microencapsulated form. To make a suitable encapsulated product, a
wet paste of enzyme is melted at 50-70°C with hydrophobic substance such as polyethyl-
eneglycol and then converted into tiny particles. These solidified spherical particles are not
hazardous when added directly to the detergent. Immobilization in fibrous polymer is another
development.
(ii) Neutral Proteases:
These proteases are excreted by Bacillus subtilis, B. cereus, B. megaterium, Pseudomonas
aeruginosa, Streptomyces griseus, Aspergillus oryzae, A. sojae and Pyricularia oryzae.
Neutral proteases are relatively unstable.
Calcium and sodium chloride must be added for maximal stability. The pH range of activity
is narrow and unstable to increased temperature. These are also quickly inactivated by
alkaline proteases which is probably the reason for their restricted industrial application.
However, they are used in leather industry and in food industry for manufacture of crackers,
bread and rolls.
(iii) Acid Proteases:
These include renin like proteases from fungi which are chiefly used in cheese production.
These enzymes have optimum pH 2-4 and used in medicine, in the digestion of soya protein
for soya sauce production and to breakdown wheat gluten in the bakery industry.
(iv) Renin:
Milk coagulating enzyme in fourth chamber of 3-4 weeks old calves stomach which is being
exploited in cheese production. Due to increased production of cheese and decline in the
number of slaughtered calves intensive research has been carried out since 1960 to develop
rennin products of microbial origin. A variety of fungi and bacteria have been isolated as
producers.
It has also been examined for required characteristics.
Alcaligenes, Bacillus, Corynebacterium, Lactobacillus, Pseudomonas, Serratia and
Streptococcus are some of the bacteria that have exhibited potential of rennin production.
Similarly species of Aspergillus, Candida, Coriolus, Endothia, Entomophthora, Irpex, Mucor,
Penicillium, Rhizopus, Sclerotium and Torulopsis are also reported to produce rennin.
Mucor pusilus var. lindt for solid substrate culture, while Endothia parasitica and Mucor
miehei produce this enzyme in submerged culture. Rennin produced by Endothia parastica
was first rennin marketed commercially in 1967. The enzyme is an acid protease stable at pH
4.0-5.5 with a molecular weight of 34,000 – 37,500 daltons and is produced in a medium
containing 3% soyameal, 1% glucose, 1% skim milk, 0.3% NaNO 3, 0.05% K2HPO4,0.025%,
MgSO4.7H2O, pH 6.8, at 25°C incubation temperature. At the end of 48 hrs incubation
period, mycelium is separated and broth which contains enzyme is concentrated and
precipitated in an evaporation process.
Similarly Mucor miehei also produces rennin in a medium containing potato starch 4%,
soyameal 3%, ground barley 10%, CaCO3 0.5%, pH 5.5, 5-6 d incubation period at an
incubation temperature of 40°C. Microbial renins are stable at high temperature and remain
active in the curd after precipitation and subsequently causing harmful proteolysis. Therefore,
complementary cDNA from calf renin is transferred into E. coli making possible the first
commercial production of the calf rennin by a microorganism.
Applications:
Proteases are employed in variety of fields such as:
1. Biological detergents — alkaline protease
2. Baking—dough modification/gluten weakening and flour improvement
3. Beer brewing —chill proofing of beer to remove protein haze
4. Leather bailing and tendering
5. Cheese manufacture — clotting of milk protein and promotion of ripening aspartic
protease, calf chymopsin, aminopeptidase etc.
6. Meat tenderization and removal of meat from bones
7. Flavour control and production in food products
8. Waste treatment- treatment of silver from spent photographic film.
3. Pectinases:
Pectinase is an enzyme complex which contains atleast six enzymes splitting pectins at
different sites of the molecule. The basic structure of pectin is α 1,4 linked galacturanic acid
with upto 95% its carboxyl groups esterified with methanol.
The methyl ester is split by pectinesterase and the glycosidic bonds of pectin or pectic acid,
and chain are split by hydrolysis with endo- polygalacturonase or exo-polygalacturonase.
Another method of splitting is transelimination by means of exo-pectate lyase or endopectate
lyase or exo-and endo-pectin lyase.
Commercial production of pectinases is carried out by using Aspergillus niger or A. wentii
and also species of Rhizopus either as surface or submerged fermentation in a medium
containing 2% sucrose and 2% pectin in a fed batch system. The pH is adjusted to 3.0 to 4.0
and incubation temperature 37°C and incubation period upto 60-80 hrs. The enzyme is
purified by separating mycelium, by filtration or centrifugation, stabilizing agents are added.
The enzyme is precipitated with organic solvents and crude protein is dried and used as
enzyme.
Applications:
Pectinases are used primarily for clarification of fruit juices, for maceration of vegetable and
fruits, for extraction of oil. By treating with pectinase, the yield of fruit juice during pressing
is considerably increased. These are also used in wine classification and coffee bean
fermentation.
4. Lipases:
Lipases (glycerolesterhydrolases) split fats (glycerolesters) into di-or monoglycerides and
fatty acids (Fig 8.5).
These are usually extracellular. Most of lipases are produced adaptively in the presence of
oils and fats. However, some organisms like Penicillium roqueforti are reported to secrete
lipase constitutively and in the presence of fats its enzymes production is repressed. Species
of Aspergillus, Mucor, Rhizopus, Penicillium, Geotrichum and yeasts (Torulopsis and
Candida) are reported to be good producers.
Similarly species of Pseudomonas, Achromobacter and Staphylococcus are good producers
of lipases. Lipases are generally bound to the cells and hence inhibit on over production, but
addition of cation such as magnesium ion liberates the lipase and leads to a higher enzyme
titer in the production process.
Applications:
Lipases are primarily marketed for therapeutic purpose as digestive enzymes to supplement
pancreatic lipases. These enzymes are used in dairy industry. The cheese ripening process
involves lipases. Lipases from Candida cylindraceae is used to hydrolyse oil in soap industry.
These are useful in the synthesis of esters from acids and alcohols in non-aqueous medium.
These are also useful in improvement of fat quality by inter-esterification (exchange of one
fatty acid by another in ester bond) lipases are useful in removal of fat in leather processing.
These enzymes are also useful in production of flavour compounds and acceleration of
ripening in dairy and meat products.
5. Cellulases:
Cellulases are the enzymes which degrade cellulose into fermentable sugars. Although
cellulose is available abundantly on the earth in the form of natural plant resources like fallen
leaves, flowers, fruits, broken stems, branches and waste wood, it is mixed with substances
such as lignin, hemicellulose, starch, proteins etc.
All these material are to be treated physically or chemically to breakdown cellulose into
fermentable sugars. This breakdown process is called as pretreatment. The pretreatment may
be done either chemically or enzymatically. In the second process cellulose enzymes are used
to breakdown cellulose into simpler fermentable sugars
Most of these enzymes are excreted out by certain microorganisms which include both
bacteria and fungi. Such enzymes are called extracellular enzymes. The bacteria include
actinomycetes and Cellulomonas and fungi include species of Trichoderma, Penicillium,
Thermoascus, Sporotrichum and Humicola. However, for commercial production of
cellulases Trichoderma viride, T. reesei, T. koningii, Penicillium funiculosum, P. enersonii,
Aureobasidium pullulans, Sporotrichum and pulverulenium are generally employed.
Cellulase is an enzyme complex containing at least three enzymes (table 8.9 and Fig. 8.8):
1. Exo- β-1, 4 glucanase, also called as cellobiodhydrolase or C.
2. Endo-β 1, 4-glucanase also called as carboxymethyl cellulase or endocellulase (Cx).
3. β-1, 4 glucosidase or cellobiase.

