Extraction of Amylase Enzyme From Yam | Experiment

Extraction of Amylase Enzyme From Yam | Experiment Amylolytic enzymes are widely distributed in plant tissues, e.g. in storage tissues such as seeds and tubers and in vegetative organs such as leaves. There exist two types of amylases in some species of plants, (E.C.3.2.1.1; 1-4-ÃŽÂ ±-D-glucan glucohydrolase) and (E.C.3.2.1.2; 1-4-ÃŽÂ ²-D-glucan maltohydrolase) amylases [Thoma, J.A., J.E. Sprandlin and S. Dygert, 1971]. Beta-amylase (-1, 4-glucan maltohyrolase, E.C.3,2,1,2) is an exoamylase that attacks the non reducing ends of starches molecules, producing a-maltose and a limit dextrin as products [Thoma, J.A., J.E. Sprandlin and S. Dygert, 1971]. In starch-enriched tissues, ÃŽÂ ²-amylase may play a role in the mobilization of starch during germination or sprouting tubers [Greenwood, C.T. and E.A. Milne, 1968]. Many reports have been demonstrated that ÃŽÂ ²-amylase has a great commercial value in food and beverage industries. The enzyme is useful in structural studies of starch and glycogen. Marshal and Whelan [Marshall, J. et al 1973] report on the removal of any contaminating ÃŽÂ ²-glucosidase. The practical interest of ÃŽÂ ²-amylase was concentrated on its capacity to produce maltose syrups from starch [Biovin, P., 1997.]. ÃŽÂ ²-amylase has previously been purified and characterized from different types of plant sources and a few of microbial origin. In higher plants, the molecular characterization of ÃŽÂ ²-amylase has been carried out on enzyme purified from the organs enriched in starch such as sweet potato tubers [Balls, A.K.,1948, et al], leaves [Vikso-Nelson, A., et al 1997], bulbs [Dicko, M.H., et al, 2000], seeds of various cereal species such as barley [Shinke, R. et al 1971], wheat [Trachuk, R. et al 1966], rice [Okamato, K. and T. Akazawa, 1978] and other higher plants such as soybean [Gertler, A. and Y. Birk, 1965]. On the other hand, much less information is available on the purification and characterization of ÃŽÂ ²-amylase from root. The present study reports the pur ification of ÃŽÂ ²-amylase from Yam (Dioscorea esculenta) root to a pure state along with its characterization. Amylase is an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. As diastase, amylase was the first enzyme to be discovered and isolated (by Anselme Payen in 1833). Specific amylase proteins are designated by different Greek letters. All amylases are glycoside drolases and act on ÃŽÂ ±-1,4-glycosidic bonds. It will start to denature at around 60C. Amylase digests not only carbohydrates but also dead white blood cells. For example, when you are low in amylase you are a candidate for abscesses (inflamed areas with pus but not bacteria). If you have a toothache and are being treated with antibiotics, but it doesnt go away, chances are you have an abscess. Amylase is involved in anti-inflammatory reactions such as those caused by the release of histamine and similar substances. The inflammatory response usually occurs in organs which are in contact with the outside world, i.e., the lungs and skin. These include skin problems such as psoriasis, eczema, hives and all types of herpes. Some lung problem including asthma and emphysema may require amylase plus other enzyme formulas depending on the particular ailment. There are many types of amylases, but of importance are: ÃŽÂ ±-amylase, ÃŽÂ ²-amylase and glucoamylase. A diagram of an amylase molecule from human saliva. 1.1 ÃŽÂ ²Ãƒâ€šÃ‚ ­ Amylase (EC 3.2.1.2) (alternate names: 1,4-ÃŽÂ ±-D-glucan maltohydrolase; glycogenase; saccharogen amylase) Another form of amylase, ÃŽÂ ²-amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, ÃŽÂ ²-amylase catalyzes the hydrolysis of the second ÃŽÂ ±-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, ÃŽÂ ²-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. Both ÃŽÂ ±-amylase and ÃŽÂ ²-amylase are present in seeds; ÃŽÂ ²-amylase is present prior to germination, whereas ÃŽÂ ±-amylase and proteases appear once germination has begun. Cereal grain amylase is key to the production of malt. Many microbes also produce amylase to degrade extracellular starches. Animal tissues do not contain ÃŽÂ ²-amylase, although it may be present in microrganisms contained within the digestive tract. 1.1.2 CARBOHYDRATE METABOLISM Digestion of carbohydrate begins in the mouth by the action of salivary ÃŽÂ ±-amylase.Only limited digestion of carbohydrate occurs, however, because salivary ÃŽÂ ±-amylase is denatured in the stomach due to the low pH. Digestion begins again in the small intestine when pancreatic ÃŽÂ ±-amylase is secreted. Starch is broken down into maltose, isomaltose, and maltotriose by ÃŽÂ ±-amylase through the hydrolysis of ÃŽÂ ±-1-4 glycosidic bonds. These products as well as any other disaccharides that were ingested must be further digested to their respective monosaccharide units by brush border enzymes (maltase, isomaltase, lactase, and sucrase) before absorption. Maltose is hydrolyzed to two glucose molecules by maltase. Isomaltose is hydrolyzed to two glucose molecules by isomaltase. Lactose is hydrolyzed to one molecule of glucose and one molecule of galactose by lactase. Sucrose is hydrolyzed to one molecule of fructose and one molecule of glucose by sucrase. After absorp tion, glucose, galactose, and fructose are transported to the liver via the portal blood. The liver can transform galactose and fructose into glucose (Gropper et al 2005). 