Enzymes

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Enzymes

  1. 1.  Enzymes work the same way as catalysts do, they can work with only one substrate and they can be used more than once. Enzymes have a structure that is called active site. Only one substance can fit into the active site to be digested, and it is the only substrate that this particular enzyme works with. The figure above shows the function of enzymes: The substrate enters the active site of the enzyme. The reaction takes place. The substrate exits the enzyme as two simpler products. You can also think of the way enzymes work as a key and a lock, the key is the substrate and the lock is the enzyme. The key should be exactly the right shape to fit in the lock, so does the substrate to fit in the active site of the enzyme. The key could only open only one lock, and the lock could be unlocked by only that key. Enzymes are two types, Builders and Breakers. Builder enzymes do the opposite of breaker enzymes. Breakers break large molecules into smaller simpler ones, builders combine smaller ones to make large molecules. Breaker enzymes are used in the digestive system to break down large insoluble molecules into simpler soluble ones to be used by the body. They are also present in cells that respire to break down sugars and oxygen into carbon dioxide, water and energy. Builder enzymes are present in plants to be used in photosynthesis, the opposite of respiration, in photosynthesis, oxygen and water are combined together to form carbon dioxide and sugars. Naming enzymes depends on the substrate they work on. For example: The sucrase enzyme works on sucrose. The maltase enzyme works on maltose. Enzymes are reusable and are only affected by the change in temperature and pH
  2. 2.  A catalyst is a substance that speeds up the chemical reaction without getting used up or altered. Are proteins by nature. Functions as biological catalyst.
  3. 3.  Affect of temperature on the enzyme’s activity: Each enzyme has an optimum temperature, this is the temperature at which the enzyme is most active, below this temperature the activity of the enzyme decreases until it becomes inactive at low temperatures, above this optimum temperature the enzyme becomes denatured and can no longer work. At low temperatures the enzyme is and the substrate are moving very slowly and collide weakly, the enzyme is said to be inactive and doesn’t work. As the temperature increases, the enzyme and substrate gain more kinetic energy and move faster colliding more, the enzyme becomes more active and the reaction takes place. When the enzyme reaches it’s optimum temperature, it is in its most active mood, if the temperature crosses the optimum the enzyme begins to die and become denatured. The enzymes become denatured when the shape of their active site changes as a result of high temperature, thus the substrate cannot fit into the active site and the enzyme is useless. Each enzyme has its own optimum temperature, enzymes in humans have optimum temperatures of around 40 degrees. Plants have enzymes with optimum temperature of about 25 degrees.
  4. 4.  As in temperature, enzymes have an optimum pH. The pH is a scale measuring the acidity or alkalinity of a substance or solution. The scale runs from 1 to 14. pH 7 is neutral, below that it is acidic and above that it is alkaline. Each enzyme has an optimum pH, if this pH changes, the shape of the active site of the enzyme is changed, thus the substrate will not be able to fit in it, and the enzyme becomes useless.
  5. 5.  `Seeds grow into plants by germinating. Seed germination involves enzymes breaking the materials stored in the seed down to be used in growth, energy and building cells. The seed contains stored substances such as: Starch: Starch is broken down by amylase enzyme into maltose, maltose is then broken down by maltase enzyme into glucose which is used in respiration. Proteins: Proteins are broken down into amino acids by Protease enzyme, amino acids are used in building up cells and growth. Fats: Fats are broken down into fatty acids by lipase enzyme, they are used in making cell membranes. In order for a seed to germinate, some conditions must be present: Water: To activate the enzymes. Oxygen: To be used for respiration. Warm Temperature: For providing the best conditions for enzymes to work and optimum temperature.
  6. 6.  Washing powders contain detergents that help in cleaning clothes by dissolving stains in water. Some stains are made of insoluble substance, these cannot be removed by normal washing powders, instead, a biological washing powder is used. Biological washing powders contain enzymes that break down the insoluble stain into smaller soluble substances, which are then dissolved in the water. For example, if your shirt gets stained by egg yolk or blood, there is an enzyme called protease in the washing powder that will break down the insoluble protein into amino acids, which are dissolved in the water and sucked away. Thus the shirt becomes clean. The best removal of stains is maintained by providing the optimum temperature for enzymes, presoaking to leave time for the enzymes to digest, putting the suitable amount of the powder.
