Novo Nordisk A/S 1874 when the Danish chemist Christian Hansen first produced rennet by extracting it from dried calves' stomachs with saline solution. Apparently this was the first enzyme preparation of relatively high purity used for industrial purposes. This significant event had been preceded by a lengthy evolutionary process. Enzymes have been used by Man throughout the ages, either in the form of vegetables rich in enzymes, or in the form of microorganisms used for a variety of purposes, for instance in brewing processes, in baking, and in the production of alcohol. It is known that enzymes were already used in the production of cheese in antiquity, and some of the earliest references to this are found in the Greek epic poems the Iliad and the Odyssey, dating from about 800 BC. In these works, text can be found suggesting that stomachs from lambs or kids, which contain the same enzymes as calves' stomachs, were used for the production of cheese. Fig sap, which contains the proteolytic enzyme ficin, was also reportedly used for the same purpose. The nature of enzymes is clarified Even though the action of enzymes has been recognized and enzymes have been used throughout history, it was not until quite recently that their inherent properties as such were realized. Enzymatic processes, particularly fermentation, were the focus of numerous studies in the 19th century and many valuable discoveries in this field were made. A particularly important experiment was the isolation of the enzyme complex from malt by Payen and Persoz in 1833. This extract, like malt itself, converts gelatinized starch into sugars, primarily into maltose, and was termed 'diastase'. The term is still used in Frenchspeaking countries as the collective name for all enzyme extracts and is not applied solely to amylases such as the enzyme complex extracted by Payen. Development progressed during the following decades, particularly in the field of fermentation physiology where the achievements of Schwann, Liebig, Pasteur and Kühne were of the greatest importance. The dispute between Liebig and Pasteur concerning the fermentation process caused much heated debate. Liebig held that fermentation resulted from a common, chemical process and that yeast was a nonviable substance continuously in the process of breaking down. Pasteur, on the other hand, argued that fermentation did not occur unless viable organisms were present. Now it is realized that neither of them was entirely right, though Liebig's 'nonvitalist' concept of fermentation turned out to be closest to the truth. The dispute was finally settled in 1897, after the death of both adversaries, when the Buchner brothers demonstrated that cellfree yeast extract could convert glucose into ethanol and carbon dioxide just like viable yeast cells. In other words, the conversion was not ascribable to yeast cells as such, but to their nonviable enzymes. In 1876, William Kühne proposed that the name 'enzyme' be used as the new term to denote phenomena previously known as 'unorganized ferments', that is, ferments isolated from the viable organisms in which they were formed. The word itself means 'in yeast' and is derived from the Greek 'en' meaning 'in' and 'zyme' meaning 'yeast' or 'leaven'. Kühne may have had similar views on fermentation to those advocated by Buchner, but he failed to provide the experimental evidence required for their validity. Early developments in Japan During the early part of this century, enzyme technology was also developing slowly but surely outside Europe. In the Far East, an ageold tradition prevailed where mould fungi called koji were (and still are) used in the production of certain foodstuffs and flavour additives based on soya protein (shoyu, miso, tempeh) and fermented beverages (sake, alcohol). Koji is prepared from steamed rice into which a mixture of mould fungi is inoculated, the composition of the mixture being passed down from generation to generation. This formed the basis on which the Japanese scientist Takamine developed a fermentation process for the industrial production of fungal amylase; the process included the culture of Aspergillus oryzae on moist rice or wheat bran. The product was called 'Takadiastase' and it is still used as a digestive aid. The method of fermentation suggested by Takamine, the 'surface culture' or 'semisolid culture', is still used in the production of certain enzymes, but now it has been almost entirely replaced by the 'submerged culture' method. Here, fermentation takes place in closed tanks containing a liquid substrate which is stirred while air is blown through it. Desizing textiles At about the same time as Takamine was developing his novel fermentation technique, another field was being opened up for the use of enzymes - the desizing of textiles. Desizing is a process by which all the starch paste is removed from the fabric after having served as a strengthening agent to stop the warp thread breaking during the weaving process. Previously, textiles were treated with acid, alkali or oxidizing agents, or soaked in water for several days so that naturally occurring microorganisms could break down the starch. However, both of these methods