Coca Cola Market Segmentation Essay

Introduction Coca cola utilizes both internal and external marketing strategies to gain a competitive advantage over its competitors becoming a successful company with great earnings. Additionally, the company claims that the innovation is at the heart of everything they do add to their success. The company has segmented its market in terms of demographics, psychographic, geographic and lifestyle. Discussions After building a strong reputation and brand image, the Coca-Cola Company changed its name from Coke to New Coke in 1985 as they continued to offer customers a better taste. Demographically, although the company seems to focus on the whole population globally, its particular target is the young generation showing the energy giving element to the customers. According to world demographics 2013, 57.4 percent of the global population lies in the age bracket of 15-54 years of age. The statistics provide a clear indicator that the Coca-Cola focus on the largest demographic in the world with potential customers thus a right strategy to ensure sustainability and growth. In regard to family size, the company serve its in different bottle sizes for families to choose according to their sizes ranging from 200ml to 2 Liters pack (Global Functional Drinks Industry Profile, 2014). The Coca-Cola Company practice geographic segmentation in terms of regions. Through the price remains constant in all parts, the brands vary according to the weather of the region. For example, in Hong Kong during summer season Coca-cola has a unique brand called freezing coke (Global Functional Drinks Industry Profile, 2014). The strategy ensures continued brand loyalty by providing all weather drinks at the same price. Further in terms of place of  consumption. Coca-Cola puts up vending machines in meeting terminus and stations to ensure access to all. Locally in states the company provides equal and continuous supply to the local shops. Low-income earners have access to returnable glass bottle with medium income earners having plastic non-returnable bottle and Coke tin for high-income earners. In psychographic segmentation, Coca-Cola distinguishes customers into different groups based on buyer’s values, lifestyle or personality. Although people share the same demographic group, they exhibit diverse psychographic profiles forcing Coca-Cola to design and manufacture products suiting personality. In terms of lifestyle, consumers portray different lifestyles. Coca-Cola Company presented a more portable packaging for their soft drinks in order to provide for the modern ever busy user. The company endows its products with brand personality in line with a particular consumer personality. Further in observing culture and especially the diet matter, Coca-Cola produced health conscious products such as Coke Zero and Diet Coke (Global Functional Drinks Industry Profile, 2014). Conclusion The Coca-Cola Company boasts of high market and business share globally. The company has continuously gained more profits through use of different marketing strategies and market segmentation. Through segmentation, the company has managed to ensure continuous customer satisfaction by providing goods and services that meet all the social classes. Sales wise, the company have continuously experienced increasing sales by increasing the benefits derived from each segment for their products and services. The trend also benefits from the ever increasing customer loyalty as a result of satisfaction. Through its franchising model, the company runs a successful business in non-alcoholic beverage industry globally. It stands to capture any new drink type in the market as it has done before. A slight decline in segment consumption attracts careful analysis from the company to dig down into the cause and innovative measures to curb such loss. References Global Functional Drinks Industry Profile. (2014). Functional Drinks Industry Profile: Global, 1-35.

Chemistry Experiment

Experiment 1: TLC Analysis of Analgesic Drugs 1/23/2011 Purpose: The goal of this experiment is to test our knowledge and understanding of TLC analysis by having us do a TLC analysis of analgesics to figure out their main chemical components. Calculations: 1. ) Rf = Distance spot traveled/ distance solvent traveled Results: Table 1: TLC Analysis Analgesic Drugs| Rf Value| Acetaminophen| 0. 323| Aspirin| 0. 597| Caffeine| 0. 081| Unknown 154 (Plate 1)| 0. 081, 0. 306, 0. 597| Ibuprofen| 0. 698|Salicyclamide| 0. 587| Unknown 154 (plate 2)| 0. 079, 0. 397, 0. 587| Discussion: The goal of this experiment was to use TLC Analysis to determine the analgesic drug present in the unknown solution 154. One observation of the unknown spotting solution was that it was cloudy. After forming the unknown spotting solution it was then heated to make it a more saturated solution thus enabling the spots on the TLC plate to contain more of the drug making it easier to compare it with the other drugs.Aft er the TLC plates were developed and visualized first through UV light and then through an Iodine chamber ( though no more spots were visualized with iodine chamber) , Rf values were then taken of each spot on the plate. The Acetaminophen had a value of 0. 323, Aspirin had a value of 0. 597, Caffeine had a value of 0. 081, Unknown 154 on the first plate had 3 different values; 0. 081, 0. 306, 0. 597, Ibuprofen had a value of 0. 698, Salicylamide had a value of 0. 587, and Unknown 154 on the second plate had three different values as well; 0. 079, 0. 97, and 0. 587. Through analysis it was determined that unknown 154 was Aspirin. This was found by comparing the Rf values of all the analgesic drugs to unknown 154. After comparing them it was seen that the Rf value for Aspirin matched one of the Rf values for unknown 154 exactly. The determination of the unknown was further supported by looking at the samples in solution. Both the unknown 154 and Aspirin were cloudy in solution thus fu rther supporting the identification of unknown 154. In this experiment there were not too many sources of error.One source could have been that the glassware being used may not have been cleaned thoroughly before using it thus possibly contaminating the unknown or the other analgesic drugs. This Experiment definitely was an efficient way to help determine what type of drug the unknown 154 was but there are a few improvements that could have been done to help better determine the unknown. One improvement could be to examine the solvent used because different solvents create different separations between spots.Another improvement could be to also run an Infrared Spectroscopy on the unknown and known compounds. Answers to Questions: 1. ) When running TLC plates three common mistakes that can be made are using a pen instead of a pencil to mark on the plate, using the wrong solvent, and touching the plates thus getting grease on them. 2. ) The Starting line and the spotting line are mark ed with pencil rather than pen because the ink from the pen would move with the solvent front thus interfering with the results. 3. ) Acetaminophen Aspirin CaffeineIbuprofen Salicylamide Pretty much all of the functional groups can determine the polarity of a compound. This includes Amides, Acids, Alcohols, Ketones, Aldehydes, Amines, Esters, Ethers, and Alkanes. Even though they all can determine the polarity of the compound Amides, Acids, Alcohols, Ketones, and Aldehydes are the most polar and therefore are the best at determining polarity in compounds. 4. ) In order of increasing polarity Ibuprofen is the least , then it is Aspirin, then Salicylamide, then acetaminophen, and Caffeine is the most polar. 5. The Acetic Acid in the TLC solvent is used to increase the polarity of the developing solvent thus reducing the amount of attraction the polar compound has for the stationary phase. 6. ) TLC is a good way to determine an unknown, but not to determine composition of an unknown. O ne good way to determine the composition of an unknown is to run an Infrared Spectroscopy which when analyzed functional groups can be identified. Another way to determine the composition of an analgesic drug could be to run a Mass Spectroscopy which when analyzed could determine the elemental composition of the drug. Chemistry Experiment Experiment 1: TLC Analysis of Analgesic Drugs 1/23/2011 Purpose: The goal of this experiment is to test our knowledge and understanding of TLC analysis by having us do a TLC analysis of analgesics to figure out their main chemical components. Calculations: 1. ) Rf = Distance spot traveled/ distance solvent traveled Results: Table 1: TLC Analysis Analgesic Drugs| Rf Value| Acetaminophen| 0. 323| Aspirin| 0. 597| Caffeine| 0. 081| Unknown 154 (Plate 1)| 0. 081, 0. 306, 0. 597| Ibuprofen| 0. 698|Salicyclamide| 0. 587| Unknown 154 (plate 2)| 0. 079, 0. 397, 0. 587| Discussion: The goal of this experiment was to use TLC Analysis to determine the analgesic drug present in the unknown solution 154. One observation of the unknown spotting solution was that it was cloudy. After forming the unknown spotting solution it was then heated to make it a more saturated solution thus enabling the spots on the TLC plate to contain more of the drug making it easier to compare it with the other drugs.Aft er the TLC plates were developed and visualized first through UV light and then through an Iodine chamber ( though no more spots were visualized with iodine chamber) , Rf values were then taken of each spot on the plate. The Acetaminophen had a value of 0. 323, Aspirin had a value of 0. 597, Caffeine had a value of 0. 081, Unknown 154 on the first plate had 3 different values; 0. 081, 0. 306, 0. 597, Ibuprofen had a value of 0. 698, Salicylamide had a value of 0. 587, and Unknown 154 on the second plate had three different values as well; 0. 079, 0. 97, and 0. 587. Through analysis it was determined that unknown 154 was Aspirin. This was found by comparing the Rf values of all the analgesic drugs to unknown 154. After comparing them it was seen that the Rf value for Aspirin matched one of the Rf values for unknown 154 exactly. The determination of the unknown was further supported by looking at the samples in solution. Both the unknown 154 and Aspirin were cloudy in solution thus fu rther supporting the identification of unknown 154. In this experiment there were not too many sources of error.One source could have been that the glassware being used may not have been cleaned thoroughly before using it thus possibly contaminating the unknown or the other analgesic drugs. This Experiment definitely was an efficient way to help determine what type of drug the unknown 154 was but there are a few improvements that could have been done to help better determine the unknown. One improvement could be to examine the solvent used because different solvents create different separations between spots.Another improvement could be to also run an Infrared Spectroscopy on the unknown and known compounds. Answers to Questions: 1. ) When running TLC plates three common mistakes that can be made are using a pen instead of a pencil to mark on the plate, using the wrong solvent, and touching the plates thus getting grease on them. 2. ) The Starting line and the spotting line are mark ed with pencil rather than pen because the ink from the pen would move with the solvent front thus interfering with the results. 3. ) Acetaminophen Aspirin CaffeineIbuprofen Salicylamide Pretty much all of the functional groups can determine the polarity of a compound. This includes Amides, Acids, Alcohols, Ketones, Aldehydes, Amines, Esters, Ethers, and Alkanes. Even though they all can determine the polarity of the compound Amides, Acids, Alcohols, Ketones, and Aldehydes are the most polar and therefore are the best at determining polarity in compounds. 4. ) In order of increasing polarity Ibuprofen is the least , then it is Aspirin, then Salicylamide, then acetaminophen, and Caffeine is the most polar. 5. The Acetic Acid in the TLC solvent is used to increase the polarity of the developing solvent thus reducing the amount of attraction the polar compound has for the stationary phase. 6. ) TLC is a good way to determine an unknown, but not to determine composition of an unknown. O ne good way to determine the composition of an unknown is to run an Infrared Spectroscopy which when analyzed functional groups can be identified. Another way to determine the composition of an analgesic drug could be to run a Mass Spectroscopy which when analyzed could determine the elemental composition of the drug.

