Introduction

• Enzymes are macromolecular biological catalysts.

• Enzymes accelerate, or catalyze, chemical
reactions.
• The molecules at the beginning of the process are
called substrates and the enzyme converts these
into different molecules, called products.
• Microbial enzymes are the biological catalysts for
the biochemical reactions leading to microbial
growth and respiration, as well as to the
formation of fermentation products.
1
2
 Types of Enzymes

ADAPTIVE
• Produced only when the need arises
Eg. When a cell is deficient of a particular
nutrient.

Constitutive
• Produced always irrespective the amount of
substrate.
3
 History
• The first enzyme produced industrially was the
fungal amylase Takadiastase which was employed
as a pharmaceutical agent for digestive disorders.
• By 1969, 80% of all laundry detergents contained
enzymes, chiefly Proteases.
• Due to the occurrence of allergies among the
production workers and consumers, the sale of
such enzyme utilizing detergents decreased
drastically.
4
• Special techniques like micro-encapsulation of
these enzymes were developed which could
provide dustless protease preparation. It was
thus made risk free for production workers
and consumers.
• Microbial rennin is also one of the most
significant enzymes. It has been used instead
of Calf’s rennin in cheese production.

5
• Location of Enzymes

6
 Intracellular enzymes

• The enzymes that act within the

cells in which they are produced are
called intracellular enzymes or
endoenzymes.
• As these enzymes catalyze most of
the metabolic reactions of the cell,
they are also referred to as
• Most of the enzymes in plants and
animals are intracellular enzymes or
endoenzymes.
• Intracellular enzymes usually break
down large polymers into smaller
chains of monomers.
• All intracellular enzymes undergo
intracellular digestion during cell
death.
 Extracellular enzymes

• The enzymes which are liberated by living cells

and catalyze useful reactions outside the cell
but within its environment are known as
extracellular enzymes or exoenzymes.
• Exoenzymes act chiefly as digestive enzymes,
catalyzing the breakdown of complex
macromolecules to simpler polymers or
monomers, which can then be readily absorbed
by the cell.
• These mostly act at the end of polymers to
break down their monomers one at a time.
• Exoenzymes are enzymes found in bacteria,
fungi, and some insectivores
like Drosera and Nepenthes.
• Extracellular enzymes, unlike intracellular
enzymes, undergo external digestion during cell
death.
11
 Mechanism of Action of
Enzymes

• The mechanism of action of enzymes in

a chemical reaction can occur by
s e v e r a l m o de s ; s u bs t r a t e b i n d i n g ,
catalysis, substrate presentation, and
allosteric modulation.
• But the most common mode of action
of enzymes is by the binding of the
substrate.
• An enzyme molecule has a specific
active site to which its substrate binds
and produces an enzyme-substrate
• The reaction proceeds at the binding site to
produce the products which remain associated
briefly with the enzyme.
• The product is then liberated, and the enzyme
molecule is freed in an active state to initiate
another round of catalysis.
• To describe the mechanism of action of
enzymes to different models have been
proposed;
• The induced fit hypothesis is a modified form of the
lock and key hypothesis proposed by Koshland in
1958.
• According to this hypothesis, the enzyme molecule
does not retain its original shape and structure.
• Instead, the contact of the substrate induces some
configurational or geometrical changes in the active
site of the enzyme molecule.
•
• As a result, the enzyme molecule is made to
fit the configuration and active centers of
the substrate completely.
• Meanwhile, other amino acid residues
remain buried in the interior of the
molecule.
• However, the sequence of events resulting in
the conformational change might be
• Some enzymes might first undergo a
conformational change, then bind the
substrate.
• In an alternative pathway, the substrate
may first be bound, and then a
conformational change may occur in the
active site.
• Thirdly, both the processes may co-occur
 Improved Prospects of Enzyme
Application
• Microbial Genetics – High yields can be obtained
by Genetic manipulation.
Example – Hansenula polymorpha has been
genetically modified so that 35% of it’s total
protein consists of the enzyme alcohol oxidase.
• Optimization of fermentation conditions (Use of
low cost nutrients, optimal utilization of
components in nutrient solution, temperature
and pH)

18
• New cell breaking methods like Homogenizer,
Bead mill, Sonication etc
• Modern purification processes like Counter
current distribution, Ion-exchange
chromatography, Molecular-sieve
chromatography, Affinity chromatography and
precipitation by using alcohol, acetone.
• Immobilization of enzymes
• Continuous enzyme production in special
reactors.

