ENZYMES 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 industrial enzymes 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. Proteases are used as the main example as this was the first enzyme 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 cellulases, are also desirable traits. In laundering, and continue to be of great benefit in automatic dishwash (ADW), proteases and amylases 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. trypsin 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 protease 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 surfactants 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 solubility 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