Death Penalty and Life Imprisonment Essay Example | Topics and Well Written Essays - 1250 words

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420 Essay Example | Topics and Well Written Essays - 250 words - 2

420 - Essay Example In particular, almost 600 sea turtles, 100 dolphins, more than 6000 birds, and lots of other mammals were found dead. Besides, the oil spill increased mortality among whales. As ecologists report, the mortality rate among dolphins increased by 50 times. According to recent researches, however, the Gulf of Mexico is gradually recovering after the catastrophe. American oceanologists claim that reef-building corals, which cannot survive in contaminated water, are currently in quite good condition. They are reproducing and growing in the ordinary course. At the same time, biologists report insufficient increase in average water temperature. Some researchers, however, voice fears concerning the impact of the  BP oil spill over the Gulf Stream, which is known to be a powerful climate forcing factor. There is an opinion that its temperature has lowered by 10 °C. What is more, the stream tends to separate into several underflows. This may be one of the prime causes of certain weather anomalies which could be observed in Europe. Heavy winter frosts may be one of these examples. However, this is just one of the theories which needs to be proven. It is hard to say how much time nature will need in order to recover after the catastrophe. There is a hope that future generations will not feel its consequences, though there is no guarantee that similar incidents will not

Homework assignment Case Study Example | Topics and Well Written Essays - 1250 words

Homework assignment - Case Study Example One customer may complain about the store’s services online and have great repercussion to the businesses sales. The presence of social networks handled to the constant connection of more customers Customer before and after sale services have a large impact on the store’s customer base due to the relationships levels. The employee’s interpersonal and communication skills have effects on the customer choice of the store. The case study has also revealed the efforts of the management to improve these skills to ensure that the store attracts more customers. The management has come up with training procedures that have will help improve employee-customer communication and relationships. It will also help interpersonal relationships between the employees that will encourage helping amongst various departments. These strategies will improve customer satisfaction ratings of the store and in the long run increase the number of sales (Lawson, 2006). Without proper customer handling skills, a firm without customer handling skill may lose all its customers. The retail store has adopted a customer service training program that will incorporate all employees. The core objectives of the employee program are to improve customer interaction through relationships and good communication skills. It will focus on the employee behavior towards customers despite the situation they are put in at the store. Knowledge will be passed to the staff through use of videos that portray the real situation that an employee may encounter. Situation behavioral approach helps control employee emotions towards customers despite the character traits of the customers (Werner and DeSimone, 2012). The videos are presented in two sessions that expose different situations depending on the employees response towards the customer. They show both good and bad customer services and ways of handling. After the

Week 4 team part of paper team a Essay Example | Topics and Well Written Essays - 750 words

Week 4 team part of paper team a - Essay Example The dangers of being sued pass through just about every single facet of the corporate world, and the enormity of prospective losses from suet can be significant. Even ignoring the indirect costs associated with lawsuits, they still represent over millions of dollars in fee opposition. Hence, the determination to report contingencies ought to be centered on the opinion of admission. For instance, as soon as the admission of an incident appends the material subject matter of the financial statements, it should be reported. Occasionally, a few variations of contingencies have the potential to result into self-fulfilling prediction. For instance, a common contend with contingency snag is the reservation of incorporating the potential loss from a litigation. The cash worth of the loss could be projected by consuming the estimated worth criteria, putting projected responsibility on the balance sheet could provide the plaintiff with supplementary confirmation of the organization’s gu ilt. Admission of contingencies may perhaps decrease stock prices and can lead to litigations. Though, overall admission of contingencies ought to be of assistance in evading litigations as they guide stakeholders in developing accurate anticipations. When a contingent liability turns into a legitimate loss, the greatest impact on stock value ought to be in those situations that admissions were unsatisfactory or there was no admission. In the incident that the litigation is lost and it is prior to the fiscal year close, however prior to the financial statements being dispersed, the loss is accrued (the aftereffect of the litigation can be used to establish the amount) and turn into a liability on the balance sheet. At which point an admission in the comments section of the financial statements is proper. In the event that the lawsuit transpires following the fiscal year end, however prior to the financial statements are distributed and the loss is substantial,

Business Strategy Assignment Example | Topics and Well Written Essays - 2000 words

Business Strategy - Assignment Example Crystal Jade is listed as one of the top market performers and they still strive to maintain their position through laying various business strategies. Limited barriers to entry inform of patents and rights. Most of foods and beverages offered by Crystal Jade are not strictly regulated through patents and legal rights making it easier for other players to sell and offer similar goods and services. No technical skills required to start up a firm. Unlike in highly controlled industries such as law, engineering, and medical where technical skills is paramount no or limited technical ability is required to start and run a restaurant successfully Competitors are high in number and equally balanced in key areas of operation. Crystal Jade is face intense competition both locally and internationally. Key among its competitors include but not limited to Old Chang Kee, Sakae Sushi, KLG, Marrybrown Restaurant, Es Teler 77, and Hoka Hoka Bento. Crystal Jade has been enjoying political stability all its areas of operation. The company currently operates at least 121 outlets in approximately 9 different countries across Asia and in the United States all of which are political stabile. However being a multinational company Crystal Jade has to deal with varied public issues such as taxation policies, different trade and labour laws and regulations which may have direct impact on the operations of the business. For instance, The US operate different labour and tax laws compared to China and Singapore and such differences may be a significant challenge to the growth and expansion of business if they are not addressed effectively. It is quite plausible that Crystal Jade is operating its business in a number of the most flourishing economies in Asian region. Key global economic bodies such as World Bank and IMF have ranked China, Singapore, Thailand and Indonesia as being among the fasted growing economies globally

Relationship Analysis Paper Research Example | Topics and Well Written Essays - 1000 words

Relationship Analysis - Research Paper Example Although these numerable theories are not a â€Å"how to† manual, but a means of getting deeper understanding into the natures of relationships between human beings. Three of these theories, offer interesting approaches as to how to understand romantic couplings and how and why people participate in interpersonal relationships. Firstly, Social Exchange Theory, as well as, two of the conceptual theories that fall under the umbrella of Social Exchange Theory, Equity Theory and Interdependence Theory. History Social Exchange Theory became popularized in the 1960s and 1970s. It works under the principle that all of human relationship behaviors are based on an exchange process. Through this they each can work to maximize rewards and avoids or minimize costs. This mental, and unconscious, a process allow the partners to measure whether their time, energy, money, and efforts balance with what they receive (Cherry, 2013). This theory has been influenced and enhanced by behavioral psyc hology, which explains that human being will repeat the behaviors that have resulted with rewards in the past. As well, as supported by Utilitarian logic, that if one receives more than the other, one will inevitably take advantage of the other (Plunkett, 2013). Simply meaning that one partner in a relationship becomes the primary giver and the other the primary receiver. Once these behaviors become habitual it will likely, not change. Equity Theory is based on the belief that people are innately drawn or desire to be treated well and valued fairly within the relationships they participate. Now, this does not necessarily mean that each partner will contribute in the same ways to the relationship, but as long as the parties involved find the relationship agreeable the a sense of balance, fairness, and equality that people wish. Unfortunately, we all do not necessarily agree on the definition of fairness is. This is when perceived imbalance can occur. When one member is not receiving positive reinforcements they may abandon the relationship. In turn, it is, also, possible for a partner who feels that the other gives much more than they do they may feel distress or guilt about this imbalance, even when that imbalance is in their favor (Deshmukh, 2011). â€Å"†¦Interdependence exists when the accomplishment of each individual’s goals is affected by the actions of others† (qtd. in Johnson & Johnson, 2013). In other words the personal successes and failures of each partner can be intertwined with one another. The theory explains that there are two types of Interdependence, positive, or cooperative, and negative, or competitive. Positive and cooperative interdependence is reflected in relationship where both partners feel that they cannot reach their goals without their partner, also, achieving their goals. Many might consider that the exact example of a committed, dedicated and equal relationship. The successes and failure are intertwined. Negativ e interdependence occurs when the partners in a relationship are competitive. One’s success can only be achieved by sacrificing the best interests of the other (Johnson & Johnson, 2013). Interestingly enough, if one partner is seeking positive interdependen