19
Methods of Enzyme Production

Semisolid
Culture

Submerged
Culture
20
 Semisolid Culture
The enzyme producing culture is grown on the
surface of a suitable semi-solid substrate
(Moistened Wheat or Rice Bran with nutrients)

Preparation of Production Medium – Bran is

mixed with solution containing nutrient salts.

pH is maintained at a neutral level. Medium is

steam sterilized in an autoclave while stirring.

21
The sterilized medium is spread
on metal trays upto a depth of 1-
10 centimeters.

Culture is inoculated either in

the autoclave after cooling or in
trays.

High enzyme concentration in a

crude fermented material.

22
Enzymes produced by Semi-solid
culture
Enzyme Micro-organisms

α- Amylase Aspergillus oryzae

Glucoamylase Rhizopus spp.

Lactase A. oryzae

Pectinase A. niger

Protease A. Niger & A. oryzae

Rennet Mucor pusillus

23
Advantages of Semi-solid culture
It involves comparatively low investment

Allows the use of substrate with high dry matter

content. Hence it yields a high enzyme concentration in
the crude fermented material.

To cultivate those moulds which cannot grow in

the fermenters due to wall growth.

Allows the moulds to develop into their natural

state.

24
Disadvantages of Semi-solid culture

Requires more space and more labour

Involves greater risk of infection

Difficult to introduce automation in such

systems

25
 Submerged Culture
• Fermentation equipment used is the same as
in the manufacture of antibiotics.
• It’s a cylindrical tank of stainless steel and it is
equipped with an agitator, an aerating device,
a cooling system and various ancillary
equipment (Foam control, pH monitoring
device, temperature, oxygen tension etc)
• Good growth is not enough to obtain a higher
enzyme yield.

26
• Presence of inhibitors or inducers should also be
checked in the medium.
Example – Presence of Lactose induces the
production of β- galactosidase.
• As the inducers are expensive, constitutive
mutants are used which do not require an inducer.
• Glucose represses the formation of some
enzymes (α-amylases). Thus the glucose
concentration is kept low.
• Either the glucose can be supplied in an
incremental manner or a slow metabolizable
sugar (Lactose or metabolized starch)

27
• Certain surfactants in the production medium
increases the yield of certain enzymes.
• Non- ionic detergents (eg. Tween 80, Triton)
are frequently used.

28
Advantages of Submerged culture

Requires less labor and space

Low risk of infection

Automation is easier

29
 Disadvantage of Submerged Culture
• Initial investment cost is very high.

30
 After fermentation
• Once fermentation is finished, the fermented liquor is
subjected to rapid cooling to about 5o C in order to
reduce deterioration.
• Separation of micro-organisms is accomplished either
by filtration or by centrifugation of the refrigerated
broth with adjusted pH.
• To obtain a higher purity of the enzyme, it is
precipitated with acetone, alcohols or inorganic salts
(ammonium or sodium sulfate).
• In case of large scale operations, salts are preferred to
solvents because of explosion hazards.

31
AMYLASE

32
 Introduction
• Amylase is an enzyme that catalyses the
hydrolysis of starch into sugars.
• Present in the saliva of humans
• Hydrolysis of Starch with amylase will first result
in the formation of a short polymer Dextrin and
then the disaccharide Maltose and finally glucose.
• Glucose is not as sweet as Fructose. Thus the
next step would be the conversion of Glucose to
Fructose by the enzyme Glucose isomerase.

33
 Types of Amylases

α- Amylase

ß- Amylase

γ- Amylase

34
 α- Amylase
• Also called as 1,4-α-D-glucan glucanohydrolase.
• Calcium metalloenzymes which cannot function
in absence of calcium ions.
• Breaks down long carbohydrate chains of
Amylose and Amylopectin.
• Amylose is broken down to yield maltotriose and
Maltose molecules.
• Amylopectin is broken down to yield Limit dextrin
and glucose molecules.

