7

DETERGENT ENZYMES

Enzymes help detergent formula-tors in their quest for environ-mentally acceptable cleaning at

low concentrations and temperatures.Yet hurdles remain for the currentcrowd of enzymes to become morefunctional. In fact, new research andmarket development may lead the wayfor enzymes nor only to solidify theirposition in laundry detergents but alsoto expand into other fields.

Enzymes are large, complex pro-tein molecules consisting of inter-twined chains of amino acids heldtogether by peptide bonds. Lurking inthe cells of all living creatures, includ-ing plants, fungi, bacteria, and single-cell microorganisms. enzymes arehighly emden! catalysts that increasethe rare of biochemical processeswhich otherwise would proceed slow-ly or nOI at all.

Detergent enzymes are a subset ofthe industrial enzyme market, whichincludes such applications as textiles,leather, and pulp and paper uses. In a1995 study, the Freedonia Group esti-mated 1994 U.S. demand for allenzyme classes to total $665 million,increasing 12% annually to reach $1.3billion in 2000 (Table I).

The international market for indus-trial enzymes was estimated to be $1.2billion in 1995, expanding 10% annu-ally. The detergent enzyme market isworth approximately $125~150 mil-lion in the United States and $500million globally. This market is splitevenly between two companies: NovoNordisk NS of Denmark and Genen-cor International Inc. of Rochester,New York (see "Small molecules, bigcompanies" artiele on page 12).

A brief historyGerman chemist OUo Rohm devel-oped the first method for washing pm-tein-stained cloth in detergents con-taining enzymes, according to NovoNordisk. Robm's 1913 patentdescribed the use of pancreatin (frompancreatic glands), which contains theenzyme trypsin. However, trypsin'sprotein-breaking effectiveness washampered by the alkaline-dissolveddetergent, and Rohm's product neverbecame a great success.

Use of enzymes in commercialdetergents did not take off until the

[ate 1960s, when a protease wasdeveloped that was stable under alka-line conditions, and new techniquesallowed production of enzymes inlarge quantities. Within six years oftheir introduction into detergents, 80%of all laundry detergent products con-tained enzymes.

Proteases, which break down pro-tein stains. were used extensively in

many detergent enzymes, all of whichhail from the hydrolase class ofenzymes. Next came amylases tobreak up starchy stains ("amylum" isLatin for starch), lipases for lipids,and finally cellulases to act on cellu-losic fibers of fabric.

"Amylases were heavily used in the1980s and then fell out of favorbecause of competing chemical alter-

Enzymes are large, complex protein moleculesconsisting of intertwined chains

of amino acids held together by peptide bonds.They lurk in the cells of all living creatures,

including plants, fungi, bacteria,and single-cell microorganisms.

the late 1960s and early 1970s, butthen safety problems became evident.The dust-generating methods of mak-ing powdered detergents at the time.along with powdery enzyme materials,allowed airborne particulate emissionsto cause allergic reactions in workersat production facilities.

"Detergent manufacturing practicehas improved significantly comparedto 20 years ago, and enzymes arc nowproduced as dust-free granules:' saidBob Petrowski, vice president of theindustrial applications group forNovo's U.S. affiliate. Novo NordiskBiochem North America.

Enzyme and detergent dusts werereduced through the development oflow-dust encapsulated enzymes,improvements in manufacturing tech-niques, and improvements in industri-al hygiene practices and procedures.Adding enzymes to detergents todayis widely considered as safe as addingany other ingredient.

All in the family"Proteases are the workhorse of theindustry," Petrowski said. "They havebeen around for a long time and arewidely accepted,"

But proteases were just the first of

natives," Petrowski said. "Now deter-gent manufacturers are more interest-ed as new amylases are introducedwith improved benefits. We wouldexpect their use to grow again in theremainder of this decade:'

While the world market for amy-lases has been slowly growing, thisenzyme has been used in greater quan-tities in Latin America for years. NovoNordisk explains that the local dietthere is high in starch, and the cold-water wash used for laundry makes itmore difficult to remove these stains.There are even some laundry deter-gents that contain amylases but notproteases. Novo Nordisk said.

