JEANNETTE BJERRE*, OLE SIMONSEN, JESPER VIND *Corresponding author Novozymes A/S Krogshoejvej 36, 2880 Bagsvaerd, Denmark

Jeannette Bjerre

Detergent enzymes – from discovery to product The power of nature harvested in eco-friendly catalysts for laundry applications

KEYWORDS: Detergent, Enzymes, Laundry, Expression, Screening, Formulation

Enzymes represent nature’s work of brilliance, affording efficient catalysis of reactions and acceleration of Abstractbiochemical processes, selectively and at a remarkable speed. The detergent industry is an important application area where are utilized to boost cleaning performance. Enzymes represent environmentally sound alternatives to the use of toxic chemicals and pollutants, with reduced generation of waste and added performance benefits as the result. The discovery, application screening, optimization, and production of industrial enzymes for detergents are presented in this article, including examples of how formulation technology and protein engineering can enhance liquid stability. are used as the main example as this was the first class to find wide-spread use in laundering, and proteases remain hallmark enzymes in household care today.

INTRODUCTION in cold water. Added performance qualities by enzymes, such as fabric care and maintenance of whiteness or Detergent enzymes have been used for a century in colour clarity by , are also desirable traits. In laundering, and continue to be of great benefit in automatic dishwash (ADW), proteases and household care today. In 1913 the first words in the story boost the cleaning of tableware (3), and enzymes are also of laundry proteases were written, when pancreatic applied in the industrial and institutional cleaning sector. was introduced as an additive in the European The workflow from discovery to product for industrial pre-soak detergent product Burnus by Röhm and Haas enzymes involves several steps. It begins at (1). In 1963, a major development in the field microorganism level with basic characterization of took place when the first bacterial subtillisin protease secreted enzymes. Promising enzymes are tested for from Bacillus licheniformis (subtilisin Carlsberg) was wash performance in detergent application testing, introduced into commercial detergent. The importance concomitantly with the use of protein engineering to of properly formulating technical enzymes became clear improve enzyme properties. In the last steps involving during the early seventies, after the discovered risk of production, the fermentation process is scaled up, allergic reactions from dusty proteases caused a major recovery and purification processes are optimized, setback for the detergent industry (2). This issue was and a suitable enzyme formulation is developed. The solved by proper formulation of the protease product novel detergent enzyme is then ready to be used within into non-dusting granulates or prills, resulting in protease laundry or ADW applications. enzyme products that are completely safe to use. A common denominator for detergent enzymes is that they target tough stains and hydrolyse the various components in the soiling into smaller more water- soluble fragments. This helps boost the mechanical removal of the stain during wash, which is also assisted by and builders in the detergent. The use of laundry enzymes results in far more efficient stain removal than that achieved by the detergent and mechanical action alone. The growing trend towards lower washing temperatures, with lower water of many stains Table 1. Commercially available detergent enzyme classes as a consequence, calls for added cleaning power by and their applications. detergent enzymes to ensure efficient stain removal even