1.1.3 REACTIONS OF BETA AMYLASE Starch + H2O in vitro breakdown of semicrystalline starch particles by beta-amylases increases significantly if they act together with glucan, water dikinase starch substrate of different sources, e.g. wheat, wheat bran, rice bran beta-amylase hydrolyzes alpha-1,4-linkage, raw starch granules from potato, wheat, rice and corn, with the granules from rice being the best substrate, beta-amylase attacks very slowly on the starch granules, hydrolyzes corn granules efficiently at 45 °C . Beta-amylase is an exo-enzyme that catalyzes the hydrolysis of the alpha-1,4-glucosidic linkage of the substrate liberating beta-maltose from the non-reducing end, Glu-172 and Glu-367 are catalytic residues, substrate recognition mechanism, enzyme structure beta-amylase is an inverting enzyme that hydrolyzes the alpha-1,4-glucosidic linkage of the substrate liberating beta-maltose from the non-reducing end, catalytic mechanism, Glu-172 acts as general acid, Glu-367 acts as general base catalyzes the hydrolysis of alpha-1,4-glucosidic linkages of soluble starch, and liberates beta-anomeric maltose from the nonreducing ends, exo-acting enzyme, composed of two functional domains, a catalytic domain: domains A and B, and starch-binding domain: domain C, beta-amylase has three carbohydrate-binding sites aside from the active site: two in domain B named Site2 and Site3, one in domain C named Site1, roles of these sites in the catalytic reaction and raw starch-binding, beta-amylase hardly h ydrolyzes raw starch from wheat, corn, potato or sweet potato, but binds to it strongly hydrolyzes the alpha-1,4-glucosidic linkage liberating beta-maltose from the non-reducing end of substrate, enzyme/domain structure, starch binding site in domain C, catalytic mechanism starch substrate of different sources, e.g. wheat, wheat bran, rice bran starch substrate of different sources. Beta-amylase hydrolyzes alpha-1,4-linkage, raw starch granules from potato, wheat, rice and corn, with the granules from rice being the best substrate, no efficient hydrolysis of raw starch granules, very slow enzymic attack catalyzes the release of maltose from soluble starch. Malbranchea sulfureastarch substrate of different sources, e.g. wheat, wheat bran, rice bran 106.9% of the activity with amylose, soluble starch, amylose and amylopectin are the most suitable substrates, some activity against native starch, exo-hydrolase that releases beta-maltose from the non-reducing end of alpha-1,4-linked poly- and oligoglucans until the first alpha-1,6-branching point along the substrate molecule is encountered, beta-amylase should be a key enzyme in starch degradation during the germination of millet seeds, enzyme activity increases during days 1-4 of germination starch substrate of different sources, e.g. wheat, wheat bran, rice bran best substrate, pure and low quality starches, maize starch, tapioca starch maltose is the major end product, traces of maltooligosaccharides, no glucose as product. Beta-amylase is involved in starch degradation during mango ripening, which is clearly triggered by detachment from the mother-plant starch enzyme induction upon a cold shock at 4 °C leads to starch-dependent maltose accumulation, which might be required for protection of the photosynthetic electron transport chain, maltose influences the carbohydrate metabolism. Of the components of starch, amylopectin presents the great challenge to hydrolytic enzyme systems. This is due to residues involved in 1,6-glycosidic branch points which constitute about 4-6% of the glucose present. Most hydrolytic enzyme are specific for 1,4-glycosidic links yet the 1,6-glycosidic links must also cleaved for complete hydrolysis of amylopectin to glucose. Some of the most impressive recent exercises in the development of new enzymes have concerned debranching enzymes. It is necessary to hydrolyse starch in a wide variety of processes which may be condensed into two basic classes; Processes in which the starch hydrolysate is to be used by microbes or man and processes in which it is necessary to eliminate starch. In the former processes, such as glucose syrup production, starch is usually the major component of reaction mixtures, whereas in the latter processes, such as the processing of sugar cane juice, small amounts of starch which contaminate non-starchy materials are removed. Enzymes of various types are used in these processes. Although starches from diverse plants may be utilized, corn is the worlds most abundant source and provides most of the substrate used in the preparation of starch hydrolysates. There are three stages in the conversion of starch Gelatinisation, involving the dissolution of the nanogram-sized starch granules to form a viscous suspension; Liquefaction, involving the partial hydrolysis of the starch, with concomitant loss in viscosity; and Saccharification, involving the production of glucose and maltose by further hydrolysis. Galatinisation is achieved by heating starch with water, and occurs necessarily and naturally when starchy foods are cooked. Gelatinized starch is readily liquefied by partial hydrolysis with enzymes or acids are saccharified by further acidic or enzymic hydrolysis (Chaplin,2004). USES OF AMYLASE Amylase enzyme finds use in bread making and to break down complex sugars such as starch (found in flour) into simple sugars. Yeast then feeds on these simple sugars and converts it into the waste products of alcohol and CO2. This imparts flavour and causes the bread to rise. While Amylase enzymes are found naturally in yeast cells, it takes time for the yeast to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long fermented doughs such as sour dough. Modern bread making techniques have included amylase enzymes (often in the form of malted barley) into bread improver thereby making the bread making process faster and more practical for commercial use. When used as a food additive, and may be derived from swine pancreas or mould mushroom. Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve starches from fabrics and dishes. Workers in factories that work with amylase for any of the above uses are at increased risk of occupational asthma. 5-9% of bakers have a positive skin test, and a fourth to a third of bakers with breathing problems are hypersensitive to amylase. An inhibitor of alpha-amylase called phaseolamin has been tested as a potential diet aid. Blood serum amylase may be measured for purposes of medical diagnosis. A normal concentration is in the range 21-101 Mol/L. A higher than normal concentration may reflect one of several medical conditions, including acute inflammation of the pancreas, macroamylasemia, perforated peptic ulcer, and mumps. Amylase may be measured in other body fluids, including urine and peritoneal fluid. Two amylases are common to the baking industry, alpha-amylase and beta-amylase also known as alpha-1,4-glucan glucanohydrolase and alpha-1,4-glucan maltohydrolase. Amylases convert starch into sugar : the ÃŽÂ ±-amylase will cleave the starch randomly (the so called 1-4 bonds in the starch) while the ÃŽÂ ²-amylase can only chop off two sugar units at the time at the end of the starch chain. Normally there is enough ÃŽÂ ²-amylase present in the flour but sometimes addition of ÃŽÂ ±-amylase is needed. The ÃŽÂ ±-amylase will cut the starch into smaller units called dextrins and the more ÃŽÂ ±-amylase activity there is, the better for the ÃŽÂ ²-amylase because there are more extremities available. So the substrate for the ÃŽÂ ²-amylase is either starch or dextrins and the product is maltose. Alpha-amylase is an endoenzyme that attacks linkages within the molecular structure. It randomly cleaves starch chains at interior a-1,4-glycosidic linkages producing short chains of glucose molecules or dextrins. Beta-amylase is an exoenzyme and cleaves maltose units from the non-reducing end of the starch molecule. In order for these enzymes to function, the starch granule must be ruptured so that the individual starch molecules are available for enzymatic action. Depending upon their origin, alpha- and beta-amylases show differences in pH and temperature optima, thermostability, and other chemical stability. They do not require co-enzymes for activity, although alpha-amylase activity is enhanced by the presence of calcium. The pH optimum for alpha-amylase is 4.5 and it is inactivated at a pH of 3.3 to 4.0. This pH dependence decreases the efficacy of this enzyme in sour doughs. Beta-amylase is active across a much broader pH range, 4.5-9.2, with a pH optimum of 5.3. Alpha-amylase is relatively thermostable up to 70 °C, whereas beta-amylase loses about half of its activity at this temperature. Fungal amylase is the least temperature stable, followed by cereal amylase, while bacterial amylase is stable at higher temperatures. New intermediate stability enzymes have been developed that are active above the gelatinization temperature of starch (60 °C), but are totally inactivated at the later stages of baking (80-90 °C). The objective is to maximize the anti-staling effect without creating a gummy, sticky product. INHIBITORS OF AMYLASE ACTIVITY Amylase inhibitors are naturally present in many plants and protect the plant from pests by not allowing the insect to break down starch and gain energy from it. Plants may contain separate protease inhibitors as well or amylase inhibitors may play a dual role and also inhibit proteases. Protein amylase inhibitors as well as non-protein amylase inhibitors exist. Amylase inhibitors may be active against a wide variety of amylases or may be specific to certain insect amylases or mammalian amylases (Franco, et al 2002). Structure of Proteinaceous Amylase Inhibitors The determination of the structure of a complex between porcine pancreatic amylase  and a protein amylase inhibitor isolated from bean (Phaseolus vulgaris) showed interaction between the pancreatic amylase active site and the inhibitor. Conformational changes were observed in the pancreatic amylase upon the binding of the inhibitor. The inhibitor was found to be a dimer with a disaccharide attached to one of the amino acid residues.  (Bompard G., et al, 1996). Amylase Inhibitors in yam tuber (Shivaraj, et al,. 1979) reported that sweet potatoes do not contain amylase  inhibitors while (Rekha, et al,1999) reported the presence of amylase inhibitors in 79 of  the 100 varieties tested. Cultivar differences as well as isolation procedure could account for  these differing results. Before performing amylase inhibitor assays, Shivaraj and others  homogenized sweet potato with water, allowed the samples to sit for 1 hour, centrifuged the  samples, collected the supernatant, and then subjected the supernatant to heat treatment  (80 °C for 10 minutes) to destroy native amylases. Rekha and others homogenized yam tuber in a sodium phosphate buffer containing polyvinyl pyrrolidone and sodium chloride,  stored the samples in the refrigerator, centrifuged the samples, and then performed  trichloroacetic acid precipitation to remove native amylases. Rekha and others chose to use  TCA precipitation rather than the heat treatment Shivaraj found heat treatment to be in effective at destroying all native amylase activity. YAM Yam is the common name for some species in the genus Dioscorea (family Dioscoreaceae). These are perennial herbaceous vines cultivated for the consumption of their starchy tubers in Africa, Asia, Latin America and Oceania. There are many cultivars of yam. Yam (Dioscorea spp., Dioscoreaceae) is classified as monocotyledonous but is considered to be closely related to dicotyledonous plants as a second cotyledon remains undeveloped in the embryo (Lawton and Lawton, 1967). The storage