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Science of Enzymes

ENZYMES are Bio-catalysts, i.e. substances which accelerate a certain chemical reaction without actually being consumed themselves. They are found in every cell of all living beings, from simple single cellular organisms to highly complex multicellular organisms, including human beings.

ENZYMES form an important part of our daily lives; from assisting in the production of the food we eat, to contributing to the care of our health by providing therapeutic agents or sensitive and specific diagnostic tests.

The most frequent biochemical reaction used when applying industrial enzymes is the hydrolytic decomposition of high molecular weight substances such as starch, proteins, cellulose, etc. ENZYMES are composed of proteins and also react as such. They occur throughout nature and control the build-up and decomposition of essential matter in vegetable and animal organisms. By "production", what is really meant is the isolation and purification of enzymes derived from micro-organisms, animal organs, or vegetable extracts.

The knowledge of some special properties is necessary for handling of industrial enzyme products. Each type of enzyme has a certain pH range in which it's affect is optimal. The activity increases with the rise of temperature, until heat inactivation takes place. Apart from a change in pH value or exposure to heat, enzymes are also inactivated by heavy metal ions and protein precipitating agents. Full activity will be maintained in dilute solutions for a few hours only. Liquid products should be stored in cool conditions, whereas dry storage is particularly important for products in powder form.

A specific enzyme will normally catalyze only the reaction for which nature has designed them, e.g. a pectinase can only degrade pectin, not starch or cellulose.

Biotechnology has assumed far greater significance in the past ten years. Several enzymes, which were available in research quantities, only are now commercially available. The opportunities for using enzymes increase daily, as does the skill required to research, develop, and market these vitally important products to a whole range of industries throughout the world.

A recent rough estimate of the world market for industrial enzymes put the sales at around 95,000 tons of commercial product. Predictions of growth of the market forecast a rise to 10 - 15% per year.

There are only about 25 enzyme-producing companies in the world. In the Western nations, almost half of all enzyme production is in Denmark, with Holland producing the further 20 %, American production is responsible for about 12%, mainly for their captive use. Japan, West Germany, France, Switzerland, Ireland, and UK account for the remainder of the enzyme production. Production in the Russian states and China are significant, but not readily quantified, since virtually none of the products are made available to the Western nations. In India, Specialty Enzymes is the only major enzyme manufacturing company.

SOURCES AND TYPES OF ENZYMES:

Around 80% of all industrial enzymes are hydrolytic in action and they are used for depolymerization ( i.e. breaking down of complex molecules in to simpler molecules.) of natural substances. Almost 60% of these are proteolytic, for use by the detergent, dairy and leather industries. The charbohydrases, used in baking, distilling, brewing, starch and textile industries, represent almost 30% of the total enzyme usage, leaving the lipases and highly specialized enzymes, such as pharmaceutical, analytic, and developmental.

There are three major sources of enzymes: Plant Enzymes, Animal Enzymes, and Microbial Enzymes.

 

PLANT ENZYMES: These are enzymes basically derived from plants. These include the well known proteases such as papain, bromelain, ficin and the amylolytic enzymes of the cereals, soybean lipoxygenase, and specialized enzymes from the citrus fruits. Most plant enzymes are available as comparatively unpurified powder extracts, although papain is notable for being recently available as a stabilized and purified liquid.

ANIMAL ENZYMES: These are enzymes which are basically derived from animal glands. These include the pancreatic trypsins, lipases, rennets, and other enzymes such as pepsin, which are produced in both ultra pure and industrial bulk qualities.

MICROBIAL ENZYMES: These are enzymes derived from micro-organisms and include fungal and bacterial amylases, diastases, etc.. Most industrial microbial enzymes are produced from no more than eleven (11) fungal, eight (8) bacteria, and four (4) yeasts.

STABILITY AND COMPOSITION:

It is not sensible to give a specific enzyme product composition, as there is a huge variation potency and physical presentation. Those produced by extraction from plant and animal tissues will contain different substances, in addition to the active enzyme, than those from microbial fermentations. Apart from noting the small proportion that is actually active enzyme protein, sugars and inorganic salts, are used as alternatives when establishing the stability of the finished product for storage and distribution, and are selected according to the acceptability in the intended application. Salts and sometimes carbohydrates such as starch, are used to dilute extracted enzymes to standard activity.

