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AMITY INSTITUTE OF BIOTECHNOLOGY

ENZYMOLOGY ASSIGNMENT-I S USED IN DETERGENTS

DATE OF SUBMISSION- 22.O4.08

SUBMITTED TO: SUBMITTED BY:: DR. S.M. BHATT MAYANK JAINN FFACULTY,ENZYMOLOGY ROLL NO.-77 AIB SECTION-U AIB

CONTENTS: PPAGE NO. HISTORY...... 3 INTRODUCTION...... 3 DETERGENT ...... 5 ...... 7 LIPASE...... 8 ...... 9 P a g e || 22

MISCELLANEOUS DETERGENTS PEROXIDASES...... 10 PULLULANASE...... 10 ENZYME FORMULATION...... 11 PRODUCTION OF ENZYME BASED DETERGENT...... 13 ENZYME STABILITY...... 14 APPLICATION OF ENZYME BASED DETERGENT...... 16 BENEFIT OF USING ENZYME IN DETERGENT...... 19 CONCLUSION ...... 20 BIBLIOGRAPHY...... 22

History The original idea of using enzyme as detergents was described in 1913 by Dr Otto Rohm, who patented the use of crude pancreatic extracts in laundry pre- soak compositions to improve the removal of biological stains. In the same year, the first enzymatic detergent named Burnus was launched, but was not popular because of its own limitations. Subsequently, Bio- 40 - a detergent containing a bacterial was produced in Switzerland and launched in the market in 1959 and it gradually became popular. In the period from 1965 to 1970, use and sale of detergent enzymes grew very fast. In 1970, the use was distorted due to dust production by formulations leading to allergies to some workers. This problem was overcome in 1975 by encapsulating the granules of enzyme. From 1980s to the 1990s, several changes took place in the detergent industry like development of softenining ththrorough the washsh, development of concentratetedd heavy-duty power detergents, development of concentrated or structured or non- aqueous liquid detergent.

Introduction Enzymes have been used to improve the cleaning efficiency of detergents for more than 35 years, and are now well accepted as ingredients in powder and liquid detergents, stain removers/laundry pre-spotters, automatic dishwashing detergents and industrial/institutional cleaning products. Deterergent enzymess account for about 30% of the total worldwide enzyme production and represent P a g e || 33 one of ththe lalargrgest and most succecessful applplicications of modern indusustrialal biotechnology. The largest segment within the global industrial enzyme market is the market for technical enzymes, estimated at around uss 980 million in 2002. In the technical enzymes category, detergent additives make up for nearly two-thirds of the market. These enzymes are used as functional ingredients in laundry detetergrgents and automated didishwashing deterergents. This articicle gigives an overview of the detergent enzymes industry and discusses its manufacturing and downstream processing.

Enzymes used in detergents are protein catalysts that consist of long chains of amino acids. They are similar to protein catalysts present in all living cells wherere ththey control metabololic procecesses, converert food nutrtrients to simplplee molecules, convert these molecules to energy and to new cell material. As catalysts; enzymes speed up specific chemical reactions, in mild conditions of tetempererataturure and pH, withthout beining alalteterered or consumed in ththe prprocesess.s. Consequentntlyly, smalall quantititities of enzyme can rerepeatatedly catatalylyze ththee breakdown of millions of molecules in minutes. Enzymes function optimally in detergents at temperatures of 20 - 60C and within a pH range of pH 7.5 - 10.5. The performance of enzymes in detergents depends on number of factors,s, inincludiding the detetergrgent’t’s compositiition, type of stains to be removed, washsh temperature, washing procedure and wash-water hardness. To help formulators optimize enzymatic detergent washing efficiency, Specialty Enzymes provides wash laboratory technical services. In our wash laboratory, customerr, basee detergents are evaluated on standard soils in both a model wash system (Terg-O- Tometer) and in full-scale household washing machines. TTable 1 Compositions of an enzyme detergent CCoonnssttiittuueenntt CCoommppoossiittiioon((%% )) Sodium tripolyphosphate (water softener, loosens dirt)a 38.0 SSooddiiuumaa llkkaanness uullpphhoonnaatte(( ssuurrffaaccttaanntt)) 2255..00 SSooddiiuum ppeerrbboorraatte tteettrraahhyyddrraatte ((ooxxiiddiissiinng aaggeenntt)) 2255..00 SSooaap(( ssooddiiuumaa llkkaannecc aarrbbooxxyyllaatteess)) 33..00 SSooddiiuumss uullpphhaatte(( ffiilllleerr,ww aatteerss oofftteenneerr)) 22..55 SSooddiiuum ccaarrbbooxxyymmeetthhyyl cceelllluulloosse ((ddiirrtt--ssuussppeennddiinng aaggeenntt)) 11..66 SSooddiiuummm eettaassiilliiccaatte(( bbiinnddeerr,ll oooosseennsdd iirrtt)) 11..00 BBaacciilllluuspp rrootteeaasse(( 33%aa ccttiivvee)) 00..88 FFlluuoorreesscceenntbbrr iigghhtteenneerrss 00..33 FFooaamm--ccoonnttrroolllliinngaa ggeennttss TTrraaccee P a g e || 44