African American Culture 5 Essay Example for Free

African American Culture 5 Essay Question# 4: African cultures, by and large, bring a similar world view to the relationship between man and the spiritual realm, one that is marked by an extremely personal interaction. In the broader African spiritual world human beings are seen to be under the constant influence of other people, their ancestors, minor deities, the Creator, and various forces of nature. As a result the African spiritual world can be described as interactive since all things are endowed with life-force. How is this idea expressed in ritual approaches to morality, wrongdoing, and spiritual empowerment? African American religion has always been heavily involved and influenced by the notion of morality, wrong doing and spiritual empowerment since the slave days if not earlier, African Americans came to embrace Protestant Christianity and adapted their own version of it which is consistent with evidence in the 19th century and a little bit of the 18th, at the time Christianity had little effect on slave society through the efforts of Anglicans, but it was not because African Americans rejected the gospel but because whites seized Christian brotherhood from blacks. As blacks in the South and in the British Caribbean struggled to develop individual and collective identities from the ideas and ways of African culture and their new conditions of life, the series of efforts by evangelicals to convert slaves eventually gave rise to a distinct African-American form of Christian theology, worship style, and religious community. The importance of religion and having their own take on it is among African Americans, as among all people, rests on fulfilling the human need for an understanding of one’s place in both the spiritual and temporal world. Although it was difficult, African Americans discovered in evangelical conversion requirements an opportunity to reassert personal authority based on their ability to communicate directly with God and to bring others to recognize the need for personal repentance and acceptance of Jesus. A perfect example that supports the connection between religious involvement and a sense of personal identity, is found in a slave woman who, back then it was not common for them to tell missionaries that her people have come from across the sea and lost their father and mother, and therefore want to know the Father. The displacement of Africans, for whom locality was critical to interactions with the spiritual world, did not strip them of their religious identity, but required them to learn the spiritual landscape of their new home and reshape their practices accordingly. â€Å"Come Shouting to Zion† details the many religious rituals that Africans preserved in the new world, especially those surrounding fundamental life events such as the birth and naming of children, marriage, burial ceremonies, and ritual dancing and singing to communicate with ancestors and deities. The influence of Africans with many diverse but fundamentally similar cultures in a strange new land encouraged slaves to form new pan-African cultures, which grew increasingly popular as later generations of slaves were born into bondage in America, establishing a distinct African-American culture. The pidgin African-English is a prime example of Africans in American creating a system of communication that was not traceable to a particular African ethnic origin, nor was it a perfect imitation of American English, but was instead shared by blacks in America. As slaves first encountered a foreign language that whites wished them to learn well enough to be more productive but not well enough to pose a threat to the race-based socioeconomic hierarchy, so they became acquainted with Christianity at the will of whites, but when given the opportunity, appropriated it for their own purposes. In the early encounters between slaves and Christianity it is without question that African, and particularly American-born slaves, sought a spirituality that would explain or show their temporal condition. Some salves looked to a theology of liberation and equality among Christians, which they could glean from 18th century evangelicals, mostly Anglicans, who tried to downplay these aspects of biblical teaching. The early period of evangelism was restricted by the fears of slave-owners that slaves who converted to Christianity would feel empowered to revolt against their bondage. Several conspired rebellions and many smaller incidents of black assertion were linked to blacks who had heard enough preaching to identify themselves with the enslaved nation of Israel. This fed the fears of whites, and Anglicans continued to complain that the planters who prohibited them from educating slaves on religious matters were the largest hindrance to saving African American souls. While racism was strengthened and slaves were unable to improve their social status by conforming to white European-American values, very few blacks found the Christian message Anglicans shared with them appealing . Anglican churches maintained strict separation of rich and poor, white and black, during services and sacraments. The high-church emphasized that learned men alone were authorized to teach and that blacks would listen without questioning and to accept the extension of their temporal message and isolation from whites into the religious sphere. Under these terms, it is I am not surprised that Christianity failed to take root as a meaningful religion, a spiritual world that Africans wanted to live in. But it is essential to recognize the role of whites in shaping the message that Africans were allowed to hear, and the role specifically of slaveholders in excluding blacks from access to Christianity. That blacks expressed their agency in rejecting this early version of Christianity offered to them. . At the same time Anglicans were confused over their lack of success in the Southern mainland, Moravians made a significant impact on blacks in the Caribbean by bringing a different vision of a Christian community. Moravians, Methodists, Separate Baptists, and a few other missionaries in the late 18th and early 19th centuries who sought out African Americans stressed spiritual, if not always worldly, equality. Africans identified with and embraced images of a savior who had suffered like they did, and joined these Christian images with African musical modes of expression to create spirituals that reminded: â€Å"Jesus been down to de mire/ You must bow low to de mire† (Stuckey, 139). However, you must finally accept Christianity as an affirmation of their lowly place in society and a divine exhortation to obedience and docility, as many white slaveholders had hoped they would. Rather, blacks found opportunities at biracial revival meetings which were meetings held at locations most often church, in which slaves and blacks were black would interpret what they heard and to share their divinely inspired interpretations of Christian faith, even from pulpits. During this critical period when a significant portion of blacks in the Caribbean and American South were first offered Christianity, they clearly adopted it and transformed it into something that was their own. After the period of revivals that first sparked wide-scale conversions in the South, many African-Americans focused on building a community in which they could support one another and worship in their own African-influenced style. Local black congregations extended their religious community, most notably with the founding of the African Methodist Episcopal Church in1816. As an institution spanning several states, the A. M. E. Church allowed blacks to take part at different levels in a collective, hierarchical social system as had never before been possible under American slavery. blacks continued to participate as minorities in biracial congregations (still with segregated seating) in most parts of the south and the expanding frontier, but found fewer opportunities to become ordained preachers or lay leaders in mixed parishes, where they were likely only to be allowed to â€Å"exercise the gift, provided they teach sound Doctrine sic† under the approval of whites (Frey Wood, 166). In the creation of their own religious communities in which no whites were present to criticize â€Å"overemotional† black forms of religious expression and persisting practices, such as polygamy and dancing, African-Americans actively designed a spirituality that fulfilled their needs in the slave societies of the Americas. African-American religiosity was then, as it is now, â€Å"centered on extended and expanding families and households, the importance of self-determination and personal dignity, mutual aid, and shared responsibility for the progress of the race† (Hortons, xi). In my opinion, African agency is most clearly supported by evidence of Africans defining their faith, modes of worship, and religious ties as part of a larger emerging African-American culture. Change was a relentless fact of life for Africans in 18th and 19th century America, most tragically present in enslavement and removal from Africa and domestic trade within the Americas that broke up families as masters bought and sold property. Outside the personal struggles of individual slaves, the changes in ideology and society wrought by the era of the American Revolution exposed Africans and their descendents to evolving external ideas about their place within American society, their rights as humans, and their needs as spiritual beings. Religion was one of the few arenas in which African-Americans could control the changes in their individual lives and their culture as a whole. Evolving religious traditions provided individuals over generations with a source of spiritual renewal and a supportive community and prepared an institution that could serve future generations. The long and turbulent transition from African forms of religiosity to African-influenced forms of Protestantism shows that black Americans created, out of all religious ideas and structures available to them, a faith that was their own. Question#3 The musical selections in this section come from Africa and the Americas. Some are examples of the preservation of traditional musical styles; others are examples of the adaptation of traditional modes of expression to modern styles. Prevalent in each performance is the use of either percussion instruments such as drums or singing in groups or by soloists. How do these musical selections exemplify a common African musical aesthetic, i. e. rhythmic syncopation, call-and-response, melodic constructions, vocal colors, in both traditional and contemporary expressions? African dance has contributed many characteristics to dance in America. We see evidence of this in many aspects of dance today. Being such a diverse nation, America has the blessing of combining original dances from different cultures to create an amazing dance repertoire. American dance as we know would be completely different, if it weren’t for the Africans. African dance began with the different rhythms of the tribes. Its roots in America began with the slave trade. The American slave trade began in 1619, (However, Africans were imported as slaves to the West Indies staring almost a century before that) with the arrival of Dutch trading ships carrying a cargo of Africans to Virginia. They were first brought over by boat to places such as Brazil, Cuba, and Haiti. Eventually different countries end up taking over those nations and slaves fall under their rule. In Brazil, the Portuguese take over, in Cuba the Spanish take over, and in Haiti, the French take over. The retaining of African culture by those in slavery was stronger in the other nations than in America, as the Spanish and French rulers adhered to the more lenient view of dancing taken by the Catholic Church. In America, the Protestant church strongly disapproved of dance. Therefore, dances that occurred in the West Indies, Brazil, Haiti and Cuba retained more of the African dance structure, than those in America did. Those dances can be classified as recreational or sacred. An example of a recreational dance is the Juba, which was a competitive dance where opponents would outdo each other in feats of skill, sometimes while balancing something on their head. Sacred dances were based on the worship of religious gods. The goal of the dance was for the dancer to become possessed by the god so that it would speak through the dancer. Two examples are voodoo and Shango dances. Traces of the African religious practice of possession, or disengaging from reality through the combined effects of music and dance, can be detected in the appeal of some forms of jazz dance. In America, the dance movement of Africa was restrained mainly by two factors: the attitude of the church towards dancing as being immoral and the restricted use of the primary African instrument (the drum). Drumming was banned in 1739 following a slave insurrection. White plantation owners responded by banning all drums and that forced slaves to search for other percussion options. They substituted with banjos, clapping hands, stomping feet, and the fiddle. Dances that occurred on the Plantations were for recreation and religious reasons also. Because of the European influence in America, the movement gave a distinct American appearance, rather than a strictly African one. Many dances imitated animals. There were also circle dances and dances for celebrations. Another category that emerged was competitive dances. The most well known one was the cakewalk. The slaves had witnessed their owners’ dancing festivities and imitated their stiff upper bodies while contrasting it with loose leg movements. The owners enjoyed watching this and gave a cake to the best dancer. The observation of African dancing by the whites led to them stereotyping the dancing slave. They began to blacken their faces and imitate them using such indigenous movements as the ‘shuffle’. The imitation dances by whites started an era of American entertainment based on the stereotype on the dancing ‘Negro’. Before the Civil War, professional dancers were mostly white, with the exception of William Henry Lane. He was also known as Master Juba and was a freeborn slave thought to be the best dancer in the World. He had lived in Manhattan where the Irish immigrants also lived. His dancing was a combination of Irish jig dancing and African rhythm, just like the slaves who were forced to compete with the Irish migrant workers aboard the ships. Both his movements and the Nigerian slaves are said to be the start of tap dance. Minstrelsy was also a popular form of entertainment in America from 1845 –1900. The Minstrel show was a group of male performers that portrayed the Negro as either slow and shuffling or sharply dressed and quick moving. The minstrel show proved prominent in spreading vernacular dances like the cakewalk and jig dancing on a wide scale. The next major change after minstrelsy came with the birth of ragtime music and ballroom dancing after 1910. A bunch of animal dances were seen in white ballrooms. Examples were the Turkey Trot, and Chicken Scratch. The invasion of ballrooms with native inspired dances set the stage for the same process to occur on Broadway. Zeigfield borrowed some of these dances for his Follies. Social dance became introduced on the theatrical stage. The big aspect being borrowed wasn’t the actual dances, but their swinging qualities. In 1921, Shuffle Along featured a jazz inspired dance called the Charleston. It left the audience with a lot of energy and a new respect towards black dancing. Tap was now also brought to white audiences and the musical comedies took on a new, more rhythmic life. In the late 1920s, jazz inspired songs replaced the popular white standards and America accepted Jazz music as its own. Louis Armstrong was a big part of the creation of swing music. It was a style of jazz music that emphasized African influenced rhythm and was played by big bands. Faster and sharper footwork came about and the Lindy was the new dance craze. It incorporated the shuffle and glide and buck and wing movements from early African dances. The Lindy was significant for starting jazz dance styles used in later musicals. It also gave the opportunity for white choreographers to experience African swing. Jazz music and dancing slowed down in popularity after WWII. Technology and music were evolving. The beat became more complex and musicians like Charlie Parker and Dizie Gillespie explored more with improve. The overall result was, jazz music became something more to listen to rather than to dance socially. The advent of Television in the 1950s also kept people at home instead of on the dance floors. African American dance became more of an artistic expression than a social means. Professional companies and dancers restored early African rhythms and the beauty and emotion of their traditional songs, including Catherine Dunham’s Shango, Alvin Ailey’s Revelations and Bill T. Jones’ Uncle Tom’s Cabin. In the past 50 years, African American dance has been rich in innovations as well as connections with the past. The definition of professional dance has broadened beyond ballet, modern, and jazz. Popular and social dances, including the urban black dance forms of break dancing and hip-hop have been recognized for their artistry and expressiveness. Dance created and performed by African Americans has become a permanent part of American dance. Every dancer and almost every person in America, in one way or another has danced steps that resemble early African polyrhythmic movements. Personally, I think the dance World in America could no have flourished as well as it did without it’s African influences. since the slave trade the drum has been used all over the world as a means of communication and self expression. Its broad variety of users includes the early African tribes, using them for ceremonial purposes. The Africans brought drums with them to the Americas and helped to develop their popularity among American musicians. In the mid 1900’s drum sets were brought about. These revolutionary collaborations of percussive pieces started off with a pair of hi-hats, a bass and snare drum, and a couple of tom toms. Later as the music progressed, so did the drum kits, completely eliminating the need for an entire drum section. With the coming of the rock and roll movement the drum kits were changing, they needed to accommodate the new music styles. They became sonically diverse and even electronic drums were brought about; making them infinitely adjustable both ergonomically and musically. With every major drum manufacturer competing to have the best product on the market drums will always be evolving. African American musicians and early slaves choose to use drums as a common form of expression because of the deep bass that was used to duplicate heart beat and thunder. The sound waves for open ended and string instruments is fairly straight forward. However, for a closed end instrument, such as a drum, the sound waves are different. A lot of the energy is dissipated through the shell of the drum, which is the reason for the variance in drum construction these days. Many different kinds of wood are used to generate different sounds, or a different amount of energy absorption. For a warmer, deeper sound maple construction is used while birch is used to get a high, resonant tone full of vibration. The heaviest wood that dissipates the most amount of energy is oak, creating a lower, flat sound. Question#1 I believe that Egypt’s economic progress over the last decade is a great example of showing how They have come a long way and are still vastly improving. Egypt is the third-largest economy in the Middle East and North Africa region (after Saudi Arabia and Israel), as well as one of the strongest, with significant potential for future economic growth and diversification. With a real commitment to economic reform, which favors a large privatization program and the encouragement of private investment and growth. The improvement in Ghana is evident in how their country has such a diverse economy. The Gold Coast was renamed Ghana upon independence in 1957 because of indications that present-day inhabitants descended from migrants who moved south from the ancient kingdom of Ghana. By West African standards, Ghana has a relatively diverse and rich natural resource base Mineralsprincipally gold, diamonds, manganese ore, and bauxiteare produced and exported. Exploration for oil and gas resources is ongoing. Timber and marine resources are important but declining resources. Agriculture remains a mainstay of the economy, accounting for more than one-third of GDP and about 55% of formal employment. Cash crops consist primarily of cocoa and cocoa products, which typically provide about one-third of export revenue, timber products, coconuts and other palm products, shear nuts , and coffee. Ghana also has established a successful program of nontraditional agricultural products for export including pineapples, cashews, and peppers. Cassava, yams, plantains, corn, rice, peanuts, millet, and sorghum are the basic foodstuffs. Fish, poultry, and meat also are important dietary staples. Ghanas industrial base is relatively advanced compared to many other African countries. Industries include textiles, apparel, steel (using scrap), tires, oil refining, flour milling, beverages, tobacco, simple consumer goods, and car, truck, and bus assembly. Industry, including mining, manufacturing, construction and electricity, accounts for about 25% of GDP. I strongly believe that since Ghana and Egypt have improved so vastly it is helping African Americans improve in general, the saying â€Å"We come from a long line of kings and queens is such a truthful statement if you look back on history. We have a lot of ancestry that lies within Ghana and Egypt. With the knowledge of the past it will help us to continue realize our past and bring us to terms with the future. We can reverse the process by not letting people hold us back and to not blame others. I also believe that strong knowledge of Ghana and Egypt and Mali, will also further our culture by being educated and not told how our past was. There are a lot of invention by many great African Americans that most people do not know that black inventors were behind the idea, not that is matters that a black or a white person constructed or came up with an idea for a patent, it is essential that we are have contributed just as many things if not more than any other culture. There have been so many contributions to society to western civilization and I feel it is so important that we surround our selves with knowledge of our ancestors because they worked hard to get us to the point today where we are able to vote and the possibility of a black president. The saying that we come from a long line kings and queens is so powerful because it shows you that black really is beautiful and if you retrace our ancestors you will find out that our people were just as important as kings and queens. Lewis Temple was the inventor of a whaling harpoon called the Temples Toggle and the Temples Iron. He was born in Richmond, Virginia in 1800 and arrived in New Bedford, Massachusetts in 1829. He worked as a blacksmith and had lots of friends that were whalers who bought harpoons and had lots of conversations with them. Granville T. Wood was known as the black Edison. Woods was born in Columbus, Ohio on April 23,1856. He never finished elementary school and he worked in a machine shop at a very young age. He moved to Missouri in 1872 at the age of sixteen. By 1881 he opened a factory in Cincinnati, Ohio and manufactured telephone, telegraph and electrical equipment. He filed for his first application for a patent in 1884 for an improved steam-boiler furnace. Woods patented the telographony , a combination of the telegraph and the telephone. He produced one of his most important inventions in 1887, it was called the Synchronous Multiplex Railway Telegraph. It enabled messages to be sent from moving trains and railways stations. In 1890 he set out to improve the lighting system by creating an efficient safe economical dimmer. It was safer and and resulted in 40% energy savings. Woods also created an overhead conducting system for electrical railways and the electrified third rail. By the time of his death in 1910 he had 150 patents awarded to him all together. Lewis H. L was a pioneer in the development of the electric light bulb. He was also the only black member of the Edison Pioneers, a group of inventors and scientists who worked with Thomas Edison. He was born in Chelsea, Massachusetts in 1848 and was raised in Boston. He enlisted in the Navy and served as a cabin boy on the U. S. S Massaoitta the age of sixteen. Latimer was given the assignment to draw plans for Alexander Graham Bells telephone patent . In 1879 Latimer went to work as a draftsman for Hiram Maxim, who invented the machine gun and headed the electric lighting company. Latimer worked on improving the quality of the carbon filament used in the light bulb. In 1882 he received a patent for an improved process for manufacturing carbon filaments. Gerrett is best remembered for his invention of the gas mask and the three way traffic signal. Mogan was born on March 4,1875 in Paris, Kentucky. He left school after fifth grade at the age of fourteen. He left Kentucky and headed for Cincinnati, Ohio and got a job as a handy man in a sewing shop. Morgan directed his attention to the frequent instances of firemen being overcome by fumes and thick smoke when they went into burning buildings. He perfected breathing device which he patented in 1914. In 1923morgan patented an automatic traffic signal which he sold to the General Electric Company for four thousand dollars. In 1963 Garrett A. Morgan died at ht age of 88 in Cleveland, Ohio after he was ill for two years. Just to name a few ,those were a couple of major contributors to the African American culture and western civilization.

