To allow enzyme-bearing products to be applied effectively in an industrial setting, one must have a basic understanding of these products' characteristics, advantages, and limitations.

Enzymes are "biological catalysts." "Biological" means the substance in question is produced or is derived from some living organism. "Catalyst" denotes a substance that has the ability to increase the rate of a chemical reaction, and is not changed or destroyed by the chemical reaction that it accelerates.

Generally speaking, catalysts are specific in nature as to the type of reaction they can catalyze. Enzymes, as a subclass of catalysts, are very specific in nature. Each enzyme can act to catalyze only very select chemical reactions and only with very select substances. An enzyme has been described as a "key" which can "unlock" complex compounds. An enzyme, as the key, must have a certain structure or multi-dimensional shape that matches a specific section of the "substrate" (a substrate is the compound or substance which undergoes the change). Once these two components come together, certain chemical bonds within the substrate molecule change much as a lock is released, and just like the key in this illustration, the enzyme is free to execute its duty once again.

Many chemical reactions do proceed but at such a slow rate that their progress would seem to be imperceptible at normally encountered environmental temperature. Consider for example, the oxidation of glucose or other sugars to useable energy by animals and plants. For a living organism to derive heat and other energy from sugar, the sugar must be oxidized (combined with oxygen) or metabolically "burned". However, in a living system, the oxidation of sugar must meet an additional condition; that oxidation of sugar must proceed essentially at normal body temperature. Obviously, sugar surrounded by sufficient oxygen would not oxidize very rapidly at this temperature. In conjunction with a series of enzymes created by the living organism, however, this reaction does proceed quite rapidly at temperatures up to 100oF (38oC). Therefore, enzymes allow the living organism to make use of the potential energy contained in sugar and other food substances.

Enzymes or biological catalysts allow reactions that are necessary to sustain life proceed relatively quickly at the normal environmental temperatures. Enzymes often increase the rate of a chemical reaction between 10 and 20 million times what the speed of reaction would be when left uncatalyzed (at a given temperature).

Nutrients locked in certain organics are complex macromolecules, or in hard-to-digest matrices may be released or predigested by a high degree of heat or concentrated acid treatment. In an alternative manner, specific enzymes can promote the predigestion of certain complex nutrients and facilitate the release of highly digestible nutrients in organics during processing without the need of excessive heat or rigorous chemical treatment.

What Comprises an Enzyme?

The majority of enzymes are purely protein in nature. Over 750 known enzymes are comprised completely of protein. Many enzymes must have other components, however, to maintain their activity and function. Some enzymes require various metals such as sodium, calcium, zinc, iron, or copper to become active. Many enzymes require other organic, non-metal components to insure their stability and/or activity. This additional organic component is known as a "prosthetic group" or "co-enzyme". Many vitamins are co-enzymes; they are necessary components for the activation of certain enzyme systems within the body of living organisms.

How Are Enzymes Named?

One researcher reports treating grain, sorghum or barley with the enzyme "gumase" while another reports the same with the enzyme "beta-glucanase". When methodologies are examined, it is discovered that both of these preparations are the same product. Unfortunately, this apparent contradiction in terms happens often.

Enzymes have been named by several methods and this fact has been known to cause confusion in their classification. For example, common or "trivial" names of enzymes, generally contain a prefix representing the name of the substance or substrate upon which they act or affect, followed by the suffix "-ase". The "ase" simply denotes or identifies that the substance is an enzyme. Examples of this system of nomenclature includes the enzyme that catalyzes the conversion of proteins into their component amino acids, the name of this enzyme is "protease" or "proteinase".

Another example is the enzyme that accelerates the breakdown of the two components of starch into sugars. The components of starch are known as "amylose" and "amylopectin", thus, the enzyme helping to break them down is called "amylase".

Confusion may exist, however, when older names of enzymes are used. Included in these older terms are ficin, pepsin, bromelin and trypsin, which are older trivial names of individual types of protease preparations, the enzymes that accelerate digestion of proteins. There are also many subclasses of enzymes. Amylases are a prime example; subclasses of amylase include: alpha-amylase, beta-amylase, and gluco-amylase, to name a few. All these enzymes do is accelerate the digestion of starch and are broadly classified as amylases, but their actions are all slightly different in nature.