PPeerrffuummee TTrraaccee Waatteerr tto110000%%

Detergent Enzymes

Presesentltlyy, deteterrgent enzyme has become an inintetegrgral part of deteterrgentnt formulation. A look at the market share of detergent enzyme indicates it to be very high in comparison with other enzyme applications. Enzymes that have to be used as detergent composite must possess the following characters:

 Stability at temperature over a broad range of 20C to 50C and even above  The optimum pH should be in alkaline or higher alkaline range  It should be detergent compatible  It should have specificity towards different proteins

Major detergent enzymes include proteaseses, amylases, lilipases, ,, miscellaneous enzymes such as peroxidases and pullulanase. A recent trend is to rereduce this phosphate contntent fofor environmentatal reasosonsns. It may be replaced by sodium carbonate plus extra protease.

Proteases

Proteases were introduced in the market in 1959 in the detergent Bio-40, produced by Schnyder Ltd in Switzerland. Most powder and liquid laundry detergents in the market, today, contain proteases. Proteases are of two types:

 Alkaline protease from Bacillus licheniformis, having optimum pH 8, for egg, liquid laundry product, (pH 7- 8.5), commercially known as Alcalase -Novonordisk Optimase- Genencor Inter . P a g e || 55

 High alkaline protease from Bacillus alkalophilus and Bacillus lentus, having an optimum pH 10. For e.g., powder laundry products, automatic dish washing formulations, known by trade names of Savinase-Novo Nordisk, Purafet- Genencor Interr. Proteases enhance the cleaning of protein-based soils, such as grass and blood by catalyzing the breakdown of the constituent proteins in these soils through hydrolysis of the amide bboonndds bbeettwweeeen ininddiivvididuuaal amiinno aaccididss. IIn ththe casse oof sseeririnnee endopeptidase, it contains a catalytic triad of amino acids at the active site;

•• An aspartyl residue containing ß-COO¯

•• A histidine containing the imidazole group

•• A serine residue with p-OH as the functional group The serine hydroxyl group functions as a potential nucleophile, where as both the aspartyl and histidine functional groups behave as general base catalysts facilitating the hydrolysis process. .

The serine group initiates the nucleophilic attack on the peptide bond to form a tetetrtrahedraral inintetermediaiatete, whicich undergrgoes an actitive hydrdrogen trtransfsferer,, facilitated by both the histidine and aspartyl residues. The net effect of the addition of water across the bond generates the original protein. The protease hydrolysis involves the transfer of electrons between the amino acids at the active site and substrate. For proteases the three-dimensional arrangement of the catalytic triad is required for the enzyme to be active. Disturbances in the confirmation are likely to afaffefect enzyme efefficacacy and thereforore cleaniningg performance.

 These were susceptible to oxygen bleaches and calcium sequestrates. But now, stable protease can be obtained .

 Oxidative attack by peroxides or per acids on the methionine residue adjacent to the catalytic serine results in nearly 90% loss of enzyme activity. However, replacing methionine with oxidatively stable amino acids like alanine improves stability of enzyme towards oxygen bleach (Boguslawski et al, 1992)

 Protease substilisin requires at least one calcium ion, which maintains three- dimensional structure of enzyme. However, calcium- sequestering agents used in many laundry procedures to control water hardness can remove this calcium resulting in the decreased thermal and autolytic P a g e || 66

stabilityy. This can be corrected by the introduction of negatively charged reresidues near the cacalcium-bibinding sisite, which increreases the bindiding affinity of enzyme for calcium and results in improved stability towards calcium sequestrants (Krawczyk et at, 1997)

 Protease has limited applications towards the detergency of wool and Constituent , because of the proteinaceous nature of these fibres.