H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 37 MICROBIAL SCREENING performing protease is found, genes for homologous proteases can be identified simply by comparing the DNA Before the catalytic power of nature can be captured or protein sequence of the high-performing protease with in the form of a technical enzyme, one must be able to sequences in the databases. These genes can be cheaply express the enzyme in a microorganism that can yield synthesized, and the used codons can be chosen to fit the commercially relevant amounts. Countless microorganisms preferences of the expression host. By optimizing the gene including the bacteria genus Bacillus, secrete several enzyme and codon to suit the host, there is an increased chance classes including proteases into their external environment. that the gene of interest will be well-expressed. Despite these Microorganisms do this to degrade surrounding biomolecules measures, there is no guarantee that a particular enzyme can into smaller entities which the microorganism can take in be expressed. Once expressed, the potential of the protease and use. The challenging part is to identify and pinpoint a is tested in application screening. microorganism which secretes a protease that can work in a detergent solution. An initial small scale screening of many different proteases from several microorganisms is used to DETERGENT APPLICATION SCREENING examine basic parameters related to the profile and activity of the protease. In the case when the secreted protease Factors influencing wash performance shows indications of being functional in detergent, the next Finding “a needle in a haystack” is a frequently used step is to ferment the microorganism to produce larger metaphor describing the search for promising enzymes in an amounts of protease for further testing in the laboratory. application screening flow comprising thousands of different Some decades ago this strain was used for the large scale molecules. To pick the right candidates, having the right and production of the enzyme, but new techniques have relevant application screening assays is absolutely essential. changed this today. The production of proteases became Plain enzyme activity measurements on simple substrates such easier in the eighties with the introduction of recombinant as the synthetic 4-nitroanilide peptides for proteases are useful DNA technology. in the early screening stages before application testing. These can be performed in high throughput using automated liquid dispenser systems and microplate readers. However, these simple activity measurements fall short in case of application purposes. This is because there is a world of difference between simple activity and the complex multi-factorial situation, which a laundry full scale wash represents, with its natural complex substrate types, mechanics, and textile load, to name just a selected few. In fact, a vast range of factors influence the observed wash performance by a laundry enzyme, such as wash cycle duration, substrate specificity, temperature, catalytic activity, water ion concentrations, enzyme stability, ballast (unsoiled textile) load, inhibitors, enzyme concentration, wash and soil load, interaction with other enzymes, impact from detergent ingredients, mechanical action in the wash machine, pH, adsorption of enzyme onto substrates, etc. Most detergent enzymes, including proteases work predominantly in-wash, while others such as lipases are also very active post-wash (5).

Stain type Detergent enzymes are typically evaluated based on their ability to remove natural and artificially produced stains on Figure 1. A model of a subtilisin serine protease showing the binding site and the amino acids of the catalytic triad textile. Cotton and polyester are the most frequent fabric (Serine-221, Histidine-64, and Aspartate-32). types used. Choice of stain type is a critical factor since most proteases are extremely effective and will often remove all measurable amounts of protein substrate in a consumer-type Once the microorganism expressing the desired proteases natural soiling. In application screening this effectiveness was identified, a library was made containing the induces the need for technically produced stain types that gene pool from the organism. The gene encoding the are particularly resilient and very difficult to completely protease of interest is cloned from the genome of the remove. The technical stain types ensure that there is a identified microorganism into another host such asBacillus window left for response measurement and performance licheniformis, which is easy to ferment, has GRAS status ranking, also with high-performing protease variants. (Generally Regarded As Safe) and can secrete enzymes at Commercially produced textiles with common soiling types commercially relevant levels (4). After the millennium, this field (e.g. milk, grass) are available in many versions, containing took a further leap forward when numerous microorganisms one or several substrates, sometimes with particulate had their genome sequenced, and the price of artificially matter added, e.g. carbon black. Another benefit of using synthesizing new genes (DNA Synthesis) has been drastically commercially produced technical stains is that these often lowered. Today many genomes from microorganisms are highly uniform. This elevates data quality when comparing have been sequenced and the information put into large thousands of molecules in a screening flow. A wide selection databases which can be mined. Thus, when a new high- of protease-relevant stain types are used (e.g. blood, grass,

