Presentation on overview of one of the most important hydrolytic enzyme -amylase.

1. AMYLASE

2. INTRODUCTION
• 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[1].
• 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) [2] .
• They belong to the glycoside hydrolase group of enzymes under
which 13 enzymes are included [3] .
[1]- Sivaramakrishnan et al., 2006 [2]- Aiyer et al.,2005 [3]- ] Windish et al.,2005

3. ENZYME NOMENCLATURE
E.C.3.-.-.- main class of enzyme - Hydrolases
E.C.3.2.-.- glycosidic bond that is hydrolyzed. Hence , they are
called Gycosylases.
E.C.3.2.1.- Glycosidases, i.e. enzymes hydrolyzing O- glycosyl
compound.
E.C.3.2.1.1 α- amylase
E.C.3.2.1.2 β- amylase
E.C.3.2.1.3 glucoamylase
ENZYME NOMENCLATURE

4. 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
bonds.
• 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
amylopectin chain[4].
[4] Muralikrishna G., Nirmala M. Cereal α-amylases—an
overview.Carbohydrate Polymers. 2005;60:163–173.

5. Various types of amylase associated with degradation of starch and
related polysaccharides structures have been detected and studied[5].
1. Enzymes that hydrolyze 𝛼-1,4 bonds e.g. 𝜶 -amylase (endoacting
amylases).
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.
glucoamylase.
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.
[5]- Van et al.,2002
TYPES OF AMYLASE

6. Mechanism of amylase activity
.
Manners et al, 1992

7. Accepted
name
α-amylase β-amylase glucoamylase
Systematic
name
1,4-α-D-glucan
glucanohydrolase
1,4-α-D-glucan
maltohydrolase
1,4-α-D-glucan
glucohydrolase
Reaction Endo hydrolysis of (1 -4)-α-D-
glycosidic linkages in
polysaccharides containing
three or more (1 4)-α-linked
D-glucose units.
Hydrolysis of (1 - 4)-α-D-
glycosidic linkages in
polysaccharides so as to
remove successive
maltose units from the
non-reducing ends of the
chains.
Hydrolysis of termin
4)-linked α-D-glucos
residues successivel
non-reducing ends of
the chains with releas
D-glucose. It is an
exoenzyme.
Comment Acts on starch related
polysaccharides and
oligosaccharides in a random
manner; reducing groups are
liberated in the α-
configuration.
Acts on starch, glycogen
and related
polysaccharides and
oligosaccharides
producing β-maltose. The
term β relates to the
initial anomeric
configuration of the free
sugar group released.
Cleaves the last α(1-
4)glycosidic linkages
nonreducing end of a
and amylopectin.
E.C number E.C.3.2.1.1 E.C.3.2.1.2 E.C.3.2.1.3
Source Bacillus licheniformis, Bacillus
stearothermophilus, Bacillus
Seeds of higher plants
and sweet potatoes.
Aspergillus oryzae,
Aspergillus niger,

8. Payan 2004.
 The human α-amylase is a classical calcium-containing
enzyme composed of 512 amino acids with a molecular
weight of 57.6 kDa[9]. .
 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
structure.
 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
catalysis [10].
 The calcium (Ca2+) is situated at B domain (Asn 100, Arg
158, Asp 167) against the wall of the barrel of Domain
A .
 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.

9. –
Characterization of α-amylase.
SOURCE Km
(mg
/ml)
Vmax
(μmol/m
g/min)
Kcat
(S-1)
Kcat/
Km
(ml
mg-1
S-1)
Inhibi
tors
Activat
or
Temperat
ure
pH Referen
ces
B.
Licheniformis
6.2 1.04 2000 3.22×
10-2
Hg2+
,Cd2+,
Mn2+,,
Ba2+,
Cu2+,
,EDTA
1500 Da
PEG,
increas
es the
enzyme
activity
by 24%
at
0.02%
w/v
85-90°C 6.5 [11]
Bacillus
megaterium
9.0 0.68 580 .64x
10-2
Hg2+
Ba2+,
,Zn2+,
Co2+,
Cr3+,
Fe3+
Titron X
increase
s the
enzyme
activity
by 34%
at
37-40°C 6.0 [12]

10. 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[13].
• 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.

11. • 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
(cubic meter)
• The hydrolytic activity of Amylase can be determined based on the
principle that starch and iodine react to form a blue colored
complex[14].
• 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

12. Microorganism Fermentation pH
optimal/stability
Temperature
optimal/stability
Reference
Bacteria
Bacillus
amyloliquefaciens
SmF 7.0 33 °C [16]
Bacillus subtilis SSF 7.0 37 °C [17]
Fungi
Aspergillus niger SSF 5.5 70 °C [18]
Aspergillus
fumigatus
SmF 6.0 30°C [19]
Amylase is ubiquitous enzyme produced by plants, animals and microbes.
In the recent past, there has been extensive research on microbial production of
Amylase.
300 tonnes of α-Amylase have been accounted to be produced from B.lichinoformis
and Aspesgillus sp. per year[15].
There are two methods widely used for production of α-Amylase on a commercially -
1) Submerged fermentation
2) Solid State fermentation

