UNIT V
Protein Engineering:
•Brief introduction to protein engineering,
•Use of microbes in industry,
•Production of enzymes-general considerations, Amylase,Catalase,
peroxidase, Lipase
•Basic principles of genetic engineering
BIOTECHNOLOGY
PRESENTED BY : SHYAM BASS
LOVELY SCHOOL OF PHARMACEUTICAL SCIENCES
B.SC. BIOTECHNOLOGY (HONS.), B.PHARMA

• Protein Engineering can be defined as the modification of the protein structure with Recombinant DNA Technology or
chemical treatments to get a desirable function for the better use in medicine, industry and agriculture.
• It the manipulation of the structures of proteins so as to produce desired properties, or the synthesis of proteins with
particular structures.
OBJECTIVES OF PROTEIN ENGINEERING:
• To develop or designing of peptides(short protein structures) that can bind selectively to target proteins.
• To produce the enzyme in larger quantity.
• To produce the compounds superior to the natural ones(synthetic peptides, synthetic drugs, etc)
• To design the peptide or to modify the protein
- with increased substrate specificity,
- with increased stability to temperature,
- with increased stability to pH etc.
BASIC STEPS OF PROTEIN ENGINEERING :
BRIEF INTRODUCTION TO PROTEIN ENGINEERING
Isolation of desired gene
sequence of that protein
Modification in the gene
sequence and development of
new gene sequence
Determination of 3D structures
of protein of interest by
using x-ray crystallography
or NMR
Expression of gene into new
protein and assess the new or
modified protein
1) 2) 3) 4)

BRIEF INTRODUCTION TO PROTEIN ENGINEERING
APPLICATIONS OF PROTEIN ENGINEERING:
• Medical application: Pre-targeted radioimmunotherapy is one of the most prominent examples of protein
engineering in the treatment of cancer. Antibodies are modified by protein engineering which is used to target
the specific antigen and the Development of peptide vaccines are few examples of the role of protein
engineering in the medical application.
• Environmental applications: Protein engineering modify the gene expression regulation of micro-organisms by
genetic methods and strategies to eliminate environmental pollutants and to survive in environmental stressed
conditions. Many organic pollutants such as phenols, organophosphorus pesticides can be detoxified using
enzymatic detoxification.
• Food industry application: There are enzymes like amylases, proteases, and lipase which are food processing
enzymes that have been improved its quality or activity using DNA recombinant technology. Example-
Microbial Protease enzymes are produced due to its low cost and high yields, Amylases is used to adjust
softness and volume in flour and bread while baking, Lipase is used in stability of dough and Cheese flavor
application.
• Application in Biopolymer production: Protein engineering is used to produce Peptide-based biomaterials
such as silk, elastin, etc.
• Detergent industry: the technique is used to produce enzymes with high activity, high Thermo and pH
stability. Example - lipase enzyme used to remove lipid stains and Protease enzyme used to remove protein
stains.

BRIEF INTRODUCTION TO PROTEIN ENGINEERING
METHODS OF PROTEIN ENGINEERING:
Basically the following are two methods of protein engineering:
CHEMICAL
MODIFICATION OF
ENZYMES
Mutagenesis:
• It is the process of generation of mutation in the gene to develop a new or modified protein molecule.
• Example- Anthranilate Synthetase enzyme in E.coli is sensitive to tryptophan inhibitor but after mutation, the
Anthranilate Synthetase enzyme in E.coli becomes insensitive to tryptophan inhibitor and helps in continuous
synthesis of tryptophan.
• According to the central dogma of life, the DNA is translated to form RNA and RNA is the transcript to form
a protein.
• Thus if the change occurs in the DNA or gene structure, the protein formed can be automatically changed.
• Therefore in the process of gene modification, there are two approaches:
• 1=> Invitro mutagenesis using synthetic oligonucleotides
• 2=> De novo synthesis of the complete modified gene.
MUTAGENESIS

BRIEF INTRODUCTION TO PROTEIN ENGINEERING
Fig 1.1 Oligonucleotide-directed mutagenesis
1=> Invitro mutagenesis using synthetic oligonucleotides
• Synthetic oligonucleotides (small DNA/RNA fragments) are used for in-vitro
(outside the body) mutagenesis.
• A small Oligonucleotide primer is synthesized with the desired modification.
• Allow the hybridization of oligonucleotide primer at the appropriate site of the
parent gene. (fig.1.1)
• Development of clone gene and replicate by using the DNA polymerase enzyme. (Fig
1.1)
Allow the expression of the gene and assess the activity.
2=> De novo synthesis of the complete modified gene.
• De novo refers to the synthesis of complex molecules from simpler molecules.
• Thus, in the same way, some cases the genes are being designed or a complete gene is
the arrangement of several Oligomers ( a molecular complex arranged in several
units ) like genes of insulin +somatostatin+interferon are ligated in correct order to
produce a complete gene. (fig 1.2)
• Therefore the sequence of synthetic genes is arranged in a particular fashion to get a
desired functional protein. (fig 1.2)
Oligomers
Biocompatible Oligomers genes
Fig 1.2 De novo synthesis of complex modified gene