were difficult to control and sometimes damaged or discoloured the material. It was therefore a major step forward when crude enzyme extracts in the form of malt extract, and later in the form of pancreas extract, were first used to carry out desizing. It was not until a heatstable bacterial amylase (derived from Bacillus subtilis) was introduced that an enzyme was found which seemed to be ideal for this purpose. Bacterial amylase was used for the first time by Boidin and Effront as early as 1917, but it was not possible to massproduce it until after the end of World War II. This is probably because bacteria are not particularly wellsuited to surface culture, and that the submerged method was not developed until after the war. Bating leather Investigations carried out by the German chemist and industrial magnate Otto Röhm before World War I were of great importance for the further development of the industrial exploitation of enzymes. Among other things, he studied the socalled 'bating' process, a step in the preparation of hides and skins prior to tanning. Bating removes some of the protein which is not essential for the strength of the leather and which otherwise might prevent leather from achieving the suppleness and the softness of touch required in numerous products. As such, bating serves to control the quality of leather; for instance, stiff leather used for soles is only lightly bated, while the soft qualities required for gloves results from intense bating. Traditionally, bating required the excrement of dogs and pigeons, a fact that did not improve the image of tanning which was understandably considered a smelly and thoroughly unpleasant job. Röhm's theory was that these excrements exerted their effect because they contained residual amounts of the animals' digestive enzymes. If this was so, it might be possible to use extracts of the pancreas directly for bating. Such extracts were tried and produced the expected positive results. Naturally, Röhm accepted this as confirmation of the correctness of his theory, but later experiments showed that it was not the animals' enzymes that were active, but rather enzymes of bacteria growing in the intestinal tract. The first detergent enzyme Parallel to his studies of the problems involved in tanning, Röhm investigated other processes where enzymes would prove even more valuable. Nevertheless, his efforts were not to score a success until 50 years later. Röhm actually developed the first method for washing proteinstained cloth in detergents containing enzymes and manufactured the first detergent preparation capable of doing this. Röhm's firm took out a patent in 1913 according to which minor quantities of enzyme were added to the detergent. The preparation was marketed until the sixties under the brand name of Burnus. The enzyme preparation used was pancreatin (extracted from pancreatic glands) which contains the proteindegrading enzyme trypsin. Burnus never became a great success because trypsin was not particularly active in the strongly alkaline liquids that were obtained by dissolving the product in water. Burnus was mainly composed of ordinary washing soda (sodium carbonate). The soda did an excellent job since it has a softening effect on water and helps to dissolve dirt on laundry. However, the effect of the trypsin was extremely limited. Research bears fruit Only moderate progress was made in developing the largescale production of enzymes for industrial purposes during the interwar period. Methods used for the extraction of enzymes from vegetable and animal materials were improved, as were methods used for the purification of extracts and for the extraction of enzymes in relatively pure form. A few new fields were discovered where enzymes could be used, for instance the baking and fruit juice industries. Microbial enzymes continued to be derived mainly from surface cultures grown on solid or liquid media, processes which made heavy demands on space and labour. On the other hand, progress in the field of research was considerable. The achievements of Willstätter and collaborators, who produced highly purified enzymes during the period 1920-1928, represented an important step forward. In fact, their achievements were indispensable for the future development of enzyme technology. At about the same time, Sumner (1926) was the first to produce an enzyme, urease, in crystalline form, and a few years later Northrop was successful in crystallizing several proteolytic enzymes. In the early years of World War II, an important goal was achieved at Novo when trypsin was produced using pancreatic tissue from which insulin had already been extracted. The development of the submerged culture technique represented a major advance in enzyme technology since it permitted the largescale production of microorganisms for industrial purposes. Penicillin drives industry forward The impetus for advancing industrial fermentation techniques stemmed from the discovery of penicillin, a discovery that had remained rather neglected since 1928. Shortly after the beginning of the war, interest in penicillin grew enormously and attempts were intensified to develop a method by which it could be produced