Use of Enzymes in Biotechnology

In this essay I will attempt to explore the uses and other important aspects of using enzymes in biotechnology, pondering over some of the most well known industries to current everyday life.

Enzymes are used in an ever widening range of industrial applications including but not limited to detergents manufacture, textile modification, leather processing, paper production, and food production. Enzymes are also used in the pharmaceutical industry for the bioprocessing of therapeutic molecules where their specificity, efficiency, and biodegradability are particularly advantageous. For instance, proteases such as trypsin and carboxypeptidase B are useful for the bioprocessing of therapeutic proteins. An example of such use is the manufacture of insulin via microbial fermentation that requires both of these enzymes in order to process the fermentation derived insulin precursor into a therapeutic entity identical to human insulin.

Biotechnology has been defined as:

The application of biological organisms, systems or processes to manufacturing and service industries.
The integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological application capabilities of micro-organisms, cultured tissue cells and parts thereof.
The application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services.
The use of living organisms and their components in agriculture, food and other industrial processes.
The integration of natural sciences and organisms, cells, parts thereof and molecular analogues for products and services.

Whatever definition is used to describe biotechnology it is clear that the subject encompasses a wide range of "traditional" or "old" industries such as brewing, baking, cheese production and mushroom cultivation, together with the more recent "modern" technologies especially in the pharmaceutical industry (antibiotic production, vaccines and

diagnostics) and other industries (food, paper, textiles and waste management).

It is important to remember that biotechnology is not exclusively genetic engineering as many biotechnological processes merely harness completely natural (or un-engineered) products or systems and simply adapt them to work in a different environment or host. Today, biotechnology remains the fastest growing industrial sector in terms of both technological advancement and market value. Many of these industrial sectors already utilise enzyme biotechnology the range of applications continues to grow rapidly. The sales of biotechnology products was estimated to be 6.4 billion US$ in 1990 and is forecasted to rise to 65 billion US$ by the year 2000.

Enzymes are put to valuable use in agriculture, the major use of utilising enzymes is in the feeding of monogastric animals ( which will be discussed later). Biological silage inoculants frequently contain enzymes in addition to lactic acid bacteria. The enzymes in such products partially breakdown some of the cell wall components of the plant material to be ensiled into soluble sugars. These liberated sugars are then metabolised by the natural or applied lactic acid bacteria such as Lactobacilli or Pediococci into lactic acid which reduces the pH and so ensiles the crop. Some enzyme preparations have been reported to improve the utilisation of feeds for ruminant animals. The use of enzymes in arable agriculture especially in the processing of some major crops and in waste disposal systems are areas which have not been fully investigated.

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The main components of animal feed are plant materials, in particular cereals such as maize, wheat and barley which provide carbohydrate energy sources and vegetable protein sources such as soya and other beans, peas, sunflower and lupins seeds. Whilst the majority of starch present in cereals is readily digested by monogastric animals, a large proportion of the energy content is present as non-starch polysachharides (NSP). The NSP's such as arabinoxylans in wheat and rye and ß-gucans in barley and oats are soluble and result in increased viscosity in the gastrointestinal tract of the animal which impaires digestion. Such NSP's are therefore frequently referred to as anti-nutritional factors (ANF). The addition of enzyme activities not present in the animal can overcome the effect of these ANF's. Feed containing predominantly wheat can be improved by the addition of xylanases to degrade the arabinoxylans and feed containing predominantly barley can be improved by the addition of ß-glucanase to degrade the ß-glucans. Such enzyme addition has become very popular. Over 90% of the poultry diets containing wheat and/or barley in the European Community are supplemented with enzymes to degrade the polysaccharide ANF's.

There is another application of enzymes in animal feeding, which is the release of phosphorus from phytic acid which is the main store of phosphorus in plant material. This addition of phytase liberates this bound phosphorus fraction, thereby reducing the need for phosphorus supplements to the feed and hence reduces the environmental impact of phosphorus excretion in animal faeces.

Bread baking is one of of the most common food processing techniques throughout the world although the bread products of different countries vary in their finished form. The basic component of all bread is wheat flour to which is added water, salt and yeast. Other ingredients are sometimes added such as sugar, fats and flavouring components. Wheat flour has naturally occuring enzymes that modify the starch, protein and fibre fractions of the flour when water is added to make the dough. The added yeast also has enzymes and ferments maltose and other sugars to produce carbon dioxide which results in the bread rise. So baking is an ancient form of fermentation by a combination of enzymes from yeast and wheat, converting the difficult to digest material into the "staff of life".

Enzymes have many roles in the pharmaceutical and diagnostic industries. There are too many applications to cover but typical applications include:

Enzymes as direct pharmaceutical products such as in the treatment of genetic disorders leading to a specific enzyme deficiency.
Extraction of medicinally important compounds such as heparin.
Manufacture of chemical pharmaceuticals where enzymes are used for the interconversion of chemical intermediates or the removal of chiral components.
Combinatorial biocatalysis.
Research and development - especially in the field of molecular biology where almost all the processes of nucleic acid manipulation are performed with enzymes (eg. restriction endonucleases and DNA polymerases).
Diagnostic procedures also frequently utilise enzymes - some examples include luciferase, glucose oxidase.

Enzymes in the fermentation industries are absolutely vital for such productions as

wine. Wine making is essentially the fermentation of grape juice. The extraction of the grape juice is however a more complicated process than other fruit juices as the extraction of flavour components and colours (in the case of red wines) is far more important.

Grapes are harvested for wine making before the fruits have fully ripened. The unripe fruit therefore contain large amounts of insoluble protopectin which can absorb large quantities of juice during pressing and also result in viscous solutions which are difficult to process. The addition of pectinases during mashing can hydrolyse the pectins which increases the yield of juice, clarifies the resulting juice and prevents the juice from gelling. Some grapes also contain large amounts of arabinoxylans which can be treated with xylanases to aid processing.