Fermentation Production:
The fungus Trichoderma viride is usually used for the commercial production of cellulose.
Inoculum of the fungus is raised from pure or mutant culture of Trichoderma viride GM 9414
by a process of repeated sub-culturing. Large fermenters of stainless steel are employed for
fermentative production of celluloses.
The fermentation is carried out at 30°C for about 140 hours. Maximum fungal growth occurs
under these conditions resulting in the maximum yield of the cellulose. However, the yield of
enzyme is dependent upon the cellulose concentration (Fig. 8.6). After the completion of the
fermentative process the products recovered from the fermenter and the fungal mycelium is
separated by filtration. Afterwards purification of the enzyme was carried out using ion
exchangers.
Applications:
1. Fruit juice and olive production and processing.
2. Wine and beer production and processing.
3. Malting-speedy modification of grains.
4. Textile processing-bio polishing of cellulose fibers.
5. Wood pulp processing.
6. Glucose Isomerase:
Glucose isomerase causes the isomerization of glucose to fructose. The reaction is reversible
and a mixture of glucose and fructose is produced. The ratio of these products depends on the
enzyme used and reaction conditions such as incubation time, pH and temperature. The
enzyme has become of commercial value because price of sugar has increased as compared to
that of starch.
In U.S. about 4 × 106 tons of isosyrup is being produced, while in Germany 10,000-12,000
tons per year. Starch can be converted to glucose by either acid hydrolysis or enzyme
hydrolysis (Fig. 8.9).

The advantage of starch hydrolysis over direct sugar production (sugar beets) is that initial
materials used such as wheat, corn, cassava and to some extent potatoes are non- perishable,
but sugar beets are available only 100 days per year.
Glucose has 70-75%, the sweetening strength of beet sugar (sucrose) but β-D-fructose
pyranose (fructose), the sweetest monosaccharide has twice the sweetening strength of
sucrose. Thus, processes for the manufacture of fructose are of considerable value. Fructose
can be produced biochemically from sucrose or starch by isomerase, glucose production
Fructose can also be produced chemically from glucose at high temperature under alkaline
conditions but byproducts are formed such as psicose which cannot be metabolized in the
human body. Thus chemical production of fructose is not acceptable to the food industry.
Fermentative Production:
Glucose isomerase is used in production of high fructose syrup (HFCs) or isosyrup must
fulfill the following criteria:
1. Low pH optimum to avoid side reactions.
2. High specific activity.
3. High temperature optima.
Some of the microorganisms producing glucose isomerase are listed in table 8.10.
Practical conversion yields are in the range of 40-50% where fructose concentration can be
increased to 55% by chromatographic enrichment. Alternatively the fructose can now be
separated from glucose (+) fructose mixture and the remaining glucose fraction isomerzed to
produce a final combined product containing upto 80% fructose. HFCs from corn starch is
cheaper than from sucrose but have same intensity of sweetening.
Consequently they have become major food ingredients particularly in North America and
annual production of HFCs is now in excess of 8 × 10 9 kg. Some of the bacteria such as
Escherichia intermedia, E. freudii and Aerobacter aerogenes can produce isomerase. Though
they are able to produce glucose isomerase which is variant and requires arsenium for its
activity, hence it cannot be used in food production.
The commercial process for production of fructose (Fig. 8.11) from glucose became feasible
only when procedures for immobilization of the enzyme were developed .
Since, glucose isomerase is formed intracellularly in most strains; many commercial
processes are carried out with immobilized cells or by addition of partly broken cells.
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2. What is meant by pharmaceutical protein and explain their role in the treatment of
diseases?
The proteins produced in the body control and mediate the metabolic processes and
help in its routine functioning. Any kind of impairment in protein production, such as
production of mutated protein or misfolded protein, leads to disruption of the pathway
controlled by that protein. This may manifest in the form of the disease. However,
these diseases can be treated, by supplying the protein from outside or exogenously.
The supply of active exogenous protein requires its production on large scale to fulfill
the growing demand. The process is complex, requiring higher protein expression,
purification, and processing. Each product needs unique settings or standardizations
for large-scale production and purification. As only large-scale production can fulfill
the growing demand, thus it needs to be cost-effective. The tools of genetic
engineering are utilized to produce the proteins of human origin in bacteria, fungi,
insect, or mammalian host . Usage of recombinant DNA technology for large-scale
production of proteins requires ample amount of time, labor, and resources, but it also
offers many opportunities for economic growth. After reading this chapter, readers
would be able to understand the basics about production of recombinant proteins in
various hosts along with the advantages and limitations of each host system and
properties and production of some of the important pharmaceutical compounds and
growth factors.
 ----------------------------------------------------------------------------------------------------
3. Write a note on therapeutic proteins?
Protein-based therapeutics are highly successful in clinic and currently enjoy
unprecedented recognition of their potential. More than 100 genuine and similar
number of modified therapeutic proteins are approved for clinical use in the European
Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs)
accounted for almost half (48%) of the sales. Based on their pharmacological activity,
they can be divided into five groups: (a) replacing a protein that is deficient or
abnormal; (b) augmenting an existing pathway; (c) providing a novel function or
activity; (d) interfering with a molecule or organism; and (e) delivering other
compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins.
Therapeutic proteins can also be grouped based on their molecular types that include
antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone
morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors,
hormones, interferons, interleukins, and thrombolytics. They can also be classified
based on their molecular mechanism of activity as (a) binding non-covalently to
target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting
activity without specific interactions, e.g., serum albumin. Most protein therapeutics
currently on the market are recombinant and hundreds of them are in clinical trials for
 therapy of cancers, immune disorders, infections, and other diseases. New engineered
proteins, including bispecific mAbs and multispecific fusion proteins, mAbs
conjugated with small molecule drugs, and proteins with optimized pharmacokinetics,
are currently under development. However, in the last several decades, there are no
conceptually new methodological developments comparable, e.g., to genetic
engineering leading to the development of recombinant therapeutic proteins. It
appears that a paradigm change in methodologies and understanding of mechanisms is
needed to overcome major challenges, including resistance to therapy, access to
targets, complexity of biological systems, and individual variations.
The 30 top-selling therapeutic proteins in 2010 (in bln US$)

