Detergent Enzymes: Recent Developments and Outlook on Entering the Twenty-First Century

Kalle Axelsen, Anders Pedersen, Keith Gibson, and Torben V. Borchert Novo Nordisk A/S, Bagsvaerd, Denmark

Introduction
This presentation deals with two important trends in the field of detergent enzymes. The first is the increased use of advanced genetic engineering for the development of new and improved enzymes, and the second is the use of enzymes for their general cleaning effect. The first industrial enzyme produced by a genetically modified microorganism, a GMO, was brought to the market nearly 15 years ago. The enzyme in question was an amylase for production of maltose syrups. By genetic engineering, the gene coding for this enzyme had been transferred from one Bacillus species to another, making it possible to produce the enzyme in high yields at commercially viable costs. Using genetic engineering in this manner to transfer genes from one species to another or to multiply genes within the same species has become the industry standard. Today, practically all detergent enzymes are based on GMO. In recent years, more advanced genetic engineering has also been applied in order to change and improve the actual enzyme molecules. This is called protein engineering and comprises everything from substituting a single amino acid by another to a complex combination of fragments of different enzyme molecules. The purpose of applying protein engineering is to improve specific performance characteristics of the enzyme. Based on value, about one-third of all detergent enzymes used today have been modified by protein engineering.

The market for detergent enzymes has grown, and is still growing, at a higher rate than the market for detergents as such (Fig. 1) (1). One of the main reasons is the increased use of multiple enzymes. More and more detergents contain, in addition to protease, amylase, and to an increasing degree, also cellulase or lipase. Apart from the ability of these detergents to remove a broader spectrum of specific stains, there is an increasing awareness of the important contribution of enzymes to general fabric care, i.e., in keeping the textiles nice and clean during their lifetime.

Advanced Genetic Engineering

Classical Protein Engineering From an enzymatic point, fabric washing is performed under conditions which are rather severe, or at least suboptimal, in terms of substrate concentration. pH, temperature, and presence of other compounds. Classical protein engineering involves the substitution of specific amino acids in the enzyme molecule. Until now this technique has been used mainly to overcome stability and compatibility problems. The development of an amylase for bleach-containing detergents will serve as an example. The most widely used amylase for detergents as well as other purposes is the heat-stable alpha-amylase from Bacillus licheniformis. This enzyme is quite robust in terms of

Fig. 1. World market for detergent enzymes. Annual value in million USD.

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temperature and pH range, but it is rapidly inactivated under oxidative stress which limits its use in a number of detergent formulations. The most oxidation-labile amino acids in proteins are cysteine and methionine, both of which contain sulfur. The B. licheniformis amylase contains no cysteines but seven methionines. It was therefore of interest to see what happened when each of these was substituted by another amino acid like leucine (2). Figure 2 shows the activity decay of the native enzyme and the leucine-substituted variants at pH 9 and 200 mM hydrogen peroxide. As can be seen, only one of the substitutions had a significant effect on the oxidation stability. The next step was then to find out which particular amino acid was the best replacement. It turned out that those with small side chains gave the best preservation of the specific activity, actually almost the same as the native enzyme. This work and a number of similar studies also addressing other properties finally resulted in a new enzyme with quite a large number of substitutions compared to the native enzyme. The new enzyme was launched in the market in 1995 and has become widely used since then. Directed Molecular Evolution As just described, classical protein engineering can be a powerful tool in addressing specific performance characteristics like oxidation stability, performance at specific temperatures, etc. It does, however, require a substantial knowledge of the biochemical/biophysical properties of the enzyme in question and also the availability of a high-resolution three-dimensional structure of the enzyme. Furthermore, the mechanisms behind certain properties of enzymes, particularly those related to activity and absolute performance, are still very complex, and attempts to improve on these properties by classical protein engineering show only modest success rates. For a long time, the ability to mimic Natures evolution process has been a desideratum. In Nature, random alterations happen, and the fitness of the individual determines to what extent the individuals genetic material is passed on to future generations. This is a constantly ongoing process.

