Amylases and pectinases

APPLICATIONS OFAPPLICATIONS OF
AMYLASES AND PECTINASESAMYLASES AND PECTINASES
Presented by:Presented by:
Sudha chibSudha chib
M.Sc. B.T. 2nd yr.M.Sc. B.T. 2nd yr.
Punjabi university PatialaPunjabi university Patiala

Saturday, May 2, 2015Saturday, May 2, 2015 22
AMYLASESAMYLASES
 Catalyses the hydrolysis of internal α-1,4-glycosidicCatalyses the hydrolysis of internal α-1,4-glycosidic
linkages in starch in low molecular weight products.linkages in starch in low molecular weight products.
 Sources: Plants, Animals and Microorganisms.Sources: Plants, Animals and Microorganisms.
 Large number of microbial amylases are available.Large number of microbial amylases are available.
 Have almost completely replaced chemical hydrolysisHave almost completely replaced chemical hydrolysis
of starch in starch processing industry.of starch in starch processing industry.
 The amylases of microorganisms have a broadThe amylases of microorganisms have a broad
spectrum of industrial applications as they are morespectrum of industrial applications as they are more
stable than when prepared with plant and animal α-stable than when prepared with plant and animal α-
amylasesamylases

STRUCTURESTRUCTURE
(α-1,4-glucan-4-glucanohydrolase(α-1,4-glucan-4-glucanohydrolase))
 Belongs to a family of endo-amylases that catalyses the initialBelongs to a family of endo-amylases that catalyses the initial
hydrolysis of starch into shorter oligosaccharides through thehydrolysis of starch into shorter oligosaccharides through the
cleavage of α-D-(1-4) glycosidic bondscleavage of α-D-(1-4) glycosidic bonds
 Has three-dimensional structure capable of binding toHas three-dimensional structure capable of binding to
substrate by the action of highly specific catalytic groups,substrate by the action of highly specific catalytic groups,
promote the breakage of the glycoside links.promote the breakage of the glycoside links.
 The human α-amylase is a classical calcium-containingThe human α-amylase is a classical calcium-containing
enzyme composed of 512 amino acids in a singleenzyme composed of 512 amino acids in a single
oligosaccharide chain with a molecular weight of 57.6 kDa.oligosaccharide chain with a molecular weight of 57.6 kDa.

CONTD..CONTD..
 The protein contains 3 domains: A, B, and C.The protein contains 3 domains: A, B, and C.
 The A domain is the largest, presenting a typical barrel shapedThe A domain is the largest, presenting a typical barrel shaped
(β/α)8 super structure.(β/α)8 super structure.
 The B domain is inserted between the A and C domains and isThe B domain is inserted between the A and C domains and is
attached to the A domain by disulphide bond.attached to the A domain by disulphide bond.
 The C domain has a β sheet structure linked to the A domainThe C domain has a β sheet structure linked to the A domain
by a simple polypeptide chain. The active site of the α-by a simple polypeptide chain. The active site of the α-
amylase is situated in a long cleft located between theamylase is situated in a long cleft located between the
carboxyl end of the A and B domains.carboxyl end of the A and B domains.
 The calcium (Ca2+) is situated between the A and B domainsThe calcium (Ca2+) is situated between the A and B domains
and act in the stabilization of the three-dimensinoal structureand act in the stabilization of the three-dimensinoal structure
and as allosteric activator.and as allosteric activator.

PRODUCTION METHODS ANDPRODUCTION METHODS AND
MICROORGANISMSMICROORGANISMS
 Submerged fermentation (SmF)Submerged fermentation (SmF)
 Solid state fermentationSolid state fermentation
Microbes utilized:Microbes utilized:
 BacteriaBacteria::
Bacillus licheniformisBacillus licheniformis,,
Bacillus stearothermophilusBacillus stearothermophilus,,
Bacillus subtilisBacillus subtilis,,
Bacillus amyloliquefaciensBacillus amyloliquefaciens
B. vulgarusB. vulgarus,,
 FungiFungi
AspergillusAspergillus andand PenicilliumPenicillium

