The three main types of enzymes based biosensors are called enzyme thermistors, enzyme transducer sensors and enzyme biochips. An enzyme thermistor measures minute changes in temperature during exothermic reactions and is sensitive to changes between 0.004 and 1.0°C. An enzyme biochip is very small, and contains immobilized enzymes on the surface of a silicone ship. A biochip can detect blood glucose levels using the enzyme glucose oxidase by measuring the amount of gluconic acid produced. An enzyme transducer sensor is an electrical device that registers chemical changes and converts then into an electrical signal. The strength of the current is directionally proportional to the amount of the substance measured. These can also measure blood glucose levels using glucose oxidase by catalyzing the reaction between oxygen and glucose to form gluconic acid and hydrogen peroxide. The enzyme urease can detect the amount of urea in blood or urine. The amount of ammonium ions is proportional to the concentration of urea in a solution.
Enzymes are usually obtained from microbial sources such as fungi and bacteria. These micoorganisms are more widely used than enzymes in plants or animals for a number of reasons. The product yield may be increased by strain selection, optimisation of conditions and by gene mutation. They are easy to manipulate genetically and be subject to gene transfer techniques. As they can occupy a great variety of habitats and extremes of conditions, their enzymes can function in an enormous range of temperature and pH. A great advantage is that they produce more enzyme molecules in relation to their mass than larger organisms.
The organisms grown must be selected so that the enzyme extracted has certain qualities, such as a wide pH tolerance as they may need to work in the presence of chemicals such as sulphur dioxide which inhibit enzyme action, and must be able to tolerate a wide temperature range, between 10-55°C. These properties prevent denaturation by extremes of temperature and pH. The microorganisms must also have simple nutritional requirements, have a high growth rate, be non-pathogenic and not produce toxins or an unpleasant smell. Most enzymes are extracellular, where they are secreted by the microorganism into their surroundings, and then can be extracted easily from the fermenter by filtration. A more complex recovery is required when intracellular enzymes are used; the cells must be broken in order to extract the enzyme from a mixture of the cell’s contents of many other enzymes and cell debris. Recent developments use isolated enzymes as there will be no wasteful side reactions and a single product is formed which are easier to isolate and purify. Also only one environmental condition needs to be considered.
A technique called enzyme immobilization prevents the wastage of enzymes. When enzymes are used in solution, it is difficult to remove the enzyme from the end-product and to purify the enzyme so that it can be used again, which is useful when the enzyme is expensive or difficult to produce. Immobilized enzymes are bound to a surface and are not allowed to dissolve in the solution containing its substrate and therefore the enzymes are held in place during the reaction and can be used again. The end-product does not have to be purified as the enzyme is held in a matrix, which must be inert as to not affect the reaction. Enzymes may be immobilized by several methods, such as being held within a semi-permeable membrane, held inside a gel e.g. silica, adsorbed onto an insoluble matrix e.g. collagen, attached to cellulose fibres, or trapped in a microcapsule, e.g. alginate beads. An advantage of using immobilization is that the enzyme is protected by the matrix with a physical barrier, and is more stable at extremes of temperature and pH. However, the reaction may be slower than if the enzymes are free in solution as some of the active sites will be embedded in the matrix and not all the enzymes will come into contact with an enzyme, and it takes time for the enzyme to percolate down the tube. This percolation is a continuous process however, and saves time and therefore money.
This technique is often used in the food industry to clear fruit juices. If the enzyme is free in the solution the enzyme must be removed at the end before it can be used. This is expensive and requires heating to denature and coagulate the enzyme which is then filtered off, which can spoil the flavour of the product. Enzyme immobilization avoids such problems.
There are many other enzymes used in the food industry. A large demand for sweeteners led to a cheaper, sucrose substitute to be produced called high fructose syrup, which requires four enzymes; α-amylase, fungal amyloglucosidase, pullulanase and bacterial isomerase. These catalyse the conversion of starch to glucose, which is then converted to a 50:50 mixture of fructose and glucose. Aspartame is an alternative sweetener sold as ‘Canderel’ or ‘Nutrasweet’ which requires the use of the enzyme aspartase.
The soft centres inside chocolates are also produced using enzymes. Originally the centre is solid containing an enzyme and polysaccharide, and coated in chocolate. The enzyme breaks down the polysaccharide, turning the hard centre soft. Lipases are used in the dairy industry to ripen blue cheeses, and proteases are used to lower the protein content of flour for biscuit production. This idea is also used when making baby foods, which are treated with proteases to break down proteins to amino acids and polypeptides so it is easier for the babies to digest the food. In low-calorie beer the sugar is broken down by amyloglucosidase.