Preservatives are generally restricted to liquid enzyme preparation.

Industrial enzymes are commonly used at levels of 0.1 to 0.5% of the substrate ( i.e. substance on which the enzyme is expected to act. ) being processed, with rare exceptions above these levels.

Generally, dry enzyme products have a longer shelf-life than liquids, and cool conditions extend their shelf-life even further. Dry products should not lose more than 15-20% of their activity over a 12 month period from the date of manufacture if stored in a cool dry place (optimal storage 23° C or below). Liquid enzymes should have a shelf life of 3 months at 25°C and approximately six months if stored at 0° to 4° C.

PROCESSING ENZYMES SELECTION: Generally, the following factors are considered while selecting an enzyme for a particular process with activities that appear appropriate.

a) SPECIFICITY : It is of prime importance to understand that enzymes are very specific in their action, i.e. one enzyme will act on only one specific substrate only to give a particular result. The generally claimed specific action of enzymes is not as sharply defined as it is often expected. Proteases are broad in the range of amino acid bonds they hydrolyze and exhibit only a degree of specificity. Among the carbohydrates, there are both highly specific enzymes, such as the components of the pectinolytic group or the lactases, and also broad acting amylases and beta-glucosidases. Within any group there are small differences of action depending upon the source and the type of enzyme, and these can be used to advantage in obtaining precise reaction products. Thus, the first question will be to determine what degree of specificity is required in the reaction.

b) pH : Each enzyme acts in a particular range of pH. Both the optimum operating value determined under analytical conditions, and the actual ability of the proposed industrial system to adjust away from the possibly unsuitable pH in relation to the enzyme stage, must be considered. Some limits will be dictated by the practicalities of the overall process, and these may influence the choice of a particular enzyme among alternatives. Again, the operation of an enzyme outside the anticipated best performance range may modify it's specificity or sensitivity to heat in both beneficial or adverse ways.

c) TEMPERATURE : When considering enzyme processes, the general rule is that the temperature quotient is between 1.8 to 2.0. The reaction rate generally increases or decreases by this order for each shift of 10° C. By using high temperatures, the reaction may be of short duration and hygienic conditions may be maintained more easily. Conversely, the use of much greater heat for thermolabile enzymes, (enzymes sensitive to heat) will be necessary to inactivate the enzymes at the end of the process. Alternatively, a significant shift of pH may be necessary to inactivate without a rise in temperature. This may not be necessary in every process.

d) ACTIVATORS AND INHIBITORS : These are usually well defined for specific enzymes and should be taken into account if they create expensive additional costs, or costly treatments to insure their elimination from reaction. Under certain circumstances, the deliberate omission of a known activator will alter the pH or temperature sensitivity of an enzyme to an extent that limits the reaction or simplifies the end process inactivation conditions.

e) ANALYSIS METHOD : This is an essential tool in process control and the detection of low levels of activity when monitoring for residual action after an inactivation treatment. Many industrial users of enzymes also maintain routine activity checks on stock enzymes to ensure good stock rotation. Therefore, when selecting an enzyme, it is helpful to have one with an established analytical method readily available.

f) TECHNICAL SUPPORT AND SERVICE: Major improvements in the range of industrial enzymes are regular occurrences, and it is important to establish that the selected enzyme is the most upto date of it's type, and that the performance data and guidance on it's application are regularly updated.

g) COST : This is a serious factor when choosing an enzyme for many processes. Clearly, no more expensive product than that necessary for the action should be selected. However, care has to be taken to establish that purity and activity are consistent and that any side activities have been identified, at least those that might influence the processing and product under consideration.

ENZYME KINETICS: The enzyme kinetics is the study of reaction rates catalyzed by enzymes, and the factors affecting them.

