Discuss the Medical and Commercial Applications of Enzyme Technology

Enzymes were first discovered towards the end of the nineteenth century by a German chemist called Edward Buchner (Roberts,1986). Like other scientific discoveries it was a fortuitous accident. He was trying to extract the yeast from a fluid used in medicine, but the extracts would soon go ‘bad’. To prevent this he tried adding sugar to it and being most surprised when the sugar turned into alcohol. This was due to fermentation. The word ‘enzyme’ literally means ‘in yeast’ so is coined from the active juice that promotes fermentation.

Enzymes act as catalyst to almost all biological reactions. Without them the human body would not be able to operate at a fast enough rate and toxins would build up and the supply of respiratory substrate would decrease, life would not be able to be maintained. Enzymes are extremely specific generally only able to bind with one specific substrate, and after one binding they can do another and another, continuously remaining unchanged.

Enzymes are made up of amino acids so are therefore a proteins. The most important part of an enzymes structure is the tertiary structure as this determines its specific action. Only one region of an enzyme is used when it combines with its specific substrate to form an enzyme/substrate complex and it is called the active site. An enzyme may become denatured if it is exposed to excessively high or low temperatures and pH levels, which would damage the weak hydrogen bonds holding the tertiary structure altering the active site making it no longer compatible to its substrate. Their performance can also be affected by inhibitors, which either block the substrate or alter the enzymes structure.

There are two main theories as to how enzymes work. One is the lock and key theory, which was suggested in 1894 by Emil Fischer (http://www.enzymes.co.uk)

who believed that the active site of an enzyme is inflexible and fits exactly to the substrate.

The other, which is now more widely accepted by biologists, is the induced fit theory. In this theory, the enzyme is a molecule whose conformation can change as the substrate approaches and starts to bind. We now know that proteins are flexible molecules, whose overall structure is maintained by weak intermolecular interactions. At any given time, these can be disrupted by small changes in their vicinity.

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Enzymes are suitable in industry due to their specific functions. This allows companies to avoid worthless by products and often speed up processes in a more achievable environment, as with enzymes such high temperatures are no longer necessary. Companies which manufacture enzymes on an industrial scale require much research and specialist equipment. One gramme of soil contains about 40,000 micro-organisms, and each of these micro-organisms produces around 100 enzymes (Discover Enzymes, 2000). Advanced screening techniques are required to find the single correct enzyme, which could range from actinoplanase to zymomarase found from spinach to snake venom (Discover Enzymes, 2000). Using computers the molecular knowledge of the enzyme structure can be combined with DNA sequence knowledge is order to ensure that it is safe. If companies need a slightly different enzyme they are able to manipulate its primary structure which is simply the sequence of amino acids. The changing of one amino acid can change the enzymes function. Once the suitable enzyme has been selected it then needs to be cultured to make more. This is done by placing it on a culture which provides all the nutrients it may need. This process is, and was, on such a large scale that petri dishes were not sufficient and industrial fermentors were used. Out of the enzymes being used industrially, over a half are from fungi and yeast, over a third from bacteria and the remainder divided between animal (8%) and plant (4%) sources (Chaplin, 2002).

The most well known use of enzymes is proteases in biological washing powder, which break down proteins. The increased use of enzymes in washing power caused a dramatic increase in the sale of industrially produces enzymes in the 1950’s. Though in the early 1980’s there was a huge drop (Anderson & Rowland, 1995). This was due to the fact that the producers and users became hypersensitive to them causing skin irritations and complains such as hay fever, and irritated eczema. This problem was luckily soon resolved as the technology was soon developed to allow the manufacturers to capsulate the enzymes in a harmless coating, creating dust-free granules about 0.5mm in diameter. The waxy coating was often polyethylene glycol with other hydrophilic binders, which due to their water loving nature dispersed in the wash. Since the late 1980’s washing powder manufacturers have also isolated fat digesting enzymes (lipases) to rid clothing of tough greasy stains. Now about 25-30% of total enzyme sales are to the detergent industry (Anderson & Rowland, 1995).