Leadership Essay Example for Free

Leadership Essay 1.Leadership as a process, is the use of no coercive influence to shape the group’s or organization’s goals, motivate behavior toward the achievement of those goals, and help define group or organizational culture; as a property, the set of characteristics attributed to individuals who are perceived to be leaders. 2.General Mandible is not a leader because he does not motivate the ants to achieve the colony’s goal, unless the goal was for all the ants to drown and die. He leaves all the ants to drown at the end of the movie. Leaders stick with the team all the way and do not try to kill their own team. 3.Zee is a leader because in a crises moment he does not get scared. At the end of the movie when all the ants are about to drown, he motivates all of them to build a ladder to the top, and his plan ends up saving all the ants. He is able to use nonaggressive force to motivate the ants. 4.The quote â€Å"Individualism makes us vulnerable† applies to ants where one ant alone is weak and small and can’t do much, but all the ants together can do anything. Like it takes all the ants to make the ladder at the end of the movie saving them from drowning, it is crucial to their survival that they stick together. This mostly applies everywhere, being in a team is better than being alone. 5.The Power Position is the physical position in the room for a business meeting, which supposedly has the most power. The leader in this position where he can see all entrances to the room and no activity is going on behind him. Three types of position power are: Legitimate Power- Power granted through the organizational hierarchy; it is the power defined by the organization that is to be accorded people occupying particular positions. Reward Power- The power to give or withhold rewards, such as salary increases, bonuses, promotions, praise, recognition, and interesting job assignments. Coercive Power- The power to force compliance by means of psychological, emotional, or physical threat. 6.A scene from the movie that illustrates the use of position power is when General Mandible is discussing about the colony with the Queen and the Queen tell him he can do whatever he likes because she trusts him that he will do everything for the good of the colony. She has legitimate power granted through the hierarchy, but then the General abuses his power and the works start to work hard because they don’t want to deal with the General because he uses Coercive Power. 7.Personal power is the power that comes from within to influence other it has nothing to do with the persons position. Two types of personal power are: Referent Power- The personal power that accrues to someone based on identification, imitation, loyalty or charisma. Expert Power- The person

Complement Serum Activity by Lysing Sheep Erythrocytes

Complement Serum Activity by Lysing Sheep Erythrocytes Introduction The Immune system is a series of complex processes which has evolved to protect the body from attack by foreign pathogens. These pathogens are able to enter our body through the skin or lining of the internal organs. The immune system is able to protect us from intracellular and extracellular organisms as well as from ourselves, stopping malignancies and autoimmune diseases from spreading in our bodies (Bastian, 1993). There are two lines of defence, the adaptive (specific) and innate (non-specific immunity), though both are united in their goal to destroy pathogens they have different ways to tackle this. Innate immunity is the 1st line of defence while adaptive immunity is the 2nds line and thus takes longer to act (Clancy, 1998). The complement system is part of the immune system and can be bought into action by the adaptive system if required. Complement is a group of proteins working together within the immune system; once stimulated by one of many triggers, proteases begin to c leave protein in the system, bringing a cascade of enzyme reactions in order to fight off foreign pathogens and activate the inflammatory response. Within the complement cascade there are many proteins that play a role but C3 is a protein critical to the effector functions of the system (Abbas, 1994). There are many paths for immune mediated lysis and the one we will be looking at is intravascular haemolyse and occurs when the complement has been triggered through the classical pathway. When the antibody binds to the antigen on the surface of the erythrocyte, a complement component triggers the membrane attack complex to form pores in the cell membrane resulting in cell lysis (Chapel, 1990). The intensity and speed at which cells lyse is dependent upon the rate at which the complement cascades to enable complete cell lysis. Experiments like these are able to provide us with an understanding of how the complement immune system functions. It can also increase our understanding of autoimmunity and perhaps lead to ways in which the effects of immunity can be prolonged or inhibited according to the disease. Systemic Lupus Erythematosus (SLE) is an autoimmune disease, in which complement is analysed, as getting SLE is dependent upon the gene which is responsible for producing MHC, a component used in haemolysis (American, 1993), patients with other immunological disorders can require their complement activity to be monitored and thus this assay would be able to show how efficiently the complement component of the immune system is working to defend their bodies. Aims To determine complement serum activity by lysing sheep erythrocytes To determine the volume of complement required for 50% lysis. Materials 20 Cuvettes 1.0ml 20 test tube plastic disposable Automatic pipette 200-1000 ÂÂ µl 6 tips Automatic pipette 0-200 ÂÂ µl 6 tips Water bath at 37Â °C Spectrophotometer Test tube rack Centrifuge Ice bucket Ice Method Wash 4ml of erythrocyte suspension three times with barbitone saline solution. Prepare a 6% stock solution of erythrocytes In one test tube mix 3.0ml of sheep anti-erythrocyte antiserum, diluted 1/50 3.0ml of the 6% SRBC Mix and gently by capping and inverting several times Incubate at 37Â °C for 15min in the water bath, mix every 5min. Set up the test tubes on ice in duplicates and label Add the reagents in order as shown in table 1 below Incubate the tubes for 60 minutes at 37Â °C mixing gently every 15minutes Place the tubes on ice and then centrifuge at 200g for 10 minutes at 4Â °C Remove the samples and put into cuvettes and read the absorbance at 541nm, with ammonia solution as blank record the results in a table. Results Discussion When carrying out the experiment raw data was recorded, and presented in table 1. However the results obtained during the practical were not used as the erythrocytes lysed before complement was added and therefore complement activity could not be observed as adding complement to lysed cells is not able to produce results, therefore the ideal data provided was used and analysed. From table 1 it is clear that absorbance levels increased as serum volume increased, this is due to the fact that as volumes of complement increase more red blood cells are lysed which in turn allows haemoglobin to be let out, this is of a dark colour and as more cells are lyses the darker the resulting sample will be, and so the absorbance as read the spectrophotometer will increase. After the guinea pig serum has been mixed with the sensitised erythrocytes, it produces anti-body coated cells with complement attaching to the antibody, and activating this attracts the MAC molecules to take action and lyse the cell (Kuby, 1994). Following the pattern seen in table one table 3 shows a progressive % lysis of cells as the volume of serum is increased, however for the 100% lysis an ammonia buffer was used to ensure that all cells are lysed during the experiment. Further to this graph 1 produced a sigmoid curve, from which it was possible to estimate CH50. However calculating the 50% lysis from this graph is not very accurate. Thus a log graph 2 was constructed, with the use of van Krogh equation to determine the actual value of 50% lysis. The equation was provided by the lecturer. Van Krogh equation: x= k[ y ]1/n 100-y Where: x= amount of complement (ml of undiluted serum) y= proportion of cells lysed k=50% unit of compliment n=inclination of graph (ideally 0.2) This resulted in table 4 giving a volume of 133.5 CH50/ml. However when calculating CH50 the x values were all in the negative. Moreover, it was not possible to compare data sets obtained against ideal data as the experiment did not yield results due to lysis of erythrocytes before complement was added. This could have occurred due to improper pipetting, handling or transporting of the cells as shaking them too much could have lysed them due to shock, as the cells were sensitized and thus prone to quick lysis. Further to this it was reported by Inglis, et al, 2007, that the use of erythrocytes from different sheep can yield inaccurate results and thus produce different CH50. Although there are many inaccuracies present within the experiment, it also gives scope to further improve the method as well as explore other area of the subject at hand such as factors which affect the performance of complement like temperature or PH. This assay is a good way to measure the activity of the immu ne system within patients, such as patients with LSE as mentioned earlier, other patients with low immunity can also be tested to see how the complement system is or isnt aiding their recovery, thus steps can be taken by medical professionals to either boost or monitor the progress of the patients immunity as fundamentally the immune system is required to work at its optimum to keep humans and animals from dying of disease(Inglis,et al, 2007). Conclusion Overall this experiment has shown how complement is important in aiding white blood cells to lyse foreign bodies. Though in the experiment carried out the blood cells lysed before complement was added the method was presented and the ideal set of data, showed what results should have been obtained. Also the hypothesis that as the complement concentration increases so will the absorbance proved positive.

Education of Boys in Victorian England :: Victorian Era

Education of Boys in Victorian England The Upper and Middle Classes * The Elementary School Act of 1870 made school compulsory up to the age of 12. * The most famous group of public schools was referred to as â€Å"the Nine Great Public Schools.† * The famous schools were Eton, Harrow, Rugby, Winchester, Shrewsbury, Charterhouse, Westminster, St. Paul's, and Merchant Taylors. * These schools were originally opened up to everyone and sustained through the donations of wealthy donors. Initially taught boys Latin and Greek grammar but in 1861 the administration was changed and more of the sciences were included. As a result, the schools became public in name only and were attended pretty much by upper class and middle class boys only. * Children of the upper and middle classes were taught at home by governesses or tutors until they were old enough to attend public schools. * Public schools were important for sons of well-off or aspiring families because schools gave them the opportunity to establish connections which could later help them out in their careers. * Most of the boys that attended these schools went off to Cambridge and Oxford and then later on to Parliament. * George Osborne was not of the upper classes but he interacted a lot with them and it was a possibility for a gain in status. * A lot of emphasize was placed on athletic games. They oftentimes even took precedence over the learning of Greek and Latin. Being a sportsman reaffirmed a man's leadership. The Lower Classes * Boys of the lower classes were excluded from attending the â€Å"public† schools of England because they did not fit into what was expected of the boys that attended those schools. * The boys attending the public schools were most often than not of well to do families, which meant they would be well-dressed, well-mannered boys. * Boys from the lower classes did not have the same upbringing and as a result did not fit into the public schools instead they attended what were often referred to as Ragged Schools. Purposes of Education * For the upper classes, the purpose of an education was to raise gentlemen and prepare them for prestigious appointments in Parliament or government.

Flag Football :: essays research papers

Whether it's running around and grabbing the opponents flag, or running around and pushing people out of the way for the touchdown, flag football is a great sport to increase adrenaline. Playing flag football gives players energy and helps keep them in shape. The constant movement helps work ones cardio and keeps one active. It is fun to play because players gat to play with friends and meet new people. It helps players work together and have competition. Everyone who likes doing a variety of exercises and likes to have fun could enjoy flag football in more ways than one. Flag football is not only a great sport, but it is also getting exercise in without one knowing it. Flag football requires moving around all the time. It keeps cardio up and keeps one on the move. Running around with a football gets the players? legs moving and other players as well because they chase the football. Flag football keeps ones arms active. Throwing a football or catching it can help the players? muscles in their arms. Most players like using their arms and legs to play which makes flag football a popular sport. With flag football being a well-known sport, most students want to try it out. Other students will most likely end up playing with people they know or can get to know. When playing with friends, players will get more active and try harder. It gets more out of them because they are excited to play. Trying to impress someone with mad throwing skills or speed could increase a player?s performance. Some players may meet new people and get new friends. Flag football is a social yet active sport that helps players get together. With friends, enemies, and other players playing flag football, most players will get along and work together. When not working together, there is even competition. Players need to throw a ball to get it across the field. They have to plan with teammates how to do that. In flag football, there is good, clean competition. To get some one tagged, all the players have to do is pull the flag without hurting the players.