organ is probably a swollen hypocotyl (Lawton and Lawton, 1969), but is often described as a swollen root. A number of species are grown widely in the humid tropics with D. rotundata and D. cayenensis being of most importance, followed by D. alata and D. esculenta (Akoroda, 1993). These are all of African or East Asian origin, with only the minor species D. trifida being of American origin (Brà ¼cher, 1989). The tubers contain about 1-3  % protein on a dry weight basis (Coursey, 1995). Yam is source of carbohydrate; carbohydrates are one of the three major food groups needed for proper nutrition. Amylase is the digestive enzyme needed to digest carbohydrates. Carbohydrates in food are an important and immediate source of energy for the body. Starch refers to carbohydrates found in plants (grains). Vegetables and fruits are a source of sugar and are broken down to sugar or glucose. Carbohydrates are present in at least small quantities in most food, but the chief sources are the sugars are the sugars and the sugars and the starches (Wright, 1993) Uses of yam Food Yams of African species must be cooked to be safely eaten, because various natural substances in raw yams can cause illness if consumed. (Excessive skin contact with uncooked yam fluids can cause the skin to itch. If this occurs, a quick cold bath will stop the itching.) Yam is consumed in various ways, but is usually boiled and eaten. This involves cutting yam into pieces, then peeling the skin, and boiling the starchy meat. This is usually consumed with palm oil (traditional way), or with other sauces. The boiled yam can also be pounded with a traditional mortar and pestle to create a thick starchy paste known as Pounded Yam. This is also eaten with traditional stews and sauces. Another method of consumption is to sun dry the raw yam pieces. When dry, the pieces turn a dark brown color. This is then milled to create a powder known as elubo in Nigeria. The brown powder can be prepared with boiling water to create a thick brown starchy paste known as amala. This is also consumed with the local stews and sauces. The most common cooking method in Western and Central Africa is cooked boiled yam. (Wikipedia 2003). In India this vegetable is also called Garadu. In central part of India people cut small slices of the vegetable, deep fry them, sprinkle lots of spices on it and eat as snacks. In southern part of India, it is eaten with fish curry and is a local favorite.(Wikipedia 2003) 1.2 TYPES OF YAM Dioscorea rotundata and D. cayenensis Dioscorea rotunda, the white yam, and Dioscorea cayenensis, the yellow yam, are native to Africa. They are the most important cultivated yams. In the past they were considered two separate species but most taxonomists now regard them as the same species. There are over 200 cultivated varieties between them. The Kokoro variety is important in making dried yam chips. They are large plants; the vines can be as long as 10 to 12 meters (35 to 40 feet). The tubers most often weigh about 2.5 to 5  kg (6 to 12 lbs) each but can weigh as much as 25  kg (60 lbs). After 7 to 12 months growth the tubers are harvested. In Africa most are pounded into a paste to make the traditional dish of pounded yam (Kay 1987). Dioscorea alata A piece of cake made with Ube (water yam).Dioscorea alata, called water yam, winged yam and purple yam, was first cultivated in Southeast Asia. Although not grown in the same quantities as the African yams, it has the largest distribution world-wide of any cultivated yam, being grown in Asia, the Pacific islands, Africa, and the West Indies (Mignouna 2003). In the United States it has become an invasive species in some Southern states. In the Philippines it is known as ube (or ubi) and is used as an ingredient in many sweet desserts. In Vietnam, it is called khoai mà ¡Ã‚ »Ã‚ ¡ and is used mainly as an ingredient for soup. In India, it is known as ratalu or violet yam. In Hawaii it is known as uhi. Uhi was brought to Hawaii by the early Polynesian settlers and became a major crop in the 1800s when the tubers were sold to visiting ships as an easily stored food supply for their voyages (White 2003). Dioscorea opposita Dioscorea opposita, Chinese yam, is native to China. The Chinese yam plant is somewhat smaller than the African, with the vines about 3 meters (10 feet) long. It is tolerant to frost and can be grown in much cooler conditions than other yams. It is now grown in China, Korea, and Japan. It was introduced to Europe in the 1800s when the potato crop there was falling victim to disease, and is still grown in France for the Asian food market. The tubers are harvested after about 6 months of growth. Some are eaten right after harvesting and some are used as ingredients for other dishes, including noodles, and for traditional medicines (Kay 1987).Air potato Dioscorea bulbifera Dioscorea bulbifera, the air potato, is found in both Africa and Asia, with slight differences between those found in each place. It is a large vine, 6 meters (20 ft) or more in length. It produces tubers; however the bulbils which grow at the base of its leaves are the more important food product. They are about the size of potatoes (hence the name air potato), weighing from 0.5 to 2  kg (1 to 5 lbs). Some varieties can be eaten raw while some require soaking or boiling for detoxification before eating. It is not grown much commercially since the flavor of other yams is preferred by most people. However it is popular in home vegetable gardens because it produces a crop after only four months of growth and continues producing for the life of the vine, as long as two years. Also the bulbils are easy to harvest and cook (Kay 1987). In 1905 the air potato was introduced to Florida