Amylases and pectinases

APPLICATIONS OF APPLICATIONS 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-glycosidic Catalyses 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 hydrolysis Have almost completely replaced chemical hydrolysis

of starch in starch processing industry. of starch in starch processing industry. The amylases of microorganisms have a broad The amylases of microorganisms have a broad

spectrum of industrial applications as they are more spectrum 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-amylases amylases


STRUCTURESTRUCTURE

(α-1,4-glucan-4-glucanohydrolase(α-1,4-glucan-4-glucanohydrolase) ) Belongs to a family of endo-amylases that catalyses the initial Belongs to a family of endo-amylases that catalyses the initial

hydrolysis of starch into shorter oligosaccharides through the hydrolysis of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glycosidic bonds cleavage of α-D-(1-4) glycosidic bonds

Has three-dimensional structure capable of binding to Has 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-containing The human α-amylase is a classical calcium-containing enzyme composed of 512 amino acids in a single enzyme 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.


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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 shaped The 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 is The 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 domain The 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 the amylase 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 domains The calcium (Ca2+) is situated between the A and B domains and act in the stabilization of the three-dimensinoal structure and act in the stabilization of the three-dimensinoal structure and as allosteric activator. and as allosteric activator.


PRODUCTION METHODS AND PRODUCTION METHODS AND MICROORGANISMSMICROORGANISMS

Submerged fermentation (SmF)Submerged fermentation (SmF) Solid state fermentation Solid state fermentation Microbes utilized:Microbes utilized: BacteriaBacteria:: Bacillus licheniformisBacillus licheniformis, , Bacillus stearothermophilusBacillus stearothermophilus, , Bacillus subtilisBacillus subtilis, , Bacillus amyloliquefaciensBacillus amyloliquefaciens B. vulgarusB. vulgarus, , FungiFungi AspergillusAspergillus and and PenicilliumPenicillium


INDUSTRAIL APPLICATIONSINDUSTRAIL APPLICATIONS

1.1. Starch conversionStarch conversion The most widespread applications of α-amylases are in the The most widespread applications of α-amylases are in the

starch industry, in the starch liquefaction process converting starch 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 in involves partial hydrolysis and loss in

viscosityviscosity Saccharification:Saccharification: involves the production of glucose and involves 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 the The most widespread applications of α-amylases are in the

starch industry, in the starch liquefaction process converting starch 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 in involves partial hydrolysis and loss in

viscosityviscosity Saccharification:Saccharification: involves the production of glucose and involves 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 and Enhances 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 the Amylases are the second type of enzymes used in the

formulation of enzymatic detergent, and 90% of all liquid formulation of enzymatic detergent, and 90% of all liquid detergents contain these enzymes. detergents contain these enzymes.

These enzymes are used in detergents for laundry and These enzymes are used in detergents for laundry and automatic dishwashing to degrade the residues of starchy automatic 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 oxidizing maintaining the necessary stability under very oxidizing conditions. conditions.

Examples of amylases used in the detergent industry are Examples of amylases used in the detergent industry are derived from derived from BacillusBacillus or or Aspergillus.Aspergillus.


FUEL ALCOHOL PRODUCTIONFUEL ALCOHOL PRODUCTION

Ethanol is the most utilized liquid biofuel. For the Ethanol is the most utilized liquid biofuel. For the ethanol production, starch is the most used substrate ethanol 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 two Starch 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 involves The bioconversion of starch into ethanol involves liquefaction and saccharification, converting it into liquefaction 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 ethanol Sugar is converted into ethanol using an ethanol fermenting microorganism such as fermenting microorganism such as yeast yeast Saccharomyces cerevisiaeSaccharomyces cerevisiae . .


FOOD INDUSTRYFOOD INDUSTRY Extensively employed in processed-food industry such as Extensively employed in processed-food industry such as

baking, brewing, preparation of digestive aids, production of baking, 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 flour Added to the dough of bread to degrade the starch in the flour into smaller dextrins, which are subsequently fermented by the into smaller dextrins, which are subsequently fermented by the yeast.yeast.

Reduce the viscosity of dough, improving in the volume and Reduce the viscosity of dough, improving in the volume and texture of the product, generates additional sugar in the dough texture 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 softness Have 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 of Amylase of Bacillus stearothermophilusBacillus stearothermophilus is used. is used.