Target/ Sale
Name Type Company Indication
mechanism s

Etanercept TNFα Fc fusion Amgen Immune 7.28

TNFR2 Wyeth diseases 7
ECD

Bevacizumab VEGF Humanize Genentech Cancer 6.97

d IgG Roche 3

Chugai

Rituximab CD20 Chimeric Genentech Cancer 6.85

IgG Biogen- 9
IDEC
Roche

Adalimumab TNFα Human Abbott Immune 6.54

IgG Eisai diseases 8

Infliximab TNFα Chimeric Centocor Immune 6.52

IgG (J&J) diseases 0
Schering-
Plough
Mitsubishi
Tanabe

Trastuzumab Her2 Humanize Genentech Cancer 5.85

d IgG Chugai 9
 Target/ Sale
Name Type Company Indication
mechanism s

Roche

Insulin glargine Insulin receptor Modified Sanofi- Diabetes 4.83

insulin Aventis 4

Epoetin alfa EPO-R Human Amgen Anemia 4.59

EPO Ortho 0
Biotech
Kyowa
Hakko
Kirin

Pegfilgrastim G-CSF receptor PEGhuma Amgen Neutropeni 3.55

n G-CSF a 8

Ranibizumab VEGF Humanize Genentech AMD 3.10

d Fab Novartis 6

Darbepoetin alfa EPO-R Modified Amgen Anemia 2.99

human Kyowa 5
EPO Hakko
Kirin

Interferon beta-1a Interferon beta Human Biogen Idec Multiple 2.51

(Avonex) receptor interferon sclerosis 8
beta-1a

Interferon beta-1a Interferon beta Human Merck Multiple 2.29

(Rebif) receptor interferon Serono sclerosis 7
beta-1a

Insulin aspart Insulin receptor Modified Novo Diabetes 2.19

insulin Nordisk 8

Rhu insulin Insulin receptor Modified Novo Diabetes 2.18

insulin Nordisk 5

Octocog alfa Factor VIII Factor Baxter Hemophilia 2.09

replacement VIII Healthcare A 5
 Target/ Sale
Name Type Company Indication
mechanism s