INDUSTRAIL APPLICATIONSINDUSTRAIL APPLICATIONS
1.1. Starch conversionStarch conversion
The most widespread applications of α-amylases are in theThe most widespread applications of α-amylases are in the
starch industry, in the starch liquefaction process convertingstarch industry, in the starch liquefaction process converting
starch into fructose and glucose syrups.starch into fructose and glucose syrups.
The enzymatic conversion of all starch includes:The enzymatic conversion of all starch includes:
 Gelatinization:Gelatinization: involves the dissolution of starch granules,involves the dissolution of starch granules,
thereby forming a viscous suspension;thereby forming a viscous suspension;
 Liquefaction:Liquefaction: involves partial hydrolysis and loss ininvolves partial hydrolysis and loss in
viscosityviscosity
 Saccharification:Saccharification: involves the production of glucose andinvolves the production of glucose and
maltose via further hydrolysis.maltose via further hydrolysis.
1.1. Starch conversionStarch conversion
The most widespread applications of α-amylases are in theThe most widespread applications of α-amylases are in the
starch industry, in the starch liquefaction process convertingstarch industry, in the starch liquefaction process converting
starch into fructose and glucose syrups.starch into fructose and glucose syrups.
The enzymatic conversion of all starch includes:The enzymatic conversion of all starch includes:
 Gelatinization:Gelatinization: involves the dissolution of starch granules,involves the dissolution of starch granules,
thereby forming a viscous suspension;thereby forming a viscous suspension;
 Liquefaction:Liquefaction: involves partial hydrolysis and loss ininvolves partial hydrolysis and loss in
viscosityviscosity
 Saccharification:Saccharification: involves the production of glucose andinvolves the production of glucose and
maltose via further hydrolysis.maltose via further hydrolysis.

ACTION OF AMYLASEACTION OF AMYLASE

DETERGENT INDUSTRYDETERGENT INDUSTRY
 Enhances the detergents ability to remove tough stains andEnhances the detergents ability to remove tough stains and
make it environmentally safe.make it environmentally safe.
 Amylases are the second type of enzymes used in theAmylases are the second type of enzymes used in the
formulation of enzymatic detergent, and 90% of all liquidformulation of enzymatic detergent, and 90% of all liquid
detergents contain these enzymes.detergents contain these enzymes.
 These enzymes are used in detergents for laundry andThese enzymes are used in detergents for laundry and
automatic dishwashing to degrade the residues of starchyautomatic dishwashing to degrade the residues of starchy
foods to dextrins and other smaller oligosaccharides.foods to dextrins and other smaller oligosaccharides.
 Amylases have activity at lower temperatures and alkaline pH,Amylases have activity at lower temperatures and alkaline pH,
maintaining the necessary stability under very oxidizingmaintaining the necessary stability under very oxidizing
conditions.conditions.
 Examples of amylases used in the detergent industry areExamples of amylases used in the detergent industry are
derived fromderived from BacillusBacillus oror Aspergillus.Aspergillus.

FUEL ALCOHOL PRODUCTIONFUEL ALCOHOL PRODUCTION
 Ethanol is the most utilized liquid biofuel. For theEthanol is the most utilized liquid biofuel. For the
ethanol production, starch is the most used substrateethanol production, starch is the most used substrate
due to its low price and easiy availability.due to its low price and easiy availability.
 Starch is solubilized and then submitted to twoStarch is solubilized and then submitted to two
enzymatic steps in order to obtain fermentable sugars.enzymatic steps in order to obtain fermentable sugars.
 The bioconversion of starch into ethanol involvesThe bioconversion of starch into ethanol involves
liquefaction and saccharification, converting it intoliquefaction and saccharification, converting it into
sugar using α-amylase, followed by fermentation.sugar using α-amylase, followed by fermentation.
 Sugar is converted into ethanol using an ethanolSugar is converted into ethanol using an ethanol
fermenting microorganism such asfermenting microorganism such as
yeastyeast Saccharomyces cerevisiaeSaccharomyces cerevisiae ..

