Purification of Fructosyl Transferase (FTase)

Purification of Fructosyl Transferase (FTase) Introduction Food products are formulated for taste, appearance, cost and convenience of the consumer. The composition of food products that confer a health benefit is a relatively new trend, and recognizes the growing acceptance of the role of diet in disease prevention, treatment and well-being. It is thus becoming increasingly clear that there is a strong relationship between the food we consume and our health. Scientific knowledge of the beneficial role of various food ingredients (nutrients) for the prevention of specific diseases is rapidly advancing. Functional foods, pharma foods and nutraceuticals are synonyms for foods that can prevent and treat diseases. Generally, a functional food can be defined as ‘any food that has a positive impact on an individual’s health, physical performance or state of mind in addition to its nutritional content’. Functional foods in addition to their basic nutritional content and natural being, will contain the proper balance of ingredients which will help to improve many aspects of human lives, including the prevention and treatment of illness and disease (Goldberg, 1994). So far, a large number of functional foods in various forms have already been introduced into the market. Many of them contain a number of characteristic functional ingredients. They include dietary fiber, oligosaccharides, sugar alcohols, peptides and proteins, prebiotics and probiotics, phytochemicals, antioxidants and polyunsaturated fatty acids (Stark and Madar, 1994). Oligosaccharides are very well recognized as ‘functional food ingredients’ because of their positive effects on human health. This research work focuses on the microbial production and purification of Fructosyl Transferase (FTase) and the production of Fructooligosaccharides (FOS) by transfructosylation using this enzyme. Oligosaccharides Oligosaccharides are usually defined as carbohydrates with a low degree of polymerization (DP) and consequently low molecular weight. They are composed of 3-10 simple sugar units linked together.The main categories of nondigestible oligosaccharides include carbohydrates in which the monosaccharide unit is fructose, galactose, glucose and xylose. Chemical differences among commercially available oligosaccharides include chain length, monosaccharide composition, degree of branching and purity. They are found naturally (small amounts) in many plants.. Plants with large amounts of oligosaccharides include  chicory root, from which most commercial inulin is extracted, and so-called Jerusalem. They are also found in onions,  leeks, garlic, legumes, wheat, asparagus, jicama, and other plant foods. Oligosaccharides are functional food ingredients that have great potential to improve the quality of many foods. In addition to providing useful modifications to food flavor and physicochemical characteristics, they possess properties that are beneficial to the health of the consumers. In general, food grade oligosaccharides are not pure products, but are mixtures containing oligosaccharides of different degrees of polymerization, the parent polysaccharide or disaccharide and the monomer sugars (Roberfroid and Slavin, 2000). The production and application of food grade oligosaccharides are increasing and their major uses are in beverages, infant milk powders, confectioneries, bakery products, yoghurts and dairy desserts. FRUCTOOLIGOSACCHARIDES FOS are chain polymers of the sugar fructose that are found in a variety of foods. The sugar units can be linked in a single straight chain or can be branched. In many cases small amounts of glucose are also contained in the chain. Chemically, FOS is difficult to define, because the length of the fructose chains can vary from source to source. Therefore a FOS analysis of a food may report the total of several closely related compounds. Inulin is an example of a longer chained compound that is considered as FOS. The shorter (lower molecular weight) compounds tend to have a sweet taste. The size and complexity of the FOS molecule desirable characteristics. Although the simple sugars fructose and glucose are quickly absorbed into the body by the intestines, FOS for the most part is indigestible and therefore acts as a non-digestible fiber in the diet. This is because humans does not have the enzymes to break down FOS as it travels down the digestive tract. When the FOS reaches the large intestine and the colon, the bacteria that are found there start to break down the FOS. These bacteria have the enzymes needed