Insulin lispro Insulin receptor Modified Eli Lilly Diabetes 2.05

insulin 4

Cetuximab EGF-R Chimeric Eli Lilly Cancer 1.79

IgG BMS 1
Merck
Serono

Peginterferon alfa- Interferon alfa PEGhuma Roche Hepatitis C 1.77

2a receptor n protein 5

Interferon beta-1b Interferon beta Human Berlex Multiple 1.66

receptor protein Bayer sclerosis 1
Schering

Eptacog alfa Initiate Human Novo Hemophilia 1.48

coagulation factor Nordisk 3
VIIa

Insulin aspart Insulin receptor Modified Eli Lilly Diabetes 1.44

insulin 5

Onabotulinumtoxin SNAP-25 Botulinum Allergan Medical 1.41

A cleavage toxin type GSK and 4
A esthetic

Epoetin beta EPO-R Human Roche Anemia 1.38

EPO 7

Rec antihemophilic F VIII substitution Human Bayer Hemophilia 1.38

factor protein Schering A 3

Filgrastin G-CSF receptor Human Amgen Neutropeni 1.28

protein a 6

Insulin detemir Insulin receptor Modified Novo Diabetes 1.27

insulin Nordisk 1

Natalizumab α 4/β1/7 integrin Humanize Biogen Idec Multiple 1.23

d IgG Elan sclerosis 0
 Target/ Sale
Name Type Company Indication
mechanism s

Insulin (humulin) Insulin receptor Human Eli Lilly Diabetes 1.08

insulin 9

Palivizumab RSV Humanize MedImmun RSV 1.03

d IgG e 8

List of Fc fusion proteins, the year denotes date of approval

1. Etanercept (TNFR2 ECD, 1998)

2. Alefacept (LFA3 ECD, 2003)

3. Abatacept (CTLA4 ECD, 2005)

4. Rilonacept (IL-1RI/IL-1RacP ECD, 2008)

5. Romiplostim (41aa thrombopoietin (TPO) analogue peptide, 2008)

6. Belatacept (CTLA4 ECD, 2011)

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4. What is meant by vaccine and classify with examples?
A vaccine is a substance that is used for the production of antidotes in the body and provides
immunity against one or a few diseases. In biological terms, a vaccine is defined as a
biological and formulated preparation to provide acquired immunity for a particular disease. 

Generally, a vaccine is an agent which contains a weakened or killed form of the disease-
causing agent, its surface, or its toxins. When this solution is introduced to the human body,
the immune system is able to identify the threat and destroy it. More than this, the human
body will recognize the threat and can initiate an appropriate response in the future also.

Definition

The process of implementing the vaccine is called vaccination. It is responsible for the
clearance of many diseases, especially infectious diseases like smallpox and chickenpox. The
word "vaccine" is derived from the Latin word "vaccines" which means "from the cows".
Invention of Vaccine

The practice of immunization of the body dates back hundreds of years, but the first official
vaccination was developed by Edward Jenner who is considered the founder of vaccinology.
In 1796, he injected a 13 year-old-boy with cowpox(vaccinia virus) and established immunity
to smallpox. In 1798, the very first smallpox was developed. During the 18th and 19th
centuries, systematic implementation of mass smallpox immunization culminated in its global
establishment in 1979.

Types of Vaccines

There are many initiations to vaccine development, but vaccines can be mainly classified by
how the antigen, active component, that produces a specific immune response against the
disease-causing organism, are prepared.

Classification of Vaccines

A. Live Attenuated Vaccines:

Attenuated vaccines are developed in many several ways. The common methods
include passing the disease-causing virus through a series of cell cultures or animal
embryos. When the vaccine virus is implemented in a human, it will be unable to
replicate enough to cause illness, but still promotes an immune response that can
protect against future infection.

B. Inactivated Vaccine:

Vaccines of this category are developed by inactivating a pathogen, typically using

chemicals or even heat such as formaldehyde or formalin. This destroys the
pathogen's ability to replicate but keeps it intact so that the immune still remembers it.

C. Toxoid Vaccine:

There are some bacterial diseases that are not directly caused by a bacteria itself, but
by producing toxins by the bacterium. For this type, immunization of pathogens can
be developed by inactivating the toxin that causes disease symptoms. As the viruses
or organisms used to kill or inactivate vaccines, this can be done through treatment
with a chemical such as formalin or by heat.

D. Subunit Vaccine:
 Subunit vaccines are only used as part of a target pathogen to promote a response
from the immune system. This can be done by isolating a specific protein from a
pathogen and presenting it as an antigen on its own.

E. Conjugate Vaccine:

Conjugate vaccines are somehow similar to recombinant vaccines, they are made up
of a combination of two different components. Conjugate vaccines, however, are
made up of using the pieces from the coat of bacteria. These coats are chemically
linked to a carrier immune protein, and this is how a combinational vaccine is used.

F. Valence Vaccine:

Vaccines may be monovalent. The monovalent vaccine is designed to be immune

against a single microorganism or single antigen. A multivalent or polyvalent vaccine
is made to immunize against two or more viruses of the same microorganism.

G. Heterotypic Vaccine:

Heterologous vaccines are also called "jennerian vaccines". These vaccines are
pathogens of different animals that either do not cause disease or cause disease or
cause mild disease in the organism being treated.

H. mRNA Vaccine: 

An mRNA Vaccine (or RNA Vaccine) is a different type of vaccine which is a

combination of nucleic acid RNA, packaged within a vector such as lipid
nanoparticles. 

Vaccination

Vaccination is a process involving introducing deactivated/ weakened disease-causing

microbes into a person, or a vaccine is administered to a person to generate immunity from
that disease. Vaccination is generally injected or administered orally. It is the drug (weak
pathogen/ inactivated) that is administered to a person to prevent the onset of a disease.

Uses of Vaccination

Vaccines are very important because they protect us from infectious diseases. In some areas
or populations, infectious diseases are endemic. For example, cholera, polio, smallpox,
hepatitis B and so on. To fight against these diseases, we need vaccines to boost our immune
systems and prevent harm.
What is an Immunization?

It is the ability of the human body to produce an immune response either naturally, or through
vaccines. These approaches develop immunity or resistance to a specific illness.
Immunization can be defined as the process by which a person is made to fight against a
particular disease by the administration of a vaccine. The basic principle of immunization is
that the human body starts to produce antibodies against disease through vaccines so that the
person is safe from the infectious disease. The body repeats the process of development of
infectious agents and memory cells that can develop antibodies immediately upon further
exposure to the infectious agent.