To help sort this out, the International Union of Biochemistry in 1961 proposed a system for enzymes' classification and naming which is finding acceptance mainly in this discussion. One example of this system, however, is the term: "alpha 1, 4-glucan glucanohydrolase" which is a name for alpha-amylase.

All these systems of nomenclature may become confusing to someone who has use for only a few types of enzymes or uses them for industrial or agricultural purposes. Therefore, the use of the more widely known terms such as "amylase" and "protease" are more or less universally in these fields. It should be remembered, however, that there are many types of enzymes that fit into these broad categories that may be more or less suitable for specific agriculturally related application. The final selection for a specific application should be made only after consulting a knowledgeable individual well-versed in the technical aspects of the particular enzyme requirements and applicable characteristics.

Where Do Enzymes Come From?

Enzymes have been isolated from every type of living organism. Many of these biological catalysts are significant only from an academic or medical standpoint, but some of the available enzymes from this vast repertoire have been utilized for agricultural and industrial purposes for years. The table below lists several of the industrially consequential enzymes and their sources in nature. It is significant to note that animals, plants, and micro-organisms all yield industrially important enzymes*. Some enzymes of animal or plant origin have been used in agricultural applications; however, those enzymes most broadly used are of microbial origin.

Where Do Enzymes Come From?

Source Enzyme

PLANT Malted grains or tubers Amylase

Pineapple Bromelin (Protease)

Fig Tree Ficin (Protease)

Papaya Papain (Protease)

ANIMAL Liver Catalase (Peroxide Breakdown)

Calf Stomach Rennet/Chymosin (Milk Clotting)

Hog Stomach Pepsin (Protease)

Hog Pancreas Pancreatic Enzymes (Several)

Digestive Tract Trypain (Protease)

MICROBIAL Fungi (Molds and Yeast) Amylase, beta glucanase, hemicellulase, protease, cellulase, pectinase, lipase, (many types of each), lactase

Bacteria Amylase, protease, isomerase, lactase (many types of each), rennet, oxidase, catalase, beta-glucanase, hemicellulase.

*While enzymes for diagnostic or medical purposes are most important for the benefit of thousands of patients yearly, their discussion is beyond the scope of this article.

One encounters many digestive or hydrolyzing enzymes in the digestive tract of human and other animals. These biological catalysts are necessary for the full utilization of foods ingested. Microorganisms, many being as small as 1/10,000th of an inch in length, are much too minute to have complicated digestive systems as animals do. Therefore, these microbes must predigest their potential foods outside of their cell boundaries so that they ca absorb the very small nutrient compounds of predigested foods.

In order to predigest the potential food sources outside their cell boundaries, many microbes excrete enzymes out through their enveloping membrane with its supportive cell wall and into the surrounding environment. Since these "extracellular enzymes" must function in the environment outside the protection of the cell's wall and membrane, they must be reasonably stable and have relatively high resistance to chemicals and must function over a relatively broad temperature range. To realize the effects of the enzymes they produce, microorganisms also must produce relatively large quantities of these catalysts. All of these factors contribute to the industrial significance and durability of extracellular microbial enzymes.

It should be noted that most of the agriculturally and industrial important enzymes, are those that catalyze the digestion or "hydrolysis" of certain large organic molecules like starch, cellulose, and protein. The enzymes actually attack these complex molecules, accelerating their digestion and yielding simpler substances. Since this process of digestion is referred to as hydrolysis, the enzymes that catalyze the process are considered to be "hydrolyzing enzymes" or "hydrolases".

The hydrolyzing enzymes include:

(1) Amylases, which catalyze the digestion of starch into small segments of multiple sugars and into individual soluble sugars.

(2) Proteases, (or proteinases), which split up proteins into their component amino acid building blocks.

(3) Lipases, which split up animal and vegetable fats and oils into their component part: glycerol and fatty acids.

(4) Cellulase (of various types) which breaks down the complex molecule of cellulose into more digestible components of single and multiple sugars.

(5) Beta-glucanase, (or gumase) which digest one type of vegetable gum into sugars and/or dextrins.

(6) Pectinase which digests pectin and similar carbohydrates of plant origin.

 
How Are Microbial Enzymes Produced and How Are Microbial Enzymes Labeled?

