In the field of biotechnology there are many industrial applications that result in biotech products that we use everyday at home. Some of these are food science applications that utilize enzymes to produce or make improvements in the quality of different foods. In the dairy industry, some enzymes are required for the production of cheeses, yogurt and other dairy products, while others are used in a more specialized fashion to improve texture or flavour. Isolation and Purification of Enzymes Enzymes are unstable molecules with a definite physico chemical organization. Even a slight change in this organization reduces the activity of enzyme and sometimes the enzyme is totally inactivated. Therefore, the enzymes have to be isolated under controlled conditions of pH, ionic strength and temperature. Since they are proteinaceous in nature, standard extraction and purification procedures for enzymes are the same as those used for proteins except that the activity of the enzyme is assayed at each of the following four steps of extraction and purification. Purification of Enzymes - Enzyme purification involves three steps, electrophoresis. These three techniques described in the following text 1.Dialysis 2.Chromatography.

1. ENZYME
TECHNOLOGY
Presented by – Sumer Pankaj
Class – Msc. EST (Sem. 3)
Roll no. – 16EST41
Institute of Science and Technology for Advanced Studies and Research
Affiliated to Sardar Patel University Recognized under section 2(f) and 12 (B) of UGC act 1956 Mota
Bazaar, Vallabh Vidyanagar, Anand, Gujarat 38812058
PG Department of Environmental Science and Technology

2. CONTENT
• Background
• Products of Enzyme Technology
• Structure of an enzyme
• Mechanism of an Enzyme
• Production of Enzymes
• Properties of Enzyme
• Sources of Enzyme
• Classification of Enzyme
• Isolation of enzymes
• Uses of Enzymes
• Application of enzymes in
various industries
• Use of Nanoparticles in
Enzyme Technology
• Enzyme immobilization
• Single cess enzyme
nanoparticle
• Enhancement of Enzyme
activity and thermos ability
• Enzyme Metallography
• A case study on
Bioluminescence of Fireflies

3. BACKGROUND
Enzyme - A biological catalyst that promotes & speeds up a chemical reaction without itself being altered in
the process, they fulfil their role by binding specific substrates at their active sites.
Enzyme technology is concerned with the application of enzymes as tools of industry, agriculture and
medicine.
The value of using enzymes over inorganic catalysts in the technological field is due to their efficiency,
selectivity and specificity.
Enzymes are able to operate at wide range of temperatures, atmospheric pressure and within normal pH ranges
– all of which create energy savings for industry.
Enzymes are biodegradable and, unlike many inorganic catalysts, cause less damage to the environment.
Catabolism
Anabolism
Functions of Enzyme
Metabolism

4. PRIMARY METABOLITES
During cell growth the nutrients of
the substrate are converted to cell
mass. The chemical compounds
produced in this process are called
“primary metabolites”.
„The cell mass itself consists
mainly of proteins, but a number
of primary waste products are also
formed, for instance carbon
dioxide, lactic acid, ethanol, etc.
Primary metabolites are produced
in parallel with the cell mass.

5. SECONDARY METABOLITES
„The formation of secondary metabolites is not
directly related to cell growth.
They are the side products of bacterial life.
In nature, they are produced in low concentration,
but through laboratory mutation and selection, cells
can be optimized to overproduce these metabolites.
Many antibiotics and vitamins are secondary
metabolites.
The formation of secondary metabolites is not
directly proportional to primary metabolism and cell
growth.

6. Micro-organisms have been
used for thousands of years
for making products such as
wine, beer, vinegar, soy sauce,
bread and cheese.
Products of Enzyme Technology
NOTE - The micro-organisms
(such as yeast) are really used as
a source of enzymes during the
manufacture of these products of
biotechnology.
Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been
ISOLATED from the micro-organisms before use
Protease
found in papaya
as meat
tenderizer

7. STRUCTURE OF AN ENZYME
ENZYMES
NON-PROTEIN
COENZYMES
( VITAMIN)
COFACTOR
(MINERAL)
PROTEIN
(Apo-enzyme)
• NOTE :- All
enzymes are
proteins but all
proteins are
not enzymes.

8. MECHANISM 0F ENZYMES - LOCK AND KEY
HYPOTHESIS
Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating
pure products free from unwanted by-products.