  7. 7.  Use Of Enzymes In Food Industry: Enzymes are often used in the manufacturing of different foods. Baking – Brewing – Cheese Making: In baking, both yeast and sugar are used. Yeast cells contain enzymes that ferment sugar by anaerobic respiration producing carbon dioxide bubbles which causes the dough to rise as in the photo. Brewing is the process of making wine or beer. In this process fermentation is Involved producing alcohol which and carbon dioxide that gives wine and beer its sparkle. In making cheese, an enzyme called rennin extracted in enzymes, helps by clotting milk. Making Juices: In fruits such as apples or oranges, a substance called pectin holds the cells together making it hard to squeeze them. An enzyme called pectinase digests pectin making it much easier to squeeze the fruit and to make the juice more clear than cloudy. Making Baby Foods: It is hard for new born babies to digest food such as high protein foods. That is why foods like that are treated with proteases to break down protein to amino acids, making it easier for newborns to absorb and assimilate them. Making Sugar: Sugar producing companies get sugar from starch by using the amylase enzyme to digest starch into maltose. For dieters a sugar called fructose is very useful because it provides a sweater taste than other sugars from a less quantity. Fructose can be obtained by using the isomerase enzyme to convert glucose to fructose. Meat And Leather Production: Proteases are used to make meat less tough and acceptable for consumers by treating cuts of meat. In leather industries hairs are removed from animal skin by digesting them using protease enzymes.
  8. 8.  Enzymes Extraction: The Enzymes used in the industries are taken from either fungi or bacteria. This takes place in a Fermenter, this is a large sterilized container with a stirrer, a pipe to add feedstock and air pipes. The following steps take place: The micro-organisms and the feedstock are added and the liquid is maintained at 26 degrees and pH of 5-6. The micro-organisms produce two types of enzymes, either extra-cellular or intra-cellular. Extra-cellular enzymes are extracted from the feedstock by filtering. Intra-cellular enzymes are extracted by filtering the micro- organisms from the feedstock, crushing them, wash them with water then extracting them from the solution.
  9. 9.  Enzymes And Antibiotics: Antibiotics are powerful medicines that fight bacterial infections. Micro-organisms are used for the production of antibiotics. Some Antibiotics, like bactericides, fight bacteria by damaging its cell walls causing them to burst and die. Other antibiotics interfere with the protein synthesis and stop the bacteria growing. Antibiotics have no effect on human cells because human cells have no cell walls and the structures involved in protein production are different than that of bacteria. Antibiotics are obtained from sources like: Bacteria (Actinobacterium Streptomyces): this bacterium produces the antibiotic strepmycin. Fungi (Penecillum fungus): penicillin, the first antibiotic discovered is produced by thing fungus. Different types of penicillin are produced by different species of the fungus. They are chemically altered in lab to make them more effective and make them able to work with different diseases. Steps of production: The Fermenting tank in filled with nutrient solution of sugar (lactose) or corn liquor which contain sugars and amino acids, Minerals are added, pH is adjusted around 5 or 6, Temperature is adjusted about 26 degrees, The liquid is stirred and air is blown through it, The micro-organisms are added and allowed to grow for a day or two in sterile conditions, When the nutrient supply is decreased, micro-organisms secrete their antibiotics, The fluid containing the antibiotic is filtered off and the antibiotic is extracted.
  10. 10.  Even for an old industry like beer brewing new industrial processes benefit from using enzymes developed from microbial sources. In the last years quality issues like flavour control, beer stability and general cost savings in the industry go hand in hand with efficient solutions of environmental problems. Future aspects focus on a wider application of enzymes to brew with high amounts of inexpensive raw materials like barley. Alternative beer processes for production of wort and beer with higher productivity and reduced amounts of waste and by-products are under development.
  11. 11.  Introduction Beer and wine are both alcoholic beverages which have been part of our social life for thousands of years. Both beverages are produced by yeast fermentation of sugars. Wine is based on grapes, and beer is traditionally based on barley. The matured grapes already contain the sugars needed for the fermentation, while barley contain starch that has to be broken down to fermentable sugars before the yeast can make alcohol. Therefore, traditional brewing contains and extra step compared with wine-making, namely malting in which enzymes needed for the degradation of starch into fermentable sugars are produced.