Detergent lipases, on the otherhand, help remove fatty food stainsconsisting mostly of hydrophobictriglycerides. Liposes decomposethese substances into morehydrophilic compounds that are moreeasily removed by detergent action.

Ironically, lipases are more activein the drying process than in the washwater itself. The breakdown of fattakes place mostly at the reducedmoisture levels thai occur during dry-ing. After the first washing and dry-

[continued on pa8~ /0)

INFORM, Vol. 8. no. I (January 199n

10

enzyme effectiveness. "Generally,those detergents that contain two ormore enzymes are limited to the pre-mium brands," Petrowski said. "Otherproducts may contain only protease toreduce formulation costs while meet-ing certain performance require-ments,"

DETERGENT ENZYMES

[continued from page 7)

ing, the fatty stains are nor significant-ly reduced, but the triglycerides havebeen partially hydrolyzed and aremore easily removed during the nextwashing.

Novo Nordisk launched the firstcellulase for the detergent industry in1987. Cellulases work on cellulosicfabrics such as cotton and blends byremoving microfibrils and small pillson the surface of the garment. Pills arethe balls of fuzz that often appear oncotton garments or blends of cottonand other fibers.

According to Novo Nordisk, cellu-lases can have several beneficialeffects. Repeated washings with cellu-lases can restore the color of cottonmaterial significantly by removing thefuzzy. greying microfibrils. CeUulasesalso help to remove din by removingthe microfibrils that attract dirt, andcan also provide a fabric softeningeffect.

What is the next enzyme in line fordetergent formulations? Research inoxidases. from the oxidoreductaseclass of enzymes, may show the way.Oxidases could replace the bleachactivator and lower the amount ofperborate needed for low-temperaturebleaching.

"An oxidase enzyme would enablebleaching by iLScatalytic activity or incombination with oxygen and novelorganic molecules," Petrowski said.

Table 1U.S. detergent enzyme market (In million dollars)

Item 1994 2000Total enzyme demand 665 1285Percentage detergent enzyme 18.8% 18.7%Detergent enzyme demand 125 240a) By fonnula:

powder detergents 80 140liquid detergents 45 100

b) 8y application:laundry detergents 105 185dishwashing and other uses 20 55

Household detergent shipments 6380 7600Enzyme cost (percentage) 2.0% 3.2%

Source: 'The Freedonia Groop. Sn;dy No. 72~.lnJustria/ and SfN'cia/ly f)t:ymts. 1995.

"We hope to find commercial applica-tions for this class of enzymes indetergent products."

The usage of enzymes commercial-ized to date varies according to type.While 65% of all detergents producedin the United States contain proteases,only 20% contain amylases. and ascant 10% contain lipases, accordingto Petrowski.

"Currently, only one major deter-gent producer is formulating cellu-lases into major brands in the UnitedStates," Petrowski said. "But wewould expect the demand for deter-gent cellulases to increase given thecurrent high interest in the industry."

The differences in enzyme usagerelate more to product pricing than

Why use enzymes?Enzyme usage is not automatic intoday's detergents, Petrowski warns.Competing technologies, such as otheractive agents, would supplantenzymes if the benefits did not war-rant the costs. Keeping the costlbene-fit rario low is a key goal in develop-ing new enzymes for the market.

[continued on /}Uge 12)

Mass-producing enzymesMost enzymes are mass-produced by the same process, but from different raw materials. Producing protease. forexample. requires carbon and nitrogen nutrients, which are found in com and soybeans. respectively. For that rea-son, protease is often manufactured at sites close to agricultural centers.

Genencor's plant at Cedar Rapids, Iowa, uses processed com. in the form of glucose. and processed soy, suchas soy flour and meal, as raw materials. These are mixed with vitamins and minerals to fonn the nutrient broth.