BRIEF INTRODUCTION TO PROTEIN ENGINEERING
Chemical Modification of enzymes:
• It is the modification of enzyme or protein by modifying the gene structure at a post-translational stage of
the central dogma. Also called a post-translational modification.
• Here the introduction of a new chemical group before translation results in the formation of modified
enzymes.
• For example- Polyethylene glycol (PEG) modification of enzyme L-asparaginase i.e PEG-L-asparaginase
formation becomes more effective than its native enzyme(L-asparaginase).
• L-asparaginase has an anti-tumor effect but toxic while modified form improves biostability and non-allergic.
• Here the gene sequence is isolated from the L-asparaginase enzyme and traced with PEG to give mutant
gene thus gene is expressed to give the PEG-L-asparaginase enzyme conjugate.

USE OF MICROBES IN INDUSTRY
S.no. Micro-organism
Enzyme/
product
Uses
1
Bacillus licheniformis,Bacillus
amyloliquefaciens
Amylase
Food, fermentation and
textile industry
2
Pseudomonas species, Staphylococcus
species
Lipase
In preparation of cheese,
detergents, leather etc
3 Aspergillus niger, Bacillus subtiles Peroxydase
Waste treatment, in ELISA
test
4 E.coli, Nocardia species Catalase
In treatment of milk, to
remove traces of H2O2 from
clothes
5 Aspergillus niger, Bacillus subtilis Protease Photographic industry
6 Penicillium notatum Penicillin Antibiotic

USE OF MICROBES IN INDUSTRY
• Bacteria, Fungi, Yeast, etc. are used commonly in the production of various fermented products like wine,
yogurt, etc.
• Production of food and dairy products. Cheese, yogurt, alcoholic beverages, coffee, tea, vitamins, etc are
some of the examples.
• Production of vaccines is another important application of industrial microbiology.
• Antibiotics are another important product produced by using microorganisms.
• Production of antibodies and enzymes from important industrial micro-organisms including fungus and
bacteria.
• Production of biofuels from algae and related organisms
• The production of biodiesel from biomass using micro-algae.
• The production of bioethanol from starch fermentation of yeast species.
Use of microbes
In industry
Fig 1.3 Use of microbes In industry

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
AMYLASE
α-AMYLASE β-AMYLASE γ -AMYLASE
• Amylase catalyses the the Hydrolysis of starch into sugars.
• Following are the types of Amylase enzyme.
• Also called as 1,4-α-D-glucan
glucanohydrolase.
• Breaks Amylose to Maltose &
Amylopectin to Dextrin and
Glucose.
• Source: Human saliva,
Pancreas, Plants, Fungi like
Ascomycetes,Aspergillus niger,
Bacteria like Bacillus
Licheneformis , Bacterium subtilis
etc.
• He enzyme is active at pH
6.7-7.0
• Also called as 1,4-α-D-glucan
Maltohydrolase.
• During ripening of fruit it
breaks starch into Maltose,
which provide sweet flavour to
fruit.
• Source: Plants , Bacteria and
fungi.
• The enzyme is active at
optimum pH 4-5.
• α and β amylase are mostly use
in brewing industry for the
preparation of beer.
• Also called as 1,4-α glucosidase
• It breaks the amylose to
Amylopectin.
• He enzyme is active at
optimum pH 3.

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
AMYLASE
Production process of Amylase enzyme is as follows:
• MEDIUM: Starch+ Corn steep liquor + Buffering agent
• INOCULUM: Bacillus licheneformis
Inoculum+Fermentation medium
Incubate at
30-40℃
Incubation causes proper growth of Inoculum
At 30-40℃
Allowed fermentation
for 100hours
This causes continuous fermentation of Amylase
Filetration
Discard the residue Filtrate- Precipitation with Acetone/
Alcohol/ Ammonium Sulphate
Wash the precipitate and dry the
content
The application of Amylase are:
• Used in liquefaction of Starch.
• Use in Brewing industry.
• Production of bread.
• Production of candy of desired softness etc.

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
CATALASE
• It protects the cell from oxidative damage.
• One molecule of Catalase can convert 40,000 molecules of H2O2(Hydrogen
Peroxide) to water and oxygen.
• Following are the types of Catalase enzyme.
Heme Catalase Manganese Catalase Peroxidase Catalase
• The enzyme along with iron. • The enzyme along with
manganese.
• The enzyme found in
peroxisomes of cell ,
neutralises the toxicity of
H2O2 .
• Catalase is Tetrameric enzyme and is present in the cells of all aerobic organisms.
• Source:Catalase is produced mainly by extraction from bovine liver and, in recent years , from Aspergillus
Niger , Micrococcus luteus , E.coli, Nocardia species etc.
• Sweet potato is a Good source of Catalase.
• The Applications Catalase enzyme are:
• H2O2 is used in textile industry fro Bleaching of clothes , Catalase enzyme is used to remove traces of
H2O2 from clothes.
• Catalase is used for treatment of milk to remove H2O2 before cheese production.
• Used to degrade from industrial effluent.