Another specific problem in winemaking is the frequent infection of the grapes by the fungus Botrytis cinerea which produces beta-glucans which pass into the wine. These large molecular glucans hinder the clarification of the wine by clogging the filtration devices. The addition of beta-glucanases can overcome this effect. Other specialist uses of enzymes in wine making include the addition of proteases to improve colour stability in red wines by reducing the binding of polymerised tannins to proteins and the use of glycosidases to hydrolyse terpenyl glycosides thereby increasing the aroma or bouquet of the wine.

Another huge industry in the biotechnological field is that of the starch industry. A considerable quantity of the sweeteners used throughout the world are derived from starch as opposed to cane or beet sugar. The enzymatic treatment of starch has become much more popular than acid hydrolysis. The treatment of starch with enzymes results in a variety of sweet syrups used thoughout the food and beverage industries. Three stages can be identified in starch modification. Firstly, amylases liberate "maltodextrin" by the liquefaction process. Such maltodextrins are not very sweet as they contain dextrins and oligosaccharides. The dextrins and oligosaccharides are further hydrolysed by enzymes such as pullulanase and glucoamylase in a process known as saccharification. Complete saccharification converts all the limit dextrans to glucose, maltose and isomaltose. The resulting syrups are moderately sweet and are frequently modified further.

Enzymes have been used in the detergent industry for a great amount of time and is probably the best known application of industrial enzymes especially in laundry products. The main enzyme activity in biological laundry detergents is protease which acts on organic stains such as grass, blood and human sweat. Although it has become apparent in recent years that a mixture of enzymes are needed, including lipases and amylases. Lipases are effective on stains from fatty products, whilst amylases help remove starchy food deposits. More recently washing powders that enhance colour have been introduced containing cellulases. It is thought that the mode of action of such cellulases is to remove detached cellulose fibrils which cause a progrssive dulling of the colour as dirt is trapped on the rough surface of the fabric.

As you would expect fruit juices require the use of enzymes as well, enzymes exist for the non-citrus juices and the citrus juices. In non-citrus fruit juices enzymes are used to maximise the production of clear juice. Nearly all fruits and berries contain pectins and other polysaccharides such as starch and aribinoxylans. Pectins hold the fruit cells together like a "glue" and result in poor liberation of juice during pulping. The presence of soluble pectins in the subsequent juice also causes hazing. The addition of pectin degrading enzymes (pectin methyl esterase, polygalacturonase and pectin lyase) at the pulping stage increases the yield of juice and helps in the clarification. Pectin degrading enzymes are particularly important in the production of fruit juice concentrates as pectins can form very viscous gels which hinder filtration and concentration to high levels of dissolved solids. Cellulases also play a role in the extraction of juice from berries where juice yield together with the extraction of colour and flavour components can be difficult.

For the processing of citrus fruits, enzymes are required in order to maximise the production of cloudy juice. The problems of extracting juice from citrus pulp and reducing the viscosity of the juice for concentration are similar to those of non-citrus fruit processing. However, citrus juices and in particular orange juice are meant to be cloudy as much or the desired flavour and colour depends on the insoluble, cloudy materials of the pressed juice. Cloud stability is controlled by careful manipulation of the pectin component of the juice. This complex process requires a balance between pectin methyl esterase which will promote cloud formation by increasing pectin / calcium complex formation and polygalacturonase which will break cloud formation by depolymerisation of the pectin before complex formation.

We have already looked at the wine making industry, which with its alcohol counterparts beer require the inclusion of enzymes to enable fermentation to exists. Beer brewing essentially involves the the production of alcohol by the action of yeasts on plant materials such as barley, maize, sorghum, hops and rice. The yeast cells are capable of converting simple sugars into alcohol and carbon dioxide. However, most of the sugar present in plant materials is in the form of complex polysaccharides such as starch and cannot be readily utilised. Traditionally these nutrients are "released" by the process of malting whereby barley is allowed to partly germinate during which endogenous enzymes are released which degrade starch and protein to simple sugars and amino acids which can be utilised by the yeast cells.

The malting process is a relatively expensive way of manufacturing enzymes and is not always easy to control. Industrial enzymes such as amylases, glucanases and proteases can be added to unmalted barley resulting in the same simple sugars and amino acids that malting would liberate but in a more controlled fashion.

Enzymes also play an important role as filtration improvers. Slow filtration of the mash or final beer often results from the presence of viscous polysaccharides such as xylans and glucans. Pretreatment with xylanases or glucanases break down these viscous polysaccharides thereby increasing filtration rates and preventing fouling of filtration membranes.

Food processing is maybe the most important of all biotechnology industries as we require food to stay alive, it’s part of our everyday life. As you would probably expect the enzymes involved in this industry are too numerous to be able to name in detail here, so a brief analysis is given. The baking industry has already been covered earlier on, that on its own belongs to the food processing industry.

Enzymes can be used to modify raw materials and aid in the processing or cooking stages. The roles of enzymes include: enhancement of flavour and aroma, removal of unwanted flavours and taints, enhancement of digestibility, modification of texture to aid processing and final product appearance, upgrading raw materials.

The main enzyme activity utilised in food processing applications is protease. However, applications utilising lipases and carbohydrate degrading activities are also becoming widespread.

Some well known specific applications include:

Cheesemaking - rennet is responsible for curd production from milk, many flavours in cheeses are a result of protease action.
Production of infant milk formulas from cows milk.
Soy sauce production.
Production of hydrolysed vegetable protein.
Gelatin hydrolysis.
Production of meat extracts.
Pet food manufacture.