Process of Immunization

 The process begins when a person is injected through the vaccine and then their
bodies begin to develop immunity to fight against the disease.
 The body generates immunity through this vaccine for the disease rabies.
 This method has proven to be very effective to prevent a number of infectious
diseases.
 Smallpox, Tetanus, measles, and other diseases have vaccines.
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5. What is antibody explain the structure and its role in the treatment of diseases?
Antibodies are the secreted form of the B-cell receptor. An antibody is identical to the
B-cell receptor of the cell that secretes it except for a small portion of the C-terminus
of the heavy-chain constant region. In the case of the B-cell receptor the C-terminus is
a hydrophobic membrane-anchoring sequence, and in the case of antibody it is a
hydrophilic sequence that allows secretion. Since they are soluble, and secreted in
large quantities, antibodies are easily obtainable and easily studied. For this reason,
most of what we know about the B-cell receptor comes from the study of antibodies.
Antibody molecules are roughly Y-shaped molecules consisting of three equal-sized
portions,loosely connected by a flexible tether. Three schematic representations
of antibody structure, which has been determined by X-ray crystallography, are
shown in . The aim of this part of the chapter is to explain how this structure is
formed and how it allows antibody molecules to carry out their dual tasks—binding
on the one hand to a wide variety of antigens, and on the other hand to a limited
number of effector molecules and cells. As we will see, each of these tasks is carried
 out by separable parts of the molecule. The two arms of the Y end in regions that vary
between different antibody molecules, the V regions. These are involved
in antigen binding, whereas the stem of the Y, or the C region, is far less variable and
is the part that interacts with effector cells and molecules.

All antibodies are constructed in the same way from paired heavy and light
polypeptide chains, and the generic term immunoglobulin is used for all such proteins.
Within this general category, however, five different classes of immunoglobulins—
IgM, IgD, IgG, IgA, and IgE—can be distinguished by their C regions, which will be
described more fully in Chapter 4. More subtle differences confined to the V region
account for the specificity of antigen binding.
Applications of antibodies

Some important applications of antibodies in medicine and biomedical research are discussed
below:

Medicine

Diagnosis

 Antibodies are highly useful in medical diagnostics. Many biochemical assays enable
the detection of specific antibodies for the diagnosis of diseases.
 Most immunodiagnostic techniques such as ELISA use multiple antibodies to detect
specific antigens capable of causing infectious diseases
 In clinical immunology, levels of different classes of immunoglobulins are helpful in
analyzing a patient’s antibody profile.
  An increase in certain immunoglobulins is also a useful indicator in the diagnosis of
many ailments. e.g., the elevation of IgM is an indication of viral hepatitis
 Antibodies that can bind to human chorionic gonadotropin are used in over the
counter pregnancy test kits.

Therapy

 Antibodies are used to treat immune deficiencies such as hypogammaglobulinemia.

Here, ready-made antibodies are administered to the patient to induce passive
immunity.
 Monoclonal antibodies are widely used to treat several diseases such as multiple
sclerosis, rheumatoid arthritis, psoriasis, and several different cancers including
colorectal cancer and breast cancer. About a dozen monoclonal antibodies for cancer
treatment have been approved by the US Food and Drug Administration so far.
Clinical trials are on for developing more monoclonal antibodies that can help treat
many more types of cancer.

Prenatal therapy

Rho(D) immune globulin antibodies are used in prenatal treatment to prevent the risk for
hemolytic disease of the newborn. In the case of an Rh-incompatible fetus and mother, any
blood mixing may cause sensitization of Rh- mother to the Rh+ antigen from the child.

If the mother is treated with anti-RhD antibodies prior to delivery of trauma, it destroys the
Rh antigen from the fetus before the antigen stimulates maternal B cells. Thus, treatment with
Rho(D) immune globulin prevents sensitization that can cause Rh disease even in future
pregnancies.

Biomedical research

The high specificity and sensitivity of antibodies is a true advantage in biomedical research
applications. Advances in biotechnology have enabled the production of antibodies in a large
scale. This section provides an overview of the applications of antibodies in biomedical
research.

Western blotting

In this technique, proteins are electrophoretically separated and then transferred to a blotting
paper, which is exposed to labeled antibodies to detect the proteins.
Immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) are highly popular techniques used to detect
and quantitate a particular antigen in blood serum. These assays exploit the high specificity of
antibodies for different target antigens. Direct ELISAs use monoclonal antibodies to detect a
specific antigen in a solution. Indirect ELISA uses a primary and secondary antibody to
detect the antigen.

Immunohistochemistry/immunocytochemistry

Immunohistochemistry and immunocytochemistry are techniques used for in situ

determination of the presence and the location of proteins. In these techniques, primary
antibodies are used to bind to target antigens and conjugated secondary antibodies are used to
detect the antigen-primary antibody complex.

Immunoprecipitation assays

In immunoprecipitation assays, antibodies help label and precipitate target antigens from an
aqueous solution. Agarose beads first bind to the Fc region of the antibody and then allow
centrifugal separation of the antibody-antigen complexes.

In vivo applications

In immunological studies, antibodies can be used in vivo to deplete specific cells for
functional analyses. Antibodies are also in vivo for neutralization of cell surface receptors to
enable binding to soluble factors.

Flow cytometry

Antibodies are widely used in flow cytometry for intracellular analysis. In this method,
single-cell suspensions are surface stained with highly specific fluorochrome-tagged
antibodies that can be detected easily.

Cells can also be labeled with multiple antibodies as advanced flow cytometers are capable of
detecting 3 or more fluorochromes at the same time. This can help detect proteins in the
cytosol, nucleus, and endosomes.

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6. Write a note on biochips?

 The first biochip was invented by an American company namely Affymetrix, and the
product of this company is GeneChip (DNA microarrays). These products comprise
the number of individual DNA sensors used for sensing defects. Biochip plays an
essential role in the field of biology research like systems biology as well as disease
biology while the number of clinical applications is rising. It is a set of microarrays
which are placed on a strong surface of a substrate to allow thousands of reactions to
be performed in less time. The development of biochip mainly includes the
combination of molecular biology, biochemistry, and genetics. Biochips are used for
analyzing organic molecules connected with a live organism.
A biochip is a set of diminished microarrays that are placed on a strong substrate that allows
many experiments to be executed at the same time to obtain a high throughput in less time.
This device contains millions of sensor elements or biosensors. Not like microchips, these are
not electronic devices. Each and every biochip can be considered as a microreactor that can
detect a particular analyte like an enzyme, protein, DNA, biological molecule or antibody.
The main function of this chip is to perform hundreds of biological reactions in a few seconds
like decoding genes (a sequence of DNA).