9. MODIFICATION – possible
application of genetic
engineering to improve
the microbial strain
LABORATORY SCALE PILOT
– to determine the optimum
conditions for growth of the
Micro-organism
PILOT PLANT – small scale
fermenter to clarify optimum
operating conditions
SCREENING – choosing an
appropriate micro-organism
for the desired enzyme
INDUSTRIAL SCALE
FERMENTATION
The Biotechnological Process of Enzyme Production

10. Pectinase is obtained
from the fungus
Aspergillus niger
Aspergillus niger
produces pectinase
as an extracellular
enzyme
Commercial Enzyme Production - An Example
PRODUCTIONOF PECTINASE
Pectin is an insoluble substance found in
the cell walls of plants
In the drinks industry, juice extracted
from fruits appears cloudy due to the
presence of pectin.
Pectinase is an enzyme that is used in the
industry to break down the pectin
The effect of pectinase is to clarify the
fruit juice and to make it flow more freely

11. • Control ripening.
• Cause food spoilage (rotting).
• Responsible for changes in flavor, color, texture
and nutritional properties.
• Can be inactivated by heat to extend storage
stability of foods.
• Control oxidation and spoilage (bioconservation)
• Increase nutritive values ( phytase , proteases etc.)
• Used for fermentation purposes in foods.
• Can be extracted and purified to a high degree.
Properties of enzymes

12. Sources of enzymes
There are three major sources of enzymes :
Plants ( 4%)
(papain, bromilain)
Animals ( 8%)
(renet)
Microorganisms
(>80%)
(yeast, fungi and
bacteria)

13. VEGETABLE COAGULANTS
• Vegetable coagulants are plant extracts which contain a mixture of proteolytic enzymes
and contribute unique characteristics to cheese.
• Vegetable coagulants are used primarily in niche products where specific flavours and
consistency are desired, or in remote regions where other coagulants are unavailable.
• Extracts of the Cardo flower (Cynara cardunculus) used in Spain and Portugal for the
production of some DOP (Denominación de Origen Protegida) cheeses, these cheeses are
often very soft or semi-liquid and may be spoonable. The flavour profile is strong, with
some bitterness.
• Extracts of the Calotropis procera plant used in Ghana for the production of Wara or
Wagashi cheese, having a hard texture and high melting point suitable for grilling.
Due to high production costs, low yield and development of off-flavors, vegetable coagulants
are not used in industrial cheese production.

14. ENZYMES EXTRACTED FROM ANIMALS
• Lipase :- Lipolytic enzymes extracted from the throat of cows, sheep or goats which
enhance cheese flavour.
• These are very expensive
• Produced from stomach of cows, enzyme chymosin (rennin)
obtained either from calf stomach or more recently from a
microorganism.
• Cut casein proteins in milk into smaller pieces
• solidify milk
Rennet

15. TAQ POLYMERASE
• Thermo stable enzyme
essential for PCR reactions
• Isolated from hot-spring
dwelling species Thermus
aquaticus
ENZYMES PRODUCED BY MICROORGANISMS

16. INVERTASE
• Derived from Saccharomyces
cerevisiae
• Also known as sucrase
• Enzyme digests sucrose into
glucose and fructose
• Used to create candies with a
soft center
• Most commonly used to make
chocolate covered cherries

18. ENZYME PRODUCTION/ISOLATION METHODS

19. PRODUCTION AND ISOLATION OF ENZYMES

20. USES OF ENZYMES
1. Analytical Applications of Enzymes
2. The Animal Feed Industry
3. The Meat and Fish Processing Industry
4. The Dairy Industry
5. The Leather Industry
6. CIP and cleaning of microfilters – Detergents
7. The modification of Fats and Oils
8. The Pulp and Paper Industry
9. The Fruit Juice Processing Industry
10.The Production of Bulk and Fine Chemicals
11.Enzyme-Replacement Therapy

21. INDUSTRIAL USE OF ENZYMES
Enzyme Source Action in food Food application
Papain Latex of unripe
papaya fruit
protein hydrolysis Meat tenderisation
Bromelain Pineapple juice
and stem
Muscle and connective
tissue protein hydrolysis
Meat tenderisation
Ficin Fig fruit latex Muscle and connective
tissue protein hydrolysis
As bromelain & papain but
not widely used due to cost
Chymosin
(rennet)
Calf abomasum Kappa casein hydrolysis Cheese making
Pepsin Bovine abomasum casein
hydrolysis in cheese
Help for rennet action