38 H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 milk), since the substrate specificity profile can vary to some (6). In the AMSA, laundry enzyme and detergent solutions extent between different proteases. Enzyme performance are placed in a microtiter plate, soiled fabric is added, is quantified by measuring the enzyme’s net contribution to and the system is sealed tightly by a lid. Mechanical colour removal from the stain measured as reflectance of action is induced by rapid oscillating movements, and the light recorded in intensity or remission units. In special cases, wash is conducted at a selected duration and controlled mechanistic studies can also be undertaken involving analysis temperature. For automatic dishwash enzymes, soiled hard of stain components using e.g. fluorescence or infrared (IR) surface melamine plates are used in the AMSA. spectroscopy, or other. Those enzymes that outshine their opponents and make it through the AMSA screening have still to undergo more trials; for laundry enzymes a range of small-to-medium scale wash assays exist. One example is the Launder-O- Meter (LOM) wash assay, which mimics European drum- type machines. The LOM uses revolving sealed beakers containing enzyme, detergent solution, soiled textile swatches, ballast, and small stainless steel balls that add mechanical action. The Terg-O-toMeter (TOM) is a medium- sized wash assay, simulating a US top loader machine. The TOM uses 1 L open metal beakers containing enzyme, detergent solution, soiled textile swatches, ballast, and mechanical action by an agitator (7). If an enzyme still delivers promising results at this stage, the next and final test in the application screening flow will usually be full scale wash trials. Analogously, for automatic dishwash enzymes, Figure 2. FT-IR spectra illustrating the protein content of home- the final evaluation typically involves washing of soiled made skim milk stains (indented image), before wash (blue), tableware in a dishwasher. Alongside the main application after wash without enzyme (red), and after wash with 80 nM of the subtilisin serine protease Savinase® (green), with CN-11 screening flow, smaller side-flows will often be incorporated, cotton textile background subtracted (TOM wash at 20°C, 30 which use custom designed assays to examine selected min., 120 rpm, 15°dH, pH 8 in liquid ). The features of enzymes, with the knowledge gained feeding catalysis by the protease results in a significant increase in directly into the main screening flow. Information gathered protein removal, seen by the reduced intensity in the peaks during application screening is used to improve the design from the amide groups in the milk protein. of promising enzyme variants in a joint learning loop with protein engineering. Detergent

When assessing stain removal by a detergent enzyme, the choice of detergent is of hallmark importance, since enzymes perform differently in different detergents. Wash solutions arising from liquid versus powder detergents differ in several ways regarding e.g. alkalinity, inclusion of bleach components etc, and these parameters all impact enzyme performance. There are also differences in the composition of powder detergents for use in laundry versus for automatic dishwash, which contain, in the case of the latter, relatively more builders, only little and a much higher enzyme load. Also the compositions of detergents may vary significantly across different regions of the world, as a consequence of local differences in water hardness, pH, washing traditions and procedures, etc.

Wash assays When a protease enters the application screening process, it engages in a series of elimination races, where the bar for satisfactory assay performance is continuously raised. Only the most promising enzymes that display desired features are taken to the next screening step, and only a selected few out of many thousands of candidates make it all the way to final full scale wash trials. Application assays are to different extents predictive of the performance seen in a full scale wash. Throughout the screening flow the assays get more advanced, complex, time- and enzyme-consuming, so a balance of data quantity versus quality must be kept. The first application assay an enzyme will be subjected to Figure 3. Terg-O-toMeter (TOM) apparatus with capacity for when it enters the detergent application screening process 12 metal beakers (1 L) with metal agitators (on tray). will often be the AMSA (automated mechanical stress assay)