Biochip
Working Principle of a Biochip:
The working of Biochip mainly includes the following steps.

1. Step1: The operator generates a low-power electromagnetic field through radio signals
2. Step2: The fixed biochip gets turn on
3. Step3: The activated chip transmits the identification code reverse to the operator through
radio signals
4. Step4: Reader strengthens the received code to change it into digital form and finally
exhibits it on LCD.
Components of BioChips
The Biochip comprises two components namely the transponder as well as reader.
 Components of BioChips
1) Transponder
Transponders are two types’ namely active transponder and passive transponder. This is a
passive transponder which means that it doesn’t contain any of its own energy or battery
whereas in passive, it is not active until the operator activates it by giving it a low electrical
charge. This transponder consists of four parts such as antenna coil, computer microchip,
glass capsule, and a tuning capacitor.

 The computer microchip stores a unique identification (UID) number that ranges from 10
digits to 15 digits long.
 The antenna coil is very small, primitive and this type of antenna is used to send and
receive the signals from the scanner or reader.
 The charging of the tuning capacitor can be done with the small signal i.e, 1/1000 of a watt
which is sent by the operator.
 The glass capsule holds the antenna coil, capacitor, and microchip, and it is made with a
biocompatible material namely soda lime glass.
2) Reader
The reader comprises of a coil namely “exciter” and it forms an electromagnetic field through
radio signals. It offers the required energy (<1/1000 of a watt) to activate the biochip.  The
reader carries a receiving coil for receiving the ID number or transmitted code sent back from
the excited implanted biochip.

Types of BioChips
There are three types of Biochips available namely DNA microarray, microfluidic chip, and
protein microarray.
 Types of BioChips
1) DNA Microarray
A DNA microarray or DNA biochip is a set of tiny DNA spots fixed to a strong surface. A
researcher utilizes to calculate the expression levels for a large number of genes. Every DNA
mark comprises picomoles of particular genes which are termed as probes. These can be a
short segment of a genetic material under high rigidity situations. Usually, probe-target
hybridization is noticed and counted by recognition of fluorophore or chemiluminescence
labeled targets to decide the relative quantity of nucleic acid series in the target. Innovative
arrays of nucleic acid were macro arrays about 9 cm X 12 cm and the initially automated icon
based analysis was published in the year 1981.

2) Microfluidic Chip
Microfluidic biochips or lab-on-a-chip are a choice to usual biochemical laboratories and are
transforming several applications like DNA analysis, molecular biology procedures,
proteomics which is known as the study of proteins and diagnostic of diseases (clinical
pathology). These chips are becoming more complex by using 1000’s of components, but
those components are designed physically called as bottom-up full-custom plan, which is a
very large workforce.

Protein Microarray
A protein microarray or protein chip method is used to follow the actions as well as
connections of proteins, and to find out their function on a large scale. The main advantage of
protein microarray is that we can track a large number of proteins in parallel. This protein
chip comprises of a surface for supporting like microtitre plate or bead, nitrocellulose
membrane, the glass slide. These are automated, rapid, economical, very sensitive, consumes
less quantity of samples. The first methodology of protein chips was introduced in antibody
microarrays of scientific publication in the year 1983. The technology behind this chip was
 quite easy to develop for DNA microarrays, which have turned into the most generally used
microarrays.

Biochips Advantages and Disadvantages

The advantages of biochip include the following.
 The biochip is used to rescue the sick
 Very small in size, powerful and faster.
 Biochips are useful in finding the lost people
 Biochips can be used to identify the persons individually
 Biochips perform thousands of biological reactions in a few seconds.
The disadvantages of biochip include the following.

 Biochips are expensive

 Biochip raises dangerous problems of individual privacy.
 Biochip marks the end of human being liberty and self-respect.
 There will be a chance of turning every person into a controlled person
 Biochips can be fixed into the human’s body without their interference.
Biochips Applications
The applications of biochip include the following.

 By using this chip we can trace a person or animal anywhere in the world.
 This chip is used to store and update the information of a person like medical financial and
demographics.
 A biochip leads to safe E-commerce systems
 These chips are effective in restoring the records of medical, cash, passport, etc.
 The biochip can be applicable in the medical field as a BP sensor, glucose detector, and
oxygen sensor.
7. Write a note on biosensors?

A biosensor is a device that measures biological or chemical reactions by generating signals

proportional to the concentration of an analyte in the reaction. Biosensors are employed in
applications such as disease monitoring, drug discovery, and detection of pollutants, disease-
causing micro-organisms and markers that are indicators of a disease in bodily fluids (blood,
urine, saliva, sweat). A typical biosensor is represented in fig ; it consists of the following
components.
 Analyte: A substance of interest that needs detection. For instance, glucose is an ‘analyte’ in
a biosensor designed to detect glucose.
 Bioreceptor: A molecule that specifically recognises the analyte is known as a bioreceptor.
Enzymes, cells, aptamers, deoxyribonucleic acid (DNA) and antibodies are some examples of
bioreceptors. The process of signal generation (in the form of light, heat, pH, charge or mass
change, etc.) upon interaction of the bioreceptor with the analyte is termed bio-recognition.
 Transducer: The transducer is an element that converts one form of energy into another. In a
biosensor the role of the transducer is to convert the bio-recognition event into a measurable
signal. This process of energy conversion is known as signalisation. Most transducers
produce either optical or electrical signals that are usually proportional to the amount of
analyte–bioreceptor interactions.
 Electronics: This is the part of a biosensor that processes the transduced signal and prepares
it for display. It consists of complex electronic circuitry that performs signal conditioning
such as amplification and conversion of signals from analogue into the digital form. The
processed signals are then quantified by the display unit of the biosensor.
 Display: The display consists of a user interpretation system such as the liquid crystal display
of a computer or a direct printer that generates numbers or curves understandable by the user.
This part often consists of a combination of hardware and software that generates results of
the biosensor in a user-friendly manner. The output signal on the display can be numeric,
graphic, tabular or an image, depending on the requirements of the end user.