Enzyme Biocatalysis

Enzyme Biocatalysis Andr? s Illanes e Editor Enzyme Biocatalysis Principles and Applications 123 Prof. Dr. Andr? s Illanes e School of Biochemical Engineering Ponti? cia Universidad Cat? lica o de Valpara? so ? Chile [email  protected] cl ISBN 978-1-4020-8360-0 e-ISBN 978-1-4020-8361-7 Library of Congress Control Number: 2008924855 c 2008 Springer Science + Business Media B. V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, micro? ming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied speci? cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper. 9 8 7 6 5 4 3 2 1 springer. com Contents Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Introdu ction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 1. 1 Catalysis and Biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2 Enzymes as Catalysts. Structure–Functionality Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 3 The Concept and Determination of Enzyme Activity . . . . . . . . . . . . . . 1. 4 Enzyme Classes. Properties and Technological Signi? cance . . . . . . . 1. 5 Applications of Enzymes. Enzyme as Process Catalysts . . . . . . . . . . . 1. 6 Enzyme Processes: the Evolution from Degradation to Synthesis. Biocatalysis in Aqueous and Non-conventional Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 2. 1 Enzyme Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2 Production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 1 Enzyme Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 2 Enzyme Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 3 Enzyme Puri? cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 4 Enzyme Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 8 16 19 31 39 57 57 60 61 65 74 84 89 2 3 Homogeneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Andr? s Illanes, Claudia Altamirano, and Lorena Wilson e 3. 1 General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3. 2 Hypothesis of Enzyme Kinetics. Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. 2. 1 Rapid Equilibrium and Steady-State Hypothesis . . . . . . . . . . . 108 v vi Contents Determination of Kinetic Parameters for Irreversible and Reversible One-Substrate Reactions . . . . . . . . . . . . . . . . . . . . . 112 3. 3 Kinetics of Enzyme Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. 1 Types of Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. Development of a Generalized Kinetic Model for One-Substrate Reactions Under Inhibition . . . . . . . . . . . . . . . . 117 3. 3. 3 Determination of Kinetic Parameters for One-Substrate Reactions Under Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 3. 4 Reactions with More than One Substr ate . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 1 Mechanisms of Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 2 Development of Kinetic Models . . . . . . . . . . . . . . . . . . . . . . . . 125 3. 4. 3 Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . 131 3. 5 Environmental Variables in Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . 133 3. 5. 1 Effect of pH: Hypothesis of Michaelis and Davidsohn. Effect on Enzyme Af? nity and Reactivity . . . . . . . . . . . . . . . . 134 3. 5. 2 Effect of Temperature: Effect on Enzyme Af? nity, Reactivity and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3. 5. 3 Effect of Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4 Heterogeneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Andr? s Illanes, Roberto Fern? ndez-Lafuente, Jos? M. Guis? n, e a e a and Lorena Wilson 4. 1 Enzyme Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 4. 1. 1 Methods of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 4. 1. 2 Evaluation of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . 166 4. 2 Heterogeneous Kinetics: Apparent, Inherent and Intrinsic Kinetics; Mass Transfer Effects in Heterogeneous Biocatalysis . . . . . . . . . . . . . 169 4. 3 Partition Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 4. 4 Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 4. 4. 1 External Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . 173 4. 4. 2 Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . 181 4. 4. 3 Combined Effect of E xternal and Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Andr? s Illanes and Claudia Altamirano e 5. 1 Types of Reactors, Modes of Operation . . . . . . . . . . . . . . . . . . . . . . . . . 205 5. 2 Basic Design of Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 1 Design Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 2 Basic Design of Enzyme Reactors Under Ideal Conditions. Batch Reactor; Continuous Stirred Tank Reactor Under Complete Mixing; Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 3. 2. 2 5 Contents vii Effect of Diffusional Restrictions on E nzyme Reactor Design and Performance in Heterogeneous Systems. Determination of Effectiveness Factors. Batch Reactor; Continuous Stirred Tank Reactor Under Complete Mixing; Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 5. 4 Effect of Thermal Inactivation on Enzyme Reactor Design and Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5. 4. 1 Complex Mechanisms of Enzyme Inactivation . . . . . . . . . . . 225 5. 4. 2 Effects of Modulation on Thermal Inactivation . . . . . . . . . . . . 231 5. 4. 3 Enzyme Reactor Design and Performance Under Non-Modulated and Modulated Enzyme Thermal Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . 234 5. 4. 4 Operation of Enzyme Reactors Under Inactivation and Thermal Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 5. 4. 5 Enzyme Reactor Design and Performance Under Thermal Inactivation an d Mass Transfer Limitations . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 6 Study Cases of Enzymatic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 6. 1 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . . . . . . . . 253 Sonia Barberis, Fanny Guzm? n, Andr? s Illanes, and a e Joseph L? pez-Sant? n o ? 6. 1. 1 Chemical Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . . 254 6. 1. 2 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . 257 6. 1. 3 Enzymatic Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . 258 6. 1. 4 Process Considerations for the Synthesis of Peptides . . . . . . . 263 6. 1. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 6. 2 Synthesis of ? -Lactam Antibiotics with Penicillin Acylases . . . . . . . 273 Andr? s Illanes and Lorena Wilson e 6. 2. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 2 Chemical Versus Enzymatic Synthesis of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 3 Strategies of Enzymatic Synthesis . . . . . . . . . . . . . . . . . . . . . . 276 6. 2. 4 Penicillin Acylase Biocatalysts . . . . . . . . . . . . . . . . . . . . . . . . . 277 6. 2. 5 Synthesis of ? -Lactam Antibiotics in Homogeneous and Heterogeneous Aqueous and Organic Media . . . . . . . . . . . . . . 279 6. 2. 6 Model of Reactor Performance for the Production of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . 282 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 6. 3 Chimiosel ective Esteri? cation of Wood Sterols with Lipases . . . . . . . 292 ? Gregorio Alvaro and Andr? Illanes e 6. 3. 1 Sources and Production of Lipases . . . . . . . . . . . . . . . . . . . . . . 293 6. 3. 2 Structure and Functionality of Lipases . . . . . . . . . . . . . . . . . . . 296 5. 3 viii Contents Improvement of Lipases by Medium and Biocatalyst Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 6. 3. 4 Applications of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 6. 3. 5 Development of a Process for the Selective Transesteri? cation of the Stanol Fraction of Wood Sterols with Immobilized Lipases . . . . . . . . . . . . . . . . . . . . . . 308 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 6. 4 Oxidoreductases as Powerful Biocatalysts for Green Chemistry . . . . 323 Jos? M. Guis? n, Roberto Fern? ndez-Lafuente, Lorena Wilson, and e a a C? sar Mateo e 6. 4. 1 Mild and Selective Oxidations Catalyzed by Oxidases . . . . . . 324 6. 4. 2 Redox Biotransformations Catalyzed by Dehydrogenases . . . 326 6. 4. 3 Immobilization-Stabilization of Dehydrogenases . . . . . . . . . . 329 6. 4. 4 Reactor Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 6. 4. Production of Long-Chain Fatty Acids with Dehydrogenases 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 6. 5 Use of Aldolases for Asymmetric Synthesis . . . . . . . . . . . . . . . . . . . . . 333 ? Josep L? pez-Sant? n, Gregorio Alvaro, and Pere Clap? s o ? e 6. 5. 1 Aldolases: De? nitions and Classi? cation . . . . . . . . . . . . . . . . . 334 6. 5. 2 Preparation of Aldolase Biocatalysts . . . . . . . . . . . . . . . . . . . . 335 6. 5. 3 Reaction Performance: Medium Engineering and Kinetics . . 339 6. 5. 4 Synthetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 6. 5. 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 6. 6 Application of Enzymatic Reactors for the Degradation of Highly and Poorly Soluble Recalcitrant Compounds . . . . . . . . . . . . . . . . . . . . 355 o Juan M. Lema, Gemma Eibes, Carmen L? pez, M. Teresa Moreira, and Gumersindo Feijoo 6. 6. 1 Potential Application of Oxidative Enzymes for Environmental Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 6. 6. 2 Requirements for an Ef? cient Catalytic Cycle . . . . . . . . . . . . . 357 6. 6. 3 Enzymatic Reactor Con? gurations . . . . . . . . . . . . . . . . . . . . . . 358 6. 6. 4 Modeling of Enzymatic Reactors . . . . . . . . . . . . . . . . . . . . . . . 364 6. 6. 5 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 6. 6. 6 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . 374 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 6. 3. 3 Foreword This book was written with the purpose of providing a sound basis for the design of enzymatic reactions based on kinetic principles, but also to give an updated vision of the potentials and limitations of biocatalysis, especially with respect to recent applications in processes of organic synthesis. The ? rst ? ve chapters are structured in the form of a textbook, going from the basic principles of enzyme structure and function to reactor design for homogeneous systems with soluble enzymes and heterogeneous systems with immobilized enzymes.The last chapter of the book is divided into six sections that represe nt illustrative case studies of biocatalytic processes of industrial relevance or potential, written by experts in the respective ? elds. We sincerely hope that this book will represent an element in the toolbox of graduate students in applied biology and chemical and biochemical engineering and also of undergraduate students with formal training in organic chemistry, biochemistry, thermodynamics and chemical reaction kinetics. Beyond that, the book pretends also to illustrate the potential of biocatalytic processes with case studies in the ? ld of organic synthesis, which we hope will be of interest for the academia and professionals involved in R&D&I. If some of our young readers are encouraged to engage or persevere in their work in biocatalysis this will certainly be our more precious reward. ? a Too much has been written about writing. Nobel laureate Gabriel Garc? a M? rquez wrote one of its most inspired books by writing about writing (Living to Tell the Tale). There he wrote â€Å"life is not what one lived, but what one remembers and how one remembers it in order to recount it†. This hardly applies to a scienti? book, but certainly highlights what is applicable to any book: its symbiosis with life. Writing about biocatalysis has given me that privileged feeling, even more so because enzymes are truly the catalysts of life. Biocatalysis is hardly separable from my life and writing this book has been certainly more an ecstasy than an agony. A book is an object of love so who better than friends to build it. Eleven distinguished professors and researchers have contributed to this endeavor with their knowledge, their commitment and their encouragement. Beyond our common language, I share with all of them a view and a life-lasting friendship.That is what lies behind this book and made its construction an exciting and rewarding experience. ix x Foreword Chapters 3 to 5 were written with the invaluable collaboration of Claudia Altamirano and Lorena Wil son, two of my former students, now my colleagues, and my bosses I am afraid. Chapter 4 also included the experience of Jos? Manuel Guis? n, e a Roberto Fern? ndez-Lafuente and C? sar Mateo, all of them very good friends who a e were kind enough to join this project and enrich the book with their world known expertise in heterogeneous biocatalysis. Section 6. is the result of a cooperation sustained by a CYTED project that brought together Sonia Barberis, also a former graduate student, now a successful professor and permanent collaborator and, beyond that, a dear friend, Fanny Guzm? n, a reputed scientist in the ? eld of peptide a synthesis who is my partner, support and inspiration, and Josep L? pez, a well-known o scientist and engineer but, above all, a friend at heart and a warm host. Section 6. 3 was the result of a joint project with Gregorio Alvaro, a dedicated researcher who has been a permanent collaborator with our group and also a very special friend and kind host. Secti on 6. is the result of a collaboration, in a very challenging ? eld of applied biocatalysis, of Dr. Guisan’s group with which we have a long-lasting academic connection and strong personal ties. Section 6. 5 represents a very challengo e ing project in which Josep L? pez and Gregorio Alvaro have joined Pere Clap? s, a prominent researcher in organic synthesis and a friend through the years, to build up an updated review on a very provocative ? eld of enzyme biocatalysis. Finally, section 6. 6 is a collaboration of a dear friend and outstanding teacher, Juan Lema, and his research group that widens the scope of biocatalysis to the ? ld of environmental engineering adding a particular ? avor to this ? nal chapter. A substantial part of this book was written in Spain while doing a sabbatical in the o Universitat Aut` noma de Barcelona, where I was warmly hosted by the Chemical Engineering Department, as I also was during short stays at the Institute of Catalysis and Petroleum Ch emistry in Madrid and at the Department of Chemical Engineering in the Universidad de Santiago de Compostela. My recognition to the persons in my institution, the Ponti? cia Universidad Cat? lica de Valpara? so, that supported and encouraged this project, particularly to o ? the rector Prof.Alfonso Muga, and professors Atilio Bustos and Graciela Mu? oz. n Last but not least, my deepest appreciation to the persons at Springer: Marie Johnson, Meran Owen, Tanja van Gaans and Padmaja Sudhakher, who were always delicate, diligent and encouraging. Dear reader, the judgment about the product is yours, but beyond the product there is a process whose beauty I hope to have been able to transmit. I count on your indulgence with language that, despite the effort of our editor, may still reveal our condition of non-native English speakers. Andr? s Illanes e Valpara? so, May 15, 2008 ? Chapter 1 Introduction Andr? s Illanes e . 