H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 39 PROTEIN ENGINEERING thereby increase the enzyme stability (14). An alternate solution can be to add protease inhibitors that bind and Protein engineering is used to improve properties of selected stabilize the protease when present in the concentrated enzymes that have shown the ability to be expressed in a host, detergent mixture. Once diluted into the wash liquor, the have shown promising results in the initial screening, and have inhibitor is diluted too, freeing the protease to perform performed well in the first rounds of application testing. Protein catalysis and consequently stain removal. The binding of the engineering can be used to change the type, number, inhibitor can be optimized by either mutating the protease or or position of some of the amino acids in the protein. This altering the molecular structure of the inhibitor (15, 16). technique is well-suited to solve issues related to detergent enzyme stability, a challenge commonly encountered with Thermostability and detergent stability non-engineered proteases. It is often more tricky to improve High temperatures and anionic surfactants such as LAS (linear protease performance by protein engineering than it is to alkylbenzene sulphonate) can denature detergent proteases increase stability, but this can also be done (8, 9). There are during storage. Improving the thermostability of a protease several strategies to improve stability, and many papers can in many cases lead to improved stability in a detergent describe how stability has been increased. Improved stability (17). A classic approach that has proven to work well in the is a feature that is often easy to demonstrate and report, case of proteases consists in the addition of disulphide bridges using a simple screen in which the temperature is increased. by introducing cysteine at the proper positions in the enzyme By contrast, it is far more challenging and costly to design and tertiary structure (18). Another possibility is to replace an conduct a performance screen which correlates with results amino acid with proline, which will increase the rigidity of the in real application. protein due to the inclusion of the pyrrolidine ring structure in the protein main chain. A third possibility is to use database Bleach stability sequences of ancestral genes as inspiration for the build-up Proteases can be inactivated during storage by bleach of a more thermostable enzyme. This is based on a theory present in the detergent. The methionine in position 221 that the earth was warmer a long time ago, and therefore (Subtilisin Carlsberg numbering), which is close to the protease ancestral enzymes had to be more thermostable. This theory active site serine, gets oxidized by the bleaching agents is being highly debated. A different approach is molecular which leads to inactivation of the protease. Simple site dynamic analysis, in which computer modelling is used to directed mutation of this methionine to other non-oxidizable deduce the most mobile regions in the protein based on the amino acids leads to improved bleach stability (10), but protein X-ray crystal structure. Amino acid substitutions can also causes reduced protease performance. It has been then be made in these particular regions with the aim of shown that protease oxidation stability can be increased by obtaining more rigidity, which in turn can increase enzyme introducing a methionine in position 216 in subtilisin Carlsberg, thermostability. An elegant feature of evolving a more likely due to the methionine 216 acting as a “suicide antioxidant” thermostable enzyme is that different mutations that confer which safeguards the important methionine 221 (11). stability to the enzyme can be combined in the same enzyme. This can boost enzyme stability further because the stabilizing Chelator sensitivity effects of the identified mutations are often additive. Chelators are added to the detergent to lower the concentrations of free ions naturally present in wash water, especially divalent calcium and magnesium ions. This is PRODUCTION relevant since calcium ions can interact with surfactants such as LAS (linear alkylbenzenesulphonate), producing LAS/ Once the needle in the haystack has been found by calcium complexes, which precipitate and lead to reduced application screening and further optimized by protein wash performance (12). Lowering the free ion concentrations engineering, the expression of the enzyme is transferred into a can also be problematic since many detergent enzymes, e.g. production-relevant microorganism (18). amylases, depend on calcium. Protein engineering can be used to improve the binding of the structural calcium ion to Mastering microbial enzyme expression the protease, thereby lowering the chelator-sensitivity of the The majority of industrial enzymes are produced in host protease. A mutation such as N76D in the Bacillus protease microorganisms such as filamentous fungi (e.g.Aspergillus Savinase® seems to improve the binding of the calcium found oryzae, Aspergillus niger or species of the Fusarium and in the structure, and makes the protease more thermostable Trichoderma genus) or gram-positive bacteria (e.g. Bacillus (10). An alternative way to improve protease storage stability licheniformis and Bacillus subtilis), using simple and cheap towards chelators is to completely remove the calcium substrates. The host microorganisms may be genetically binding site from the protease. One example is the BPN´ manipulated in order to further improve their characteristics protease, which after multiple rounds of protein engineering was for the industrial level fermentation, e.g. for removal of evolved into a calcium free, stable and active subtilisin (13). undesired enzyme activities that could interfere with the function or stability of the intended product. The fermentation Proteolytic stability process is developed and optimized with regard to Since enzymes are proteins, proteases can potentially be fermentation time, temperature, pH, addition of substrates their own target. Apart from auto-proteolysis, proteases etc., and subsequently scaled up (19). can hydrolyse other enzymes, e.g. lipases, which can be degraded by the action of a protease included in a Recovery and purification detergent formulation. By means of protein engineering, it In parallel to the expression the enzyme recovery/purification is possible to mutate those sites which are highly prone to process is developed, optimized, and scaled up too. The proteolytic cleavage to less protease-sensitive sites, and main purpose of recovery is to remove non-enzymatic