on an industrial scale. Commercial production did, indeed, soon begin, but it was based on the surface culture of mould fungi growing in flatbottomed glass flasks. Because of the workload involved, largescale production could not be established and it was not until the submerged culture technique was introduced that penicillin could be produced economically in sufficient quantities and achieve the widespread use it enjoys today. The initial steps towards an improvement of the submerged culture technique for fermentation were taken during wartime, and efforts were intensified after the war ended. This technique proved to be of value not just for the production of penicillin and other antibiotics, but also for the production of enzymes. Such a technique was introduced into Novo's laboratories early in the fifties at a time when the production of bacterial amylases for the textile industry began. Very soon other microbial enzymes were also produced, submerged fermentation being used throughout. Breakthrough in detergents In 1959, there was a significant new development in the detergent industry; the Swiss chemist Dr. Jaag, who worked for the detergent company Gebrüder Schnyder in Biel, developed a new product called Bio 40 containing a bacterial protease instead of trypsin. Even though this bacterial protease was more fit for the purpose than trypsin, it was not ideal. When Novo launched Alcalase® in 1962, it was the first enzyme product that could readily satisfy all the demands made of it. The action of this alkaline protease was relatively unaffected by other components of washing powder and it worked at the desired temperatures. The first great marketing success for a detergent formulated with enzymes was Biotex, which contained Alcalase and was made by the Dutch firm Kortman & Schulte (now Kortman Intradal) in collaboration with Gebrüder Schnyder. The tremendous success of this product marked the real breakthrough for detergent enzymes. The use of enzymes for industrial purposes progressed rapidly after 1965, due mainly to the increasing use of enzymes in detergents. However, there was a temporary setback in the early 1970s when it was ascertained that enzymes - like all other proteins, incidentally - could cause allergic reactions. On the initiative of the U.S. Food and Drug Administration (FDA), the National Academy of Science (NAS) made a thorough investigation of this issue. In its report from 1971, NAS concluded that detergent enzymes are not only harmless to consumers, but provide definite technological advantages. The problem of allergenicity was soon overcome by the introduction of dustfree preparations. The detergent industry is still the largest outlet for industrial enzymes, and new enzyme products are constantly being developed for use in detergents. Indeed, a major breakthrough in this field by Novo Nordisk was the successful introduction in 1988 of a fatdegrading lipase enzyme called Lipolase®. More sugars from starch A very important field in which enzymes have proved to be of great value over the last 15-20 years is the starch industry. As early as the 1950s, fungal amylase was used in the manufacture of specific types of syrup, i.e. those containing a range of sugars which could not be produced by conventional acid hydrolysis. The real turning point was reached early in the 1960s with the launch of the first enzyme preparation, amyloglucosidase, that could completely break down starch into glucose. Within a few years, almost all glucose production was reorganized and enzyme hydrolysis was used instead of acid hydrolysis because of the obvious benefits provided by the former, such as greater yield, a higher degree of purity and easier crystallization. The process was further improved by the introduction of a new technique used for the enzymatic pretreatment (liquefaction) of starch; a suitable enzyme, bacterial amylase, was already available. A highly successful and fully enzymatic starch hydrolysis was achieved when the bacterial amylase Termamyl® which is stable at high temperatures, was introduced by Novo in 1973. Since then, the interest of the starch industry has focused mainly on an enzyme of a quite different nature, glucose isomerase. Using this enzyme, glucose containing an aldehyde group can be converted into the corresponding ketone sugar, fructose. This has the same calorific value as glucose, but its sweetening effect is approximately twice as high. Hence, starting from pure glucose it is now possible to use glucose isomerase to produce a mixture of glucose and fructose with the same calorific content and sweetening effect as ordinary cane or beet sugar, which themselves consist of equal amounts of glucose and fructose. Considering that starch is much more universally available and frequently cheaper than cane sugar and beet sugar, it makes economic sense to use the new product 'High Fructose Corn Syrup' (HFCS) or 'isosyrup' in many products where sugar has hitherto predominated. The succes and importance of using enzymes in a variety og modern industrial processes is illustrated by the applications described under "industrial applications". Novo Nordisk A/S questions please contact enzymes.novo.dk