35
• Found in saliva and pancreas.
• Found in plants, fungi (ascomycetes and
basidiomycetes) and bacteria (Bacillus)
• Because it can act anywhere on the substrate,
α-amylase tends to be faster-acting than β-
amylase.
• In animals, it is a major digestive enzyme, and
its optimum pH is 6.7–7.0

36
 ß- Amylase
• Also called as 1,4-α-D-glucan maltohydrolase.
• Synthesized by bacteria, fungi, and plants.
• Working from the non-reducing end, β-amylase
catalyzes the hydrolysis of the second α-1,4
glycosidic bond, cleaving off two glucose units
(maltose) at a time.
• During the ripening of fruit, β-amylase breaks
starch into maltose, resulting in the sweet flavor
of ripe fruit.
• The optimum pH for β-amylase is 4.0–5.0

37
 γ- Amylase
• Also termed as Glucan 1,4-α-glucosidase.
• Cleaves α(1–6) glycosidic linkages, as well as
the last α(1–4) glycosidic linkages at the
nonreducing end of amylose and amylopectin,
yielding glucose.
• The γ-amylase has most acidic optimum pH of
all amylases because it is most active around
pH 3.

38
 Effects of α-Amylases

• Break down the starch

Starch- polymer but does not
Liquefying give free sugar

• Gives free sugars

Saccharogenic

39
 Producing strains
• Bacteria – B. cereus, B.subtilis, B.
amyloliquefaciens, B. polymyxa, B.
licheniformis etc
• Fungi – Aspergillus oryzae, Aspergillus niger,
Penicillum, Cephalosporin, Mucor, Candida
eetc.

40
 Applications
• Production of sweeteners for the food industry.
• Removal of starch sizing from woven cloth
• Liquefaction of starch pastes which are formed
during the heating steps in the manufacture of
corn and chocolate syrups.
• Production of bread and removal of food spots in
the dry cleaning industry where amylase works in
conjunction with protease enzymes

41
LIPASES

42
 Introduction
• Lipases are also called as Glycerol ester
hydrolases
• They are a subclass of esterases
• It splits fats into mono or di- glycerides and
fatty acids.
• They are extracellular enzymes
• Mainly produced by Fungi
Eg: Aspergillus, Mucor, Rhizopus, Peniciilum
etc
43
• Bacteria producing lipases include species of
Pseudomonas, Achromobacter and
Staphylococcus.
• Yeasts like Torulopsis and Candida are also
commercially used.

44
Mode of Action

45
• Enzyme production must be induced by
adding oils and fats.
• But in some cases the fats have effect on the
lipase production.
• Glycerol, a product of lipases action, inhibits
lipase formation.
• Lipases are generally bound to the cells and
hence inhibit an overproduction but addition
of a cation such as magnesium ion liberates
the lipase and leads to a higher enzyme titer
in the production medium.

46
 Applications
• Primarily marketed for therapeutic purposes
as digestive enzymes to supplement
pancreatic lipases.
• Since free fatty acids affect the odor and taste
of cheese, and the cheese ripening process is
affected by lipases, microbial affects during
the aging process can be due to lipase action.
• In the soap industry, lipases from Candida
cylindraceae is used to hydrolyze oils.

47
Pectinases

48
 Introduction
• Pectinase is an enzyme that breaks down
pectin, a polysaccharide found in plant cell
walls.
• Pectic enzymes include Pectolyase, Pectozyme
and Polygalacturonase.
• Pectin is the jelly-like matrix which helps
cement plant cells together and in which other
cell wall components, such as cellulose fibrils,
are embedded.

49
• Basic structure of a pectin consists of α-1,4
linked Galactouronic acid with upto 95% of it’s
carboxyl groups esterified with methanol.
• Pectinase might typically be activated at 45 to
55 °C and work well at a pH of 3.0 to 6.5.

50
Mode of Action

51
 Production Strains
• Aspergillus niger, A. wentii, Rhizopus etc
• Fermentation with Aspergillus Niger runs for
60-80 hours in fed batch cultures at pH 3-4
and 37o C using 2% sucrose and 2% pectin.

