The Application of Enzymes in Industry and Medicine.

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Samantha Fulcher         12B2        Jan 2004

The Application of Enzymes in Industry and Medicine.  

                                   

Enzymes are Biological catalysts, allowing the chemical reactions of metabolism to take place, controlling the speed of the reaction. They are found in all living cells and are divided into two main groups, intracellular and extra cellular. Intracellular are found and work inside the cells, therefore are secreted inside the cell membrane, from where they control metabolism.  The cells will also produce the extracellular, but these only achieve their full affect outside of the cell, so are secreted outside the cell membrane. Examples of extracellular enzymes include digestion enzymes such as pepsin.                                                              

 Enzymes are complex globular proteins. Their long peptide chains of amino acids linked by peptide bonds are wound, folded and bonded into a precise 3D structure, owing their activity to this particular shape. They are compounds of high molecular weight.

See Figure 1. (www.worthington-biochem.com)

Hydrogen, ionic, and disulphide bonds as well as hydrophobic interactions all hold the chain in its three-dimensional spherical form. Each enzyme has a unique shape. The precise shape of the active site (the place at which the substrate binds) is so because the enzyme is specific to one substrate-specificity. Thus meaning that the active site of the enzyme has a distinct chemical configuration to which only one substrate has the correct complimentary chemical configuration. This is known as the ‘lock and Key’ hypothesis. An enzyme works by combining with the substrate molecules to form an enzyme-substrate complex. With their various bonds held in relation with each other, the substrate molecule then reacts to form an enzyme-product complex. This then splits into the unchanged enzyme and a product. The enzyme is then free to be used elsewhere. This can be repeated causing no change to the enzyme, and as often as 100,000/second.

A more up to date version of this hypothesis is the induced fit theory. It is similar, but doesn’t require such a precise connection being made between the enzyme and substrate at the active site. It states that the active site is able to change in order to enfold with the substrate molecule. It takes up its most catalytic shape having bonded with the substrate so the shape of the enzyme is affected by the substrate. The rate of reaction is lowered as the distorted enzyme molecule distorts the substrate weakening the bonds, thus making it less stable and lowering the potential energy. When the reaction forms the product, it can no longer bind with the active site so moves away. The flexible enzyme then returns to its original state ready for the next substrate molecule.

The human body contains at least 1000 different enzymes. Less than 200 enzymes are used out of the roughly 2500 which have been isolated and described so far. Only around 10% in turn come from nature sources of microorganisms, such as fungi, yeast species and bacteria species. The chosen microorganisms are usually cultured in large fermentation chambers under controlled conditions to maximise yield. Large numbers of microbes are grown in specific growth media. The growth medium is previously super heated to make it free of microbes other than those that are to be grown in the sterile conditions. Often the useful products are produced naturally by their normal metabolism. Through genetic engineering specific genes can be added to DNA of the microorganisms so they produce designer enzymes naturally made by other organisms.

 (Oxford Advanced Biology)

Enzymes are usually needed in pure form for applications, so have to be isolated from the microbe cells and the whole mixture of metabolic products. In obtaining an intracellular enzyme, the microbe cells are harvested from the culture and broken open. The resultant mixture is then centrifuged to remove large cell fragments, and the enzyme is precipitated from the solution by a salt or an alcohol. The enzyme can then be purified by techniques such as electrophoresis or column chromatography. Extracellular enzymes are present in culture outside the microbial cells. They are usually soluble so can be extracted from the culture medium and made pure. Extracellular enzymes are more useful for isolation on an industrial scale because the cells do not need to be broken down as they are always being secreted by the cell. This is therefore faster, easier and more economical than isolating intracellular enzymes.  Extracellular enzymes are also more stable as they are subjected to harsher conditions out side of the cell. In some cases it is not possible to use isolated enzymes in some applications, where whole cells are used instead. During instances such as limited budget, too difficult to be extracted and purified, or when two or more enzymes are needed to work together. When using whole cells, the substrate has to diffuse into the cells before the reaction commences. The product may then be extracted and purified.                        (Mr Price class notes)

Reactions don’t take place until certain energy levels are reached in order to activate the reaction-activation energy. Enzymes lower this activation energy (Ea). The enzyme is thought to reduce the ‘path’ of a reaction. This shortened path would require less energy for each molecule of substrate converted to product. Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present (the shortened ‘path’) than when it is absent. Hence, the reaction is said to go faster in a given period of time. A theory to explain the catalytic action of enzymes was proposed by a Swedish chemist called Savante Arrhenius in 1888, it closely ties with the lock and key hypothesis as previously explained.

(www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/01t.html)

Enzymes function best in optimum temperature, pH, and substrate/enzyme concentration or with a non-protein substance called a cofactor. However, ample temperature, pH extremes, incubation time and enzyme inhibitors can have an adverse affect.

Heating increases the rate of reaction in most chemical reactions. Heating a substance gives it greater kinetic energy, thus making the substance move around more increasing its chance of collision to react. The optimum temperature for most enzymes is around 45°C, a little higher than body temperature but each enzyme has evolved to its surroundings. Increasing the temperature of an enzyme –controlled reaction will increase the rate of reaction but only up to its optimum temperature. Any increase in temperature increases the energy of a single atom. This means that in an enzyme, the bonds between the polypeptide chain, would begin to be affected. This rise would mean the bonds would begin to vibrate. Eventually the vibrating would get so fast, the bonds would break-after the enzymes optimum temperature. The bonds that hold the precise three-dimensional shape will be broken, and so the precise shape will be lost. Denaturation of the enzyme structure would give rise to a distorted active site upon which the substrate would no longer be able to bond. Any damage to an enzyme is permanent and it becomes inactive, even when cooled.

Most enzymes also have an optimum pH at which the reaction rate is fastest. As we know the three-dimensional shape of the enzyme is vital for it to function properly. The most abundant chemical bonds found in the enzyme are the hydrogen bonds. Small changes in the pH (or hydrogen concentration) can affect the rate of reaction without denaturing the enzyme. At the extremes of its pH range, an enzyme can become unstable and denatured. Acidity and alkalinity can affect the active site of an enzyme. Free hydrogen or hydroxyl ions can affect the charges on the amino acid side chains of the enzymes active site. This will also affect the hydrogen bonds. If the bonds break due to a change in charge and deviations from the optimum pH the three-dimensional shape will be lost, thus changing the shape of the active site. The substrate will no longer fit into the active site and form an enzyme substrate complex. Meaning the enzyme looses its activity and the rate of reaction falls. This is often why Phosphate buffers are used in applications of enzymes, because they maintain a specific, constant pH concentration. Many enzymes have an optimum pH of around 7, but again some have evolved to suit their surroundings.

Providing conditions such as pH and temperature are normal for that specific enzyme, and there is an excess amount of substrate, then the rate of reaction will be directly proportional to the enzyme concentration.  Increasing the amount of enzyme will increase the rate of reaction, providing there is still excess substrate. If the amount of enzyme stays the same, the rate of reaction will increase with the addition of substrates up to a point. When the active sites are all working as quickly as they can, adding more substrate would bring about no further increase in the reaction rate.

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Some enzymes only work in the presence of a non-protein substance called a cofactor. A cofactor tightly bound into its enzyme is called a prosthetic group. They may be organic (carbon containing) or inorganic. Inorganic cofactors are named activators, they include metal atoms and may attach on to active sites to make its shape more efficient. Organic cofactors are named coenzymes, many of which are vitamins or vitamin compounds. Some transfer chemical groups, atoms of electrons from one enzyme to another.

Incubation time is the length of time over which a reaction has taken place. As the incubation time increases ...

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