to break down FOS. Bifidobacteria have been reported to use FOS. It is believed that foods that promote bifidobacterial growth are good for the health. Of all the oligosaccharides known so far, FOS has attracted special attention. FOS is a common name for fructose oligomers that are mainly composed of 1- Kestose (GF2), 1 Nystose (GF3) and 1F – Fructofuranosyl nystose (GF4) in which fructosyl units (F) are bound at the ÃŽ ² 2, 1 position of sucrose (GF). Structure of Fructooligosaccharides Occurrence FOS derived from sucrose occur in many higher plants as reserve carbohydrates. They are found in a variety of edible plants, including banana, barley, garlic, honey, onion, rye, brown sugar, tomato, asparagus root, Jerusalem artichoke, wheat and triticale (Fishbein et al, 1988). The concentration of FOS in these foods is diagrammatically represented in Figure 1.2 (derived using data from the Environmental Protection Agency’s Dietary Risk Evaluation system EPA, 1984). FOS like 1- kestose, neokestose, 6- kestose and their derivatives have also been isolated from the plant Agave vera cruz (Satyanarayana, 1976). FTase producing organisms FTase by transfructosylation activity helps in the production of FOS. The enzyme source of FOS synthesis can be divided into two classes; one is plants such as asparagus, sugar beet, onion, Jerusalem artichoke etc; the other consists of bacteria and fungi such as Arthrobacter sp., Fusarium sp., Aspergillus sp., Aureobasidium sp. Functional properties of FOS Low calorific value The ÃŽ ²- configuration of the anomeric carbon, C2 in their fructose monomers make FOS nondigestible by human digestive enzymes which are mostly specific for ÃŽ ±-osidic linkages and hence they are not utilized as an energy source in the body. However, due to colonic fermentation, they have an energy contribution to food of about 1.5 kcal/ g. This property makes them suitable for use in sweet, low-calorie diet foods and safe for consumption by individuals with diabetes. In the case of very sweet foods, they may be used as bulking agents in conjunction with intense artificial sweeteners such as aspartame, phenylalanine or sucralose, thereby masking the aftertaste produced by some of these intense sweeteners (Crittenden and Playne, 1996). Non cariogenicity Unlike starch and simple sugars, FOSs are not utilized by oral microflora like Streptococcus mutans to form acids and insoluble ÃŽ ²-glucans, that serve as a matrix for plaque formation and are the main culprits in causing dental caries (Oku, 1994). Hence, FOSs are presently used as non – cariogenic sugar substitutes in confectionery, chewing gums, yoghurts and drinks. Dietary fibre effect ‘Dietary fibre is the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine’ (AACC Report, 2001). The nondigestible quality of FOS makes them act similar to dietary fibre, and thus prevent constipation. However, excessive consumption of FOS may cause flatulence or diarrhea, the minimum dose being 40-50 g/day. The advantages FOS has over dietary fibre are that they have a smaller daily requirement, do not cause diarrhea in recommended doses, are slightly sweet, have neither bad texture nor bad taste, are completely water soluble, do not build viscosity, do not bind minerals, are physically stable, and are easier to incorporate into processed foods and drinks (Tomomatsu H, 1994). Prebiotic effect In recent years, the ability of FOS to promote the proliferation of bifidobacteria in the colon has been recognized. These intestinal bacteria metabolize FOS readily and produce large amounts of short chain fatty acids resulting in an acidic pH in the lumen of the large intestine. The beneficial bacteria such as Bifidobacterium sp. and Lactobacillus sp. are resistant to the acidic pH, whereas the harmful bacteria such as Clostridium sp. are sensitive to the acidic conditions. Therefore the proliferation of useful bacteria is stimulated and that of harmful bacteria is suppressed. Subsequently, FOS has been described as one of several prebiotics, which can be defined as ‘a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and / or the activity of one or a limited number of bacteria in the colon and thus improves host health’ (Gibson and Roberfroid, 1995). FOS have been demonstrated to increase bifidobacteria numbers in the colon at doses of Lipid lowering effect FOS in diet has been found to decrease the levels of triglycerides, serum cholesterol and lipids. Hypotriglyceridemia is due to decrease in the hepatic synthesis of triglycerides and hypocholesterolemia is likely to result from the antagonistic effect of