Characterstics of biosensors

Selectivity
Selectivity is perhaps the most important feature of a biosensor. Selectivity is the ability of a
bioreceptor to detect a specific analyte in a sample containing other admixtures and
contaminants. The best example of selectivity is depicted by the interaction of an antigen
with the antibody. Classically, antibodies act as bioreceptors and are immobilised on the
surface of the transducer. A solution (usually a buffer containing salts) containing the antigen
is then exposed to the transducer where antibodies interact only with the antigens. To
construct a biosensor, selectivity is the main consideration when choosing bioreceptors.

Reproducibility

Reproducibility is the ability of the biosensor to generate identical responses for a duplicated
experimental set-up. The reproducibility is characterised by the precision and accuracy of the
transducer and electronics in a biosensor. Precision is the ability of the sensor to provide alike
results every time a sample is measured and accuracy indicates the sensor's capacity to
provide a mean value close to the true value when a sample is measured more than once.
Reproducible signals provide high reliability and robustness to the inference made on the
response of a biosensor.

Stability

Stability is the degree of susceptibility to ambient disturbances in and around the biosensing
system. These disturbances can cause a drift in the output signals of a biosensor under
measurement. This can cause an error in the meas-ured concentration and can affect the
precision and accuracy of the biosensor. Stability is the most crucial feature in applications
where a biosensor requires long incubation steps or continuous monitoring. The response of
transducers and electronics can be temperature-sensitive, which may influence the stability of
a biosensor. Therefore, appropriate tuning of electronics is required to ensure a stable
response of the sensor. Another factor that can influence the stability is the affinity of the
bioreceptor, which is the degree to which the analyte binds to the bioreceptor. Bioreceptors
with high affinities encourage either strong electrostatic bonding or covalent linkage of the
analyte that fortifies the stability of a biosensor. Another factor that affects the stability of a
measurement is the degradation of the bioreceptor over a period of time.

Sensitivity
The minimum amount of analyte that can be detected by a biosensor defines its limit of
detection (LOD) or sensitivity. In a number of medical and environmental monitoring
applications, a biosensor is required to detect analyte concentration of as low as ng/ml or
even fg/ml to confirm the presence of traces of analytes in a sample. For instance, a prostate-
specific antigen (PSA) concentration of 4 ng/ml in blood is associated with prostate cancer
for which doctors suggest biopsy tests. Hence, sensitivity is considered to be an important
property of a biosensor.

Linearity

Linearity is the attribute that shows the accuracy of the measured response (for a set of
measurements with different concentrations of analyte) to a straight line, mathematically
represented as y=mc, where c is the concentration of the analyte, y is the output signal,
and m is the sensitivity of the biosensor. Linearity of the biosensor can be associated with the
resolution of the biosensor and range of analyte concentrations under test. The resolution of
the biosensor is defined as the smallest change in the concentration of an analyte that is
required to bring a change in the response of the biosensor. Depending on the application, a
good resolution is required as most biosensor applications require not only analyte detection
but also measurement of concentrations of analyte over a wide working range. Another term
associated with linearity is linear range, which is defined as the range of analyte
concentrations for which the biosensor response changes linearly with the concentration.

Applications of biosensors

Biosensors have a very wide range of applications that aim to improve the quality of life.
This range covers their use for environmental monitoring, disease detection, food safety,
defence, drug discovery and many more. One of the main applications of biosensors is the
detection of biomolecules that are either indicators of a disease or targets of a drug. For
example, electrochemical biosensing techniques can be used as clinical tools to detect protein
cancer biomarkers .Biosensors can also be used as platforms for monitoring food traceability,
quality, safety and nutritional value . These applications fall into the category of ‘single shot’
analysis tools, i.e. where cost-effective and disposable sensing platforms are required for the
application. On the other hand, an application such as pollution monitoring requires a
biosensor to function from a few hours to several days. Such biosensors can be termed ‘long-
term monitoring’ analysis tools. Whether it is long-term monitoring or single shot analysis,
biosensors find their use as technologically advanced devices both in resource-limited
settings and sophisticated medical set-ups: e.g. with applications in drug discovery for the
detection of a number of chemical and biological agents that are considered to be toxic
materials of defence interest for use in artificial implantable devices such as pacemakers and
other prosthetic devices and sewage epidemiology A range of electrochemical, optical and
acoustic sensing techniques have been utilised, along with their integration into analytical
devices for various applications. Fig  indicates different areas of research where biosensors
have been used.

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8. Write briefly about biofuels?