  12. 12.  Malt is germinated barley or other cereals like wheat and sorghum: First the grains are "steeped" bringing the water content from about 12% to 45%, then they are allowed to germinate for 4-6 days and finally the germination is stopped by heating (kilning) reaching a final moisture content of about 4%. Some enzymes are already present in the barley, e.g. β-amylases, but the majority of enzymes are produced during the germination, e.g. α-amylases and proteases, and in the final malt all the enzymes needed for the conversion of "grains" into a fermentable liquid (wort) is present (Figure 1 and 2)
  13. 13.  In former days, production of malt was an integrated part of every brewery, but to day most malt is produced outside the brewery in large malt factories, and malt has become a purchased raw material, like other raw materials. This means that the breweries to day are more flexible in the use raw materials, and for that matter for the source of enzymes. The malt enzymes do have some limitations. They can only work at certain temperatures, pH values etc., and the activities might be too low to do a proper job in proper time. In contrast, commercial exogenous enzymes can be designed to work at preferred temperatures and pH values, to have more enzymatic power, or to express wanted enzyme activities that are not present in malt. Addition of exogenous enzymes at various steps during the brewing process can therefore make brewing easier, faster and more consistent. It gives the brewmasters extra flexibility in the choice of raw materials due to less dependence on malt enzymes, as well as providing opportunity to create new products, which is not possible to make with malt enzymes alone. Also the possibility to improve beer quality by avoiding off-flavours is possible with commercial enzymes. The increasing concern on resources and CO2- emission has also put the use of commercial enzymes within the brewing industry in focus. By the use of exogenous enzymes more can be extracted from the raw materials, more local raw materials can be used, and more unmalted grains can be used, saving significant amounts of energy and transport
  14. 14.  The brewing process Traditionally, beer is produced by mixing crushed barley malt and hot water in a mash copper to perform the mashing. Besides malt, other starchy cereals such as maize, sorghum, rice and barley, or pure starch itself, can be added to the mash. These are known as adjuncts. The standard mashing for pilsner type beer consists of several temperature steps, each favouring different malt enzyme activities. The lowest temperature (45 ºC) is the optimal temperature for cell wall degrading enzymes, β-glucanases. The proteases works best at 52 ºC, the β-amylase best at 63 ºC and the α-amylase at 72°C. The last step in the mashing is inactivation of the enzymes at 78 ºC (Figure 3). Figure 3: The traditional mashing temperature profile is determined by the temperature optima for the various malt enzymes. Larger version If β-glucan and protein are properly broken down during malting, single temperature mashing at 65- 71°C has shown to be sufficient, as in the case of traditional ale brewing. During mashing the starch is degraded to dextrin and fermentable sugars. α-amylase liquefy the gelatinized starch by hydrolysis of the α-1,4 linkages at random. β-amylases are exo-enzymes which attack the liquefied starch chains resulting in successive removal of maltose units from the non- reducing end. After mashing, the mash is sieved in a lauter tun or on a mash filter. The resulting liquid, known as sweet wort, is then transferred to the copper, where it is boiled with hops. The hopped wort is cooled and transferred to the fermentation vessels, where yeast is added. In normal wort 2/3 of the carbohydrates are fermentable sugars. After fermentation, the so-called ‘green beer is matured before final filtration and bottling. Fig. 4 shows a diagram of the brewing process and where external enzymes are used for process aids.
  15. 15.  Commercial enzymes from exogenous sources The traditional source of enzymes used for the conversion of cereals into beer is barley malt. If too little enzyme activity is present in the mash, there will be several undesirable consequences: the extract yield will be too low; wort separation will take too long; the fermentation process will be too slow; too little alcohol will be produced; the beer filtration rate will be reduced; and the flavour and stability of the beer will be inferior. Exogenous enzymes are used to supplement the malts own enzymes in order to prevent these problems. Furthermore, industrial enzymes are used to ensure better adjunct liquefaction, to produce low-carbohydrate beer (‘light beer), to shorten the beer maturation time, and to produce beer from cheaper raw materials. The various steps of the brewing operations, where microbial enzymes are occasionally added, are shown in table 1. Enzymes, enzymic action and their functions are summarized.
  16. 16.  Enzymes at work Quality and supply constraints on malt, and doubling of malt prices have given increased interest for enzyme solutions in 2007 and 2008. Many breweries has run programs within the last two years in order to increase efficiency and optimize raw material usage, and many of them have focused on commercial enzymes to shorten the production time, increase capacity, and to allow use of raw material alternative to malt. Three important examples are mentioned: Exchanging part of the malt with barley has been popular because using barley in combination with commercial enzymes gives the same beer quality as with malt. Introducing a higher content of starch hydrolysing enzymes offer the possibilities of producing "light beer" also called "low calorie beer". An enzyme solution for diacetyl control after fermentation improves vessel utilization, save energy and ensures a high beer quality after a reduced maturation time
  17. 17.  Enzymes to improve fermentation Small adjustments in fermentability can be achieved by adding amyloglucosidase alone or in combination with debranching enzymes at mashing-in or a fungal α-amylase at the start of fermentation. To describe to which extent the extracted sugars are fermentable brewers define degree of attenuation, which is synonymously with degree of fermentation or fermentability. Figure 6. Total fermentable sugar production with different dosages of Attenuzyme® (kg per ton malt) and extended mashing at 63°C Beer types with very high attenuation ("light beer" or "low calorie beer") are most often produced using amyloglucosidase alone. Extended mashing at 63°C and high dosages of enzymes is necessary to produce extremely high attenuated beer (see figure 6). Fungal α-amylases are used to produce mainly maltose and dextrins whereas amyloglucosidase produces glucose from both linear and branched dextrins

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