BP605 T. PHARMACEUTICAL BIOTECHNOLOGY Unit-I

a) Brief introduction to Biotechnology with reference to Pharmaceutical Sciences.
b) Enzyme Biotechnology- Methods of enzyme immobilization and applications.
c) Biosensors- Working and applications of biosensors in pharmaceutical Industries.
d) Brief introduction to Protein Engineering.
e) Use of microbes in industry. Production of Enzymes- General consideration
Amylase, Catalase, Peroxidase, Lipase, Protease, Penicillinase.
f)
Basic principles of genetic engineering.

1. Pharmaceutical
Biotechnology
UNIT-1
Prepared And Presented By
Mr. Sandip R. Bhoi
Assistant Professor
KVPS’S I.P.E. College, Boradi

2. introduction to Biotechnology
Theword"biotechnology"wasfirstusedin1917todescribe
processesusinglivingorganismstomakeaproductorruna
process,suchasindustrial
fermentations.
Biotechnologymeansanyscientificapplicationthatuses
biologicalsystems,livingorganisms,orderivativesthereof,
toproduceoralterproductsorprocessesforparticularuse.
Theutilizationoflivingorganisms,systems,orprocesses
constitutesbiotechnology.

4. 10,000 years ago: People began using biological
processes to improve their lives, starting with the first
agricultural communities.
6,000 years ago: Humans started using
microorganisms’ biological processes to make bread,
alcoholic beverages, cheese, and to preserve dairy
products.
1960s and '70s: The term “biotechnology” was first
widely used to describe molecular and cellular
technologies that were emerging during this time.
HISTORY OF BIOTECHNOLOGY

5. Mid-to late 1970s: The “biotech” industry began to
form, led by Genentech, a pharmaceutical company
established in 1976 by Robert A. Swanson and
Herbert W. Boyer. They aimed to commercialize the
recombinant DNA technology pioneered by Boyer,
Paul Berg, and Stanley N. Cohen.
Early biotech companies: Companies like
Genentech, Amgen, Biogen, Cetus, and Genexstarted
by manufacturing genetically engineered substances
mainly for medical and environmental uses

6. 1.1919: Karl Erekycoins “biotechnology”.
2.1928: Alexander Fleming discovers penicillin.
3.1943: Oswald Avery shows DNA carries genetic information.
4.1953: James Watson and Francis Crick discover DNA’s double helix structure.
5.1960s: Insulin synthesized.
6.1969: First in vitro enzyme synthesis.
7.1973: Herbert Boyer and Stanley Cohen develop genetic engineering.
8.1980s: First biotech cancer drugs developed.
9.1982: FDA approves first genetically engineered product, a form of insulin.
10.1983: First genetically modified plant introduced.
11.1997: First mammal cloned.
12.2010: First synthetic cell created.
13.2013: First bionic eye created.
14.2020: MRNA vaccine and monoclonal antibody technology
used to treat SARS-CoV-2 virus.

7. ApplicationsOf Biotechnology

8. 1.Enzymes are biological catalysts that speed up a reaction without
being used up during the process. They are protein molecules folded
into complex shapes where smaller molecules, also called substrates,
can fit into them. The place where the substrate binds is called the
active site.
2.Enzyme has five important properties, they are protein molecules,
reusable, affected by temperature, affected by pH and they are
“specific”.
ENZYMEBIOTECHNOLOGY

9. Starting with enzymes being protein molecules, most of enzymes are
protein molecules; however, not all the enzymes are purely protein
molecules. Cofactors are non-protein bits in enzymes that are necessary for
enzymes to work. If enzymes don’t have their cofactor, the enzymes won’t
work. Inactive protein molecules are called, “Apoenzymes” and protein
molecules with the right cofactors that are working properly, the enzymes
are called, “holoenzymes”.
https://youtu.be/pVoytz_3H_s?si=d--kkZJP46O39kkL
https://www.rcsb.org/3d-view/1ckn

10. Factors that Affect the Rate of Enzyme Reactions
1.Temperature
2.pH
3.Enzyme concentration
4.Substrate concentration
5.Inhibitors
a .competitive inhibitor
b. non-competitive inhibitor
6.Feedback Inhibition
(Allosteric Effectors)