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
CATALASE
Production process of Catalase enzyme is as follows:
• MEDIUM: Sucrose/Lactose/Fructose + Peptone/Beef extract + Ferrous sulphate/
Ammonium sulphate.
• STOCK SOLUTION: Nutrient broth+E.coli kept for 24hours at 30℃.
• INOCULUM: E.coli.
Fermentation medium + Inoculum
Kept at 37 ℃ for 24hours
And centrifuged at 12000 rpm
at 4℃ for 10min.
SupernatantDiscard the residue
Treated with
Ammonium Sulphate
Precipitates are
washed , dried and
Obtained the product.

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
PEROXIDASE
• Peroxidase are the group of enzymes that catalyses Oxidation-Reduction
reactions.
• It is also called as Oxidoreductase.
• Peroxidase ise H2O2 as electron acceptor for catalysing different oxidative
reaction.
• Source: Fungi- Aspergillus niger , Bacteria- Bacillus subtilis.
Production process of Catalase enzyme is as
follows:
1st method
• MEDIUM: Glucose +yeast extract+Ammonium Nitrate +
Magnesium sulphate +Dipotassium phosphate
• INOCULUM: Aspergillus niger
• STOCK: Stock culture is prepared by inoculating A.niger
on potato dextrose agar slants at 4℃ for 24hours.
Fermentation medium + Inoculum
Incubate at 22dgree celcius on a
rotatory shaker at 160 rpm
For 7 days followed by Filterartion
Discard the residue Filterate
Centrifuge the filtrate at 500rpm for 15min at
4℃
Discard the pellets Supernatant
Added Ammonium sulphate
Collected the ppt , washed , dried and collected the product

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
PEROXIDASE Production process of Catalase enzyme is as follows:
2nd method
• MEDIUM: Glucose +yeast extract+ Dipotassium Hydrogen phosphate
• INOCULUM: Bacillus subtilis
Fermentation medium + Inoculum
37 ℃ on a
rotatory shaker incubator
for 48 hours
followed by Centrifugation
Discard the pellets Supernatant
Added Ammonium sulphate
Collected the ppt , washed , dried and collected the product
Centrifuge the fermentation
medium at 500 rpm for 10min
The applications of Peroxidase are:
• Mostly use for treatment of waste material
• Used in ELISA for detection of Antigen-Antibody (Ag-Ab)
Reaction.

PRODUCTION OF ENZYMES AND GENERAL CONSIDERATIONS
LIPASE Lipase is also called as Glycerol ester hydrolase.
Lipase converts fat to mono/di - glycerides and fatty acids
Source: Fungi: Aspergillus species, Penicillium species & Bacteria- Pseudomonas
species, Staphylococcus species.
Production process of Lipase enzyme is as follows:
• MEDIUM: Peptone +yeast extract+ olive oil +Dipotassium Hydrogen
phosphate+ Manganese chloride + Ammonium sulphate + Calcium chloride
• INOCULUM: Pseudomonas aeruginosa
• pH maintained at 7.2
Fermentation medium + Inoculum
Incubate at 35℃
with constant shaking at
125 rpm for 3days
followed by Centrifugation
Discard the residue
at bottom
Supernatant fluid
Added Ammonium sulphate
Collected the ppt , washed , dried and collected the product
Centrifugation at 10,000 at 4℃for
20min
The applications of Lipase are:
• Used in dairy preparation like cheese , butter etc.
• Used in preparation of detergents to remove greasy stain.
• Also used in leather industry to clean leather.

BASIC PRINCIPLE OF GENETIC ENGINEERING
• Genetic Engineering involves manipulation of genetic material , this is alternately called Recombinant DNA
technology or Gene Cloning.
• Manipulation or Alteration of gene
• Artificial cutting a piece of DNA from one organism and going this piece of DNA into DNA of another organism.
• The basic principles are as follows:
Fig 1.4 The principles of genetic engineering.
A bacterial cell receives a human gene so it makes a human protein-
The Hormone Insulin
1) DNA fragment of
interest is obtained
by cleaving
chromosomes by
Restriction
endonuclease.
2) Cloning vector is
cleaved with
Restriction
endonuclease.
3) Fragments are
ligated to the
prepared cloning
vector.
4) Recombinant
vector DNA is
introduced into the
host cell
5) Propagation
(cloning) produces
many copies of
recombinant DNA
6) The gene is
extracted and
harvested the
product.

• https://www.slideshare.net/AkshayParmar22/application-of-protein-engineering
• https://nuclineers.com/microbes-used-industrial-biotechnology/
• https://www.hindawi.com/journals/er/2011/615803/
• https://www.biotecharticles.com/Biotech-Research-Article/Basic-Principles-of-Genetic-
Engineering-827.html
• https://www.tandfonline.com/doi/abs/10.3109/03639048509055598?journalCode=iddi20
REFERENCES

THANK YOU
PRESENTED BY : SHYAM BASS
LOVELY SCHOOL OF PHARMACEUTICAL SCIENCES
B.SC. BIOTECHNOLOGY (HONS.), B.PHARMA