Certain proteases have been used in food processing for many centuries. Rennet, obtained from the fourth stomach of an unweaned calf has been used traditionally in the production of cheese (Anderson & Rowland, 1995). Pectinase acts upon pectin, a substance that is found in cell walls helping to hold the structure together, it can therefore be used to partially digest fruit and vegetables in baby food, or to help extract the juices (Chaplin, 2002). The enzymes in yeast are essential to the baking of bread and brewing of beer, aswell as hundreds of other food and drink products. Not only has the food and drink industry benefited from the use of enzymes but so has the textile industry. When treating animal skins to make leather a chemical mixture of calcium hydroxide and sodium sulphide was once used to remove the hair. Although this method was effective many unpleasant side effects followed as it produced hydrogen sulphide, a poisonous gas. Enzymes posed the perfect solution. An alkaline protease enzyme is now used as it partially digests the keratin in the base of the hair, making it easy to remove. A more acidic enzyme, pancreatic enzymes can then be used for softening (baiting) the leather to make it more pliable. Due to the widespread use of protein-digesting enzymes the consumption of sodium sulphide has reduced by about 40% and pollution dramatically reduced (Indge, Rowland & Baker, 2000).

There is a vast worldwide demand for sweeteners, most commonly for confectionary and soft drinks. Originally sucrose was used which was obtained from sugar beet or sugar cane, but now a money saving, sucrose substitute alternative has arrived, high fructose syrup. Fructose is about ten times as sweet as sucrose and can be made from starch, a relatively cheap and abundant foodstuff; it is often a waste product from other areas of the food production industry.

Four enzymes are required for the production of high fructose syrup. These are bacterial α-amylase, fungal amyloglucosidase, pullulanase and bacterial glucose isomerase (Indge, Rowland & Baker, 2000). The bacterial and fungal enzymes are indeed obtained from bacteria and fungi, along with over 50% of enzymes produced industrially. The first three listed catalyse the reaction producing glucose from starch. Glucose isomerase then converts the glucose to about a 50:50 mixture of glucose and fructose. The end product is high fructose syrup. Sweeteners on the market such a canderel are aspartames, a dipeptide one hundred and eighty times sweeter than sucrose. The production of aspartame involves the enzyme aspartase. Also the soft centres inside chocolates are only possible because of the action of enzymes. To start with, the now runny centre was solid, a mixture of polypeptides and an enzyme. One the chocolate coating has set the enzymes get to work breaking down the polysaccharides providing the consumer a gloopy runny filling on consumption.

Enzymes have also been applied to medicine, and in some cases vanity, for example papain, a protease is used to remove stains from false teeth, and certain enzymes are used in hair dyes so less damage is done to the hair (Indge, Rowland & Baker, 2000). The research and development of enzymes for medicinal use has been at least as extensive as those for industrial applications. The magnitude for potential rewards are what keeps funds flowing as finding a cure for diseases, such as cancer would be a medical breakthrough. Pancreatic enzymes are one of the earliest know used of enzymes as medicine in the nineteenth century for the treatment of digestive disorders. Nowadays there are many important therapeutic enzymes such a asparginase for acute lymphocytic leukaemia sufferers (Chaplin, 2002). Every care has to be made when developing and testing new medicinal enzymes as some may be recognised by the users body as foreign proteins and could elicit a severe or life threatening allergic response.

In the 1970’s the immobilisation of enzymes became common (Anderson & Rowland, 1995). It involved a technique in which the required enzyme is bound immovably to a surface and not able to dissolve in the substrate solution. Various methods are used from entrapment in a gel micro-capsule, bound to cellulose fibres to being absorbed in a collagen matrix. This was a huge benefit as the enzymes were kept purer and could easily be removed afterwards to be used over and over again preventing the wastage of enzymes left in the end product. Also the matrix surrounding it makes the enzymes more stable at extremes of temperature and pH.

Enzymes seem to have been a blessing enabling the production and development of many areas of industry at a huge rate. The fact that enzymes are specific in their reactions, reacting with a single substrate means we can use them guaranteeing a specific product, so are unlikely to produce unwanted and wasteful by-products. Although enzymes are highly sensitive to changes in their physical and chemical environment meaning that the condition that they are exposed to have to be stringently controlled to avoid them being denatured. In addition the equipment put in contact with the enzymes must be scrupulously clean to avoid contamination, which may hinder the reactions. The conditions at which enzymes function at their optimum are easily obtained as they work in moderate temperatures and neutral pH and normal atmospheric pressure saving energy that would otherwise be essential without enzymes. Another disadvantage is that enzymes are difficult to purify meaning that the cost may be rather expensive but they are organic therefore biodegradable saving on pollution and if immobilised can be repeatedly used.

Immobilised enzyme technology is still developing very rapidly and it seems likely there will be many new applications for immobilised enzymes in industry, medicine and waste disposal. New uses are discovered everyday. We are only at the beginning of enzyme technology.