12. 1.Enzyme immobilization is a strategy to improve the stability of an
enzyme, but also a strategy for easily re-using the enzymes.
2.Enzyme immobilization technology refers to the natural enzyme
limited within a certain space or attached to a solid structure.
3.Immobilization is a common, effective, and convenient means for
enzymatic modification to improve its catalytic activity and
stability.
4.Enzyme immobilization is the process by which the enzyme
catalyst is trapped at the bio-anode or bio-cathode surface.
5.Immobilized enzymes are the enzymes that are fixed to inert and
insoluble carriers.
ENZYME IMMOBILISATION

13. Properties of carrier molecules
1.Inert and Insoluble
2.Stable at all pH
3.Carrier should be stable at all ionic strength
4.Should be stable in a particular solvent at a particular condition(neither
should be unstable nor insoluble)
Types of carriers:
A. Organic natural carriers
Favourable compatibility with proteins.
Example: chitosan, starch, agar
B . Inorganic carriers
High-pressure stability and may undergo abrasion.
Example: mineral material-clay, celite, centonite. Poroursglass, silica.
C. Organic synthetic carriers
High chemical and mechanical stability.
Examples: polystyrene, polyvinyl acetate, and acrylic polymers.

14. Advantages/significance of enzyme immobilisation:
1.Prevents deactivation/degradation of enzymes.
2.The enzymes can be recovered at the end of the reaction and can be
reused.
3.Easily separated from the products.
4.Increases the stability of the enzyme.
5.Better control of reaction
6.Potential in preparation of medicine and other products in the food
and detergent industry.

15. Enzyme Immobilization Methods

16. 1. Adsorption
1.Enzymes are immobilized by adsorbing onto the surface of carrier material.
2.This technique is reversible, enzymes can easily be desorbed from carrier molecules
due to the change in substrate and ionic strength.
3.In this technique, enzymes are going to attach with the carrier molecule by hydrogen
bonding and Van der Waal’s force of attraction.
2. Entrapment
1.By this technique, the enzymes are immobilized by entrapping within the pores of
the carrier matrix.
2.The matrix material like polyacrylamide gel, cellulose derivatives, silica, and
calcium alginate.
3.Immobilization by this method can be done in two ways:
1) Inclusion in gel eg.polyacrylamide gel
2) Inclusion in fiber eg.cellulose derivatives

17. 3) Encapsulation
1.By this technique the enzymes are immobilized by encapsulating within the semi-permeable
membrane of the carrier.
2.Carrier used for micro-encapsulation include cellulose derivatives, polystyrene, nylon, etc
4) Covalent bonding
1.By this technique, the enzymes are immobilized by forming covalent bonds with the carrier
matrix.
2.The functional group of matrix-like carboxylic acid, alcoholic group, amino group, tyrosyl
group, etc. attaches with an enzyme for immobilization.
3.The functional group of a carrier participates in Covent coupling but does not affect the
activity of the enzyme.
There are three methods by which immobilization is done:
1) formation of Diazotization
2) Formation of peptide bond-bond is formed between the amino/carboxylic group of the carrier
by that of the enzyme.
3) By multifunctional/ Di-functional agents-a bond is formed between amino groups by that of
the enzyme.

18. 5) Cross-linking (Polymerization)
1.By this technique the enzymes are immobilized by cross-linking with multi-
functional agents which lead to the formation of a 3D network of enzymes.
2.Examples of multifunctional agents are Glutaraldehyde and diazonium salts are used
in industrial techniques for enzyme immobilization.
3.An increase in the concentration of these agents can cause enzyme denaturation.

19. APPLICATIONS
I.Immobilized enzymes are widely used in the diagnosisand treatmentof many
diseases.
II.Immobilized enzymes can be used to overcome inborn metabolic disorders by the
supply of immobilized enzymes.
III.Immobilization techniques are effectively used in drug delivery systems,
especially to oncogenic sites.
IV.The use of immobilized enzymes allows researchers to increase the efficiency of
different enzymes such as Horse Radish Peroxidase (HRP) in blotting experiments
and different Proteases for cell or organelle lysis.
V.Widely used in the commercial production of antibiotics, beverages, amino acids
secondary metabolites, etc. of industrial-grade
VI.Immobilized enzymes are most commonly used in fast diagnostic kits like ELISA
and the treatment of many pathogenic diseases.