In relation to enzymes, the first attempt to simulate this process in the laboratory includes the construction of a library of variant enzymes containing random alterations in their protein structure. The library is subjected to a relevant screening and selection procedure in order to identify the best variant. This variant is isolated and used as the starting point for the next library of random variants, and this process is continued until the desired result is obtained. Nature also utilizes another process, recombination, in order to speed up the development. This can also be mimicked in the laboratory, andactually much faster and more efficiently than in Nature. The principle is that not only the best candidate in the variant library is used, but rather all improved candidates are randomly recombined in a second library in order to identify the optimal combinations of beneficial mutations. This process is also carried out for as many times as needed to reach the desired result. The total process of random mutagenesis, recombination, and screening carried out in as many rounds as needed is called Directed Molecular Evolution (Fig. 3). Various formats for this approach are now available including in vitro DNA shuffling (3) as well as in vivo systems developed at Novo Nordisk. The most recent trend in Directed Molecular Evolution is to generate the first library by random recombination of homologous genes, all encoding proteins with the same function. This family shuffling approach thus expands diversity from starting material that has already been optimized in Nature (4). The first examples indicate that this technology will be superior for many enzyme development projects. Such systems were used to develop a new high-performance lipase that is currently being introduced into the market. As seen in Figure 4, the change in performance is significant. Compared to the parent enzyme, the new lipase shows a much more significant effect, also during the first wash. Another recent example of the application of advanced protein engineering is the field of proteases. The target for this work was to develop a protease with superior performance under typical Japanese washing condition, i.e., very low temperature and very soft water.

Fig. 2. Oxidation stability of Bacillus licheniformis alpha-amylase and leucine-substituted variants.

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Figure 5 shows the performance of the resulting enzyme compared to the standard commercial enzyme (Savinase, Novo Nordisk Als, Bagsvaerd, Denmark) from which it was derived. The improvement, on enzyme protein basis, is a factor 3 or higher. This new enzyme, was introduced into the Japanese market in late 1997 and was, for obvious reasons, very well received. Directed Molecular Evolution is indeed a strong tool for improvement of very basic enzyme characteristics. But it is also a new tool and its full potential is still to be realized.

Cleaning Effect of Enzymes Fabric-Care Considerations. One aspect of detergent enzymes receiving increased attention is their contribution to fabric carethe goal being to maintain the attractive, new appearance of textiles even after multiple wash-wear cycles. Focusing on cotton, enzymatic fabric-care benefits are obtained in two areas: (i) reduced microfibrils and fuzz, resulting in improved surface properties and visual color and (ii) Reduced residual soil, resulting in improved whiteness and prevention of dingy appearance.

Fig. 3. Simplified illustration of Directed Molecular Evolution.

Fig. 4. Performance of native and variant lipase. European detergent compact powder detergent, 3.3 g/L. Stain: lard/sudan red on cotton. Launder-o-meter, 20 steel balls, 1530C: 8 min., 30C: 52 min, pH 9.9, 18dH water hardness.

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Fig. 5. Performance of native and full-scale Japanese laundry machine, 15C, 3dH water hardness.

The first of these two benefit areas has been described in detail (5,6). Color clarification and fuzz prevention are convincingly achieved by use of cellulases, such as the commercially available EG V component of the Humicola insolens complex. The second fabric-care benefit arearelated t o reduced trace residues of soilcan involve several different classes of enzymes, including enzymes that were originally developed for their stain-removal effect. In this application, the concern is no longer solely the ability of enzymes to speed up the initial degradation of high concentrations of their substrate, such as might be found in localized spots and concentrated stains. Rather, the concern is with their ability to do the job completely and achieve removal of the last traces of soil. This change of focusfrom stain removal to removal of the last traces of soilcalls for some underlying changes in the detergency mechanisms involved. In particular there will be more emphasis placed on: removal of soil bound directly onto the fiber surface and redeposition effects. Because of these mechanistic considerations, the shift of emphasis is not trivial, and there is no reason to expect that the enzyme performance profiles are necessarily the same as those found with heavier levels of soiling. Before illustrating these ideas by reference to effects of selected enzymes, some generalizations can be presented summarizing important leads from the evaluation of trace soil removal from cotton fabrics: (i) Particulate soils are important. Visible effects are obtained with extremely low levels of some particulate soils. Relevant examples are carbon particles and clay particles, both of which are widely distributed in practice, which can have intense color and are nonbleachable. (ii) Particulate soil binding is influenced by fiber surface properties and by the presence of nonvisibie contaminations on the fibers. (iii) Redeposition effects arc important. Redeposition of soil during washing and soiling during wear must be considered as equally relevant. (iv) Multiple soil-wash cycles can lead to a dingy appearance on white cot-