TEXTILE INDUSTRYTEXTILE INDUSTRY Used in textile industry for desizing process. Sizing agents like Used 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 which Desizing involves the removal of starch from the fabric which

serves as the strengthening agent to prevent breaking of the serves 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 attack The α-amylases remove selectively the size and do not attack the fibres. the fibres.

Amylase from Amylase from BacillusBacillus strain was employed in textile strain 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 the Used 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 to It 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 partially The viscosity of the natural starch is altered by partially

degrading the polymer with α-amylases in a batch or degrading 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 in Examples of amylases obtained from microorganisms used in

paper industry includes paper industry includes Amizyme®Amizyme® (Fermentation Products, (Fermentation Products, Peoria, USA), Peoria, USA), BAN®BAN® (Novozymes, Denmark) and (Novozymes, Denmark) and α-amylase α-amylase G9995®G9995® (Enzyme Biosystems, USA). (Enzyme Biosystems, USA).


MANUFACTURING HIGH MANUFACTURING HIGH FRUCTOSE SYRUPFRUCTOSE SYRUP

High fructose containing syrups (HFCS) 42 F (Fructose High fructose containing syrups (HFCS) 42 F (Fructose content = 42%) is prepared by enzymic isomerization of content = 42%) is prepared by enzymic isomerization of glucose with glucose isomerase. glucose with glucose isomerase.

The starch is first converted to glucose by enzymic The starch is first converted to glucose by enzymic liquefaction and saccharificationliquefaction and saccharification


TREATMENT OF STARCH TREATMENT OF STARCH PROCESSING WASTE WATERPROCESSING WASTE WATER

Starch is also present in waste produced from food processing Starch 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 can Biotechnological treatment of food processing waste water can produce valuable products such as microbial biomass protein produce valuable products such as microbial biomass protein and also purifies the effluent.and also purifies the effluent.


PRODUCTION OF “ALO PRODUCTION OF “ALO MIXTURES”MIXTURES”

““Alo mixture” is a mixture of isomaltose, panose, Alo mixture” is a mixture of isomaltose, panose, isomaltotriose and branched oligosaccharide isomaltotriose 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 to The ‘Alo mixture’ has properties that are favorable to food industry such as: food industry such as:

Mild sweetness, low viscosity, high moisture Mild sweetness, low viscosity, high moisture retaining capacity and low water activityretaining capacity and low water activity

These are convenient in controlling microbial These 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 and amylase. The reaction mixture is finally purified and concentrated to 25% moisture.concentrated to 25% moisture.


PECTINASESPECTINASES Pectinases are today one of the upcoming enzymes of Pectinases are today one of the upcoming enzymes of

the commercial sector. the commercial sector. Responsible for the degradation of pectin that occur Responsible for the degradation of pectin that occur

as structural polysaccharides in the middle lamella as 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 and Pectinases are now an integral part of fruit juice and textile industries as well as having various textile industries as well as having various biotechnological applications. biotechnological applications.

By 2005, the whole world market for industrial By 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 a Complex colloidal acid polysaccharides, with a

backbone of galacturonic acid residues linked backbone of galacturonic acid residues linked by by 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 compositions It 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 it Pectin in native form is located in the cell wall and it may be interlined with other structural may be interlined with other structural polysaccharides and proteins to form insoluble polysaccharides and proteins to form insoluble protopectin. protopectin.


CLASSIFICATIONCLASSIFICATION

Depolymerizing enzymesDepolymerizing enzymes: : These are the enzymes These are the enzymes hydrolyzing glycosidic linkages.hydrolyzing glycosidic linkages.

They include:They include: Polymethylgalacturonases (PMG) :Polymethylgalacturonases (PMG) : Catalyze the hydrolytic Catalyze the hydrolytic

cleavage of cleavage of αα-1,4-glycosidic bonds. -1,4-glycosidic bonds. They may be:They may be:1.1. Endo-PMGEndo-PMG:: causes random cleavage of causes random cleavage of αα-1,4-glycosidic -1,4-glycosidic

linkages of pectin.linkages of pectin.2.2. Exo-PMGExo-PMG:: causes sequential cleavage of causes sequential cleavage of αα-1,4-glycosidic -1,4-glycosidic

linkage of pectin.linkage of pectin.


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Polygalacturonases (PG):Polygalacturonases (PG): Catalyze hydrolysis of Catalyze 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 of glycanohydrolase, 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 sequential galacturonohydrolase, catalyzes hydrolysis in a sequential fashion of fashion of αα-1,4-glycosidic linkages on pectic acid.-1,4-glycosidic linkages on pectic acid.


