• Amylases are starch degrading enzymes.
• It was first isolated by French chemists Anselme and Jean François
from germinating barley and was named as "diastase" in 1833.
• These enzymes act by hydrolyzing glycosidic bonds-α-1,4 glycosidic
bonds and α-1,6 glycosidic bonds between adjacent glucose units,
yielding progressively smaller polymers composed of glucose units
(characteristic of the particular enzyme involved)  .
• They belong to the glycoside hydrolase group of enzymes under
which 13 enzymes are included  .
- Sivaramakrishnan et al., 2006 - Aiyer et al.,2005 - ] Windish et al.,2005
E.C.3.-.-.- main class of enzyme - Hydrolases
E.C.3.2.-.- glycosidic bond that is hydrolyzed. Hence , they are
E.C.3.2.1.- Glycosidases, i.e. enzymes hydrolyzing O- glycosyl
E.C.220.127.116.11 α- amylase
E.C.18.104.22.168 β- amylase
MAJOR SUBSTRATE FOR AMYLASE -STARCH
Starch is a polymer of glucose
linked to another one through the
glycoside bond. Two types of
glucose polymers are present in
starch : amylose and amylopectin.
• Amylose is a linear polymer
consisting of up to 6000 glucose
units with α-1,4 glycosidic
• Amylopectin consists of short α-
1,4 linked to linear chains of
10–60 glucose units and α-1,6
linked to side chains with 15–
45 glucose units.
• Amylase is able to cleave these
glycosidic bonds present in the
inner part of the amylose or
 Muralikrishna G., Nirmala M. Cereal α-amylases—an
overview.Carbohydrate Polymers. 2005;60:163–173.
Various types of amylase associated with degradation of starch and
related polysaccharides structures have been detected and studied.
1. Enzymes that hydrolyze 𝛼-1,4 bonds e.g. 𝜶 -amylase (endoacting
2. Enzymes that hydrolyze 𝛼 -1,4 e.g. -𝜷 amylase (exoacting amylases
producing maltose as a major end product).
3. Enzymes that hydrolyze terminal 1,4 linked 𝛼 D-glucose residues. e.g.
4. Enzymes that hydrolyze only 𝛼 -1,6 linkages e.g. pullulanase .
5. Enzymes that hydrolyze preferentially 𝛼 -1,4 linkages in short chain
oligosaccharides produced by the action of other enzymes on amylose
and amylopectin e.g. - 𝜶 glucosidases.
- Van et al.,2002
TYPES OF AMYLASE
Mechanism of amylase activity
Manners et al, 1992
α-amylase β-amylase glucoamylase
Reaction Endo hydrolysis of (1 -4)-α-D-
glycosidic linkages in
three or more (1 4)-α-linked
Hydrolysis of (1 - 4)-α-D-
glycosidic linkages in
polysaccharides so as to
maltose units from the
non-reducing ends of the
Hydrolysis of termin
non-reducing ends of
the chains with releas
D-glucose. It is an
Comment Acts on starch related
oligosaccharides in a random
manner; reducing groups are
liberated in the α-
Acts on starch, glycogen
producing β-maltose. The
term β relates to the
configuration of the free
sugar group released.
Cleaves the last α(1-
nonreducing end of a
E.C number E.C.22.214.171.124 E.C.126.96.36.199 E.C.188.8.131.52
Source Bacillus licheniformis, Bacillus
Seeds of higher plants
and sweet potatoes.
The human α-amylase is a classical calcium-containing
enzyme composed of 512 amino acids with a molecular
weight of 57.6 kDa. .
The protein contains 3 domains: A, B, and C.
The A domain (residues 1-99, 169-404 )is the largest,
presenting a typical Tim barrel shaped (β/α)8 super
The B domain (residues 100-168) is the smallest
domain is attached to the A domain by disulphide
bond. The C domain (residues 405-512) is made up of
anti-parallel beta-structure and is only loosely
associated with Domains A and B.
The active site of the α-amylase is situated in a cleft
located between the carboxyl end of the A and B
domains. Asp206, Glu230 and Asp297 participate in
The calcium (Ca2+) is situated at B domain (Asn 100, Arg
158, Asp 167) against the wall of the barrel of Domain
Chloride ion is present at A domain (Arg 195, Asn 298,
and Arg 337)
These ions are required for the stabilization of the
three-dimensional structure .
STRUCTURAL CHARACTERISTICS OF α-AMYLASE
Structure human α-amylase.
Characterization of α-amylase.
6.2 1.04 2000 3.22×
85-90°C 6.5 
9.0 0.68 580 .64x
37-40°C 6.0 
DETERMINATION OF AMYLASE ACTIVITY
• Amylase activity was estimated by measuring either the appearance of one of the
products or the disappearance of the substrate over time.
• The Enzyme – substrate reaction can be determined by measuring the increase in
reducing sugars using the 3, 5 Dinitro salicylic acid reagent.
• The pale yellow colored the 3, 5- dinitro salicylic acid undergo reduction in
presence of reducing sugar to yield orange colored 3- amino -5-nitrosalicylic acid.
• The absorbance of resultant solutions is read at 540nm. The intensity of color
depends on the concentration of reducing sugars produced.
Lever et al.,1972.
• One unit of amylase activity is defined as the amount of enzyme that
produces 1 μmol of reducing sugar per minute under specific conditions.
Enzyme activity =
U/ml incubation time(min) X volume of starch ( ml)X Volume of cubette
• The hydrolytic activity of Amylase can be determined based on the
principle that starch and iodine react to form a blue colored
• On hydrolysis of starch this complex changes. The absorbance can be
read after the enzyme substrate reaction has been terminated.
Conc. Of reducing sugar(µmol) volume content
Obtained from standard graph X in tube X dilution factor
Microorganism Fermentation pH
SmF 7.0 33 °C 
Bacillus subtilis SSF 7.0 37 °C 
Aspergillus niger SSF 5.5 70 °C 
SmF 6.0 30°C 
Amylase is ubiquitous enzyme produced by plants, animals and microbes.
In the recent past, there has been extensive research on microbial production of
300 tonnes of α-Amylase have been accounted to be produced from B.lichinoformis
and Aspesgillus sp. per year.
There are two methods widely used for production of α-Amylase on a commercially -
1) Submerged fermentation
2) Solid State fermentation
• Amylases constitute a major class of industrial enzymes which alone form 25% of
the enzyme market covering industrial processes.
saccharification of starch
Converting starch in dough to smaller
Degrade the residues of starchy foods
such as potatoes, gravies, custard,
chocolate, etc. to dextrins and other
smaller oligosaccharides .
Textile Industry Bacillus sp Used in removal of starch sizing agent
from woven fabric.
Fuel Production E.coli, B.subtilis Converting starch in to smaller
fermentable sugars which are acted upon
by yeast to produce ethanol.
Paper industry Bacillus sp Viscosity of the natural starch is too high
for paper sizing and this can be altered by
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