ton. Such tests can be used to study improved whiteness and dinge prevention benefits of various enzymes. Amylases and Removal of Starch Traces. A-mylases have been used in household detergents for many years. Their contribution to the removal of starchy food stains can be easily demonstrated by use of test fabrics that are stained with colored food products. Useful examples are sauces, curry, chocolate desserts, and other similar food formulations that contain gelatinized starch in order to control the rheoiogical properties. Starch has a strong affinity for cellulosic fibers. Starch stains on cotton fabrics may, therefore, be considered in two partsthe bulk of the starchy material, and the fraction of the starchy material that is directly bound as a film on the fiber surface. Removal of the bulk of a starch-based food stain can leave behind a residual starch film contamination. Trace contamination of cotton fibers by starch can also result from redeposition. Starch redeposition occurs readily under typical household washing conditions. Thus, inclusion of starch-soiled items in a washload can result in starch contamination of other items in the load. Starch films are applied deliberately as part of the size used during preparation of cotton yarns for woven fabrics. Although most of this starch is removed again during the fabric finishing processes, the removal is not always complete and starch can frequently be detected as a contaminant on new cotton textiles. A low level of starch contamination will not in itself give a significant color or any other visual change to the fabric appearance. But even trace starch contamination can have a major visual impact by increasing the binding of particulate soils. Figure 6 shows remission measurements on bleached cotton fabric onto which known concentrations of gelatinized starch have been applied. The starch itself has no detectable effect (top line) even at the highest concentrations. But when these cotton samples are soiled, the soil-binding effect of the starch becomes very obvious. In these examples, the soil is

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Fig. 6. Remission values for cotton with applied starch, before and after particulate soiling.

either carbon particles (bottom curve) or clay taken from a tennis court (middle curve). Even at the lowest starch concentrations of only 20 mg per kg of cotton, there is a significant and visible effect. Since residual starch traces will often be distributed quite evenly on laundry itemsas a consequence of the deposition mechanisms that contribute to the presence of the starchthe result of particulate soil binding is often seen as a relatively even, overall discoloration of the fabric. Model examples with particulate carbon soil are greyish. Examples with red clay as the predominant soil are yellow-red. Avoidance of nonbleachable dingy soiling has received considerable attention in recent years, and this focus is expected to continue. Progress depends on understanding the soiling and soil-binding mechanisms involved. In this case the mechanism is clearstarch film formation with associated soil binding. An important property of a starch film on cotton is its resistance to detergent washing. Under typical household conditions starch filmsand the particulate soil they bind persist even after multiple wash cycles. With the inclusion of amylases, this picture changes. Amylases efficiently increase starch-film removal by household detergents. Amylases also effectively avoid the risk of new starch-film formation during washing, where starchcontaminated items are included in the load. In cases where particulate soils have been included, the elimination of residual starch traces results in a visible reduction in the dingy discoloration. Presentation of practical results which demonstrate more complete removal of trace soil is always difficult because the differenceswhile visually importantare obviously relatively small and not well suited to slides and photographs. In Figure 7 an attempt is made to show socks that were spotted with a colorless starch solution and then washed four times using Asian household machine wash conditions, in detergent without or with amylase. Between washing with detergent, these items were soiled with a suspension of tennis court clay. The starch spot, which was no

longer visible already after the first detergent wash, is clearly still present even after the four wash cycles. The inclusion of amylase has had a marked effect on reducing the binding of the clay soil by providing a more complete starch removal. Thus, the amylase has provided a whiter wash result. Cellulases and Soil Reduction Effects Fabric-care cellulases were mentioned earlier in connection with color maintenance and fuzz prevention. Mechanistically, these benefits are explained in terms of a selective hydrolysis of amorphous cellulose which provides a sufficient weakening of the microfibril attachment to result in microfibril removal (5,6). This in turn results in reduced development of visible fuzz and pilling and an overall improvement of the visual color impression after multiple wash cycles. Cellulases can also contribute to soil removal from cotton fabrics. On heavily soiled test fabrics, the same cellulases that give color-care benefits also contribute to soil removal. These two effects may be similar mechanistically, with the soil-release effect also being related to removal of soiled microfibrils. A different type of soil reduction benefit by cellulase can be observed if the evaluation is conducted under trace soil conditions. Again the target fabric can be white cotton which has been exposed to a low level of particulate soiling. Conditions are chosen such that after a series of wash cycles, an accumulation of residual soil can be observed and the white cotton takes on a gray, dingy appearance. Under suitable conditions, cellulases give a clear reduction in the accumulation of dingy soil on cotton. Figure 8 illustrates this anti-graying effect. The results are taken from a full-scale multi-cycle wash trial under European 40C conditions using a commercial European bleach-containing detergent. The washload was predominantly white cotton, and particulate soil was introduced by including a small quantity of carbon-soiled test fabric in each wash. This soiling leads to a clear graying effect on the white washload seen here by comparison with the reference without the soil