and has since become an invasive species in much of the state. Its rapid growth crowds out native vegetation and is very difficult to remove since it can grow back from the tubers, and new vines can grow from the bulbils even after being cut down or burned (Schultz 1993). Dioscorea esculenta Dioscorea esculenta, the lesser yam, was one of the first yam species cultivated. It is native to Southeast Asia and is the third most commonly cultivated species there, although it is cultivated very little in other parts of the world. Its vines seldom reach more than 3 meters (10 feet) in length and the tubers are fairly small in most varieties. The tubers are eaten baked, boiled, or fried much like potatoes. Because of the small size of the tubers, mechanical cultivation is possible; which, along with its easy preparation and good flavor, could help the lesser yam to become more popular in the future (Kay 1987). Dioscorea trifida Dioscorea trifida, the cush-cush yam, is native to the Guyana region of South America and is the most important cultivated New World yam. Since they originated in tropical rain forest conditions their growth cycle is less related to seasonal changes than other yams. Because of their relative ease of cultivation and their good flavor they are considered to have a great potential for increased production (Kay 1987). Dioscorea dumetorum Dioscorea dumetorum, the bitter yam, is popular as a vegetable in parts of West Africa; one reason being that their cultivation requires less labor than other yams. The wild forms are very toxic and are sometimes used to poison animals when mixed with bait. It is said that they have also been used for criminal purposes (Kay 1987). 1.3 Nutritional value Yams are high in vitamin C, dietary fiber, vitamin B6, potassium, and manganese; while being low in saturated fat and sodium. Vitamin C, dietary fiber and vitamin B6 may all promote good health. Furthermore, a product that is high in potassium and low in sodium is likely to produce a good potassium-sodium balance in the human body, and so protect against osteoporosis and heart disease. Yam products generally have a lower glycemic index than potato products, which means that they will provide a more sustained form of energy, and give better protection against obesity and diabetes. Aim and objective The objective of this experiment is to extract the amylases mainly ÃŽÂ ²-amylase from yam tuber and determine the enzymatic activities of the enzymes. At the end of the experiment, the amylase extracted from yam tuber can be made use of in the industries like; the pharmaceutical, plastic and textile industries among others in place of barley commonly made used of. This is even favorable considering the land mass covered by yam and also large usefulness of the yam. CHAPTER TWO 2.0 MATERIALS AND METHODS 2.1 MATERIALS Yam (Dioscoreaceae esculenta),used was from Oja Oba Market in Iwo Osun State, Ethanol, Soluble starch, 3,5-dinitrosalicylic acid, sodium hydroxide, sodium potassium tertarate, Sephadex G200 was obtained from Pharmacia fine chemicals, Uppsala, Sweden, disodium hydrogen phosphate were products of British Drug House(BDH), poole England. The distilled water was obtained from the Department of Biochemistry, Obafemi Awolowo University, Ile Ife. 2.2 EQUIPMENTS Water incubator manufactured by Grant Instruments(Cambridge) Ltd, Weighing balance made in Switzerland, Centrifuge manufactured by microfield instruments England. Spectrophotometer. 2.2 Preparation of buffer and Reagents 2.2.1 preparation of 0.016 M sodium acetate buffer, pH 4.8 To prepare acetate buffer for, 73.10 g of sodium acetate was dissolved in 900 ml of distilled water, 4.2 ml of acetic acid was added and then made up to 1 Litre in volumetric flask. 2.2.2 Preparation of 2 N sodium hydroxide To 8 g of sodium hydroxide pellets was dissolved in 100ml of distilled water. 2.2.3 Preparation of colour reagent Dinitrosalicyclic acid colour reagent, was prepared by dissolving 1.0 g of 3,5-dinitrosalicyclic acid in 50 ml of distilled water. 30.0 g sodium potassium tartrate tetrahydrate was added slowly with 20 ml of 2N sodium hydroxide. It was diluted to 100 ml with distilled water. 2.2.4 Preparation of 1% starch Prepared by dissolving 1.0 g of soluble starch in 100 ml of 0.016 M sodium acetate buffer pH 4.8. It was boiled to dissolve and cooled, diluted to 100ml with distilled water. 2.3 Method The rate at which maltose is released from starch is measured by its ability to reduce 3,5-dinitrosalicylic acid according to Bernfold(1955) . One unit releases one micromole of ÃŽÂ ²-maltose per minutes at 25ËÅ ¡C and pH 4.8 under the specified conditions. 2.3.1 Mashing and Extraction of Enzymes With the use of mortar and pestle, 434.52 g of yam were ground and 400ml of homogenization buffer (i.e 0.016 M sodium acetate pH) was added and stirred, it was kept in a refrigerator for 1 hour with intermittent stirring at 10 minutes interval. It was then centrifuged at 4000 rpm for 10 minutes into components. Assay for protein and enzyme activity was then carried out after it has being stored in 70% of ammonium sulphate(212.4 g/L). 2.3.2 Purification Purification of ÃŽÂ ²-amylase: All enzymes purification steps were carried out at room temperature. Enzyme Precipitation: The crude extract was initially fractioned by 70% (v/v) ammonium sulphate. After centrifugation at 4000 rpm for 10 minutes, the precipitated pellets were collected and re-suspended in of cold buffer. The solution was dissolved in 0.016 M sodium acetate buffer of pH 4.8 and layered on a Sephadex G-200 Colum (1 x 40 cm). Fractions of 5 ml were collected. The fraction was monitored for protein at 280 nM. Elution was in 0.016 M sodium acetate buffer, pH4.8.