1 Catalysis and Biocatalysis Many chemical reactions can occur sponta neously; others require to be catalyzed to proceed at a signi? cant rate. Catalysts are molecules that reduce the magnitude of the energy barrier required to be overcame for a substance to be converted chemically into another. Thermodynamically, the magnitude of this energy barrier can be conveniently expressed in terms of the free-energy change. As depicted in Fig. 1. 1, catalysts reduce the magnitude of this barrier by virtue of its interaction with the substrate to form an activated transition complex that delivers the product and frees the catalyst.The catalyst is not consumed or altered during the reaction so, in principle, it can be used inde? nitely to convert the substrate into product; in practice, however, this is limited by the stability of the catalyst, that is, its capacity to retain its active structure through time at the conditions of reaction. Biochemical reactions, this is, the chemical reactions that comprise the metabolism of all living cells, need to be catalyze d to proceed at the pace required to sustain life. Such life catalysts are the enzymes. Each one of the biochemical reactions of the cell metabolism requires to be catalyzed by one speci? enzyme. Enzymes are protein molecules that have evolved to perform ef? ciently under the mild conditions required to preserve the functionality and integrity of the biological systems. Enzymes can be considered then as catalysts that have been optimized through evolution to perform their physiological task upon which all forms of life depend. No wonder why enzymes are capable of performing a wide range of chemical reactions, many of which extremely complex to perform by chemical synthesis. It is not presumptuous to state that any chemical reaction already described might have an enzyme able to catalyze it.In fact, the possible primary structures of an enzyme protein composed of n amino acid residues is 20n so that for a rather small protein molecule containing 100 amino acid residues, there are 201 00 or 10130 possible School of Biochemical Engineering, Ponti? cia Universidad Cat? lica de Valpara? so, Avenida Brasil o ? 2147, Valpara? so, Chile. Phone: 56-32-273642, fax: 56-32-273803; e-mail: [email  protected] cl ? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Science + Business Media B. V. 2008 1 2 Trasition State A. Illanes Catalyzed Path Uncatalyzed PathFree Energy Ea Ea’ Reactans ? G Products Reaction Progress Fig. 1. 1 Mechanism of catalysis. Ea and Ea are the energies of activation of the uncatalyzed and catalyzed reaction. ?G is the free energy change of the reaction amino acid sequences, which is a fabulous number, higher even than the number of molecules in the whole universe. To get the right enzyme for a certain chemical reaction is then a matter of search and this is certainly challenging and exciting if one realizes that a very small fraction of all living forms have been already isolated.It is even more promising when one considers the possibility of obtaining DNA pools from the environment without requiring to know the organism from which it comes and then expressed it into a suitable host organism (Nield et al. 2002), and the opportunities of genetic remodeling of structural genes by site-directed mutagenesis (Abi? n et al. 2004). a Enzymes have been naturally tailored to perform under physiological conditions. However, biocatalysis refers to the use of enzymes as process catalysts under arti? cial conditions (in vitro), so that a major challenge in biocatalysis is to transform these hysiological catalysts into process catalysts able to perform under the usually tough reaction conditions of an industrial process. Enzyme catalysts (biocatalysts), as any catalyst, act by reducing the energy barrier of the biochemical reactions, without being altered as a consequence of the reaction they promote. However, enzymes display quite distinct properties when compared with chemical catalysts; most of these properties are a consequence of their complex molecular structure and will be analyzed in section 1. 2.Potentials and drawbacks of enzymes as process catalysts are summarized in Table 1. 1. Enzymes are highly desirable catalysts when the speci? city of the reaction is a major issue (as it occurs in pharmaceutical products and ? ne chemicals), when the catalysts must be active under mild conditions (because of substrate and/or product instability or to avoid unwanted side-reactions, as it occurs in several reactions of organic synthesis), when environmental restrictions are stringent (which is now a 1 Introduction Table 1. 1 Advantages and Drawbacks of Enzymes as Catalysts Advantages High speci? ity High activity under moderate conditions High turnover number Highly biodegradable Generally considered as natural products Drawbacks High molecular complexity High production costs Intrinsic fragility 3 rather general situation that gives biocatalysis a distinct advantage over alternative technologies) or when the l abel of natural product is an issue (as in the case of food and cosmetic applications) (Benkovic and Ballesteros 1997; Wegman et al. 2001). However, enzymes are complex molecular structures that are intrinsically labile and costly to produce, which are de? ite disadvantages with respect to chemical catalysts (Bommarius and Broering 2005). While the advantages of biocatalysis are there to stay, most of its present restrictions can be and are being solved through research and development in different areas. In fact, enzyme stabilization under process conditions is a major issue in biocatalysis and several strategies have been developed (Illanes 1999) that include ? chemical modi? cation (Roig and Kennedy 1992; Ozturk et al. 2002; Mislovi? ov? c a et al. 2006), immobilization to solid matrices (Abi? n et al. 2001; Mateo et al. 2005; a Kim et al. 2006; Wilson et al. 006), crystallization (H? ring and Schreier 1999; Roy a and Abraham 2006), aggregation (Cao et al. 2003; Mateo et al. 2004 ; Schoevaart et al. 2004; Illanes et al. 2006) and the modern techniques of protein engineering (Chen 2001; Declerck et al. 2003; Sylvestre et al. 2006; Leisola and Turunen 2007), namely site-directed mutagenesis (Bhosale et al. 1996; Ogino et al. 2001; Boller et al. 2002; van den Burg and Eijsink 2002; Adamczak and Hari Krishna 2004; Bardy et al. 2005; Morley and Kazlauskas 2005), directed evolution by tandem mutagenesis (Arnold 2001; Brakmann and Johnsson 2002; Alexeeva et al. 003; Boersma et al. 2007) and gene-shuf? ing based on polymerase assisted (Stemmer 1994; Zhao et al. 1998; Shibuya et al. 2000; Kaur and Sharma 2006) and, more recently, ligase assisted recombination (Chodorge et al. 2005). Screening for intrinsically stable enzymes is also a prominent area of research in biocatalysis. Extremophiles, that is, organisms able to survive and thrive in extreme environmental conditions are a promising source for highly stable enzymes and research on those organisms is very active at present (Adams and Kelly 1998; Davis 1998; Demirjian et al. 001; van den Burg 2003; Bommarius and Riebel 2004; Gomes and Steiner 2004). Genes from such extremophiles have been cloned into suitable hosts to develop biological systems more amenable for production (Halld? rsd? ttir et al. 1998; o o Haki and Rakshit 2003; Zeikus et al. 2004). Enzymes are by no means ideal process catalysts, but their extremely high speci? city and activity under moderate conditions are prominent characteristics that are being increasingly appreciated by different production sectors, among which the pharmaceutical and ? ne-chemical industry (Schmid et al. 001; Thomas et al. 2002; Zhao et al. 2002; Bruggink et al. 2003) have added to the more traditional sectors of food (Hultin 1983) and detergents (Maurer 2004). 4 Fig. 1. 2 Scheme of peptide bond formation between two adjacent ? -amino acids R1 + H3N CH C OH O A. Illanes H R2 + H N CH COO? H2O R1 H2O H R2 H3N CH C N CH COO? O + 1. 2 Enzymes as Cataly sts. Structure–Functionality Relationships Most of the characteristics of enzymes as catalysts derive from their molecular structure. Enzymes are proteins composed by a number of amino acid residues that range from 100 to several hundreds.These amino acids are covalently bound through the peptide bond (Fig. 1. 2) that is formed between the carbon atom of the carboxyl group of one amino acid and the nitrogen atom of the ? -amino group of the following. According to the nature of the R group, amino acids can be non-polar (hydrophobic) or polar (charged or uncharged) and their distribution along the protein molecule determines its behavior (Lehninger 1970). Every protein is conditioned by its amino acid sequence, called primary structure, which is genetically determined by the deoxyribonucleotide sequence in the structural gene that codes for it.The DNA sequence is ? rst transcribed into a mRNA molecule which upon reaching the ribosome is translated into an amino acid sequence a nd ? nally the synthesized polypeptide chain is transformed into a threedimensional structure, called native structure, which is the one endowed with biological functionality. This transformation may include several post-translational reactions, some of which can be quite relevant for its functionality, like proteolytic cleavage, as it occurs, for instance, with Escherichia coli penicillin acylase (Schumacher et al. 986) and glycosylation, as it occurs for several eukaryotic enzymes (Longo et al. 1995). The three-dimensional structure of a protein is then genetically determined, but environmentally conditioned, since the molecule will interact with the surrounding medium. This is particularly relevant for biocatalysis, where the enzyme acts in a medium quite different from the one in which it was synthesized than can alter its native functional structure. Secondary three-dimensional structure is the result of interactions of amino acid residues proximate in the primary structure, ma inly by hydrogen bonding of the amide groups; for the ase of globular proteins, like enzymes, these interactions dictate a predominantly ribbon-like coiled con? guration termed ? -helix. Tertiary three-dimensional structure is the result of interactions of amino acid residues located apart in the primary structure that produce a compact and twisted con? guration in which the surface is rich in polar amino acid 1 Introduction 5 residues, while the inner part is abundant in hydrophobic amino acid residues. This tertiary structure is essential for the biological functionality of the protein.Some proteins have a quaternary three-dimensional structure, which is common in regulatory proteins, that is the result of the interaction of different polypeptide chains constituting subunits that can display identical or different functions within a protein complex (Dixon and Webb 1979; Creighton 1993). The main types of interactions responsible for the three-dimensional structure of proteins are (Haschemeyer and Haschemeyer 1973): †¢ Hydrogen bonds, resulting from the interaction of a proton linked to an electronegative atom with another electronegative atom.A hydrogen bond has approximately one-tenth of the energy stored in a covalent bond. It is the main determinant of the helical secondary structure of globular proteins and it plays a signi? cant role in tertiary structure as well. †¢ Apolar interactions, as a result of the mutual repulsion of the hydrophobic amino acid residues by a polar solvent, like water. It is a rather weak interaction that does not represent a proper chemical bond (approximation between atoms exceed the van der Waals radius); however, its contribution to the stabilization of the threedimensional structure of a protein is quite signi? ant. †¢ Disulphide bridges, produced by oxidation of cysteine residues. They are especially relevant in the stabilization of the three-dimensional structure of low molecular weight extracellular protein s. †¢ Ionic bonds between charged amino acid residues. They contribute to the stabilization of the three-dimensional structure of a protein, although to a lesser extent, because the ionic strength of the surrounding medium is usually high so that interaction is produced preferentially between amino acid residues and ions in the medium. Other weak type interactions, like van der Waals forces, whose contribution to three-dimensional structure is not considered signi? cant. Proteins can be conjugated, this is, associated with other molecules (prosthetic groups). In the case of enzymes which are conjugated proteins (holoenzymes), catalysis always occur in the protein portion of the enzyme (apoenzyme). Prosthetic groups may be organic macromolecules, like carbohydrates (in the case of glycoproteins), lipids (in the case of lipoproteins) and nucleic acids (in the case of nucleoproteins), or simple inorganic entities, like metal ions.Prosthetic groups are tightly bound (usually covale ntly) to the apoenzyme and do not dissociate during catalysis. A signi? cant number of enzymes from eukaryotes are glycoproteins, in which case the carbohydrate moiety is covalently linked to the apoenzyme, mainly through serine or threonine residues, and even though the carbohydrate does not participate in catalysis it confers relevant properties to the enzyme. Catalysis takes place in a small portion of the enzyme called the active site, which is usually formed by very few amino acid residues, while the rest of the protein acts as a scaffold.Papain, for instance, has a molecular weight of 23,000 Da with 211 amino acid residues of which only cysteine (Cys 25) and histidine (His 159) 6 A. Illanes are directly involved in catalysis (Allen and Lowe 1973). Substrate is bound to the enzyme at the active site and doing so, changes in the distribution of electrons in its chemical bonds are produced that cause the reactions that lead to the formation of products. The products are then rele ased from the enzyme which is ready for the next catalytic cycle.According to the early lock and key model proposed by Emil Fischer in 1894, the active site has a unique geometric shape that is complementary to the geometric shape of the substrate molecule that ? ts into it. Even though recent reports provide evidence in favor of this theory (Sonkaria et al. 2004), this rigid model hardly explains many experimental evidences of enzyme biocatalysis. Later on, the induced-? t theory was proposed (Koshland 1958) according to which he substrate induces a change in the enzyme conformation after binding, that may orient the catalytic groups in a way prone for the subsequent reaction; this theory has been extensively used to explain enzyme catalysis (Youseff et al. 2003). Based on the transition-state theory, enzyme catalysis has been explained according to the hypothesis of enzyme transition state complementariness, which considers the prefc erential binding of the transition state rather than the substrate or product (Benkovi? and Hammes-Schiffer 2003).Many, but not all, enzymes require small molecules to perform as catalysts. These molecules are termed coenzymes or cofactors. The term coenzyme is used to refer to small molecular weight organic molecules that associate reversibly to the enzyme and are not part of its structure; coenzymes bound to enzymes actually take part in the reaction and, therefore, are sometime called cosubstrates, since they are stoichiometric in nature (Kula 2002). Coenzymes often function as intermediate carriers of electrons (i. e. NAD+ or FAD+ in dehydrogenases), speci? c atoms (i. e. oenzyme Q in H atom transfer) or functional groups (i. e. coenzyme A in acyl group transfer; pyridoxal phosphate in amino group transfer; biotin in CO2 transfer) that are transferred in the reaction. The term cofactor is commonly used to refer to metal ions that also bind reversibly to enzymes but in general are not chemically altered during the reaction; c ofactors usually bind strongly to the enzyme structure so that they are not dissociated from the holoenzyme during the reaction (i. e. Ca++ in ? -amylase; Co++ or Mg++ in glucose isomerase; Fe+++ in nitrile hydratase).According to these requirements, enzymes can be classi? ed in three groups as depicted in Fig. 1. 