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Fig. 7. Socks, spotted with 50 mg starch, washed four times, soiled with clay.

addition (left and center columns)and graying increases with the number of wash cycles. Inclusion of a cellulase (right columns) decreases graying. In this case the reduction is approximately 50%. Not all cellulases are equally effective. A particularly useful cellulase in this application is the EG I component of the H. insolens complex. This is in clear contrast to the performance ranking when judged as color-care cellulases, in which case the EG V component of the same complex gives a far greater benefit. Soil-reduction effects do depend on washing conditions and detergent. The most useful results are typically obtained under European washing conditionsan observation that implies that anti-redeposition mechanisms are important. Alternative

interpretations (7) invoke an action of the cellulase on hydrated amorphous cellulose in or on the cotton fibers, reducing the ability to bind strongly colored particulate soils and lipids.

Conclusions and Future Outlook

Genetic engineering is a well-established technology today, certainly in the production of enzymes and, to an increasing degree, also in the development of new and improved enzymes. The technology is subject to constant development. This presentation took a glance at the most recent progress: the Directed Molecular Evolution. To be able, in a high-speed mode, to mimic Natures process of evolution opens a world of opportunities. Where

Fig. 8. Average remission values of 14 cotton garments and test fabrics, from a full-scale multi-cycle 40C wash trial, using a European powder detergent with bleach, at 5 g/L.

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the limits are, is yet unknown. But in relation to enzymes, it would be a fairly conservative prediction to say that in 5 years, the technology will have been applied to all the major enzyme classes in use in the detergent industry. As a result, proteases, amylases, cellulases, and lipases will become available with significantly improved performance compared not only to the native enzymes, but also compared to todays protein engineered enzymes. The trend of using enzymes for their fabric-care effects is predicted to continuenot to the extent that all detergents will include enzymes beyond proteases, but perhaps to the extent that all premium brands will include enzymes for fabric care, be it for preservation of clear color or for prevention of dinge. The trend will be supported by the development of improved color-care enzymes and special anti-dinge enzymes and, in the longer term, also by changes in washing conditions. In Europe, a reduction of wash-water volumes has been implemented successfully. It can be expected that this reduction eventually will also take place globally. When it does, adequate control of residual soil and soil redeposition becomes a more severe hurdle. And enzymes appear to be a key to deal effectively with this hurdle.

From an enzyme manufacturers point of view, the future outlook is bright. Some important basic technologies are in place and at work producing a steady stream of significantly improved single enzymes. The detergent manufacturers respond positively to these developments, and they also pay increased attention to the enzymes contribution to general fabric care. All in all, this translates into the expectation for a continued growth of the detergent enzyme market for the next 510 years.

References
1. Novo Nordisk, Annual Reports (19931997). 2. Borchert, T.V., Lassen, S.F., Svendsen, A., and Frantzen, H.B, Progress in Biotechnology 10, Carbohydrate Bioengineering, Elsevier, 1995, pp. 175179. 3. Stemmer, W.P.C., Nature 370:389391 (1994). 4. Crameri A., Raillard, S.A., Bermudez, E., and Stemmer, W.P.C., Ibid. 391:288291 (1998). 5. Jakobsen, T.S., Lindegaard, P., and Chan, M., INFORM 9:788 792 (1998). 6. Sejr Olsen, H., and Falholt, P., J. Surf. Deterg. 1:555567 (1998). 7. Murata, M., Hoshino, E., Yokosuka, M., and Suzuki, A., J. Am. Oil Chem. Soc. 68:553558 (1991).

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