short chain fatty acids, especially propionate on cholesterol metabolism. Propionate has been found to be an inhibitor of HMG CoA reductase, an important enzyme in cholesterol synthesis (Roberfroid and Slavin, 2000). Effect on mineral absorption FOS have been found to enhance the absorption of Ca2+ and Mg2+ ions and maintain balance of iron and Zn2+ ions. This has been found to be due to the osmotic effect, acidification of the colonic content due to fermentation and production of short chain carboxylic acids, formation of calcium and magnesium salts of these acids and hypertrophy of the colon wall (Roberfroid and Slavin, 2000). Anticancer effect FOS has an indirect effect on prevention of cancer in human beings due to its prebiotic properties. This is due to immunity enhancements by the cells, cell wall components and extracellular components of bifidobacteria (Tomomatsu H, 1994). Production of nutrients The presence of a good colonic environment with high bifidobacterial count leads to the production of Vitamins B-1, B-2, B-6, B-12, nicotinic acid and folic acid. Bifidobacteria – fermented dairy products also improve lactose tolerance, calcium absorbability and digestibility (Tomomatsu H, 1994). Antidiabetic effect FOS have been claimed to have no effect on blood glucose levels in patients with type 2 diabetes, possibly due to effects of short chain fatty acids produced during fermentation. (Luo et al, 2000). PROBLEM STATEMENT Normally, pure sucrose is expensive and the cost to produce FTase by using sucrose is high. There are some by-product like cereal bran, corn-products, sugarcane molasses and by-products of coffee and tea processing industries were used as a substrate to produce FTase from microorganism (Sangeetha et al., 2003). This by-product can lower the cost of production of FTase. Before FOS can be produced, the enzyme FTase must be undergone a series of purification. The purification of FTase must be done in order to get high yield of FOS. Many researchers have reported the purification and characterization of FTase from various sources and FTase has been found to differ in their molecular weight and properties from one source to another (Lateef et al., 2006, Sangeetha et al., 2003). FTase exhibits hydrolytic activity which can dominate the process (Delphine et al., 2007). This fact will lead to lower production yields and to a contamination of the final product with glucose and fructose (Delphine et al., 2007). Purification of FTase is important for batch production of FOS. By doing so, the nature of its hydrolytic activity can be studied, improve the understanding its mode of operation and be able to classified which type of enzyme should it belong to (Là ¢Ã¢â€š ¬Ã… ¸Hocine et al., 2000). RESEARCH OBJECTIVES The main objective of this research is to purify FTase enzyme from microfungi Aspergillus oryzae isolated from CFTRI. To enhance the production of FOS as a food additive. SIGNIFICANCE OF STUDY In Malaysia, the FTase enzyme used to be imported from other country such as Japan, India and United States (Sangeetha et al., 2005). Normally, the cost for production of FOS from commercialized enzyme is high, so by producing and purifying the enzyme, we can reduce the cost. Normally, crude enzyme will produce lower yield of FOS than purified enzyme. In order to fulfill high customer demand on the functional food such as FOS, the production of FOS must be increased. In order to produce high yield of FOS, the enzyme need to be purified. By doing this, the activity of enzyme will be increased and so do the production of FOS. Purification and characterization of the enzyme (FTase) are necessary steps to obtain the product (FOS) rapidly, with high purity, to improve our understanding of its mode of action and the nature of activity. Efforts have been made to purify the FTase enzyme to get high specific activity and fold of purification but the reaction mechanism involved in FOS production does not lead to higher yields than the theoretical maxima of 56 – 58 % due to inhibition caused by accumulation of glucose. Nevertheless, the time involved in obtaining maximum FOS yield has been considerably reduced by using the purified FTase in comparison to the crude FTase. The product FOS formed has also been characterized for its physicochemical properties and structure using HPLC or LC-MS. Studies on the prebiotic effects of FOS are required to establish its functional properties and to demonstrate its use in product development. The present research work has also focused on the scaling up of both the produ ction of FTase as well as FOS.