20. “A self-contained integrated device which is capable of providing specific
quantitative or semiquantitative analytical information using a biological
recognition element which is in direct spatial contact with a transducer
element.”
biosensors

21. 1. BIO-ELEMENT
It is a typically complex chemical system usually extracted or derived
directly from a biological organism.
• Enzymes • Antibiotics • Tissue • Nucleic acid
Function
• To interact specifically with a target
• The ability of a bio-element to interact specifically with the target
compound (specifically) is the basis for biosensors.
2. TRANSDUCER
To convert the biological response into an electrical signal.
• Electrochemical
• Optical
• Mass bases

23. APPLICATIONS OF BIOSENSORS
1. Biosensors in Medical Diagnostics medium:
2. Biosensors at the Food and Agriculture level:
3. Biosensors and Pathogen detection
4. Environmental Monitoring:
5. Food and Fermentation:
6. Biosensors for cardiac biomarkers detection
7. DiseaseDiagnosis.
8. Cancerdiagnosis
9. Biosensors: Industrial Biotechnology:

24. •Protein engineering can be defined as the modification of protein
structure with recombinant DNA technology or chemical treatment to get
a desirable function for better use in medicine, industry, and agriculture.
•Protein engineering is the process of developing useful valuable
proteins.
The objectives of protein engineering are as follows
•To create a superior enzyme to catalyze the production of high value
specific chemicals.
•To produce enzymes in large quantities.
•To develop useful valuable proteins
•To get a desirable function for better use in medicine, industry and
agriculture.
Protein engineering

25. •Elimination of allosteric Regulations
•Improved kinetic properties
•To produce biological compounds (including synthetic peptides,
storage proteins, and synthetic drugs) superior to natural ones.
•Enhanced substrate and reaction specificity
•Increased thermostability.
•Alteration in optimum pH
•To speed up the process( rate of reaction)
•Increase Protein/ Enzymes' Shelf Life
•To get high-quality of Product Suitability face

26. three techniques of protein engineering
1.Rational Protein Design: This is like an architect designing a
building. They know exactly what they want the building to look like
and what it should do, so they create detailed plans to achieve this.
Similarly, in rational protein design, scientists use their knowledge
of a protein’s structure and function to make specific changes.
2.Directed Evolution: This is more like nature’s way of creating new
species through evolution. Random changes (mutations) occur,
and if they are beneficial, they are kept. In the lab, scientists
introduce random mutations into a protein and then select the ones
that have the desired traits. It’s a bit like trial and error, but guided
by the scientist’s goals.
3.De Novo Design: Involves designing and producing novel protein
structures from scratch. It’s effective when the 3D structure and
mechanism of the protein are well known.

28. Protein engineering applications:
1.Industrial Use: Engineered proteins can be used where natural proteins
might not work efficiently
.
2.Medical Applications: They are used for developing antiviral peptides,
antibody engineering, and designing therapeutics specific to diseases.
3.Research: Protein engineering helps in probing protein sequence-function
relationships.
4.Agriculture: Engineered proteins can improve crop yield and resistance.
5.Environmental Sciences: They can be used for bioremediation and pollution
control.
6.Nanobiotechnology: Engineered proteins can be used to develop
nanostructures and devices.

29. Industrial applications of the microbial enzymes

30. Production of Enzymes

31. submerged fermentation
•The medium in submerged
fermentation is liquid which
remains in contact with the
microorganism.
•A supply of oxygen is essential in
submerged fermentation.
•There are four main ways of
growing microorganisms in
submerged fermenters: batch
culture, fed-batch culture,
perfusion batch culture, and
continuous culture.

32. •Solid State Fermentation is
defined as fermentation involving
solids in the absence of free water,
although the substrate
must possess sufficient moisture to
support microbial growth
and metabolism.
•The process of SSF is performed
on a solid substrate with a
low moisture content, with the
advantages of a high product
concentration but only a relatively
low energy being required.

34. Genetic engineering (also known as genetic modification or
genetic manipulation) is the process of direct manipulation of an
organism's genes using recombinant DNA (rDNA) technology to
alter the genetic makeup of an organism.
INTRODUCTION TO GENETIC ENGINEERING

36. https://youtu.be/4YKFw2KZA5o?si=Lzmn9qJXy_HwWv6n
APLICATION OF GANETIC ENGENIRING
1.Medicine: It’s like a factory for making medicines.For
example, we can make bacteria produce insulin, a hormone
that helps regulate sugar in our body.
2.Research: It’s a tool for understanding life.By changing the
genes in bacteria or other organisms, we can learn what those
genes do.
3.Industry: It’s a way to make useful stuff.For example, we can
engineer bacteria to produce proteins that can be used in food,
fuels, or even to make biodegradable plastics.
4.Agriculture: It’s a method to improve crops.We can make
plants that are resistant to pests, diseases, or harsh weather

37. THE END