40 H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 components from the enzyme fermentation broth. Most Protease inhibitors industrial enzymes produced today are extra-cellular Traditionally Borax (Na2B4O7·10 H2O, a sodium salt of enzymes, i.e. the enzymes are secreted into the medium boric acid) has been used as a protease inhibitor in by the producing microorganism and not confined within liquid detergents. However, Borax is a weak inhibitor (KI the cell. The first recovery step is often the removal of whole in the 10 mM range) such that large amounts, typically cells and other debris by centrifugation and/or filtration. around 2%, are needed in the detergent formulation Ultrafiltration is increasingly used to remove water, salts, and to provide a sufficient degree of inhibition. This high other low molecular weight impurities after the first recovery percentage causes problems in complex detergent step. Precipitation of the enzymes by use of salts, polymers, or solutions, such as reduced solubility of surfactants etc.. organic solvents is another simple method employed for the Moreover, Borax has also been found to be reprotoxic. recovery and purification of enzymes. Vacuum evaporators Therefore, a good incentive exists to develop and are often used to remove water and concentrate the explore new efficient inhibitors, which should be custom enzyme. In some cases spray-drying may also be used if a designed for the specific protease enzyme to be most powdery intermediate product is desired. The concentrates effective. There is a delicate balance between the need to be completely free from genetically modified use of too strong inhibitors, which may not release the organisms. The colour and odor of the concentrate should protease to a sufficient extent during the wash causing not negatively affect the final detergent and enzyme lowered wash performance, and too weak inhibitors, concentrates for liquid detergents need to be clear. which may need a high dosing resulting in added costs and cause problems related to solubility and the physical stability of the detergent. One example FORMULATION of a solution for liquid detergents is the inhibitor 4-formylphenyl boronic acid (4-FPBA) in the Ultra brand In the final step of the process, the concentrated enzyme by Novozymes, which is about 100 times more efficient formulations are developed, which are divided into solid than Borax on a weight basis and is co-formulated into products for powder detergents and liquid products for concentrated enzyme products (22). liquid detergents. The final product will have to live up to a multitude of quality parameters (20).

Powder For powder detergents, an enzyme granulate is developed which is typically 0.3-1.2 mm in diameter. The main quality parameters are particle size, dissolution rate, stability in detergent, dust, colour, and flowability. The largest challenge for enzyme stability in powder detergents is bleach, which in combination with humidity can oxidize the enzyme (21). This is typically handled by inclusion of antioxidant/reducing agents and protective coatings, such as those used in the Novozymes Evity® detergent enzyme range, developed to deliver consistent performance after storage in detergent even at harsh conditions.

Liquid Stability is very challenging in liquid detergent products as the enzyme can diffuse in the solution and is not separated from other detergent components. This can lead the highly efficient proteases to auto-proteolysis or to digest other detergent enzymes. This issue can be pronounced for low temperature proteases that are highly active below and at Figure 4. Docking of 4-formylphenyl boronic acid into the room temperature which is a typical storage condition. binding pocket of a subtilisin serine protease.

A neat solution is to mitigate the proteolytic attack using reversible protease inhibitors. The inhibitor (I) works by In the future, non-boron based ingredients are inhibiting the protease (P) when present in high concentration expected to play an increasingly important role in in the detergent, thus forming an inactive inhibited protease enzyme formulations. The demand for non-boron based complex (PI): inhibitors is predicted to rise accordingly, with one recent example being the second generation inhibitor under the Evity® brand by Novozymes, which is far more efficient than 4-FPBA, and not based on boron. Upon dilution with water in the washing machine the Improvements in liquid stability solutions and formulation chemical equilibrium is pushed towards free protease (P), pave the way for further growth in the application of which then becomes active in the wash liquor. The effect of industrial enzymes. The liquid Evity® products are based an inhibitor is typically given by its inhibition constant KI, the on an inhibitor which was specifically designed for dissociation constant for the enzyme-inhibitor complex (PI). the Novozymes protease family. This delivers improved

Small KI values correspond to strong inhibitors. stability in liquid detergent both of the protease and

H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 41 other enzymes added to the detergent thus enabling a REFERENCES AND NOTES more widespread use of enzymes.