The manufacturing process begins in a flask that contains the desired organism. Nutrients are added to theflask to make the organisms grow rapidly, The flask is emptied into a small tank. where more nutrients are added,and finally into a larger fermentation reactor for full-scale production.

Computers control parameters such as temperature. pH. air flow. and mixing rates in the aerobic and exother-mic fermentation process.

The recovery process involves extracting the product from the broth with separation and fittration equipment.Recovered enzymes are then shipped to the customer in tank trucks or a range of smaller packages.

The total time between injecting microbes into the first flask and the beginning of enzyme recovery is only afew days. It takes less than a day to fully recover the enzymes from the fermentation reactor.

INFORM. Vol. 8. M. 1 (January 1997)

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DETERGENT ENZYMES

Small molecules, big companiesThe smoke has cleared from the battlefield of the detergent enzyme industry and two combatants have survived.primarily by consuming the competition. Based in Europe and the United States. the rivals are now concentratingon recuperating and consolidating their gains in the global enzyme game.

Fanned in 1990, Genencor lntemational lnc. is a joint venture of Eastman Chemical Co. of Kingsport. Ten-nessee. and Cultor Ltd. of Helsinki, Finland. In 1995, Genencor acquired the detergent, starch processing, tex-tiles. and pulp and paper enzyme businesses from Gist-brocades BSn. Genencor bought the industrial enzymebusiness of Solvay SA in 1996. The company has also completed a $66 million research facility in Palo Alto,California. complete with a pilot-scale manufacturing plant.

Novo Nordisk AlS, meanwhile, has invested $120 million in expanding its Franklinton, North Carolina, facili-ty to become the largest enzyme plant in North America. The company also is building a $150 million enzymefacility in Tianjin (90 miles east of Beijing), China, to be completed by 2000. In 1996, Novo Nordisk acquiredthe industrial enzymes technology and patents of Showa Denko. the third and smallest detergent enzymes pro-ducer behind Novo and Genencor.

"The detergent enzyme industry has gone through a period of fierce competition, with a resultant consolida-tion of companies," according to Bob Petrowski, vice president of the industrial applications group for Novo'sU.S. affiliate, Novo Nordisk Biochem North America. ''That has dropped pricing, on proreases in particular."

According to Petrowski. both companies have survived by developing technologies to deliver new value-addedproducts. Now that the market has consolidated, both rivals hope to build on this.

"Commodity products must return to price levels that ensure profitability and help sustain our ability to fundcontinued research and development of new products:' Petrowski said. "Ultimately, we 'must give the customersthe best value for their money, and that will come from the discoveries made through continued R&D."

Still. about half of total enzyme sales are to only two detergent companies: Procter & Gamble Co. andUnilever NV. If one of these decided to produce its own enzymes, then Novo Nordisk and Genencor might facewhat occurred to starch-conversion enzymes, which became commodity products after the entry of key comrefiners to the market.

tcominued fmm page 10) "Wash temperatures all over theworld have been decreasing," saidSeren Hansen, vice president ofenzyme sales for Novo Nordisk AlS."When you wash at low temperatures.it helps to add enzymes, as surfacrantshave trouble removing alt stains atlower temps."

Enzymes function wetl between30-70·C, but lose some activity in the10-20'C range more common in Asia.where cold-water washes are used.

"Proteases work best around55-60'C, but still have very positivebeneficial effects down to the range of20·C;' Hansen said. "At temperaturesbelow this, enzymes in general do notwork at their optimum. Part of ourongoing research is to provideenhanced activity at these low temper-atures."

detergent ingredients and cannot reactwith each other. Once diluted in thewash water, interaction of detergentingredients becomes less of a prob-lem.