Biofuels are being promoted as a low-carbon alternative to fossil fuels as they could
help to reduce greenhouse gas (GHG) emissions and the related climate change
impact from transport. However, there are also concerns that their wider deployment
could lead to unintended environmental consequences. Numerous life cycle
assessment (LCA) studies have considered the climate change and other
environmental impacts of biofuels. However, their findings are often conflicting, with
a wide variation in the estimates. Thus, the aim of this paper is to review and analyse
the latest available evidence to provide a greater clarity and understanding of the
environmental impacts of different liquid biofuels. It is evident from the review that
the outcomes of LCA studies are highly situational and dependent on many factors,
including the type of feedstock, production routes, data variations and methodological
choices. Despite this, the existing evidence suggests that, if no land-use change (LUC)
is involved, first-generation biofuels can—on average—have lower GHG emissions
than fossil fuels, but the reductions for most feedstocks are insufficient to meet the
GHG savings required by the EU Renewable Energy Directive (RED). However,
second-generation biofuels have, in general, a greater potential to reduce the
emissions, provided there is no LUC. Third-generation biofuels do not represent a
feasible option at present state of development as their GHG emissions are higher
than those from fossil fuels. As also discussed in the paper, several studies show that
reductions in GHG emissions from biofuels are achieved at the expense of other
impacts, such as acidification, eutrophication, water footprint and biodiversity loss.
The paper also investigates the key methodological aspects and sources of uncertainty
in the LCA of biofuels and provides recommendations to address these issues.
 iofuels can be differentiated according to a number of key characteristics, including
feedstock type, conversion process, technical specification of the fuel and its use.
Owing to this multitude of possible distinctions, various definitions are in use for
biofuel types. Two commonly used typologies are ‘first, second and third generation’
and ‘conventional and advanced’ biofuels. Biofuels produced from food or animal
feed crops are referred to as first-generation biofuels. Since first-generation biofuels
are produced through well-established technologies and processes, such as
fermentation, distillation and transesterification, they are also commonly referred to as
‘conventional biofuels'. A key characteristic for second-generation biofuels is that
they are derived from non-food feedstocks, such as dedicated energy crops
(e.g. Miscanthus, switchgrass, short rotation coppice (SRC) and other lignocellulosic
plants), agricultural residues, forest residues and other waste materials (e.g. UCO and
municipal solid waste). Biodiesel produced from microalgae through conventional
transesterification or hydro-treatment of algal oil is commonly known as third-
generation biofuel. Second- and third-generation biofuels are often referred to as
‘advanced biofuels’ as their production techniques or pathways are still in the research
and development, pilot or demonstration phase. In this paper, the terminology ‘first,
second and third generation’ has been selected and followed throughout. 

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9. Explain transgenic animals and plants?
Transgenic Animals

The transgenic animals are successfully used for production of recombinant proteins. Protein
production poses great risk in terms of safety as transmission of infectious agents, allergic
responses, immune reactivity, and autoimmune responses might occur.

ATryn was the only approved (approved in 2006 by European medical agency and in 2009 by
FDA) recombinant biopharmaceutical using transgenic animals. It contains human
antithrombin (432AA) with 15 % glycosylated moieties and is secreted into milk of
transgenic goats.

Rhucin intended for acute attacks of angioedema in patients with congenital C1 inhibitor
activity deficiency, obtained from transgenic rabbit, was denied approval. Transgenic plants
are being explored as recombinant protein producers for research and diagnostic uses.
 Transgenic Plants

Obtaining therapeutic proteins from their natural source poses threat for spread of diseases.
Therefore, alternative systems for the production of therapeutic agents have their own
benefits. Molecular farming in plants has been widely explored for production of
recombinant pharmaceutical proteins. Their advantages are low cost, high mass production,
scale-up, lack of human pathogens, and addition of eukaryotic PTMs. The first recombinant
protein obtained in 1986 from tobacco plants was human growth hormone. However,
sometimes plant-specific PTMs might result in adverse immune reactivity.

Production of recombinant heterologous proteins in plants is simple and is used for

production of non-naturally occurring proteins as single chain Fv fragments (ScFvs). High
yield of recombinant protein is the main goal of production system in transgenic plants.
Therefore appropriate expression vectors and constructs are designed to achieve high yields
of the engineered gene products .

Protein Production in Plant System 

The transgene in expression construct is chimeric structure as it is surrounded by various

active regulatory elements. Polyadenylation sites play an important role for the high level of
expression of transgene. Cauliflower mosaic virus (CaMV) 35S promoterworks well with
dicots. It is a strong constitutive promoterthat is made more active by duplicating the
enhancer region. However, in monocots maize ubiquitin-1 promoter is the preferred
promoter. The presence of an intron in the 5′-untranslated region (5′-UTR) enhances
transcription in monocots.

For obtaining high yield of the protein, several factors may be appropriately considered:

 The incorporation of polyadenylation sites may be from CaMV 35S transcripts or

the Agrobacterium tumefaciens nos gene or the pea ssu gene.
 The yield can be controlled by placing the gene under the control of the promoterwhich is
active in a particular tissue or developmental stage or particular environment (e.g., rice
glutelin, pea legumin).
 Usage of inducible promoter (e.g., tomato hydroxyl-3-methylglutaryl CoA reductase-2
(HMGR2)) which has mechanical gene activation system developed by Cramer (Crop Tech
 Corp., Virginia, USA). Transcription starts when harvested tobacco leaves are sheared during
processing.
 Codon bias in the host plant may be overcome by engineering of transgene at positions,
which might lead to truncation and/or misincorporation, or slowing the process.
 Subcellular targeting is also very important factor which affects the process of folding,
assembly, and posttranslational modification and can be efficiently achieved by inclusion of
an N-terminal signal peptide.
 Position of transgene integration.
 Structure of transgene locus.
 Gene copy number.
 Presence of truncated or rearranged transgene copies.
 Affinity tags as His or the FLAG epitopes can be used to ease the process of purification;
however, these modifications not only affect the primary structure but also the properties of
the protein.

Figure shows the general vector pCAMBIA (it is small in size (7–12 kb) maintained in high
copy number with pVS1 replicon that imparts chloramphenicol or kanamycin resistance and
high stability in Agrobacterium) that is used for transgene expression in the plants. The
modified vector has shown success in insulinproduction.
The figure shows the structure of pCAMBIA vector (cambia.org) used for plant
transformation. The vector has CaMV35S promoter, multiple cloning site, and reporter gene
(GUS or GFP may be used). The vector can be modified to express genes for insulin (tomato)
or Hep-B surface antigen (HBsAg) for recombinant therapeutics

Nowadays plant system is efficiently engineered to produce human growth hormone, human
serum albumin, erythropoietin, α-interferon, antibodies and ScFvs, toxins, subunit vaccines,
and insulin .

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