3: (i) those that do not require of an additional molecule to perform biocatalysis, (ii) those that require cofactors that remain unaltered and tightly bound to the enzyme performing in a catalytic fashion, and (iii) those requiring coenzymes that are chemically modi? ed and dissociated during catalysis, performing in a stoichiometric fashion. The requirement of cofactors or coenzymes to perform biocatalysis has profound technological implications, as will be analyzed in section 1. 4.Enzyme activity, this is, the capacity of an enzyme to catalyze a chemical reaction, is strictly dependent on its molecular structure. Enzyme activity relies upon the existence of a proper str ucture of the active site, which is composed by a reduced number of amino acid residues close in the three-dimensional structure of 1 Introduction Fig. 1. 3 Enzymes according to their cofactor or coenzyme requirements. 1: no requirement; 2: cofactor requiring; 3: coenzyme requiring S 1 7 P E E CoE 2 S E-CoE P E CoE 3 E CoE’ E P S E-CoE the protein but usually far apart in the primary structure.Therefore, any agent that promotes protein unfolding will move apart the residues constituting the active site and will then reduce or destroy its biological activity. Adverse conditions of temperature, pH or solvent and the presence of chaotropic substances, heavy metals and chelating agents can produce this loss of function by distorting the proper active site con? guration. Even though a very small portion of the enzyme molecule participates in catalysis, the remaining of the molecule is by no means irrelevant to its performance.Crucial properties, like enzyme stability, are very muc h dependent on the enzyme three-dimensional structure. Enzyme stability appears to be determined by unde? ned irreversible processes governed by local unfolding in certain labile regions denoted as weak spots. These regions prone to unfolding are the determinants of enzyme stability and are usually located in or close to the surface of the protein molecule, which explains why the surface structure of the enzyme is so important for its catalytic stability (Eijsink et al. 2004). These regions have been the target of site-speci? c mutations for increasing stability.Though extensively studied, rational engineering of the enzyme molecule for increased stability has been a very complex task. In most cases, these weak spots are not easy to identify so it is not clear to what region of the protein molecule should one be focused on and, even though properly selected, it is not clear what is the right type of mutation to introduce (Gaseidnes et al. 2003). Despite the impressive advances in th e ? eld and the existence of some experimentally based rules (Shaw and Bott 1996), rational improvement of the stability is still far from being well established.In fact, the less rational approaches of directed evolution using error-prone PCR and gene shuf? ing have been more successful in obtaining more stable mutant enzymes (Kaur and Sharma 2006). Both strategies can combine using a set of rationally designed mutants that can then be subjected to gene shuf? ing (O’F? g? in 2003). a a A perfectly structured native enzyme expressing its biological activity can lose it by unfolding of its tertiary structure to a random polypeptide chain in which the amino acids located in the active site are no longer aligned closely enough to perform its catalytic function.This phenomenon is termed denaturation and it may be reversible if the denaturing in? uence is removed since no chemical changes 8 A. Illanes have occurred in the protein molecule. The enzyme molecule can also be subjected to chemical changes that produce irreversible loss of activity. This phenomenon is termed inactivation and usually occurs following unfolding, since an unfolded protein is more prone to proteolysis, loss of an essential cofactor and aggregation (O’F? g? in 1997). These phenomena de? e what is called thermodynamic or cona a formational stability, this is the resistance of the folded protein to denaturation, and kinetic or long-term stability, this is the resistance to irreversible inactivation (Eisenthal et al. 2006). The overall process of enzyme inactivation can then be represented by: N U ? > I where N represents the native active conformation, U the unfolded conformation and I the irreversibly inactivated enzyme (Klibanov 1983; Bommarius and Broering 2005). The ? rst step can be de? ned by the equilibrium constant of unfolding (K), while the second is de? ed in terms of the rate constant for irreversible inactivation (k). Stability is not related to activity and in many cases they have opposite trends. It has been suggested that there is a trade-off between stability and activity based on the fact that stability is clearly related to molecular stiffening while conformational ? exibility is bene? cial for catalysis. This can be clearly appreciated when studying enzyme thermal inactivation: enzyme activity increases with temperature but enzyme stability decreases. These opposite trends make temperature a critical variable in any enzymatic process and make it prone to optimization.This aspect will be thoroughly analyzed in Chapters 3 and 5. Enzyme speci? city is another relevant property of enzymes strictly related to its structure. Enzymes are usually very speci? c with respect to its substrate. This is because the substrate is endowed with the chemical bonds that can be attacked by the functional groups in the active site of the enzyme which posses the functional groups that anchor the substrate properly in the active site for the reaction to take p lace. Under certain conditions conformational changes may alter substrate speci? city.This has been elegantly proven by site-directed mutagenesis, in which speci? c amino acid residues at or near the active site have been replaced producing an alteration of substrate speci? city (Colby et al. 1998; diSioudi et al. 1999; Parales et al. 2000), and also by chemical modi? cation (Kirk Wright and Viola 2001). K k 1. 3 The Concept and Determination of Enzyme Activity As already mentioned, enzymes act as catalysts by virtue of reducing the magnitude of the barrier that represents the energy of activation required for the formation of a transient active complex that leads to product formation (see Fig. . 1). This thermodynamic de? nition of enzyme activity, although rigorous, is of little practical signi? cance, since it is by no means an easy task to determine free energy changes for molecular structures as unstable as the enzyme–substrate complex. The direct 1 Introduction 9 conseq uence of such reduction of energy input for the reaction to proceed is the increase in reaction rate, which can be considered as a kinetic de? nition of enzyme activity. Rates of chemical reactions are usually simple to determine so this de? nition is endowed with practicality.Biochemical reactions usually proceed at very low rates in the absence of catalysts so that the magnitude of the reaction rate is a direct and straightforward procedure for assessing the activity of an enzyme. Therefore, for the reaction of conversion of a substrate (S) into a product (P) under the catalytic action of an enzyme (E): S ? > P v=? ds dp = dt dt (1. 1) E If the course of the reaction is followed, a curve like the one depicted in Fig 1. 4 will be obtained. This means that the reaction rate (slope of the p vs t curve) will decrease as the reaction proceeds.Then, the use of Eq. 1. 1 is ambiguous if used for the determination of enzyme activity. To solve this ambiguity, the reasons underlying this beh avior must be analyzed. The reduction in reaction rate can be the consequence of desaturation of the enzyme because of substrate transformation into product (at substrate depletion reaction rate drops to zero), enzyme inactivation as a consequence of the exposure of the enzyme to the conditions of reaction, enzyme inhibition caused by the products of the reaction, and equilibrium displacement as a consequence of the law of mass action.Some or all of these phenomena are present in any enzymatic reaction so that the catalytic capacity of the enzyme will vary throughout the course of the reaction. It is customary to identify the enzyme activity with the initial rate of reaction (initial slope of the â€Å"p† versus â€Å"t† curve) where all the above mentioned Product Concentration e e 2 e 4 Time Fig. 1. 4 Time course of an enzyme catalyzed reaction: product concentration versus time of reaction at different enzyme concentrations (e) 10 A. Illanes phenomena are insigni? a nt. According to this: a = vt>0 = ? ds dt = t>0 dp dt (1. 2) t>0 This is not only of practical convenience but fundamentally sound, since the enzyme activity so de? ned represents its maximum catalytic potential under a given set of experimental conditions. To what extent is this catalytic potential going to be expressed in a given situation is a different matter and will have to be assessed by modulating it according to the phenomena that cause its reduction. All such phenomena are amenable to quanti? ation as will be presented in Chapter 3, so that the determination of this maximum catalytic potential is fundamental for any study regarding enzyme kinetics. Enzymes should be quanti? ed in terms of its catalytic potential rather than its mass, since enzyme preparations are rather impure mixtures in which the enzyme protein can be a small fraction of the total mass of the preparation; but, even in the unusual case of a completely pure enzyme, the determination of activity is unavoida ble since what matters for evaluating the enzyme performance is its catalytic potential and not its mass.Within the context of enzyme kinetics, reaction rates are always considered then as initial rates. It has to be pointed out, however, that there are situations in which the determination of initial reaction rates is a poor predictor of enzyme performance, as it occurs in the determination of degrading enzymes acting on heterogeneous polymeric substrates. This is the case of cellulase (actually an enzyme complex of different activities) (Montenecourt and Eveleigh 1977; Illanes et al. 988; Fowler and Brown 1992), where the more amorphous portions of the cellulose moiety are more easily degraded than the crystalline regions so that a high initial reaction rate over the amorphous portion may give an overestimate of the catalytic potential of the enzyme over the cellulose substrate as a whole. As shown in Fig. 1. 4, the initial slope o the curve (initial rate of reaction) is proportio nal to the enzyme concentration (it is so in most cases). Therefore, the enzyme sample should be properly diluted to attain a linear product concentration versus time relationship within a reasonable assay time.The experimental determination of enzyme activity is based on the measurement of initial reaction rates. Substrate depletion or product build-up can be used for the evaluation of enzyme activity according to Eq. 1. 2. If the stoichiometry of the reaction is de? ned and well known, one or the other can be used and the choice will depend on the easiness and readiness for their analytical determination. If this is indifferent, one should prefer to measure according to product build-up since in this case one will be determining signi? ant differences between small magnitudes, while in the case of substrate depletion one will be measuring small differences between large magnitudes, which implies more error. If neither of both is readily measurable, enzyme activity can be determine d by coupling reactions. In this case the product is transformed (chemically or enzymatically) to a ? nal analyte amenable for analytical determination, as shown: E S P A X B Y C Z 1 Introduction 11 In this case enzyme activity can be determined as: a = vt>0 = ? ds dt = t>0 dp dt = t>0 dz dt (1. 3) t>0 rovided that the rate limiting step is the reaction catalyzed by the enzyme, which implies that reagents A, B and C should be added in excess to ensure that all P produced is quantitatively transformed into Z. For those enzymes requiring (stoichiometric) coenzymes: E S CoE CoE P activity can be determined as: a = vt>0 = ? dcoe dt = t>0 dcoe dt (1. 4) t>0 This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i. e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase (? galactosidase; EC 3. 2. 1. 23). The enzyme catalyzes the hydrolysis of lactose according to: Lactose + H2 O > Glucose + Galactose Glucose produced can be coupled to a classical enzymatic glucose kit, that is: hexoquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD), in which: Glucose + ATP ? > Glucose 6Pi + ADP Glucose 6Pi + NADP+ ? ? ? ?