AMYLASE

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

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

MAJOR SUBSTRATE FOR AMYLASE -STARCHStarch 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.

Various types of amylase associated with degradation of starch and related polysaccharides structures have been detected and studied[5]. 1. Enzymes that hydrolyze -1,4 bonds e.g. -amylase (endoacting amylases).2. Enzymes that hydrolyze -1,4 e.g. - amylase (exoacting amylases producing maltose as a major end product).3. Enzymes that hydrolyze terminal 1,4 linked D-glucose residues. e.g. glucoamylase.4. Enzymes that hydrolyze only -1,6 linkages e.g. pullulanase .5. Enzymes that hydrolyze preferentially -1,4 linkages in short chain oligosaccharides produced by the action of other enzymes on amylose and amylopectin e.g. - glucosidases.

[5]- Van et al.,2002

TYPES OF AMYLASE

Mechanism of amylase activity

.

Manners et al, 1992

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 terminal (1-4)-linked α-D-glucose residues successively from non-reducing ends ofthe chains with release of β-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 at the nonreducing end of amylose 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 amyloliquefaciens Aspergillus oryzae and Aspergillus niger.

Seeds of higher plants and sweet potatoes.Bacillus polymyxa,B.cereus

Aspergillus oryzae, Aspergillus niger,Rhizopus nevius

References [6] [7] [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.

–Characterization of α-amylase.

SOURCE Km (mg/ml)

Vmax (μmol/mg/min)

Kcat (S-1)

Kcat/Km(ml mg-1 S-

1)

Inhibitors

Activator

Temperature

pH References

B. Licheniformis

6.2 1.04 2000 3.22×10-2

Hg2+ ,Cd2+,Mn2+,,Ba2+,Cu2+, ,EDTA

1500 Da PEG,

increases the

enzyme activity by 24%

at 0.02%

w/v

85-90°C 6.5 [11]

Bacillus megaterium

9.0 0.68 580 .64x10-2

Hg2+

Ba2+,,Zn2+,Co2+,Cr3+,Fe3+

Titron Xincreases the enzyme activity by 34% at 0.02% w/v

37-40°C 6.0 [12]

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.

• 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

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

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 Reference

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 degrading the polymer with amylases .

[25]

REFERENCES.[1] Sivaramakrishnan S., Gangadharan D.,Madhavan K., Ricardo C., Pandey A. α-Amylases from

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.

[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.

[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.

[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.

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