This document discusses the production of the enzyme amylase. It begins by defining amylase and its types - alpha, beta, and gamma amylase. It then discusses the major production methods of solid state fermentation and submerged fermentation. Key parameters that affect amylase production are described such as carbon and nitrogen sources, temperature, pH, moisture, and duration of fermentation. The document outlines the steps for recovery and purification of amylases. Finally, it briefly mentions methods to determine amylase activity and industrial applications of amylase in baking, textiles, fuel production and more.

1. BY :
ROHINI YADAV
FERMENTATION TECHNOLOGY

2. Production of Amylase

3. Contents
• Introduction
• Types of amylase
• Production of amylase
• Recovery and purification
• Determination of enzyme activity
• Industrial application

4. Amylase
• Amylase is an enzyme that catalyses the hydrolysis of starch
into sugars.
• Amylase is present in the saliva of humans and some other
mammals, where it begins the chemical process of digestion
• These ezymes randomly cleave internal glycosidic linkages
in starch molecules to hydrolyze them and yield dextrins and
oligosaccharides

5. Types of Amylase:
α-Amylase
• α-Amylase is a hydrolase enzyme that catalyses the hydrolysis of
internal α-1, 4-glycosidic linkages in starch to yield products like
glucose and maltose.
• It is a calcium metalloenzyme i.e. it depends on the presence of a
metal co factor for its activity.
• There are 2 types of hydrolases: endo-hydrolase and exo-
hydrolase. Endo- hydrolases act on the interior of the substrate
molecule, whereas exo-hydrolases act on the terminal non
reducing ends .

6. • The substrate that α-amylase acts upon is starch. Starch is a
polysaccharide composed of two types of polymers – amylose and
amylopectin.
• Amylose constitutes 20-25% of the starch molecule. It is a linear
chain consisting of repetitive glucose units linked by α-1,4-
glycosidic linkage. Amylopectin constitutes 75-80% of starch and
is characterized by branched chains of glucose units.
• The optimum pH for activity is found to be 7.0.

7. β – Amylase
• β-Amylase is an exo-hydrolase enzyme that acts from the
nonreducing end of a polysaccharide chain by hydrolysis of α-1, 4-
glucan linkages to yield successive maltose units.
• It is unable to cleave branched linkages in branched polysaccharides
such as glycogen or amylopectin, the hydrolysis is incomplete and
dextrin units remain.
• During ripening of fruits, β-Amylase breaks down starch into maltose
resulting in the sweetness of ripened fruit.
• The optimal pH of the enzyme ranges from 4.0 to 5.5.

8. γ – Amylase
•
• γ-Amylase cleaves α(1-6)glycosidic linkages, in addition to
cleaving the last α(1-4)glycosidic linkages at the nonreducing end
of amylose and amylopectin, unlike the other forms of amylase,
yielding glucose.
• γ- amylase is most efficient in acidic environments and has an
optimum pH of 3.
•

9. Production of α-Amylase

10. Sources
• α-Amylase can be isolated from plants, animals or microorganisms.
• The enzyme has been isolated from barley and rice plants .
• It has been found that cassava mash waste water is a source of α-
Amylase which is active in wide range of pH and temperature .
• The most widely used source among the bacterial species is the
Bacillus spp. B. amyloliquefaciens and B. licheniformis
• Fungal sources of α-Amylase are mostly Aspergillus species and only
few species of Penicillium, P. brunneum , penicillium fellutanum

11. Production Methods :
There are mainly two methods which are used for production of α-Amylase
on a commercial scale.
These are :
1) Solid State fermentation.
2) Submerged fermentation

12. • Method used for microbes which require less moisture content for
their growth.
• The solid substrates commonly used in this method are, bran,
bagasse, and paper pulp.
• The main advantage is that nutrient-rich waste materials can be easily
recycled and used as substrates in this method.
1. Solid State Fermentation [SSF]

13. • employs free flowing liquid substrates, such as molasses and
broths.
• The products yielded in fermentation are secreted into the
fermentation broth. The substrates are utilized quite
rapidly,hence the substrates need to be constantly replenished
• SmF is primarily used for the extraction of secondary metabolites
that need to be used in liquid form .
2. Submerged Fermentation
[SMF]

14. Composition (g/l)
• Na2HPO4. 6g
• KH2PO4 3g
• NH4Cl 1g
• NaCl 0.5g
• CaCl2 0.15g
• MgSO4.7H2O 0.25g
• Casein hydrolysate 0.20g
• Yeast extract. 0.10g
• Starch 20g
Amylase Production Media

15. 1. Carbon Source:
Common carbon sources used as substrates include maltose,
sucrose and glucose. A. tamarii actively synthesizes α-Amylase
when cultured on maltose, starch and glycogen under static
conditions. . The aim is to use substrates that are waste or by
products of other processes in order to make the process of enzyme
production environment friendly. One such substrate is oil cake.
Wheat bran has been used as substrate for α-Amylase production
by B.licheniformis and A.niger.
Substrate

16. 2. Nitrogen Source
The nitrogen source used for production of α-Amylase may be organic or
inorganic. Few of the inorganic nitrogen sources include ammonium
sulphate, ammonium chloride and ammonium hydrogen phosphate. Most
commonly used organic sources of nitrogen include peptone, yeast
extract and soyabean meal.