Comparing Ulysses and American Beauty :: comparison compare contrast essays

Ulysses and American Beauty      Ã‚   In the "Nausicaa" chapter of James Joyce's Ulysses, a virginal exhibitionist, Gerty McDowell, flashes her "knickers. . .the wondrous revealment, half-offered like those skirt-dancers" at Leopold Bloom, igniting his sexual fireworks on a beach in Dublin (366). In a film set almost 100 years later in an American suburb, another virginal seductress flips her dance skirt, giving admirers a peek at her panties, and inspires Bloom's modern incarnation, Lester Burnham, into a similar burst of auto-eroticism.    The "metempsychosis" of Leopold Bloom into Lester Burnham isn't the only astonishing similarity between Ulysses and American Beauty. When screenwriter Alan Ball accepted the 2000 Golden Globe and Academy Awards for his screenplay of American Beauty, he owed a substantial debt--albeit universally unnoticed and, as he claimed in a telephone interview, "unintended"--to Joyce's masterpiece, the book chosen just months earlier by the Modern Library editorial board as the "best novel" of the Twentieth Century.    Yes, the ending of American Beauty represents a major departure from the plot of Joyce's novel--but an explicable one in a modern update of the Ulysses saga. Late twentieth-century audiences, who have become desensitized to escalating media violence over the past 100 years and have, in fact, developed an appetite for gore, require a bloody resolution. Despite the ending, we are left with striking reincarnations of Irish urbanites into suburban American personalities.    Consider other parallels: heroes Leopold Bloom and Lester Burnham (same initials, LB) are both middle-aged, middle-class, mediocre, unappreciated admen (Lester describes himself as "a whore for the advertising industry"[49], neither of whom has had sex with their wives in years . Ultimately both Bloom and Lester yearn to regain the past unity and warmth of their homes.    Bloom muses, "I was happier then" and fantasizes he could "somehow reappear reborn" to his marriage bed with wife Molly (728) while Lester tells us, "That's my wife Carolyn. . . . We used to be happy" and vows, "It's never too late to get it back" (2, 5). Both also feel displaced by a growing estrangement from their teenage daughters: Bloom's surviving child, Milly, and Lester's only child, Jane.    To compensate for their non-existent sex lives, both Leopold and Lester turn first to solo sex in the bath (or in Lester's case, the shower) and both enjoy adulterous, guilty dreams of unorthodox sexual practices, often accompanied by flower imagery.

English Poem Analysis Essay

In this poem, Pablo Neruda is talking about life, and how he feels that it is an insignificant business. He talks about life after death, saying that nobody keeps what they have and says that life is nothing but a ‘borrowing of bones’. The best thing he claims to have learnt from life is that one should not have too much of either joy or sadness, but experience both in equal quantities. He feels that his being happy was a punishment, a condemnation that caused him to plunge into the sorrows of others, and to share with them their sorrow. He says that he did not do this for fame or for money, but because he could not live in the shadows, the shadows of other people. He says that we can heal our own wounds by weeping and singing, but in front of us lie thousands of others who are in constant suffering. He feels that his business on earth was to fulfil his spirit, the happiness he felt with the sum of all his actions. It gave him great joy to bathe in the sea under the sun, and in the very foam of the sea, his heart which lay dying was seeped into the sand. The poet makes use of immense imagery in all his works. There is no poem written by Pablo Neruda, which is lacking imagery. It is one of the most common literary devices used by the poet. And this is one such poem where he has used various kinds of imagery to illustrate his thoughts.