Women in Combat Rough Draft

Auriel Payan April 7, 2013 English 2010 Rough Draft Women fighting in combat is a topic that has stirred a lot of controversy in the last few months. This comes about from the uplifting of the ban that did not let women fight in combat. Though the Women’s Armed Services Integration Act in 1948, created a corps for women in every branch of military, it was also the beginning of when women would not be included in combat. (Mackenzie 1,) Whenever the topic of war comes to mind, the one’s usually pictured in war are men. People tend to forget that men are not the only ones involved in war or the military. In January 1994, a memorandum from then Secretary of Defense Les Aspin rescinded the â€Å"risk rule† barring women from any positions that could expose them to direct combat, hostile fire, or capture; the rule was replaced by the â€Å"direct ground combat assignment rule,† which more narrowly tailored the restriction to frontline combat positions. † ( Mackenzie, 1) This would completely change the way women in the military would be. Though not as big in number, women still do have a significant role in the military today. They can join the Military but they can have no part in combat.This limits the kinds of jobs that women can have. Woman in the United States want to feel equal to the man and the stigma that war is a â€Å"man’s thing â€Å" has created a lot of controversy, especially in the society that we live in now. Women in our society are breaking away from the boundaries set from the past generations. Women are now taking and seeking roles that were once considered only for men. Just like in our society, women are trying to advance in the military. Advancing in the military, not only has to be being able to fight in combat, but also reaching some of the highest military rankings.The leap for women being able to fight in combat came this past January, when the United State’s Department of Defense, uplifted the ban on women being able to fight in combat. A woman representing her country by joining the military is something that not only takes great sacrifice, but courage as well, and they deserve the utmost respect. If a woman is willing to risk her life for the sake of her country, just like the men, should be allowed to partake in combat. War is much physical as it is mental. Opponents argue that women are physically not capable to succeed out in the battlefield.If a soldier is not physically capable to perform, they could possibly endanger themselves or their fellow soldiers. Soldiers out on the battle field carry various items like electronic equipment, weapons, ammunition, heavy body armor, and enough water, weighing anywhere between fifty to hundred pounds. There is no denying that women on average do have forty to fifty percent less upper-body strength compared to men, but it is not one hundred percent certain that certain women are not fully capable. The statistic that women a re a lot weaker in their upperbody comes up a lot, but it is stretched from the truth. The physical fitness argument, which tends to focus on differences between average male and female bodies, is also undermined by the fact that women who join the military tend to be more fit than the average American. Additional training and conditioning further decrease the gap between female and male service members, and evidence indicates that women usually benefit substantially from fitness-training programs. † (Mackenzie, 1) To join the military there is this mental and physical toughness that one has to have or has to overcome by the time that they are about to be deployed.Yes, there is some women that are not physically capable to perform in the military, but at the same time, there are many men as well. Needless to say, there is a certain kind of person that is needed when joining the military. This goes beyond male and female. Character has a lot more to do with the success of an in dividual than the sex does. Regardless of this, the opposition still argues that women are still not physically capable and this could seriously put people in jeopardy.Here in the United States, opponents of allowing women fight in combat, strongly believe that women are not physically capable, but in other Countries this has been disproven. â€Å"In the 1970s, the Canadian military conducted trials that tested women's physical, psychological, and social capacity for combat roles. The results informed the final decision of the Canadian Human Rights Tribunal to remove Canada's female combat exclusion. After similar tests, Denmark also lifted its combat ban in the late 1980s. (Mackenzie, 1) If other countries have dismantled this theory that women are not physically capable, why has the United State’s not followed? Qualifications for being able to fight on the battle field should be tested on whether or not the individual is physically and mentally fit, the sex of the individu al should not be the determining factor for why they cannot be out in combat. The United States has had a rich history of patriotism. Citizens, for the most part, are proud of the nation that they live in. When one looks outside for the fourth of July, all one sees is red, white and blue.A lot of Americans have a great sense of pride in the nation that they live in. Most Americans have a sense of recognition for the sacrifices that some people have made in order to make this a very prosperous country. Among those that Americans feel that deserve the recognition are ones that serve in the military. There is the upmost respect and pride that comes along when an American sees a soldier that is fighting for their country. According to Pew Research Center for the People and Press, â€Å"Two-thirds (66%) support allowing women in the military to serve in ground units that engage in close combat, while just 26% are opposed. More than half of Americans are in support of the uplift of this ban. As an