 Proteases are added in an encapsulated or granulated form, which protects ththem from otother detetergrgent iningredidient and eleliminates ththe prproblem of autotolylysisis or prprototeolylysisis of otother enzymes. In aqueous deteterrgentnt formulations, protease inhibitors show a preventive effect of avoiding contact of the protease molecules with each other as well as other enzyme molecules. This effect gets nullified on dilution and enzyme molecules are free to act on stains (Krawczyk et al, 1997)

Amylases Amylases facilitate the removal of starch-based food soils, by catalyzing the hydrolylysisis of glglycososidic linkages in starcrch polylymers. Generarally, starcrch-h- containinining stainins are of chocolalatete, gravy, spaghetti, cocoa, puddining, etc.. Amylases can be classified as: : a-amylases: These enzymes catalyze the hydrolysis of the amylose fractions of the starch under hydrolysis of the glycosidic bonds in the interior of the starch chaiain. The fifirsrst ststep in ththe rereactition is calllled as endorereactition & leleads toto ololigosasacchararides, wherere shshort chain wateter- sosoluble dextxtririns arare prproduceced.d. . ß-amylases: Thesese enzymes actoton dextxtririns frfrom rereducicing end and foformss maltose units.

Pullulanases or isoamylases: These degrade starch directly into linear dextrins fofor ththey also atattack cici-1-1,6 glycosidic bonds.s. . . Amyloglycosidases: These enzymes act on the dextrin or maltose units and forms glucose units. . a-amylases are mostly used for detergents, although recently other carbohydrate cleaving enzymes such as pullulanases or isoamylases have also been described P a g e || 77 for this application. a-amylases bring about the primary hydrolysis of starch into the oligosaccharides and dextrins. Currently, these enzymes are produced from bacteteriria. Bacicillllus subtbtililisis. Bacicillllus amylylololiqiquefafaciciens, and Bacicillllus licheniformis. These are available under the trade names Maxamyl- Genencor Int or Termamyl -Novo Nordisk Lipases

Tomato-based sauces, butter, edible oils, chocolate and cosmetic stains are very difficult to remove as they form due to greasy food stains. Body soils, sebum and sweat on collars, cuffs and underarms, are generally composed of a mixture of proteins starch pigments and lipids. Lipases hydrolyze the water insoluble ttririgglylycceerriiddees ccomppoonneenntts iinntto tthhe mmoorre wwaatteerr--solluubblle pprroodduuccts aass monoglycerides, diglycerides, free fatty acids and glycerol. The Novo Nordisk launched the first lipase product in 1987. They transferred the lipolase gene into the Asper6yillus oryzae for industrial production, Genencor followed in 1993 with lumafast (Pseudomonas menocina) and Gist-Brocades in 1995 with Lupomax. .

Currently, the known sources of lipases include mammalian lipases (human pancreases/colipases), fungal (Rhizomucor mehei, Humicola lanuginose, etc), yeasast (Candidida rugososa, Candidida antatarticaca), bacteririal lipasase (P(Pseseudomonass glumae, Pseudomonas aeroginosa, Chrobacterium viscosum) (Ishida et al, 1995) . .

Lipases possess a catalytic triad that is similar to the serine proteases of and subtilisin type. Hence, these are also called as serine hydrolysate lysate. Lipases can decompose a fatty stain up to 25%, which then can be removed very easily because of the hydrophilic character (Dorrit et al, 1991). It is generally thought that lipases get adsorbed on to the hydrophobic stain during the washing period. And, during the drying cycle when the water content is decreased, the enzyme is activated and can hydrolyze triglycerides in the stain. This facilitates the removal of stain in the next wash cycle (Dorrit et al 1991). The enzyme also has stability over a broad range of temperature 30C to 60C. These novel alkaline lipases also retained 100% activity in the presence of strong oxidants.

Effofortrts arare on to mmanufufactuture enzymes ththat can work belolow ththe normalal temperature range of 30C to 40C to save energy. It has been observed that energy consumption per wash in household washing machine (3 kg clothes) at lolow tetemmperaratuture (3(30C) is leless ththan 1 % of ththe enerergy used at a hihigher temperature (60C) (Edvardetal, 1991). . P a g e || 88

Cellulases

These enzymes introduced in the late 1980s were described for the first time in a Japanese patent filed by Murata. These enzymes are used in UK and US since 1991. Cellulases remove microfibrils from cotton and cotton-blended fabrics. These microfibrils stick out from the main fibre of cotton and are formed during use and repeated washing condition of the tissue. This makes garments and household textiles unusable. The cellulases can be used as softening agents, to remove soil particles and to revive colours. . .

The overall structure has two types of region; one that has a higher ororder of cryststallininity is cacalled crcrysystalline reregion. The otother tytype has lelessss structured order, and hence is called as amorphous regions. The activating but not hydrolytically acting enzyme was named as C i-activity. According to this concept, microorganisms that are able to degrade crystalline cellulose have C i-i- activity. This enzyme is not present in that microorganism that attack only substituted cellulose like carboxymethyl cellulose as they have Cx-activity.