FOOD INDUSTRYFOOD INDUSTRY
 Extensively employed in processed-food industry such asExtensively employed in processed-food industry such as
baking, brewing, preparation of digestive aids, production ofbaking, brewing, preparation of digestive aids, production of
cakes, fruit juices and starch syrups.cakes, fruit juices and starch syrups.
 Added to the dough of bread to degrade the starch in the flourAdded to the dough of bread to degrade the starch in the flour
into smaller dextrins, which are subsequently fermented by theinto smaller dextrins, which are subsequently fermented by the
yeast.yeast.
 Reduce the viscosity of dough, improving in the volume andReduce the viscosity of dough, improving in the volume and
texture of the product, generates additional sugar in the doughtexture of the product, generates additional sugar in the dough
improving the taste, colour and toasting qualities of the bread.improving the taste, colour and toasting qualities of the bread.
 Have anti-staling effect in bread baking, improving softnessHave anti-staling effect in bread baking, improving softness
of baked goods and increase the shelf life of products.of baked goods and increase the shelf life of products.
 Amylase ofAmylase of Bacillus stearothermophilusBacillus stearothermophilus is used.is used.

TEXTILE INDUSTRYTEXTILE INDUSTRY
 Used in textile industry for desizing process. Sizing agents likeUsed in textile industry for desizing process. Sizing agents like
starch are applied to yarn before fabric production.starch are applied to yarn before fabric production.
 Desizing involves the removal of starch from the fabric whichDesizing involves the removal of starch from the fabric which
serves as the strengthening agent to prevent breaking of theserves as the strengthening agent to prevent breaking of the
warp thread during the weaving process.warp thread during the weaving process.
 The α-amylases remove selectively the size and do not attackThe α-amylases remove selectively the size and do not attack
the fibres.the fibres.
 Amylase fromAmylase from BacillusBacillus strain was employed in textilestrain was employed in textile
industries for quite a long time.industries for quite a long time.

PAPER INDUSTRYPAPER INDUSTRY
 Used for the modification of starch of coated paper, i.e. for theUsed for the modification of starch of coated paper, i.e. for the
production of low-viscosity, high molecular weight starch.production of low-viscosity, high molecular weight starch.
 It makes the surface of paper sufficiently smooth and strong toIt makes the surface of paper sufficiently smooth and strong to
improve the writing quality of the paper.improve the writing quality of the paper.
 The viscosity of the natural starch is altered by partiallyThe viscosity of the natural starch is altered by partially
degrading the polymer with α-amylases in a batch ordegrading the polymer with α-amylases in a batch or
continuous processes.continuous processes.
 Enhances the stiffness and strength in paper.Enhances the stiffness and strength in paper.
 Examples of amylases obtained from microorganisms used inExamples of amylases obtained from microorganisms used in
paper industry includespaper industry includes Amizyme®Amizyme® (Fermentation Products,(Fermentation Products,
Peoria, USA),Peoria, USA), BAN®BAN® (Novozymes, Denmark) and(Novozymes, Denmark) and α-amylaseα-amylase
G9995®G9995® (Enzyme Biosystems, USA).(Enzyme Biosystems, USA).

MANUFACTURING HIGHMANUFACTURING HIGH
FRUCTOSE SYRUPFRUCTOSE SYRUP
 High fructose containing syrups (HFCS) 42 F (FructoseHigh fructose containing syrups (HFCS) 42 F (Fructose
content = 42%) is prepared by enzymic isomerization ofcontent = 42%) is prepared by enzymic isomerization of
glucose with glucose isomerase.glucose with glucose isomerase.
 The starch is first converted to glucose by enzymicThe starch is first converted to glucose by enzymic
liquefaction and saccharificationliquefaction and saccharification

TREATMENT OF STARCHTREATMENT OF STARCH
PROCESSING WASTE WATERPROCESSING WASTE WATER
 Starch is also present in waste produced from food processingStarch is also present in waste produced from food processing
plants. It causes pollution problems.plants. It causes pollution problems.
 Biotechnological treatment of food processing waste water canBiotechnological treatment of food processing waste water can
produce valuable products such as microbial biomass proteinproduce valuable products such as microbial biomass protein
and also purifies the effluent.and also purifies the effluent.