When considering the industrial uses of enzymes, the following questions arise:

How much enzyme is needed?
How long is the reaction time?
What are the concentrations of substrates required?
What physical conditions of temperature, pH, and ionic strength should be used for optimal reaction?
How much is it going to cost?
Enzyme kinetics provides some useful answers to all these questions. The cost-effectiveness of an enzyme-catalyzed is derived directly from the relationship between the additional cost of the enzyme system involved and either the final added value of the product obtained, the cost saving achieved by overall cheaper processing, or the higher product yield. In reality this reduces to the relationship between the enzyme concentration required, the physical conditions operating, and the degree of conversion obtained.

Industrially used enzymes are characterized by the nature of the reaction they catalyze and their catalytic activity. The qualitative description of the chemical reactions they catalyze forms the basis for their classifications, while their catalytic activity gives an indication of the magnitude of their effect, and on unit weight basis, an indication of their value. Since nearly all industrial enzymes are of relatively low chemical purity, they are primarily characterized as specific catalysis rather than defined active proteins.

Enzymes are sold primarily on activity basis-that is, a quoted cost for a specified activity. Secondary features such as degree of purity, extent of modification (stabilization, activation, or physical form) and microbiological specifications can modify this cost. Enzymes for analytical and medical purposes are often in a state of medium to high purity, and are sold in terms of numbers of enzyme units per lot, whilst those for industrial processing are quoted on a unit weight basis for a standardized product of guaranteed activity per unit weight. This applies to most conventionally produced solid and liquid products.

The quantitative activity of enzymes gives an indication of how much enzyme should be used to achieve a required effect (product yield) and forms the basis for comparison of several similar enzyme products. As enzymes are catalysts, and therefore reformed at the end of reaction, a minute amount of enzyme can transform any amount of substrate. However, as is the case with any chemical catalyst, there is a practical and finite activity and concentration of substrate acted upon. This governs the speed of product formation, and is described by enzyme kinetics. Activity also serves as a basis for estimation of added product cost, and as a guide in assessing investment and maintenance costs, since the activity of an enzyme will have an important influence on plant throughout and hence plant size for a defined product target production rate.

ACTIVITY UNITS:

The catalytic effect of an enzyme is quantitatively expressed in terms of units of activity. These have, naturally enough, been defined under (near) optimal, or idealized, conditions so as to give a set of favorable standard for comparison.

One unit (U) of enzyme is defined as that amount which will catalyze the transformation of one micromole of substrate per minute under defined conditions.

Wherever possible, the reaction temperature should be 25° C. The other conditions including pH and substrate concentration, should where practical, be optimal and defined. Where more than one bond of each substrate molecule is attacked, one micro equivalent of the group concerned is considered. From this basic definition, two derived expressions are suggested;

Specific Activity - units of enzyme per mg protein for (N times 6.25)

Molecular Activity - units of activity per micromole of enzyme

THE TIME COURSE OF AN ENZYME REACTION:

Although industrial application of enzymes is primarily concerned with maximizing the yield of product, clearly, reaction times must be realistic. Enzymes used on an industrial scale currently operate at reaction times varying from a few minutes (e.g. liquefaction of starch) to several days (i.e. saccharification of liquefied starch) up to overall reactions, but this has to be balanced by reactions due to processing and scale-up conditions, and must also be economically justifiable. Generally, three phases for the overall time course of an enzyme reaction are recognized. These are:

Phase 1: The transient initiation phase - a few seconds duration.

Phase 2: A steady state phase - the initial reaction rate.

Phase 3: A non liner phase - the main reaction, up to completion.

Industrial applications of enzymes are thus primarily concerned with phase 3, although phase 2 is important, especially when measuring enzyme activity, or designing new enzyme processes. Phase 1 is not without interest, as it provides the model on which the enzyme reaction can be considered to proceed.

THE OPTIMA CONCEPT: A

When defined the proposed unit of activity for any enzyme, the reaction conditions should be specified and optimal. This implies that enzyme activities are only valid within a range of physical properties. The properties of enzymes, like those of all proteins, are modified by prevailing physical conditions, and only within a fairly narrow range of conditions is any one property maximal or optimal.

A given enzyme will exhibit its catalytic property only within the specified ranges of physical conditions. These ranges of conditions are the optimal conditions for the enzyme and the conditions under which it expresses its maximum effect. Outside these ranges enzymes performance is either considerably reduced, or completely obliterated.