The Articles of the Confederation and the American Revolution essays

The Articles of the Confederation and the American Revolution essays The Articles of Confederation and the American Revolution significantly changed the constitutional history of the American people. The American Revolution was based on the transition of the British colonists' to American citizens. The three challenges consisted of the cultural and political alterations needed by British Colonist's, apprehension of a new identity, and the formation of a new institution. The transition required the British colonists to change their way of thinking related to their cultural and political beliefs. The Colonist's always had a sense of their independence, but they were adapted to a controlled source of stability because they were under one ruler, allowing the feeling of safety. The continental congress represented the colonists' by ending his reign. Before the American Revolution, British colonists' did not accept the new found American Government in fear of losing legislative control. The King formally announced that he would no longer protect the colonies, leaving his people in danger in the midst of war, the delegates of the Continental Congress "brought about the end of British Government in the colonies." During the transition period, the Articles of Confederation held the states together until Americans were ready for a stronger, united union. The fear of a new lifestyle was extremely difficult for the Colonists'. The articles of confederation al lowed the thirteen colonies to maintain its dominion and all rights to govern, except the rights specifically granted to Congress, providing a sense of relief and eased the transition from British colonists' to American citizens. Between the years of 1763 and 1776, the British colonists' felt fairly untaxed, watched over like children, and ignored in their attempts to address their feelings of injustice and inequality. British Colonists' were taken advantage of, due to the Stamp tax, which placed a tax on everyday consumer goods. At this point, British co...

Secret Methods of Writing an Argumentative Essay

Secret Methods of Writing an Argumentative Essay Most students note that analytical essays take too much time and effort. Its one of the most challenging tasks. Along with that, if you write a blueprint of the structure, and figure out in what order its better to sort your claims, such a process will become much simpler. We suggest that you dont like to stare at the blank Word page for hours, so you can check various online sources devoted to argumentative writings and get some creative ideas. However, many sources of such a kind provide you with complicated advices which are hard to follow. This is a reason why we decided to describe a secret method that can improve your writing with no extra effort. Generally, it is based on analogies. Analogies help you understand the more general meaning of your entire work. Here is an example of a good analogy: your readers are a cat who wants you to feed it with tasty and fresh information. Now imagine it and get some cat food with our help. Introduction Most writers agree that the introduction is extremely important. Thats not a surprise because this is the first thing that your readers see, and the impression from the introduction affects their impression of the entire paper. To feed your cat with the best food, pay your attention to the all details, especially to the hook. It is the first sentence of your essay, and thats what can help you get some fish for your cat. There are several types of hooks, but they have one thing in common good hooks grab your readers attention and motivate them to keep reading. Lets take a look at the most common hook types: Quotes; Questions; Analogies; Philosophical statements; Facts; Anecdotes; Stories from the first person. Choose any one of these types, and just make sure that it fits your topic and your writing style. Once you hooked your readers, you must provide them with the background information on your subject, to make sure that your audience understands the context of your paper and is familiar with all the specific details. One of the most common mistakes is assuming that your readers know as much about your topic as you do. Dont provide them with detailed and sophisticated data in the introduction section, just write a necessarily brief summary, and go further. You also have to explain why your topic is important, and how your paper may help others who are interested in this subject. Usually, introductions end with a thesis statement its a backbone of your paper, all your arguments and claims must be directly related to this idea. Obviously, its not really easy to write a good thesis statement, but if you take into account our advice, you wont feel any troubles with it. Our advice is simple dont make your thesis too broad. The broader your thesis statement, the more information you need to prove and support it. Nobody wants to read a huge text which is basically about nothing in particular. Introducing your key statement, you can also give a brief description of arguments that youre going to use, to help your readers understand why you choose this point of view. Sometimes, a thesis statement may be even longer than one sentence. Just make sure that it expresses your point clearly, and its not too long. Build Your Argumentation A practice of using three arguments to support your point is quite old and common, because it really works. If you have three paragraphs, each one of which provides readers with a strong argument described in details, your point will look persuasive and strong. This is exactly what you need. At the same time, dont forget that you shouldnt be focused only on your point. We suggest mentioning a few strongest counterarguments, to let your readers understand why an opposite point is wrong. Here is an effective method: describe advantages and disadvantages of the idea. Introduce an opposite point, and rewrite your thesis statement right after that. Another extremely important thing is that if you write something, it must be supported by good evidence. Never count on your readers to just believe you. Prepare for the critical perception, and make all that you can to overcome it. Your evidence must be informative, credible, and relevant. To get enough facts, we suggest preparing for writing and collecting as much information as possible. Along with this, avoid common mistakes that can destroy your paper: Making up facts this is probably the worst thing you can do. Dont even try to fool your audience. All of your readers and instructors can check any fact any time they want, so dont fail because of the lie. Being highly emotional. Emotions are good for narrative essays, but not for argumentative essays. Rely on facts only, and dont be emotional. We also suggest moving from one thought to another through transitional phrases. Such an approach will allow your readers to follow your line of thoughts naturally, without missing any logical details. Dont be afraid to remind them your thesis statement a couple times, it helps stay focused on general ideas of your paper and on thoughts written in any paragraph in particular. Conclusion Once you provided your readers with all necessary information, you have to push them to conclusions. All information must be systematized, so that your audience could get your point clear and have something to think about, after reading. Essays that motivate readers to think are always successful, so make sure that your conclusion section is really good, and your entire paper makes a strong impression. To do it well, follow three simple rules: Repeat your thesis once again. It will help you explain why your claims are really important. Explain what will happen if people dont take your side regarding this issue. This is a really good thing in case youre writing about a controversial issue that is related to the society, politics, health, environment, and so on. Summarize your arguments and explain how they prove your point. Your essay must be persuasive, and all arguments must be tied to your general idea. If you follow our simple advice and do everything right, your essay will get highest grades. Dont be afraid of such a kind of writing task, because everyone is able to cope with it. All you need is practice and a proper writing method.