PRODUCTION OF “ALOPRODUCTION OF “ALO
MIXTURES”MIXTURES”
 ““Alo mixture” is a mixture of isomaltose, panose,Alo mixture” is a mixture of isomaltose, panose,
isomaltotriose and branched oligosaccharideisomaltotriose and branched oligosaccharide
composed of 4 and 5 glucose residues.composed of 4 and 5 glucose residues.
 The ‘Alo mixture’ has properties that are favorable toThe ‘Alo mixture’ has properties that are favorable to
food industry such as:food industry such as:
Mild sweetness, low viscosity, high moistureMild sweetness, low viscosity, high moisture
retaining capacity and low water activityretaining capacity and low water activity
 These are convenient in controlling microbialThese are convenient in controlling microbial
contamination.contamination.
 Starch is dextrinized using thermostable bacterial α-Starch is dextrinized using thermostable bacterial α-
amylase. The reaction mixture is finally purified andamylase. The reaction mixture is finally purified and
concentrated to 25% moisture.concentrated to 25% moisture.

PECTINASESPECTINASES
 Pectinases are today one of the upcoming enzymes ofPectinases are today one of the upcoming enzymes of
the commercial sector.the commercial sector.
 Responsible for the degradation of pectin that occurResponsible for the degradation of pectin that occur
as structural polysaccharides in the middle lamellaas structural polysaccharides in the middle lamella
and the primary call walls of young plant cells.and the primary call walls of young plant cells.
 Pectinases are now an integral part of fruit juice andPectinases are now an integral part of fruit juice and
textile industries as well as having varioustextile industries as well as having various
biotechnological applications.biotechnological applications.
 By 2005, the whole world market for industrialBy 2005, the whole world market for industrial
enzymes is expected to be $1.7–2 billion.enzymes is expected to be $1.7–2 billion.

STRUCTURESTRUCTURE
 Complex colloidal acid polysaccharides, with aComplex colloidal acid polysaccharides, with a
backbone of galacturonic acid residues linkedbackbone of galacturonic acid residues linked
byby αα (1–4) linkages.(1–4) linkages.
 The side chains of the pectin molecule consist of L-The side chains of the pectin molecule consist of L-
rhamnose, arabinose, galactose and xylose.rhamnose, arabinose, galactose and xylose.
 It is mixture of widely differing compositionsIt is mixture of widely differing compositions
containing pectinic acid as the major component.containing pectinic acid as the major component.
 Pectin in native form is located in the cell wall and itPectin in native form is located in the cell wall and it
may be interlined with other structuralmay be interlined with other structural
polysaccharides and proteins to form insolublepolysaccharides and proteins to form insoluble
protopectin.protopectin.

CLASSIFICATIONCLASSIFICATION
 Depolymerizing enzymesDepolymerizing enzymes:: These are the enzymesThese are the enzymes
hydrolyzing glycosidic linkages.hydrolyzing glycosidic linkages.
They include:They include:
 Polymethylgalacturonases (PMG) :Polymethylgalacturonases (PMG) : Catalyze the hydrolyticCatalyze the hydrolytic
cleavage ofcleavage of αα-1,4-glycosidic bonds.-1,4-glycosidic bonds.
They may be:They may be:
1.1. Endo-PMGEndo-PMG:: causes random cleavage ofcauses random cleavage of αα-1,4-glycosidic-1,4-glycosidic
linkages of pectin.linkages of pectin.
2.2. Exo-PMGExo-PMG:: causes sequential cleavage ofcauses sequential cleavage of αα-1,4-glycosidic-1,4-glycosidic
linkage of pectin.linkage of pectin.

CONTD..CONTD..
 Polygalacturonases (PG):Polygalacturonases (PG): Catalyze hydrolysis ofCatalyze hydrolysis of αα-1,4--1,4-
glycosidic linkages in pectic acidglycosidic linkages in pectic acid
They are also of two types:They are also of two types:
1.1. Endo-PGEndo-PG:: also known as poly (1,4-also known as poly (1,4-αα-D-galacturonide)-D-galacturonide)
glycanohydrolase, catalyzes random hydrolysis ofglycanohydrolase, catalyzes random hydrolysis of αα-1,4--1,4-
glycosidic linkages in pectic acid.glycosidic linkages in pectic acid.
2.2. Exo-PGExo-PG:: also known as poly (1,4-also known as poly (1,4-αα-D-galacturonide)-D-galacturonide)
galacturonohydrolase, catalyzes hydrolysis in a sequentialgalacturonohydrolase, catalyzes hydrolysis in a sequential
fashion offashion of αα-1,4-glycosidic linkages on pectic acid.-1,4-glycosidic linkages on pectic acid.