American, one has the god given right to choose if they want to defend their country. Upon deciding this, a lot of courage and pride has to come for one to say that they want to join the military. Though women only make up fifteen percent of the United State’s military, or roughly two hundred and fifteen thousand women currently serving, they still deserve the same amount of respect given to the men. Women joining the military are aware of the disadvantages that they are going to face just because of the simple fact that they are women.They realize at a very early start that war for men and war for women are two distinct things. It is a given that women are much more susceptible to negative aspects of war like sexual assault and sexual harassment. The fifteen percent of women that make up the military have chosen to overlook these negativities and have chosen to join the military regardless. These women are aware of the risks and dangerous and still have chosen to join the military. The courage, dedication and willingness of women in the military alone, should be the reason for why they should be allowed to fight in combat.The debate on women fighting in combat has a similar history to the debate on the policy â€Å"Don’t Ask Don’t Tell. † In 1950, President Truman signed the Uniform Code of Military Justice. This policy sets up discharge rules for homosexual members or anyone seen acting out in homosexual ways (Cristina Rivero, 1). This was the beginning of disallowing openly gays from joining the military. During Ronald Reagan’s presidency, it was made clear from his staff that â€Å" Homosexuality is incompatible with military service. Those that were believed to act in homosexual ways or were gay were discharged because of the fear that homosexuals would disrupt the order of the military. According to the supporters of this ban, the disruption was going to be so great, that the military as a whole was in jeopa rdy. Many people felt, that this â€Å"would create an unacceptable risk to the high standards of morale, good order and discipline, and unit cohesion that are the essence of military capability. † (Wiki, 1) In essence, military officials were scared that soldiers would end up betraying or not performing to their peak if there were an openly gay member in their unit.This would all change In 1993 when President Clinton, ordered that a bill be passed that military members would not be asked their sexual orientation. This would become to be known as â€Å"Don’t Ask, Don’t tell. † In simple terms, if one was gay and was in the military, one could not openly express themselves as being gay. This stirred a lot of controversy because people believed that one should be able to express their sexual orientation without repo caution or fear of being discriminated against. Even though in 2003, President Clinton would want the â€Å"Don’t ask, don’t tel l† policy removed, it would not come about until years later.In 2011, don’t ask, don’t tell would come to an end. As the end of don’t ask, don’t tell came, a lot of military officials came out. Several high ranking military officials decided it was the appropriate time to finally come out. For years, the fear that openly gay members would be detrimental to the military kept many military men and women from being open about their sexuality. There was such a great fear when in reality there was minimal or even no consequences from openly gay members serving in the military. This is much like the debate on women fighting in combat today.Women have been serving in the military for decades. Hundreds of women have been hurt and even killed while proudly serving their country. With the proper training, any military member should be able to perform at their peak. If someone is not physically, mentally ready to be out on the battlefield, one should not be ou t there. There have been gay members in the military that have had the proper training leading to successful military careers. The fact of their sexual orientation did not limit or hurt them while serving in the military. Like men, women have been serving their country with honor and pride for decades.But unlike men, the women in the military do not tend to receive as much recognition for their service to their country. Women may only make up fifteen percent of the military, but they have been excluded from twenty percent of the jobs in the military. â€Å"According to the Department of Defense, ending the ban will open up 237,000 positions to women. † (Hallman, 1) With the uplift of this ban, more opportunities will be opened for women not only in the types of jobs that they can have, but the rankings that they could possibly reach. Sexual assaults in the military towards women tend to go unreported.By allowing women to reach higher-rankings, there could be the possibility t hat sexual assaults go down or more likely to go reported. â€Å"Recent surveys of female veterans found that close to a third were victims of rape or assault while they were serving, which is double the rate in the civilian population. † (Hallman, 1) More often than not, sexual assaults go unreported in the military. By having women as higher-ranking officials, the sexual assault epidemic in the military may start being addressed properly. The proper steps in reporting sexual assaults may take place.There is always this fear that there will be severe repo cautions if women come forward. Women may feel a lot more comfortable reporting sexual assault to a women high-ranking official. Allowing women to fight in combat will be a positive movement that may even get rid or limit the negative aspect of the military. During war, soldiers encounter a variety of difficult tasks, mishaps and dangerous situations. All of these things are inevitable and are a part of war. Soldiers are pu t through situations that are not only physically but also mentally exhausting. Because of this a lot of soldiers develop PTSD or