The aqueous environment of liquiddetergents, however, challengesenzymes from the start. Whcn theingredients are mixed and packaged,proreases break down proteins, includ-ing proteases and other enzymes. in acannibalistic attack called autodiges-tion. Besides proteolytic degradation,the chemical environment in liquiddetergents also can damage enzymesduring storage.

This would not be such a problemexcept that liquids comprise roughly40% of the detergent market.Enzymes must be useful in liquid for-mulations to fully realize their marketpotential.

Boric acid or polyols such as glyc-erine have been used to inhibit pro-tease activity and reduce autodiges-tion. Once in the wash water. boricacid becomes diluted and enzymeactivity increases. Formulating with

In its 1995 report on industrialenzymes, The Freedonia Group saidthat growth in the detergent market isbased on three main factors. First,detergent manufacturers arc eager toaddress environmental issues. Second,enzymes are being developed to with-stand harsh conditions in detergentformulations. Third, new products areexpanding enzyme applications in thedetergent market. where several prod-ucts are almost considered commodi-ties now.

Enzymes are widely used becausethey are multifunctional, providingstain removal, anti redeposition, white-ness retention. color maintenance, andfabric softening. Space efficiency isalso a main selling point-enzymesare typically used at less than I% byweight in detergent.

In addition, starch and fat stains areeasier 10 remove in hot water. Aswash-water temperatures drop.enzymes that aid in their removalbecome more important.

Liquid detergentsLiquid detergents present uniqueproblems for enzymes.

In powder detergents, enzymes arestable in the absence of water. Granu-lar enzymes are not degraded by other

INFORM. Vol. 8. no. 1 (January 1997)

13

nnsm about the use of enzymes in liq-uids. Frecdonia predicted that enzymeusc in liquid detergents would more(hun double from 1994 to 2000.

amphoteric surfuctants rather thananionics can help, as does loweringthe pH to a range of 7-9. Theseoptions, however. limit the detergentformulator's options.

"For liquid detergents. it is a bal-ancing act to find the right stabiliza-tion agents." Petrowski said. "Thereare a myriad of ways 10 do this. butwe strive to be better. to use new tech-nology to deliver enhanced stability.We are working on novel ways to pro-teet enzymes with the goal of provid-ing 100% storage stability in liquiddetergents,"

Other research efforts focus on thestability of enzymes in liquid deter-gents thai contain bleaching agents.such as hydrogen peroxide. As thepopularity of bleach in detergentgrows. so does the attack on enzymeadditives. According to Petrowski.there are currently no liquid heavy.duty detergents that contain both

bleach and enzymes."This is very difficult, as enzymes

are easily oxidized by these agents,"Petrowski said. "We are now workingto modify protein structures toimprove enzyme compatibility withbleaches."

According to Hansen, one solutionis to use a nonaqueous base for theliquid detergent. Another is to usestructured liquids that contain morethan one phase of liquid.

"You could have an enzyme in onephase, and another enzyme or agent inanother phase of the liquid:' Hansensaid.

Another way to tackle these prob-lems is by encapsulating the enzymeprotein to physically separate it fromtile liquid detergent. The enzymewould then remain protected in thedetergent, but go to work once dilutedin the wash water.

Regardless, there is increasing opti-

Dlshweshmg optionsThe use of enzymes in automatic dish-washing detergent (ADD) is gainingground. Several industry trends areleading formulators to consider enzy-matic ADDs: lower wash tempera-tures, shorter wash cycles, less waterconsumption, and lower pH.

Perhaps more important. though.are the pressures to change key ADDingredients. Metasilicates, importantas corrosion inhibitors, also providehigh alkalinity, and can lead to ADDswith a pH value above II. As rnetasil-icates give way to disilicates, the highalkalinity that hampered enzyme per-fonnance is dropping.

Although phosphates were left inADDs long after being stripped from

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INFORM. Vol. 8, no. I (Januory 1997)

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DETERGENT ENZYMES

Still, dishwashers can be found inless than 5% of domestic householdsin Japan. Automatic dishwashers arcused in 57% of all U.S. households,but in only half that level in Europe.