1. Malmos H., Chemistry and Industry, 6, 183-186 (1990). 2. Basketter D. et al., Regul. Toxicol. Pharmacol., 64, 117-123 (2012). 3. Enzymes at Work, Novozymes A/S, Bagsvaerd, Denmark, 12-13 (2008). 4. Falch E.A., Biotech. Adv., 9, 643-658 (1991). 5. Aaslyng D., Gormsen E., Malmos H., J. Chem. Tech. Biotechnol., 50, 321-330 (1991). 6. Bechmann G.R.T. et al., patent WO2002042740 (2002). 7. Harris J.C., Brown E.L., J. Am. Oil Chem. Soc., 27(12), 564-570 (1950). 8. Knötzel J.C.F. et al., patent WO2011036263 (2011). 9. Draborg H., Minning S., Nielsen V.S., patent WO2007006305 (2007). 10. Osten C. et al., J. Biotechnol., 28, 55-68 (1993). 11. Vojcic L. et al., Biol. Chem., 394, 79-87 (2012). Figure 5. Protease stability in a liquid laundry detergent at 30 °C 12. Lund et al., J. Surfact. Deterg. 15, 265-276 (2012). with and without non-boron protease inhibitor system. 13. Strausberg et al., Biochemistry, 44, 3272-3279 (2005). 14. Boguslawski G. et al., patent US 5543302. CONCLUSIONS 15. Ganz et al., Protein Eng., Design & Selection, 17, 333-339 (2004). 16. Pedersen & Nørregaard-Madsen, patent WO200218588 (2002). 17. Mansfeld & Ulbrich-Hofmann, Biotechnol. Bioeng., 97, 672-679 The discovery, development, a nd production of novel (2007). industrial enzymes is all about harvesting the power and 18. Bryan P., Biochim. Biophys. Acta, 1543, 203-222 (2000). catalytic potential of nature and adapting this for use 19. Enzyme Applications, Industrial, Chapter 10, 248-317, in Kirk- in detergents or other industries. Trends move towards Othmer Encycl. Chem. Technol., John Wiley & Sons Inc., New lower wash temperatures, and demands increase for Jersey, USA (2005). reduced use of toxic chemicals, added performance 20. Enzymes in Detergency, Surfactant Sci. Ser., 69, Edited by van qualities, and eco-friendly solutions. Detergent enzymes Ee J.H., Misset O. and Baas E.J., Marcel Dekker, New York, USA help meet all these requirements and the continued (1997). search for improved industrial enzymes is highly relevant. 21. Lagnemo H., Simonsen O., Mechanism of deactivation of enzyme and sodium carbonate in HDP, in 40th WFK The quest for improved catalytic proteins is facilitated International Detergency Conference Proceedings (2001). by recent scientific progress in biotechnology, screening 22. Nielsen L.N. and Simonsen O., Design of Liquid Enzyme Products assays, and formulation. Together, these technological with Built-in Liquid Detergent Stabilization System, Chapter 5, advances facilitate the advent of new sustainable in. Chemical Product Design, Comput.-Aided Chem. Eng., detergent ingredients in the form of nature’s favourite 23, Edited by Ka N.G., Gani R. and Dam-Johansen K., Elsevier, workhorse: Enzymes. Amsterdam, The Netherlands (2007).

Find out more... in some of our past monographic supplement series! Visit our website www.teknoscienze.com