Graduate Association in Sourcing and Procurement Essay

Graduate Association in Sourcing and Procurement - Essay Example Overall Improvement: The individual will also be responsible to ensure the overall improvements of the products and services and will need to work with other team heads to ensure that the needs of the stakeholders are met and adhered too. Â  Products and Studios Management: Develop the funding plans for the company and ensure that the company receives higher levels of discounts minimum of 15% - 20% and pays lowered levels of interest on the funds, i.e. almost pay only 10%. Â  Pricing and Price Management: Get all the costing and budgeting details and plan the prices keeping in mind the various possible changes in the environment and the changes in terms of choice. This can be based on the trends of the markets and the historical data. Â  Overall Improvement: Ensure that the shareholders receive a higher rate of return on their investments. Currently, the company provides 10.3% on the return on equity (MSN Money, 2010). This can be increased to 12%. In terms of the employees make sure that the employees are paid based on their work and this will help keep a tab on the costs of the company as well. Â  Rating the performance of an intern is highly strict and more focused and this rating is based on the hours worked. In the case of a traditional employee, the rating is based on the hours worked along with the initiatives and the overall interaction and contribution of the person to the company. The rating of the performance of a Disney employee is very different from that of any other industry as the processes and the working styles are very different and hence here in Disney the possible rating systems that might work may not work in other industries. Â  

Take Home Final Exam Research Paper Example | Topics and Well Written Essays - 1750 words

Take Home Final Exam - Research Paper Example The limits of closeness are defined in one’s life through the relationships that were established. According to Marshall (2003), â€Å"a relationship is the mutual sharing of life between two or more persons† (p. 163). In explaining the nature of relationships, Marshall (2003) actually begun explaining the concept of mutuality, as a crucial facet in defining explicit contribution of knowledge, skills, or abilities to the interaction. One strongly believes that the aspect of mutuality and the class or category of the relationship would define the extent of closeness or intimacy that is applied. For instance, the classes of relationships, as discussed by Marshall (2003) distinguished between instrumental or cooperative, versus social or consummatory (p. 167). Likewise, the characteristics of relationships clearly included aspects of intimacy or closeness; and scope or extent (Marshall, 2003, p. 167). One is convinced that in social or consummatory relationships, there are more opportunities to establish greater intimacy and closeness. Interpersonal relationships that have been effectively established in one’s life include relationships with family members (parents and siblings), peers (friends and acquaintances), special loved ones (third parties who one developed close or intimate relationships with during one’s life span), and colleagues in the work setting. Within these relationships, one could assert leadership skills and related specifically defined limits of closeness, depending on the role in the relationship that is assumed. For instance, within the family circle, one could assume the role of an eldest sibling and therefore evidentenly assume a legitimate power by virtue of one’s position of birth. As such, one could exemplify leadership role as an authority in delegating tasks or in providing guidance and direction to the next siblings. Likewise, the limits of closeness in this type of social relationship is extended on ly insofar as familial bonds are established. Thus, as a sibling and a child to one’s parents, it is expected that there is a comfortable level of closeness where private information are assumed to be known and exchanged only within the members of the nuclear family. At the same token, despite sharing similar information regarding family matters or relevant issues concerning each family member, each member also expects a level of privacy and confidentiality that is still maintained. Thus, although a certain level of closeness and intimacy is a given fact within the family circle, there is still a limit to the closeness in terms of matters pertaining each of the member’s personal life. The role of relationships within the work setting is more challenging in terms of establishing and defining limits of closeness or intimacy. This concept was clearly explained by Marshall (2003) when he asserted that â€Å"leaders are therefore generally advised not to have personal frie nds among their congregation, because such friendships cause problems† (p. 173). Leaders or managers at work are expected to delineate their role as a superior to that of being a personal friend, for instance, to one or two of office colleagues. Any preferential attention or affectionate behavior manifested by the leader to a subordinate could be construed as an action of favoritism and is therefore a violation of the policy of avoiding conflicts of interests. Thus, there should be a limit to the closeness that is exhibited within a work environment to abide by the standards of professionalism, as well as conform to ethical, moral and legal standards. It was therefore appreciated from Marshall’s (2003) affirmation that â€Å"the question of intimacy in personal relationships can be something of a