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
   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

   The nature of enzymes is
   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

   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

   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

   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

   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

   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
   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

   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

   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

   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

   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
   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

   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

   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

   The succes and importance of
   using enzymes in a variety
   og modern industrial
   processes is illustrated by
   the applications described
   under "industrial

Novo Nordisk A/S
questions please contact