CONTD..CONTD..
 ProtopectinaseProtopectinase
 This enzyme solubilizes protopectin forming highlyThis enzyme solubilizes protopectin forming highly
polymerized soluble pectin.polymerized soluble pectin.
 Pectinesterases (PE)Pectinesterases (PE)
Pectinesterases also known as pectinmethyl hydrolase,Pectinesterases also known as pectinmethyl hydrolase,
catalyzes deesterification of the methoxyl group ofcatalyzes deesterification of the methoxyl group of
pectin forming pectic acid.pectin forming pectic acid.

IMPORTANT PRODUCERSIMPORTANT PRODUCERS
 On the bases of their applications,On the bases of their applications,
pectinases are mainly of two types:pectinases are mainly of two types:
acidic pectinases and alkalineacidic pectinases and alkaline
pectinasespectinases
 The important producers of theseThe important producers of these
pectinases are given in the tablepectinases are given in the table
ProducerProducer
( Acidic( Acidic
pectinase)pectinase)
Type ofType of
pectinasepectinase
AspergillusAspergillus
nigerniger
Endo-Endo-
pectinase,pectinase,
Exo-Exo-
pectinasepectinase
MucorMucor
pusiluspusilus
EndopolygalEndopolygal
acturonaseacturonase
SclerotiumSclerotium
rolfsiirolfsii
EndopolygalEndopolygal
acturonaseacturonase

CONTD..CONTD..
Producer (AlkalineProducer (Alkaline
pectinases )pectinases )
Type of pectinaseType of pectinase
Bacillus polymyxaBacillus polymyxa PolygalacturonatePolygalacturonate
AmucolaAmucola sp.sp. Pectate lyasePectate lyase
Penicillium italicumPenicillium italicum Pectin lyasePectin lyase
Bacillus subtilisBacillus subtilis Pectate lyasePectate lyase

APPLICATIONSAPPLICATIONS
 Acidic pectinasesAcidic pectinases
Acidic pectic enzymes used in the fruit juice industries andAcidic pectic enzymes used in the fruit juice industries and
wine making often come from fungal sources, especiallywine making often come from fungal sources, especially
fromfrom Aspergillus nigerAspergillus niger..
The juices produced by these industries commercially include:The juices produced by these industries commercially include:
(A)(A) Sparkling clear juicesSparkling clear juices, (apple, pear and grape juices,, (apple, pear and grape juices,
(B)(B) Juices with cloudsJuices with clouds (citrus juices, prune juices, tomato(citrus juices, prune juices, tomato
juice and nectars),juice and nectars),
(C)(C) Unicellular productsUnicellular products to preserve the integrity of the plantto preserve the integrity of the plant
cells by selectively hydrolyzing the polysaccharides of thecells by selectively hydrolyzing the polysaccharides of the
middle lamella.middle lamella.

CONTD..CONTD..
 Sparkling clear juicesSparkling clear juices
Enzymes are added to increase juice yield during pressing ofEnzymes are added to increase juice yield during pressing of
juice and to remove suspended matter to give sparkling clearjuice and to remove suspended matter to give sparkling clear
juices.juices.
 Cloudy juicesCloudy juices
Pectin enzymes containing high levels of polygalacturonasePectin enzymes containing high levels of polygalacturonase
activity are added to fruit juices to stabilize the cloud of citrusactivity are added to fruit juices to stabilize the cloud of citrus
juices and nectars.juices and nectars.
 Unicellular productsUnicellular products
Formed by transformation of organized tissues into aFormed by transformation of organized tissues into a
suspension of intact cells, resulting in products used as basesuspension of intact cells, resulting in products used as base
material for pulpy juices, baby foods, puddings and yogurt.material for pulpy juices, baby foods, puddings and yogurt.
Enzymes used for this purpose are referred to asEnzymes used for this purpose are referred to as “macerases”“macerases”

CONTD..CONTD..
 Alkaline pectinasesAlkaline pectinases
Alkaline pectinases are mainly used in the degumming andAlkaline pectinases are mainly used in the degumming and
retting of fiber crops and pretreatment of pectic wastewaterretting of fiber crops and pretreatment of pectic wastewater
from fruit juice industries.from fruit juice industries.
These enzymes come mostly from bacterial sources.These enzymes come mostly from bacterial sources.
In the industrial sector, alkaline pectinases, mainlyIn the industrial sector, alkaline pectinases, mainly
fromfrom BacillusBacillus spp. are applied.spp. are applied.