13. APPLICATION
• Amylases constitute a major class of industrial enzymes which alone form 25% of
the enzyme market covering industrial processes.
Industrial
application
Microbial
source
Role Refere
nce
Starch
conversion
B.amyloliquefaciens,
B.licheniformis
gelatinization, liquefaction,
saccharification of starch
[20]
Bakery Bacillus
stearothermophilus
Converting starch in dough to smaller
fermentable sugars.
[21 ]
Detergent
Industry
Bacillus sp
Aspergillus sp
Degrade the residues of starchy foods
such as potatoes, gravies, custard,
chocolate, etc. to dextrins and other
smaller oligosaccharides .
[ 22]
Textile Industry Bacillus sp Used in removal of starch sizing agent
from woven fabric.
[23 ]
Fuel Production E.coli, B.subtilis Converting starch in to smaller
fermentable sugars which are acted upon
by yeast to produce ethanol.
[24 ]
Paper industry Bacillus sp Viscosity of the natural starch is too high
for paper sizing and this can be altered by
[25]

14. REFERENCES.
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Microbial Sources. Food Technol. Biotechnol. 2006; 44 (2):173-184.
[2] Aiyer, P.V. Amylase and their applications. African Journal of Biotechnology.2005; 4(13): 1525
- 1529.
[3] Windish, W., Mhatre, N.S. Microbial amylases. Advances in applied microbiology,.2005;7:273
- 304.
[4] Van M., Leemhuis H, Dijkhuizen L.Properties and applications of starch converting enzymes
of the α-amylase family, J. Biotechnol. 2002; 94:137-155.
[5] Manners, D.J. Enzymatic synthesis and degradation of starch and glycogen. Adv. Carbohydr.
Chem. (1992);17:371–430.
[6] Gangadharan, D., Nampoothiri, K. M., Soccol, C. R., & Pandey, A. α-Amylases from Microbial
Sources. Food Technology & Biotechnology.2006.,44(2):23-27.
[7] Kaplan F., Dong S., Charles L. “Roles of β-amylase and starch breakdown” .International
Journal on Plant Physiology. 2006., 126:120–128.

15. [8] Reddy N.S., Nimmagadda A., Sambasiva K.R.S. An overview of the microbial
amylase family. Afr. J. Biotechnol. 2003;2:645–648.
[9] Payan F. Structural basis for the inhibition of mammalian and insect alpha-
amylases by plant protein inhibitors. Biochem Biophys Acta. 2004;1696:171–180.
[10] Muralikrishna G., Nirmala M. Cereal α-amylases—an overview. Carbohydrate
Polymers. 2005;60:163–173.
[11] Saptadip S., Das A., Kumar H., Jana A. Thermodynamic and kinetic
characteristics of an α-amylase from Bacillus licheniformis SKB4. Acta Biol
Szeged.2014; 58(2):147-156 .
[12] Tanaka, A and Hoshino, E. 2002. Calcium binding parameter of Bacillus
amyloliquefaciens amylase determined by inactivation kinetics. Biochemistry
Journal, 364: 635 – 639.
[13] Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing
sugar, Anal. Chem.1959; 31:426-428.

16. [14] Hamilton, L. M., Kelly, C. T., & Fogarty, W. M. Carbohydrate Research.1998; 314, 251–257.
[15] Chi Z., Liu G., Wang F., Ju L., Zhang T. Saccharomycopsis fibuligera and its applications in
biotechnology. Biotechnol Adv. 2009;27:423–431.
[16] Gupta R., Gigras P., Mohapatra H., Goswami V.K., Chauhan B. Microbial α-amylases: a
biotechnological perspective. Process Biochem. 2003;38:1599–1616.
[17] Tanyildizi M.S., Ozer D., Elibol M. Production of bacterial α-amylase by B. amyloliquefaciens
under solid substrate fermentation. Biochem. Eng. J. 2007;37:294–297.
[18] Baysal Z., Uyar F., Aytekin C. Solid state fermentation for production of α-amylase by a
thermotolerantBacillus subtilis from hot-spring water. Process Biochemistry. 2003;38:1665–
1668.
[19] Uguru G.C., Akinyauju J.A., Sani A. The use of yam peel for growth of locally
isolated Aspergillus niger and amylase production. Enzyme Microb. Technol. 1997;21:46–51.
[20] Jin B., Leeuwen H.J., Patel B., Yu Q. Utilisation of starch processing wastewater for
production of microbial biomass protein and fungal α-amylase by Aspergillus oryzae. Bioresour.
Technol. 1998;66:201–206.

17. [20] Reddy N.S., Nimmagadda A., Sambasiva Rao K.R.S. An overview of the microbial α-amylase
family. Afr. J. Biotechnol.2003;2:645–648.
[21] Couto S.R., Sanromán M.A. Application of solid-state fermentation to food industry- A
review. Journal of Food Engineering. 2006;76:291–302.
[22] Hmidet N., El-Hadj Ali N., Haddar A., Kanoun S., Alya S., Nasri M. Alkaline proteases and
thermostable α-amylase co-produced by Bacillus licheniformis NH1: Characterization and
potential application as detergent additive. Biochemical Engineering Journal. 2009;47:71–79.
[23] Feitkenhauer H. Anaerobic digestion of desizing wastewater: influence of pretreatment and
anionic surfactant on degradation and intermediate accumulation. Enzyme Microb. Technol.
2003;33:250–258.
[24] Moraes L.M.P., Filho S.A., Ulhoa C.J. Purification and some properties of an α-amylase
glucoamylase fusion protein from Saccharomyces cerevisiae. World J. Microbiol. Biotechnol.
1999;15:561–564.
[25] Bruinenberg P.M., Hulst A.C., Faber A., Voogd R.H. A process for surface sizing or coating of
paper. In: European Patent Application. 1996.

18. THANK YOU