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ProtopectinaseProtopectinase This enzyme solubilizes protopectin forming highly This 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 of catalyzes 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 alkaline acidic pectinases and alkaline pectinasespectinases

The important producers of these The important producers of these pectinases are given in the tablepectinases are given in the table

ProducerProducer

( Acidic ( Acidic pectinase) pectinase)

Type of Type of pectinasepectinase

Aspergillus Aspergillus nigerniger

Endo-Endo-pectinase, pectinase, Exo-Exo-pectinase pectinase

Mucor Mucor pusiluspusilus

EndopolygalEndopolygalacturonase acturonase

Sclerotium Sclerotium rolfsiirolfsii

EndopolygalEndopolygalacturonase acturonase


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Producer (Alkaline Producer (Alkaline pectinases )pectinases )

Type of pectinaseType of pectinase

Bacillus polymyxaBacillus polymyxa Polygalacturonate Polygalacturonate

AmucolaAmucola sp. sp. Pectate lyase Pectate lyase

Penicillium italicumPenicillium italicum Pectin lyase Pectin lyase

Bacillus subtilisBacillus subtilis Pectate lyase Pectate lyase


APPLICATIONSAPPLICATIONS

Acidic pectinasesAcidic pectinases Acidic pectic enzymes used in the fruit juice industries and Acidic pectic enzymes used in the fruit juice industries and

wine making often come from fungal sources, especially wine making often come from fungal sources, especially from from 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 plant to preserve the integrity of the plant

cells by selectively hydrolyzing the polysaccharides of the cells by selectively hydrolyzing the polysaccharides of the middle lamella.middle lamella.


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Sparkling clear juicesSparkling clear juices Enzymes are added to increase juice yield during pressing of Enzymes are added to increase juice yield during pressing of

juice and to remove suspended matter to give sparkling clear juice and to remove suspended matter to give sparkling clear juices.juices.

Cloudy juicesCloudy juices Pectin enzymes containing high levels of polygalacturonase Pectin enzymes containing high levels of polygalacturonase

activity are added to fruit juices to stabilize the cloud of citrus activity 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 a Formed by transformation of organized tissues into a

suspension of intact cells, resulting in products used as base suspension 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 as Enzymes used for this purpose are referred to as “macerases”“macerases”


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Alkaline pectinasesAlkaline pectinases Alkaline pectinases are mainly used in the degumming and Alkaline pectinases are mainly used in the degumming and

retting of fiber crops and pretreatment of pectic wastewater retting 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, mainly In the industrial sector, alkaline pectinases, mainly

from from BacillusBacillus spp. are applied. spp. are applied.


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Retting and degumming of fiber crops:Retting and degumming of fiber crops: Pectinolytic enzymes are involved in the retting and Pectinolytic enzymes are involved in the retting and

degumming of jute, flax, hemp, ramie and coir from coconut degumming of jute, flax, hemp, ramie and coir from coconut husks.husks.

Retting is a fermentation process in which certain bacteria Retting 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.


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Treatment of pectic wastewater:Treatment of pectic wastewater:

The wastewater from the citrus-processing industry contains The wastewater from the citrus-processing industry contains pectinaceous materials barely decomposed by microbes during pectinaceous 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-pectate secrets 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 by Alkaline pectinase produced by BacillusBacillus sp. and sp. and Erwinia Erwinia

carotovora carotovora has been used for retting of bast. has been used for retting of bast. These retted basts have been used for the preparation of These 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 that The 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 and The 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 aqueous Pectinase are used to extract vegetable oil in an aqueous

process by liquefying the structural cell wall components of process 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 is The enzymes are added during grinding of the seeds the oil is released easily in the separation techniques. This increases the released easily in the separation techniques. This increases the yield. yield.

Olivex,Olivex, an enzyme preparation derived from an enzyme preparation derived from A. aculeatusA. aculeatus, , contains pectinolytic activity and give good oil extraction and contains pectinolytic activity and give good oil extraction and better stability when stored. The oil also shows increased better 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.


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Coffee and tea fermentation:Coffee and tea fermentation:

Pectinases play an important role in coffee and tea Pectinases play an important role in coffee and tea fermentation. fermentation.

Fermentation of coffee using pectinolytic microorganisms is Fermentation 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

http://www.scielo.br/

http://www.sciencedirect.com/

http://blog.targethealth.com/

Amylases and pectinases

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