1
A BRIEF HISTORY OF ENZYMOLOGY

KEY LEARNING POINTS

• Enzyme reactions have been used since antiquity in the production of bread, cheeses, and alcoholic beverages. Early references to the action of enzymes can be found in Homer's Iliad and the Codex of Hammurabi.
• Systematic studies of enzyme activity began in the eighteenth century in studies of food digestion from gastric juices taken from the stomachs of birds and mammals (including humans).
• In the late nineteenth and early twentieth centuries, mathematical models for enzyme activity began to be derived and published.
• From these early studies, the modern science of enzymology has developed. Today, enzymology is studied as basic science but is also widely studied in a variety of commercial applications, including medicine, agriculture, food and beverage making, cleaning products, and much more.

Life depends on a well-orchestrated series of chemical reactions. Many of these reactions, however, proceed too slowly on their own to sustain life. Hence, nature has designed catalysts, which we now refer to as enzymes, to greatly accelerate the rates of these chemical reactions. The catalytic power of enzymes facilitates life processes in essentially all life forms from viruses to man. Many enzymes retain their catalytic potential after extraction from the living organism, and it did not take long for mankind to recognize and exploit the catalytic power of enzymes for commercial purposes. In fact, the earliest known references to enzymes are from ancient texts dealing with the manufacture of cheeses, bread, and alcoholic beverages, and for the tenderizing of meats. Today enzymes continue to play key roles in many food and beverage manufacturing processes and are ingredients in numerous consumer products, such as laundry detergents (which dissolve protein-based stains with the help of proteolytic enzymes). Enzymes are also of fundamental interest in the health sciences since many disease processes can be linked to the aberrant activities of one or a few enzymes. Hence, much of modern pharmaceutical research is based on the search for potent and specific inhibitors of these enzymes. The study of enzymes and the action of enzymes has, thus, fascinated scientists since the dawn of history, not only to satisfy erudite interest but also because of the utility of such knowledge for many practical needs of society. This brief chapter sets the stage for our studies of these remarkable catalysts by providing a historic background of the development of enzymology as a science. We shall see that while enzymes are today the focus of basic academic research, much of the early history of enzymology is linked to the practical application of enzyme activity in the industry.

1.1 ENZYMES IN ANTIQUITY

The oldest known reference to the commercial use of enzymes comes from a description of winemaking in the Codex of Hammurabi (ancient Babylon, c. 2100 B.C.). The use of microorganisms as enzyme sources for fermentation was widespread among ancient people. References to these processes can be found in writings not only from Babylon but also from the early civilizations of Rome, Greece, Egypt, China, and India. Ancient texts also contain a number of references to the related process of vinegar production, which is based on the enzymatic conversion of alcohol to acetic acid. Vinegar, it appears, was a common staple of ancient life, being used not only for food storage and preparation but also for medicinal purposes.

Dairy products were another important food source in ancient societies. Because in those days fresh milk could not be stored for any reasonable length of time, the conversion of milk to cheese became a vital part of food production, making it possible for the farmer to bring his product to distant markets in an acceptable form. Cheese is prepared by curdling milk via the action of any of a number of enzymes. The substances most commonly used for this purpose in ancient times were ficin, obtained as an extract from fig trees, and rennin, as rennet, an extract of the lining of the fourth stomach of a multiple-stomach animal, such as a cow. A reference to the enzymatic activity of ficin can, in fact, be found in Homer's classic, the Iliad:

As the juice of the fig tree curdles milk and thickens it in a moment though it be liquid, even so instantly did Paeeon cure fierce Mars.

The philosopher Aristotle likewise wrote several times about the process of milk curdling and offered the following hypothesis for the action of rennet:

Rennet is a sort of milk; it is formed in the stomach of young animals while still being suckled. Rennet is thus milk which contains fire, which comes from the heat of the animal while the milk is undergoing concoction.

Another food staple throughout the ages is bread. The leavening of bread by yeast, which results from the enzymatic production of carbon dioxide, was well known and widely used in ancient times. The importance of this process to ancient society can hardly be overstated.

Meat tenderizing is another enzyme-based process that has been used since antiquity. Inhabitants of many Pacific islands have known for centuries that the juice of the papaya fruit will soften even the toughest meats. The active enzyme in this plant extract is a protease known as papain, which is used even today in commercial meat tenderizers. When the British Navy began exploring the Pacific islands in the 1700s, they encountered the use of papaya fruit as a meat tenderizer and as a treatment for ringworm. Reports of these native uses of the papaya sparked a great deal of interest in eighteenth-century Europe, and may, in part, have led to some of the more systematic studies of digestive enzymes that ensued soon after.

1.2 EARLY ENZYMOLOGY

While the ancients made much practical use of enzymatic activity, these early applications were based purely on empirical observations and folklore, rather than any systematic studies or appreciation for the chemical basis of the processes being utilized. In the eighteenth and nineteenth centuries, scientists began to study the actions of enzymes in a more systematic fashion. The process of digestion seems to have been a popular subject of investigation during the years of the enlightenment. Wondering how predatory birds manage to digest meat without a gizzard, the famous French scientist Réaumur (1683-1757) performed some of the earliest studies on the digestion of buzzards. Réaumur designed a metal tube with a wire mesh at one end that would hold a small piece of meat immobilized, to protect it from the physical action of the stomach tissue. He found that when a tube containing meat was inserted into the stomach of a buzzard, the meat was digested within 24 hours. Thus, he concluded that digestion must be a chemical rather than a merely physical process, since the meat in the tube had been digested by contact with the gastric juices (or, as he referred to them, "a solvent"). He tried the same experiment with a piece of bone and with a piece of a plant. He found that while the meat was digested and the bone was greatly softened by the action of the gastric juices, the plant material was impervious to the "solvent"; this was probably the first experimental demonstration of enzyme specificity.

Réaumur's work was expanded by Spallanzani (1729-1799), who showed that the digestion of meat encased in a metal tube took place in the stomachs of a wide variety of animals, including humans. Using his own gastric juices, Spallanzani was able to perform digestion experiments on pieces of meat in vitro (in the laboratory). These experiments illustrated some critical features of the active ingredient of gastric juices: by means of a control experiment in which meat treated with an equal volume of water did not undergo digestion, Spallanzani demonstrated the presence of a specific active ingredient in gastric juices. He also showed that the process of digestion is temperature dependent and that the time required for digestion is related to the amount of gastric juices applied to the meat. Finally, he demonstrated that the active ingredient in gastric juices is unstable outside the body; that is, its ability to digest meat wanes with storage time.

Today we recognize all the foregoing properties as common features of enzymatic reactions, but in Spallanzani's day, these were novel and exciting findings. The same time period saw the discovery of enzyme activities in a large number of other biological systems. For example, a peroxidase from the horseradish was described, and the action of a-amylase in grain was observed. These early observations all pertained to materials-crude extract from plants or animals-that contained enzymatic activity.

During the latter part of the nineteenth century, scientists began to attempt fractionations of these extracts to obtain the active ingredients in pure form. For example, in 1897, Bertrand partially purified the enzyme laccase from tree sap, and Buchner, using the "pressed juice" from rehydrated dried yeast, demonstrated that alcoholic fermentation could be performed in the absence of living yeast cells. Buchner's report contained the interesting observation that the activity of the pressed juice diminished within 5 days of storage at ice temperatures. However, if the juice was supplemented with cane sugar, the activity remained intact for up to 2 weeks in the ice box. This is probably the first report of a now well-known phenomenon-the...