22. Lysozyme Hen egg white Hydrolysis of
bacterial
cell wall
polysaccharides
Prevention of late
blowing
defects in cheese by
spore-forming bacteria
Lactoperoxidase Cheese whey:
bovine colostrum
Oxidation of thiocyanate
ion to bactericidal
Hypothiocyanate
Cold sterilisation of milk
Aminopeptidase Lcictococcus lactix
Axpergillux spp.
Rhizopux oryzae
Releases free amino acids
from N-terminus
of proteins and peptides
Releases free amino acids
from N-terminus
of proteins and peptides
Lipase/
esterase
Gullet of
goat&lamb: calf
abomasum:
pig pancreas
Triglyceride (fat)
hvdrolvsis
Flavour enhancement in
cheese products:

24. Enzymes break down specific components within
fruit & vegetables such as pectin, starch, proteins and
cellulose which results in increased yields, shortening
of processing time and improving sensory
characteristics.
Some examples:
Pectinases and Cellulases are used to break down cell
walls in fruit and vegetables, resulting in improved
extraction and increase in yield. They can also be
used to decrease the viscosity of purees or nectars,
and to provide ‘cloud stability’ and texture in juices.

25. Serial
no.
Enzyme Purpose/Function
1. Cellulases, beta-glucanases, alpha
amylases, proteases, maltogenic
amylases
For liquefaction, clarification
and to supplement malt
enzymes
2. Amyloglucosidase Conversion of starch to sugar

26. Serial
no.
Enzyme Purpose/Function
1. Alpha-amylases Breakdown of starch, maltose production
2. Amyloglycosidases Saccharification
3. Maltogen amylase (Novamyl) Delays process by which bread becomes stale
4. Protease Breakdown of proteins
5. Pentosanase Breakdown of pentosan, leading to reduced gluten
production

27. Sr.
no.
Industry Enzyme Purpose/Function
1. Wine &
fruit Juice
Pectinase
Glucose oxidase
Increase of yield and juice
clarification
Oxygen removal
2. Meat Protease Meat tenderising
3. Protein Proteass, trypsin,
aminopeptidases
Breakdown of various
components
4. Starch Alpha amylase,
glucoamylases,
hemicellulases,
amylases,
Modification and
conversion (eg to dextrose
or high fructose syrups)

28. Alpha-amylase: Converts starch to dextrins in producing corn syrup. Solubilizes carbohydrates found in
barley and other cereals used in brewing.
Glucoamylase: Conversion of dextrins to glucose in the production of corn syrup. Conversion of residual
dextrins to fermentable sugar in brewing for the production of "light" beer.
Beta-glucanase: Breakdown of glucans in malt and and other materials to aid in filtration after mashing in
brewing.
Lipase: Enhancing flavor development and shortening the time for cheese ripening. Production of specialty fats
with improved qualities. Production of enzyme-modified cheese/butter from cheese curd or butterfat.
Papain: Used as meat tenderizer. Used in brewing to prevent chill-haze formation by digesting proteins that
otherwise react with tannins to form insoluble colloids.
Chymosin: Curdling of milk by breaking down kappa-caseins in cheese making.
Microbial proteases: Processing of raw plant and animal protein. Production of fish meals, meat extracts,
texturized proteins, and meat extenders.
Pectinase: Treatment of fruit pulp to facilitate juice extraction and for clarification and filtration of fruit juice.

29. Lactase:Additive for dairy products for individuals lacking lactase. Breakdown of lactose
in whey products for manufacturing polyactide.
Acetolactate decarboxylase:Reduction of maturation time in wine making by
converting acetolactate to acetoin.
Glucose oxidase: Conversion of glucose to gluconic acid to prevent Maillard reaction in
products caused by high heat used in dehydration.
Cellulase: Conversion of cellulose waste to fermentable feedstock for ethanol or single-
cell protein production. Degradation of cell walls of grains, allowing better extraction of cell
contents and release of nutrients.