17. The optimum process control parameters vary depending on the
microbial source, desired end product, method of fermentation
employed and many other such factors.
Process Parameters:
1.Temperature
• There are two temperatures that need to be in optimum
range during production.
• They are temperature for the growth of the microbial
source and optimum temperature at which maximum
production of enzyme takes place.

18. • The optimum temperatures for growth and α-Amylase
production were found to be 45°C to 46 °C and 50 °C,
respectively.
2. pH
• Optimum pH is a critical factor for the stability of enzyme produced.
Enzymes are pH sensitive and hence care must be taken to control
the pH of the production process.
• Pyrococcus furiosus produces α-Amylase which shows activity at
an optimum pH of 6.5–7.5

19. • Optimum levels of initial moisture content may vary depending on
the microbial source used.
• For fungal sources the moisture content required is less whereas
bacterial sources need more moisture content for high yield of the
enzyme.
• production of α-Amylase by Penicillium janthinellum the moisture
content was varied within a range of 20- 80% by varying the amount
of salt solution used in moistening the substrate particles.
Moisture

20. • This is a crucial factor in fermentation process.
• The enzyme activity increases with increase in incubation time till it
reaches the optimum duration. In most cases, the production of
enzyme begins to decline if the incubation time is further increased.
This could be due to the depletion of nutrients in the medium or
release of toxic substances.
• Bacillus subtilis, show high yield of alpha amylase after 48 hours of
fermentation
• Penicillium fellutanum isolated from mangrove rhizosphere soil. The
culture when incubated at 96 h, showed the maximum activity.
Duration of fermentation

21. • The desired enzyme produced may be excreted into the culture
medium (extracellular enzymes) or may be present within the cells
(intracellular enzymes).
• Depending on the requirement, the commercial enzyme may be crude
or highly purified. Further, it may be in the solid or liquid form.
• The steps involved in downstream processing i.e. recovery and
purification steps employed will depend on the nature of the enzyme
and the degree of purity desired.
Recovery and purification of
enzymes

22. • In general, recovery of an extracellular enzyme which is present in the
broth is relatively simpler compared to an intracellular enzyme. For the
release of intracellular enzymes, special techniques are needed for cell
disruption. Microbial cells can be broken down by physical
means(sonication, high pressure, glass beads)
• The recovery and purification steps will be the same for both
intracellular and extracellular enzymes, once the cells are disrupted
and intracellular enzymes are released. The most important
consideration is to minimise the loss of desired enzyme activity.
• Removal of cell debris:
• Filtration or centrifugation can be used to remove cell debris.

23. • Removal of nucleic acids : Nucleic acids interfere with the recovery
and purification of enzymes. They can be precipitated and removed by
adding poly-cations such as polyamines, streptomycin and
polyethyleneimine.
• Enzyme precipitation: Enymes can be precipitated by using salts
(ammonium sulfate) organic solvents (isopropanol, ethanol, and
acetone). Precipitation is advantageous since the precipitated enzyme
can be dissolved in a minimal volume to concentrate the enzyme.
• Liquid-liquid partition : Further concentration of desired enzymes can
be achieved by liquid-liquid extraction using polyethylene glycol or
polyamines.

24. • Separation by chromatography: there are several
chromatographic techniques for separation and purification of
enzymes. These include ion-exchange, size exclusion, affinity,
hydrophobic interaction and dye ligand chromatography. Among
these, ion- exchange chromatography is the most commonly used
for enzyme purification.
• Drying and packing: The concentrated form of the enzyme can be
obtained by drying. This can be done by film evaporators or freeze
dryers (lyophilizers). The dried enzyme can be packed and
marketed.

25. 1. Dinitrosalicylic Acid Method (DNA)
2. Nelson-Somogyi (NS) method
3. Iodine Activity
Determination of enzyme activity

26. • Hydrolysis of starch into the extreme forming a less viscous starch
suspension.
• In bakers industry, converting starch in two smaller fermentable
sugars.
• In textile industry, used to removal of starch sizing agent for woven
fabric.
• Fuel alcohol production, converting starch into smaller fermentable
• sugars which are acted upon by East to produce alcohol.
Application of amylase enzyme

27. Thank you