post traumatic stress disorder. Post-traumatic stress disorder (PTSD) is a mental health condition that's triggered by a terrifying event. Symptoms may include flashbacks, nightmares and severe anxiety, as well as uncontrollable thoughts about the event. † (Mayo Clinic) From 2009, post-traumatic stress disorder diagnoses have increased in women from fifteen percent up to twenty-two percent. Men have increased from nine percent to fourteen percent in the diagnosis of PTSD. (Anderson, 1) This alone raises a lot of questions whether women are mentally stable enough to be on the front lines.Though it is true that women are being diagnosed at a much higher rate than men, there are probably a lot of men that are not willing to come forward and admit that they have post-traumatic stress disorder. Men in general tend to not want to come forward because of t he fear that they will be looked as less of a man or less honorable. Women on the other hand are much more likely than men to come forward and because of this, the statistic may not be as accurate and be swayed. Women are traditionally viewed as these caring, emotional, and even soft individuals when compared to men.But realistically, in the military all of these characteristics of traditional women are dropped. It takes a certain kind of individual, man or woman, to join the military. The women that do decide to join the military are not like the traditional women. These women are tough, rigid individuals that are not much different to their male counterparts. The military lifestyle is a lot different than the civilian lifestyle. Yes it is true that women officially may not be allowed to fight on the frontlines, but that does not mean that women are not exposed to the same dangers as the men who are.USA Today States, â€Å"since the start of the wars in Afghanistan and Iraq, 292,0 00 women have served in those combat zones out of a total of almost 2. 5 million, Pentagon records show. In both wars, 152 women have died from combat or noncombat causes, records show, and 958 have been wounded in action. † Much like the men, some women that have served for their country have died. This just goes to show that even though women typically aren’t in the battlefield, the risk of them dying is significant. The military should be black and white not male or female.A lot of people are calling the uplifting of women fighting in combat an â€Å"experiment†. Is it really an experiment or just an official document that allows women to fight in combat? Women in the military are exposed to the same dangers as much as the men are. With the uplifting of this ban, men and women will now receive the same training essential for the front lines. Both men and women should receive this training in order to guarantee that both will be successful. If a soldier is not prepared mentally or physically, they should not be allowed to fight in combat.There should be rigorous testing on each individual soldier determining if they are combat ready. The sex of the soldier should not be the deciding factor whether or not someone is combat ready. There are plenty of men that are physically and mentally ready, but there is also plenty of women as well. Women are becoming more equal in our society vastly due to the fact that they are fully capable of doing any task that men can perform. Citations Mackenzie, M. H. (2012). Let Women Fight. Foreign Affairs, 91(6), 32-42. Tom V. B. and Jim M, (2013, January 23).USA Today http://www. usatoday. com/story/news/nation/2013/01/23/panetta-women-combat-pentagon/1859221/ http://en. wikipedia. org/wiki/Don't_ask,_don't_tell Mackenzie, M. H. (2013,January 23) http://www. foreignaffairs. com/articles/138200/megan-h-mackenzie/let-women-fight Hallman, L. (2013, March 6) End of Combat Ban Will Give Women Recognition They Dese rvehttp://www. foreignaffairs. com/articles/138200/megan-h-mackenzie/let-women-fight Pew Research (2013, January 29) http://www. people-press. org/2013/01/29/broad-support-for-combat-roles-for-women/1/

Conflict Between Hamlet And Hamlet - 1125 Words

In William Shakespeare’s, Hamlet, Hamlet experiences conflict with those around him, as well as a battle within himself. This is represented in three distinct ways throughout the play. Inward conflict is represented between Hamlet and himself, whereas outward conflict is represented between Hamlet vs. the surrounding society, and Hamlet vs. Claudius. The use of conflict in both an inward and outward sense allows for progression and a climatic build within the play as Hamlet works towards a resolution. The most obvious conflict is the inner conflict between Hamlet and himself. Hamlet recently lost his father; now his mother has betrayed him and married his uncle, his father’s brother. This strikes upon the inward conflict where Hamlet contemplates suicide. â€Å"O, that this too too-solid flesh would melt, Thaw, and resolve itself into a dew! Or that the everlasting had not fix’d His canon ‘gainst self-slaughter!†(1.2.129-132) By Hamlet contemplating his suicide, it represents a moral dilemma in which he has to deal with by himself. As the play progresses, Hamlet continues to contemplate his suicide as he discovers that King Claudius was the one who murdered his father. He is wondering whether or not is it worth living on earth as he says, â€Å"To be, or not to be, that is the question- whether tis nobler in the mind to suffer the slings and arrows of outrageous fortune, or to take arms against a sea of troubles and by opposing end them?†(3.1.57- 61) This is a continuation of theShow MoreRelatedConflict Between Hamlet And Laerte By William Shakespeare1564 Words   |  7 PagesThroughout Hamlet, several characters die. For most, this happens through combat, such as the duel between Hamlet and Laerte, which kills them both. In the play, however, two characters die in quick, seemingly careless ways. 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