31. •Nanotechnology is the study of manipulating matter on an atomic scale.
•Nanotechnology refers to the constructing and engineering of the functional
systems at very micro level or we can say at atomic level.
•A Nanometer is one billionth of a meter, roughly the width of three or four atoms.
The average human hair is about 25,000 nanometers wide.
Use of Nanoparticles in Enzyme Technology
• The first ever concept was presented in 1959 by the famous professor of physics Dr.
Richard P.Feynman.
• Invention of the scanning tunneling microscope in 1981 and the discovery of
fullerene(C60) in 1985 lead to the emergence
of nanotechnology.
• The term “Nano-technology" had been coined by Norio Taniguchi in 1974

32. ENZYME IMMOBILISATION
• Application of nanomaterials as novel supporting materials for enzyme
immobilisation has generated incredible interest in the biotechnology
community.
• These robust nanostructured forms, such as nanoparticles, nanofibres,
nanotubes, nanoporous, nanosheets, and nanocomposites, possess a high
surface area to volume ratios that can cause a high enzyme loading and
facilitate reaction kinetics, thus improving biocatalytic efficiency for
industrial applications.
• The current status of versatile nanomaterial support for biofuel production
employing cellulases and lipases is described in details
• nanomaterials will become an integral part of sustainable bioenergy
production.

33. How nanoparticles helps
enzyme in increasing the
catalytic activity ???

34. SINGLE ENZYME NANOPARTICLES
• SEN (single enzyme nanoparticles )
• Enzyme lead short and brutal lives ,to increase the enzymes
longevity and versatility, a a team at department of Energy’s
Pacific Northwest , National Laboratory in Richlad caged single
enzyme to create a new class of catalysts called SENs
• The nanostructure protects the catalyst, allowing it to remain
active for several months
• Kim and Grate , working in te W.R Wiley Environmental molecular
sciences laboratory modified a common protein splitting enzyme
called alpha chymotrypsin

35. ENHANCEMENT OF ENZYME ACTIVITY AND
THERMOSTABILITY
Study on Impaired Pectate Lyase from AttenuatedMacrophomina phaseolina in
Presence of Hydroxyapatite Nanoparticle
 Hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting
thermostability) but can serve as a synthetic enhancer of activity of an isolated
extracellular pectate lyase (APL) with low native state activity.
The purified enzyme showed feeble activity at 50°C and pH 5.6. However, on
addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased
27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to
untreated APL.
The upper critical temperature for such compensation was elevated from 50°C to
90°C in presence of NP.

36. ENZMET (ENZYME METALLOGRAPHY)
• EnzMet (Enzyme Metallography) is a new biological labeling and
staining method developed at Nanoprobes.
• It uses a targeted enzymatic probe with a novel metallographic
substrate to provide a quantum leap in staining clarity over
conventional chromogenic and fluorescent substrates.
• EnzMet™ has proven highly sensitive both for in situ hybridization
(ISH), where it readily visualizes endogenous copies of single genes,
and immunohistochemistry (IHC) detection.

37. NANOTECHNOLOGY TO HARNESS THE NATURAL
LIGHT PRODUCED BY FIREFLIES
• By designing a way to chemically attach genetically manipulated
luciferase enzymes directly to the surface of nanorods, scientists at
Syracuse University found a new way to harness the natural light
produced by fireflies.
• Fireflies produce light through a chemical reaction between
luciferin and it’s counterpart, the enzyme luciferase.
• In Maye’s laboratory, the enzyme is attached to the nanorod’s
surface; luciferin, which is added later, serves as the fuel.
A Case Study

38. CONTINUE….
• The energy that is released when
the fuel and the enzyme interact is
transferred to the nanorods,
causing them to glow. The process
is called Bioluminescence
Resonance Energy Transfer
(BRET).
• The nanorods are composed of an
outer shell of cadmium sulfide and
an inner core of cadmium
seleneide.

39. REFRENCES
• http://www.azonano.com/article.aspx?ArticleID=736
• http://omicsonline.org/2153-0777/2153-0777-2-e114.pdf
• http://www.nanoprobes.com/products/EnzMet-SISH-enzyme-metallography-for-ISH-and-IHC.html
• http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0063567
• http://www.che.udel.edu/research_groups/wilfred/Current%2013%20final.pdf
 www.journals.elseveir.com/biological-control/
http://microbewiki.kenyon,edu.index.php/Biocontrol
www.sciencedirect.com/science/article/pii/0261219491900385
• J. Srilakshmi, ;J. Madhavi, Lavanya S and Ammani (2014)
Commercial Potential of Fungal Protease: Past, Present and Future Prospects , Journal of Pharmaceutical,
Chemical and Biological Sciences ,2(4)p.218-234.
• Sumantha (2006).Food-Grade Proteases, journal of Food Technology & Biotechnology. 44 (2), p.211–220
• Riddhi Sawant and Saraswathy Nagendran (2014),protease: an enzyme with multiple industrial application,
www.wjpps.com .

40. THANK YOU…