If the United States has the largestADD market, then why are enzymesnor as widely used as in other, smallermarkets overseas? According to NovoNordisk, several "biological" ADDsthat included enzymes but not bleachwere introduced in the 19705. butfared so poorly they were discontin-ued. Detergent companies havebecome wary of turning from provenformulations, even those with chlo-rine, unless there is good reason.

Still, Freedonia claimed that deter-gents will remain the largest marketfor enzymes in the United States,accounting for about 19% of totalenzyme demand and over 25% ofindustrial enzyme sales (Figure I).Growth will be spurred by the contin-ued penetration of lipases and cellu-lases into laundry detergents as wellas the introduction of enzymes intoother cleaners, such as ADDs, draincleaners, and industrial and institu-tional cleaners.

"The industry is undergoing enor-mous transformation," said PeterPlanck, senior applications chemist atGenencor. •

laundry detergents. they are slowlybeing phased out as other viable alter-natives are proven. A common substi-tute for phosphate in laundry deter-gents is zeolite, but it tends to build upon surfaces when used in ADDs.Curates, polyacrylates, and otherpolycarboxylates can be used, butwith decreased performance. Enzymeshelp to bring performance back up tothe level of phosphate ADDs.

Lynn Tierney of Genencor present-ed data at the 1996 AOCS annualmeeting in Indianapolis describing anenzymatic ADD that removed up to60% more egg soil and 10% more ricesoil than conventional ADDs did. Theenzymes also reduced filming andspotting significantly.

Chlorine-releasing bleach com-pounds, such as hypochlorite, havealways been deemed essential toremove tough stains on dishes. Butchlorine will degrade enzymes even atvery low concentrations. Use of oxy-gen-releasing alternatives such asperborure and percarbonate hasoffered the hope of storage stabilityand compatibility with enzymes.

"Enzyme-based ADDs are widelyused in Europe, where chlorine use isbeing minimized," Petrowski said. "Inthe United States, none of the majorretail manufacturers currently has a

8evenlge,..Rgurel. U.S.enzyme dttrnIindby market (1994 cUlt.)

national brand of this type. We wouldhope that the European experiencesare transferred over to create similaropportunities for enzymatic ADDs inthe U.S. market:'

Enzymatic ADDs were launched inseveral European countries in 1990.but Japan has used enzymes in ADDssince 1986, when dishwashers werefirst introduced. The delicate chinaand wooden utensils used in Japancould not have withstood the harshformulations traditionally found inEurope and the United States.

Engineering takes center stageGenetic engineering is an important key in developing enzymes.

"Genetic engineering is taking a gene coding for the production of a specific protein from one organism andtransferring it to a production organism," according to Bob Petrowski of Novo Nordisk Biochem North America."There are no changes in the protein produced; production efficiency is the issue."

In 1988, Novo Nordisk released Lipolase, the first detergent enzyme to be produced through genetic engineer-ing. The product also was the first commercial detergent lipase.

In a 1995 study, The Freedonia Group said the use of genetic engineering had revitalized the enzyme indus-try. promoting gains even in mature markets such as detergents and food processing. Extracting enzymes fromplants and animals is expensive and does not yield the volumes needed for industrial applications, the consult-ing group remarked. Genetic engineering allows enzyme manufacturers to produce large quantities of almostany enzyme.

According to Petrowski, protein engineering is different from genetic engineering. With protein engineering.the characteristics of a protein molecule are changed so that the ideal enzyme for a specific purpose is created.Protein engineering techniques allow researchers to take a single amino acid out of a complex protein moleculeand substitute another so that the protein produced will be structurally and functionally altered.

The process is known as site-directed mutagenesis. Production is not the issue. but rather a particular trait.such as enhanced bleach stability or low-temperature performance.

"It is now a common tool used in producing new enzymes," Petrowski said.

INFORM. VOl. 8. no. 1 (January 199n