CONTD..CONTD..
 Retting and degumming of fiber crops:Retting and degumming of fiber crops:
Pectinolytic enzymes are involved in the retting andPectinolytic enzymes are involved in the retting and
degumming of jute, flax, hemp, ramie and coir from coconutdegumming of jute, flax, hemp, ramie and coir from coconut
husks.husks.
Retting is a fermentation process in which certain bacteriaRetting is a fermentation process in which certain bacteria
((ClostridiumClostridium,, BacillusBacillus) and certain fungi) and certain fungi
((AspergillusAspergillus,, PenicilliumPenicillium) decompose the pectin of the bark) decompose the pectin of the bark
and release fiber.and release fiber.

CONTD..CONTD..
 Treatment of pectic wastewater:Treatment of pectic wastewater:
The wastewater from the citrus-processing industry containsThe wastewater from the citrus-processing industry contains
pectinaceous materials barely decomposed by microbes duringpectinaceous materials barely decomposed by microbes during
the activated-sludge treatment.the activated-sludge treatment.
A soft-rot pathogen,A soft-rot pathogen, Erwinia carotovoraErwinia carotovora secrets endo-pectatesecrets endo-pectate
lyase in the pretreatment of pectinaceous wastewater.lyase in the pretreatment of pectinaceous wastewater.

CONTD..CONTD..
 Production of Japanese paper:Production of Japanese paper:
Alkaline pectinase produced byAlkaline pectinase produced by BacillusBacillus sp. andsp. and ErwiniaErwinia
carotovoracarotovora has been used for retting of bast.has been used for retting of bast.
These retted basts have been used for the preparation ofThese retted basts have been used for the preparation of
Japanese paper.Japanese paper.
The strength of the pulp from bacterial retting is as high as thatThe strength of the pulp from bacterial retting is as high as that
obtained by the conventional soda-ash cooking method.obtained by the conventional soda-ash cooking method.
The paper sheets prepared from this pulp are very uniform andThe paper sheets prepared from this pulp are very uniform and
soft to touch.soft to touch.

CONTD..CONTD..
 Oil extraction:Oil extraction:
Pectinase are used to extract vegetable oil in an aqueousPectinase are used to extract vegetable oil in an aqueous
process by liquefying the structural cell wall components ofprocess by liquefying the structural cell wall components of
the oil-containing crop.the oil-containing crop.
The enzymes are added during grinding of the seeds the oil isThe enzymes are added during grinding of the seeds the oil is
released easily in the separation techniques. This increases thereleased easily in the separation techniques. This increases the
yield.yield.
Olivex,Olivex, an enzyme preparation derived froman enzyme preparation derived from A. aculeatusA. aculeatus,,
contains pectinolytic activity and give good oil extraction andcontains pectinolytic activity and give good oil extraction and
better stability when stored. The oil also shows increasedbetter stability when stored. The oil also shows increased
content of polyphenols and vitamin E, which stabilizes the oil.content of polyphenols and vitamin E, which stabilizes the oil.

CONTD..CONTD..
 Coffee and tea fermentation:Coffee and tea fermentation:
Pectinases play an important role in coffee and teaPectinases play an important role in coffee and tea
fermentation.fermentation.
Fermentation of coffee using pectinolytic microorganisms isFermentation of coffee using pectinolytic microorganisms is
done to remove the mucilage coat from the coffee beans.done to remove the mucilage coat from the coffee beans.

BIBLIOGRAPHYBIBLIOGRAPHY
 www.scielo.brwww.scielo.br
 www.sciencedirect.comwww.sciencedirect.com
 http://blog.targethealth.comhttp://blog.targethealth.com
 Toxnet.nlm.nih.comToxnet.nlm.nih.com

Amylases and pectinases

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