52
 Applications
• Pectinase enzymes are commonly used in
processes involving the degradation of plant
materials, such as speeding up the extraction of
fruit juice from fruit, including apples.
• Pectinases have also been used in wine
production since the 1960s
• Helps to clarify fruit juices and grape must, for
the maceration of vegetables and fruits and for
the extraction of olive oil.
• By treatment with pectinase, the yield of fruit
juice during pressing is considerably increased.
53
Proteases

54
 Introduction
• Protease (Mixture of Peptidases and
Proteinases) are enzymes that perform the
hydrolysis of Peptide bonds.
• Peptide bonds links the amino acids to give
the final structure of a protein.
• Proteinases are extracellular and Peptidases
are endocellular.
• Second most important enzyme produced on
a large scale after Amylase
55
Mode of Action

56
Classification Based upon the residues
in the Catalytic site
Serine Protease

Threonine Protease

Aspartate Protease

57
Cysteine Protease

Glutamatic acid Protease

Metalloproteases eg: Zinc

58
Classification Based upon the pH in
which the Proteases are Active
Alkaline serine Proteases

Acid Proteases

Neutral Proteases

59
 Alkaline Serine Proteases
• pH of the production medium is kept at 7.0 for
satisfactory results.
• Have serine at the active site
• Optimum temperature maintained is 30o to
40o C.
• Important producers are B. licheniformis, B.
amyloliquefaciens, B. firmus, B. megaterium,
Streptomyces griseus, S. fradiae, S. rectus and
fungi like A. niger, A. oryzae, A.flavus.
60
• Enzymes used in detergents are chiefly
proteases from bacillus strains
(Bacillopeptidases)
• Best known proteases are Subtilisin Carlsberg
from B. licheniformis and Subtilisin BPN and
Subtilisin Novo from B. amyloliquefaciens.
• These enzymes are not inhibited by EDTA
(Ethylene diamine tetraacetic acid) but are
inhibited by DFP (Di isopropyl
fluorophosphate)

61
 Proteases for the Use in Detergent
industries
• Stability at high temperature
• Stability in alkaline range (pH- 9 to 11)
• Stability in association with chelating agents
and perborates
• But shelf life is affected in presence of surface
active agents.

62
 Screening
• Because the enzymes should be stable in
alkaline conditions, screening for better
producers is done by using highly alkaline
media.
• It was found that B. licheniformis and B.
subtilis showed growth is the range of pH 6-7
by new strains were found to grow even in pH
10-11.
• Genetic Manipulation can also be carried out.

63
 Fermentation Process

Cultures are stored in the lyophilized state or

under Liquid nitrogen.

Initial cultures are carried out in shaken flasks and

small fermenters (40-100 m3) at 30-37o C

Fed-Batch culture is generally used to keep down

the concentration of ammonium ions and amino
acids as they may repress protease production

64
High oxygen partial pressure is
generally necessary for optimal
protease titers

Time span for fermentation is

48-72 hours depending upon the
organism

Proteases must be converted in

a particulate form before they
are added to detergents..

65
• To prepare a suitable encapsulated product, a
wet paste of enzyme is melted at 50-70o C
with a hydrophobic substance such as
polyethylene glycol and then converted into
tiny particles.

66
 Neutral Proteases
• They are relatively unstable and calcium,
sodium and chloride must be added for
maximal stability.
• Not stable at higher temperatures
• Producing organisms are B. subtilis, B.
megaterium etc
• They are quickly inactivated by alkaline
proteases.

67
 Acid Proteases
• Similar to Mammalian pepsin
• It consists of Rennin like proteases from fungi
which are chiefly used in cheese production
• They are used in medicine, in the digestion of
soy protein for soya sauce production and to
break down wheat gluten in the baking
industry

68
 Applications
• Textile industry to remove proteinaceous
sizing.
• Silk industry to liberate silk fibers from
naturally occurring proteinaceous material in
which they are embedded.
• Tenderizing of Meat
• Used in detergent and food industries.

69
70
 Taq polymerase

• Thermostable enzyme essential for PCR reactions

Isolated from hot-spring dwelling species
Thermus aquaticus
Species is a thermophile (heat-loving) because
of its ability to grow thrive under extreme
heat
Several companies have permission to inspect
geysers in Yellowstone
 Cellulase Enzyme

• produced by E. coli that degrades

cellulose
• Widely used in the biotech industry, including
• Making animal food more easily digested
• Makes faded jeans by digesting cellulose fibers
in cotton
• Processing of coffee beans
• Fermentation processes to create biofuels
•
 Subtilisin
• Derived from Bacillus subtilis
• Protease (breaks down protein)
• Valuable component of laundry detergents
• Degrades removes protein stains from clothing
•