> 6PiGluconate + NADPH where the initial rate of NADPH (easily measured in a spectrophotometer; see ahead) can be then stoichiometrically correlated to the initial rate of lactose hydrolysis, provided that the auxiliary enzymes, Hx and G6PD, and co-substrates are added in excess.Enzyme activity can be determined by a continuous or discontinuous assay. If the analytical device is provided with a recorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay; in that case, the course of react ion should be monitored discontinuously by sampling and assaying at predetermined time intervals and samples should be subjected to inactivation to stop the reaction.This is a drawback, since the enzyme should be rapidly, completely and irreversibly inactivated by subjecting it to harsh conditions that can interfere with the G6PD Hx 12 A. Illanes analytical procedure. Data points should describe a linear â€Å"p† versus â€Å"t† relationship within the time interval for assay to ensure that the initial rate is being measured; if not, enzyme sample should be diluted accordingly. Assay time should be short enough to make the effect of the products on the reaction rate negligible and to produce a negligibly reduction in substrate concentration. A major issue in enzyme activity determination is the de? ition of a control experiment for discriminating the non-enzymatic build-up of product during the assay. There are essentially three options: to remove the enzyme from the r eaction mixture by replacing the enzyme sample by water or buffer, to remove the substrate replacing it by water or buffer, or to use an enzyme placebo. The ? rst one discriminates substrate contamination with product or any non-enzymatic transformation of substrate into product, but does not discriminate enzyme contamination with substrate or product; the second one acts exactly the opposite; the third one can in rinciple discriminate both enzyme and substrate contamination with product, but the pitfall in this case is the risk of not having inactivated the enzyme completely. The control of choice depends on the situation. For instance, when one is producing an extracellular enzyme by fermentation, enzyme sample is likely to be contaminated with substrate and or product (that can be constituents of the culture medium or products of metabolism) and may be signi? ant, since the sample probably has a low enzyme protein concentration so that it is not diluted prior to assay; in this ca se, replacing substrate by water or buffer discriminates such contamination. If, on the other hand, one is assaying a preparation from a stock enzyme concentrate, dilution of the sample prior to assay makes unnecessary to blank out enzyme contamination; replacing the enzyme by water or buffer can discriminate substrate contamination that is in this case more relevant.The use of an enzyme placebo as control is advisable when the enzyme is labile enough to be completely inactivated at conditions not affecting the assay. An alternative is to use a double control replacing enzyme in one case and substrate in the other by water or buffer. Once the type of control experiment has been decided, control and enzyme sample are subjected to the same analytical procedure, and enzyme activity is calculated by subtracting the control reading from that of the sample, as illustrated in Fig. . 5. Analytical procedures available for enzyme activity determinations are many and usually several alternati ves exist. A proper selection should be based on sensibility, reproducibility, ? exibility, simplicity and availability. Spectrophotometry can be considered as a method that ful? ls most, if not all, such criteria. It is based on the absorption of light of a certain wavelength as described by the Beer–Lambert law: A? = ?  · l  · c where: A? = log I I0 (1. 5) (1. 6) The value of ? an be experimentally obtained through a calibration curve of absorbance versus concentration of analyte, so that the reading of A? will allow the determination of its concentration. Optical path width is usually 1 cm. The method is based on the differential absorption of product (or coupling analyte or modi? ed 1 Introduction 13 Fig. 1. 5 Scheme for the analytical procedure to determine enzyme activity. S: substrate; P: product; P0 : product in control; A, B, C: coupling reagents; Z: analyte; Z0 : analyte in control; s, p, z are the corresponding molar concentrations oenzyme) and substrate (or co enzyme) at a certain wavelength. For instance, the reduced coenzyme NADH (or NADPH) has a strong peak of absorbance at 340 nm while the absorbance of the oxidized coenzyme NAD+ (or NADP+ ) is negligible at that wavelength; therefore, the activity of any enzyme producing or consuming NADH (or NADPH) can be determined by measuring the increase or decline of absorbance at 340 nm in a spectrophotometer. The assay is sensitive, reproducible and simple and equipment is available in any research laboratory.If both substrate and product absorb signi? cantly at a certain wavelength, coupling the detector to an appropriate high performance liquid chromatography (HPLC) column can solve this interference by separating those peaks by differential retardation of the analytes in the column. HPLC systems are increasingly common in research laboratories, so this is a very convenient and ? exible way for assaying enzyme activities. Several other analytical procedures are available for enzyme activity determination.Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (? avin adenine dinucleotide) and FMN (? avin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less ? exible and the equipment not so common in a standard research laboratory.Enzymes that produce or consume gases can be assayed by differential manometry by measuring small pressure differences, due to the consumption of the gaseous substrate or the evolution of a gaseous product that can be converted into substrate or product concentrations by using the gas law. Carboxylases and decarboxylases are groups of enzymes that can be conveniently assayed by differential manomet ry in a respirometer. For instance, the activity of glutamate decarboxylase 14 A. Illanes (EC 4. 1. 1. 15), that catalyzes the decarboxylation of glutamic acid to ? aminobutyric acid and CO2 , has been assayed in a differential respirometer by measuring the increase in pressure caused by the formation of gaseous CO2 (O’Learys and Brummund 1974). Enzymes catalyzing reactions involving optically active compounds can be assayed by polarimetry. A compound is considered to be optically active if polarized light is rotated when passing through it. The magnitude of optical rotation is determined by the molecular structure and concentration of the optically active substance which has its own speci? rotation, as de? ned in Biot’s law: ? = ? 0  · l  · c (1. 7) Polarimetry is a simple and accurate method for determining optically active compounds. A polarimeter is a low cost instrument readily available in many research laboratories. The detector can be integrated into an HPL C system if separation of substrates and products of reaction is required. Invertase (? -D-fructofuranoside fructohydrolase; EC 3. 2. 1. 26), a commodity enzyme widely used in the food industry, can be conveniently assayed by polarimetry (Chen et al. 2000), since the speci? optical rotation of the substrate (sucrose) differs from that of the products (fructose plus glucose). Some depolymerizing enzymes can be conveniently assayed by viscometry. The hydrolytic action over a polymeric substrate can produce a signi? cant reduction in kinematic viscosity that can be correlated to the enzyme activity. Polygalacturonase activity in pectinase preparations (Gusakov et al. 2002) and endo ? 1–4 glucanase activity in cellulose preparations (Canevascini and Gattlen 1981; Illanes and Schaffeld 1983) have been determined by measuring the reduction in viscosity of the corresponding olymer solutions. A comprehensive review on methods for assaying enzyme activity has been recently published ( Bisswanger 2004). Enzyme activity is expressed in units of activity. The Enzyme Commission of the International Union of Biochemistry recommends to express it in international units (IU), de? ning 1 IU as the amount of an enzyme that catalyzes the transformation of 1  µmol of substrate per minute under standard conditions of temperature, optimal pH, and optimal substrate concentration (International Union of Biochemistry).Later on, in 1972, the Commission on Biochemical Nomenclature recommended that, in order to adhere to SI units, reaction rates should be expressed in moles per second and the katal was proposed as the new unit of enzyme activity, de? ning it as the catalytic activity that will raise the rate of reaction by 1 mol/second in a speci? ed assay system (Anonymous 1979). This latter de? nition, although recommended, has some practical drawbacks. The magnitude of the katal is so big that usual enzyme activities expressed in katals are extremely small numbers that are har d to appreciate; the de? ition, on the other hand, is rather vague with respect to the conditions in which the assay should be performed. In practice, even though in some journals the use of the katal is mandatory, there is reluctance to use it and the former IU is still more widely used. 1 Introduction 15 Going back to the de? nition of IU there are some points worthwhile to comment. The magnitude of the IU is appropriate to measure most enzyme preparations, whose activities usually range from a few to a few thousands IU per unit mass or unit volume of preparation.Since enzyme activity is to be considered as the maximum catalytic potential of the enzyme, it is quite appropriate to refer it to optimal pH and optimal substrate concentration. With respect to the latter, optimal is to be considered as that substrate concentration at which the initial rate of reaction is at its maximum; this will imply reaction rate at substrate saturation for an enzyme following typical Michaelis-Mente n kinetics or the highest initial reaction rate value in the case of inhibition at high substrate concentrations (see Chapter 3).With respect to pH, it is straightforward to determine the value at which the initial rate of reaction is at its maximum. This value will be the true operational optimum in most cases, since that pH will lie within the region of maximum stability. However, the opposite holds for temperature where enzymes are usually quite unstable at the temperatures in which higher initial reaction rates are obtained; actually the concept of â€Å"optimum† temperature, as the one that maximizes initial reaction rate, is quite misleading since that value usually re? cts nothing more than the departure of the linear â€Å"p† versus â€Å"t† relationship for the time of assay. For the de? nition of IU it is then more appropriate to refer to it as a â€Å"standard† and not as an â€Å"optimal† temperature. Actually, it is quite dif? cult to de? ne the right temperature to assay enzyme activity. Most probably that value will differ from the one at which the enzymatic process will be conducted; it is advisable then to obtain a mathematical expression for the effect of temperature on the initial rate of reaction to be able to transform the units of activity according to the temperature of operation (Illanes et al. 000). It is not always possible to express enzyme activity in IU; this is the case of enzymes catalyzing reactions that are not chemically well de? ned, as it occurs with depolymerizing enzymes, whose substrates have a varying and often unde? ned molecular weight and whose products are usually a mixture of different chemical compounds. In that case, units of activity can be de? ned in terms of mass rather than moles. These enzymes are usually speci? c for certain types of bonds rather than for a particular chemical structure, so in such cases it is advisable to express activity in terms of equivalents of bonds b roken.The choice of the substrate to perform the enzyme assay is by no means trivial. When using an enzyme as process catalyst, the substrate can be different from that employed in its assay that is usually a model substrate or an analogue. One has to be cautious to use an assay that is not only simple, accurate and reproducible, but also signi? cant. An example that illustrates this point is the case of the enzyme glucoamylase (exo-1,4-? -glucosidase; EC 3. 2. 1. 1): this enzyme is widely used in the production of glucose syrups from starch, either as a ? al product or as an intermediate for the production of high-fructose syrups (Carasik and Carroll 1983). The industrial substrate for glucoamylase is a mixture of oligosaccharides produced by the enzymatic liquefaction of starch with ?-amylase (1,4-? -D-glucan glucanohydrolase; EC 3. 2. 1. 1). Several substrates have been used for assaying enzyme activity including high molecular weight starch, small molecular weight oligosaccharid es, maltose and maltose synthetic analogues (Barton et al. 1972; Sabin and Wasserman 16 A. Illanes 1987; Goto et al. 1998). None of them probably re? cts properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities; however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a transesteri? cation reaction that produces thiophenol as one of the products (Han et al. 004); in this case, a method based on a reaction leading to a ? uorescent adduct of thiophenol is a good system to assess the transesteri? cation potential of such proteases and is to be preferred to a conventional protease as say based on the hydrolysis of a protein (Gupta et al. 1999; Priolo et al. 2000) or a model peptide (Klein et al. 1989). 1. 4 Enzyme Classes. Properties and Technological Signi? cance Enzymes are classi? ed according to the guidelines of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) (Anonymous 1984) into six families, based on the type of chemical reaction catalyzed.A four digit number is assigned to each enzyme by the Enzyme Commission (EC) of the IUBMB: the ? rst one denotes the family, the second denotes the subclass within a family and is related to the type of chemical group upon which it acts, the third denotes a subgroup within a subclass and is related to the particular chemical groups involved in the reaction and the forth is the correlative number of identi? cation within a subgroup. The six families are: 1. Oxidoreductases. Enzymes catalyzing oxidation/reduction reactions that involve the transfer of electrons, hydroge n or oxygen atoms.There are 22 subclasses of oxido-reductases and among them there are several of technological signi? cance, such as the dehydrogenases that oxidize a substrate by transferring hydrogen atoms to a coenzyme (NAD+ , NADP+ ,