Microbial enzymes are manufactured by growing the microbial cells under specialized conditions so that these cells produce their maximal amount of active enzymes. It is important to control environmental conditions during productions so that a high percentage of these active catalysts are preserved intact.

After the microbial cells have finished growing and producing their enzymes, they may be inactivated and harvested along with the enzymes, or the material may be processed in various ways in order to reach several stages of purificatory of the enzymes. Where other concerns, like solubility or sprayability of the final enzyme-containing product enter, the use of a semi-purified solubles or extracted, soluble products are desirable.

Depending upon the products' degree of processing and selectivity of enzymes contained, the products are listed as follows in the ingredients statements:

Dried _________________ Fermentation Extract

Dried _________________ Fermentation Solubles

Dried _________________ Fermentation Product

Liquid ________________ Fermentation Product

 
The scientific name of the microorganism used to produce the enzymatic ingredient would be substituted for the blank provided. The AFFCO Official Publication specifies that enzyme activity will be guaranteed for those products that represent themselves as having enzyme activity.

What Affects the Activity and Stability of Enzymes?

Waste processing levels usually dictate some variation in physical conditions under which the enzyme products must function. In order to utilize enzymes to their optimal potential in catalytic ability, we must be familiar with the basic principles that can affect the activity and stability of these enzymes. Enzymes, being biological compounds and being comprised of a high percentage of protein, are subject to many environmental effects. Although the following principles hold true for most biological enzymes produced for commercial agricultural use:

The pH of the environment has a profound affect on enzyme activity and stability. Activity optimal for pHs of various enzymes vary; however, the optimal pH's for the biological catalysts produced by most commercial strains of microorganisms lies between pH 4.0 and 7.5. This range is from moderately acidic to mildly alkaline in nature. These are the pH levels normally encountered. Figure 1, indicates a difference in activity levels that various enzymes exhibit at varying pH levels.

Another major affect of enzyme activity and stability is temperature. Since enzymes are biochemical catalysts, made up at least partially of protein, they are sensitive in varying degrees to heat. Raising temperatures of the environment generally multiplies the degree of activity by the enzyme. Once an optimum temperature has been reached, however, even higher temperatures cause rapid degradation of the enzyme with concurrent and irreversible loss in activity (See Figure 2). Optimal temperatures generally range from 98oF to 140oF (37oC to 60oC) for most hydrolytic enzymes. High temperatures (over 150oF, 66oC) generally have detrimental effects on the enzymes. However, there is broad variation in resistance and sensitivity to heat among the enzymes' types. Bacterial enzymes such as those from Bacillus subtilin are less sensitive to heat than are the fungal enzymes of A. oryzae. Some amylase preparations prepared by the fermentation of Bacillus species can withstand even boiling for short periods and have optimal activities in the 158o-176oF (70o-80oC) range. Our laboratory has determined that approximately 85% of the activity from B. subtilin/licheniformis alpha-amylase survives high heat. A. oryzae amylases, however, showed a greater than 90% loss activity in high heat. When the enzyme-bearing, dried fermentation products of these two microorganisms are kept dry, they are much more resistant to environmental temperature stress than if they are moistened. In fact, very few stability problems are encountered with most enzymes in typical situations.

Basic Knowledge of Enzymes Applied

Making use of general knowledge about enzymes including how they act, under what conditions they perform, and how to preserve their activity is important in applying the technology of enzymes to organic waste digestion.

 

HELPFUL REFERENCES ON ENZYMES:

Aunstrup, K. 1979. In Enzyme Technology, L.B. Wingard, Jr., E. Katchalek-Katzir, L. Goldstein (ed). Academic Press, Inc., New York.

Boyer, P.D. (ed). 1971. The enzymes, 3rd, ed. Vol. 5. Academic Press, Inc., New York.

Fogarty, W. M. 1974. Enzyme technology - projects and developments. In Projects and prospects in industrial fermentation: proceedings of meeting held in Holly Royde. A. J. Powell and J. D. Bu'Lock (ed). U. of Manchester. Manchester, England.

Godfrey, T. and J. Reichelt (eds). 1983. Industrial enzymology: the application of enzymes in industry. Nature Press. New York.

Kulp, K. 1975. Carbohydrases and other enzymes.

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