Accordrding to a rerececent reresesearch, bibiodegrgradation of cecellulolose requiuireres ththee interaction of three different hydrolytes or at least the first two enzymes to attack simultaneously. These include:

·· Cellobiohydrolase is alalso calllled as exocelellululalase (C i-i-actitivivityty)) . . ·· Endoglucanases is aallso ccaalllleed aas EEnnddoceelllluulalasses (C ii-a-accttiivvitityy)) . . ·· ß-Glucosidase iis aallsso ccaalllleed aas cceelllloobbiiasasee . .

It was observed that sebum in the interior of cotton fibres cannot be removed by ordinary detergents satisfactorily, although they readily remove sebum on the exterior of the fibres. Alkaline cellulase interacts selectively with cellulose in interfiber spaces in the interior of fibre, and selectively removes the sebum soil. The removal of the soil is by the hydrolysis of amorphous regions (Murata et al, 1991).

Cellulases can be chemically modified to have greater stability and efficiency in alkaline medium. It can be done by treating the acid cellulases with reagents lliikke mmaalleeiic aannhhyyddrriidde ((BBuunnd aannd SSiinngghhaal eet aall, 22000022)).. P a g e || 99

.

Currently, the cellulases used in detergents are manufactured from bacteria and fungi. Bacterial cellulases have been in use since 1987, for example, Biotex. Somme genetiticalllly engigineerered ststrarainins, wwhihich arare widely used ininclcludee StStreptomyces spsp. KSM-2, Bacicillus KSM-635. The fufungal cecellulase fromm Humicola isolens DSM1800 is active under mild alkaline conditions.

Miscellaneous detergent enzymes

Peroxidases:

These are one of the newest classes of enzymes that have been included in detergent formulations. Peroxidases are subclass of general oxidoreductases and are very popular and commercially available for manufacturing detergents. Novo Nordisk produces this under the brand name Guardzyme obtained from mushroom Corprinus cinereus. It is a heme containing protein, which in the presence of H2O2 can mediate the oxidation of fugitive dyes in solution and inhibits the dye transfer.

Pullulanases:

In recent years, pullulanases (Pullulan 6- glucanohydrolase) a debranching enzyme has been gaining importance due to its efficiency of starch hydrolysis by cleaving a-1, 6 linkages. Pullulanases with other amylolytic enzymes are used in detergents for improved stain removal and enhanced overall cleaning performance. This enzyme was first isolated in Klebsiella pnuemoniae (Shaw et al, 1995). Manufacturing and downstream processing Nearly all-detergent enzymes, which are used and marketed today, are produced through large-scale fermentation of microorganisms. Most of enzymes "are obtained from the bacterial or fungal strains. As low cost enzymes are needed to support the requirements of the global detergents business, enzyme manufacturers should consider the following points to ensure lower costs: P a g e || 10

 The enzyme-producing micro-organism must be capable of secreting the enzyme extra-cellularly in the bulk fermentation broth, as the cost in terms of both money and time to recover enzyme from the fermentation broth is very high  The production organism should be able to produce highest possible yields. Strain optimisation can be accomplished either through classical mutagenesis and screening methods or using genetic engineering · The number of steps in the downstream processing should be kept to a minimum to be economical and also to avoid yield losses  The production organism should produce the desired enzyme in a highly pure state without any contaminating side activities or proteins. This can be done by deleting the genes, which codes for unwanted enzymes and proteins

Enzyme formulations . Enzymes are formulated mainly in two forms, as a liquid product or as a granular product .

1.1. Liquid product formulation . The highly concentrated liquids of the evaporator or the ultra filtration unit can be used for the manufacturing of the liquid formulations. The liquiuids, whicich arare to be inincorporated into ththe formulation, must be sterilized against microbial growth. Stabilizing agents like borax, organic bororic acacid derivatatives, alalkali salts, etetc shoululd be added along withth preservatives like urea, propel glycol, diglycol, and sorbitol. The current trend is to formulate these liquid formulations as structured liquids with the help of salts and polymers, so that all remains in the structured liquid and enzymes remain in the aqueous phase. (Hermann et al, 1997) 2.2. Granular enzyme products . Highly ulultrtra fifiltlterered and didialalysed enzyme solulutition is subjbjecteted toto adjustment of pH, turbulences, and temperature in the suitable range, when the enzyme crystallises out. It can be precipitated at high salt concentrationsns. The follolowing four tytypes of granulalation prprocess araree employed:

i.i. Enzyme pulling: : The enzyme (dry) is dispersed into a molten wax, non-ionic surfactant, or polymer matrix, and then sprayed in a cooling tower to form solid, spherical, molten water-soluble or water dispersible material with a