Lemongrass as Mosquito Repellent

Introduction Nowadays, our country is very rampant with mosquitoes that carry various diseases and one of the most popular diseases that are caused by a mosquito is â€Å"DENGUE†. Because of this disease, a lot of Filipinos died. Dengue is a disease caused by any one of the four related viruses transmitted by mosquitos. One of the easiest ways to prevent mosquitoes is to use Mosquito Repellent. This is the reason why the researchers want to make an alternative Mosquito Repellent.There are many commercial Mosquito Repellent available in the market. The commonly used commercial Mosquito Repellent in the Philippines is quite unaffordable and use chemical that may cause irritation. The researchers planned to use natural ingredients. Since the ingredient to be use is natural, the Mosquito Repellent to be made is more affordable and environmental friendly. The ingredient that the researchers are going to use is Lemongrass.Lemongrass has natural anti-microbial properties, is an antis eptic, suitable for use on various types of skin infections, usually as a wash or compress, and is especially effective on ringworm, infected sores, acne and athlete’s foot. Lemongrass is also used as a Mosquito Repellent. In using this product, you will spray it over all exposed skin areas. Avoid spraying near your eyes. You may reapply after washing. If irritation occurs, wash of immediately and discontinue use. The very most importance of this product is to avoid or prevent the spread of various diseases caused by mosquito bites.Mosquito Repellent is a substance skin, clothing, or other surfaces which discourages insects from landing or climbing on that surface. Mosquito Repellent also helps prevent and control the outbreak of insect borne diseases such as Malaria, Lyme disease, Dengue fever, Bubonic plague, and West Nile Fever. Theoretical Framework Lemongrass herb is very popular plant found commonly in India and used for medicinal, food and insect/mosquito repellent pro ducts.The lemongrass oils are also used in cosmetics, soaps, perfumes, dyes and dorizes along with thousands of other products. (www. newtechbio. com/articles/Lemongrass-as-an-insect-repellent. htm) Lemongrass oil has a fresh, strong, lemon-like, and pungent odor and is used in deodorants, herbal teas, skin care products, fragrances, and insect repellents, and for aromatherapy. (http://www. naturalstandard. com/index-abstract. asp? create-abstract=patient-lemongrass. asp&title=Lemongrass) Significance of the Study People nowadays can be easily infected with diseases caused by mosquitoes.Diseases that can kill people. The people who are easily infected with this diseases are those people living near canals, dump sides, and rivers. Mosquitoes are dangerous pest in the whole world. Nowadays, people encountered various diseases such as malaria and dengue. Due to economic crises the researcher found a solution to get rid of mosquitos that exist in locality. However the following product is tested by the following stake holders such as client, teachers, students, and workers.The researchers find the study significant because there is an increasing number of mortality due to mosquito bites, although there are alternative solutions to this problem. Filipinos cannot deny that some of their brothers and sisters cannot afford to buy organic insect repellent made out of lemongrass. Scope and Limitations The researcher’s product is only limited to minimize the amount of mosquitoes in a certain household. This is to see how effective the product will be before applying it somewhere else.

Microbiology 311 Lab Report

Rebekah Worley February 21, 2012 Mitchell Section 4 Biol 311 Staining and Identifying Unknown Bacteria Introduction: The microbiology lab up to this point has been used to teach the students how to stain and identify bacteria. There are several types of staining through which the bacteria can be identified based on the color and shape. The staining methods used in the lab are Gram Staining, Capsule Staining, Endospore Staining, and Acid Fast staining. One of the most significant method of staining is the Gram Staining, as it is highly dependent (McCarthy, 25).In the specific experiment that was done, Gram Staining was used and the bacteria that was found was purple and round (cocci) shaped. Through this the bacteria was identified as Staphylococcus epidermis. Material and Methods: The first step to identifying the bacteria was to heat fix it to the slide. The materials used were a slide, water, a Bunsen Burner, bibulous paper and clothes pin. The unknown bacteria was in a vial in sol id form. The steps on page 19 and 20 of the Customized Biol 311 General Microbiology Laboratory Manual were followed to heat fix the bacteria.After this gram staining was used to identify the unknown bacteria. The materials used for gram staining include the slide the was heat fixed, bibulous paper, crystal violet, distilled water, Gram's iodine, 95% ethyl alcohol, safranin, oil and a microscope. The steps on page 26 of the Customized Biol 311 General Microbiology Laboratory Manual were used to stain the bacteria. Several changes were made in the procedure. The crystal violet was on the slide for 1 minute rather than 20 seconds. The decolorizing step was used with alcohol for 10 seconds rather than 20 seconds.The only other change was that the safranin was on the slide for 1 minute instead of the recommended 20 seconds. The slide was put under the microscope at 1000x magnification using oil immersion. Results: When looking under the microscope the bacteria was found to be purple and cocci shaped. Because of the specific color and shape of the bacteria it was easily identifiable as Staphylococcus epidermis. From this it is seen that only a Gram stain was necessary to identify the bacteria. Discussion: From this experiment it is seen that bacteria is easily identified when stained correctly.Going through the procedure with accuracy is vital, and when done right the bacteria is clear and concise. When the bacteria was stained in this experiment the color was difficult to determine at first. After exploring the bacteria on the slide it was seen to be mainly purple. If the staining had not been done properly it would have been a lot more difficult to distinguish between bacteria. This was an important thing to learn because staining is so vital in identifying unknown bacterium. Works Cited McCarthy, Charlotte M and Harold Benson. Customized Biol 311 General Microbiology Laboratory Manual. 2nd. ed. New York. McGraw-Hill 2002 Print.

Architecture Essays – Baroque Architecture Characteristics

Baroque Architecture CharacteristicsIntroductionThe Baroque period took the humanist Roman position of Renaissance architecture and showed it in a new rhetorical, theatrical and sculptural manner they expressed the victory of absolutist church and province. The chief position of Baroque architecture was more concerned about colour, visible radiation and shadiness, sculptural values and strength which could be seen in its features. Baroque is defined periods in literature every bit good as music ; nevertheless critics perceived it to be unstructured, over ornamented, theatrical and grotesque. Although many things influenced the Baroque period it was besides influenced by faith every bit good as the states political relations. Architects were interested in the infinite of the unfolding existence every bit good as the workings of the head and attempted to portray passions of the psyche through facial looks.FeaturesThe characteristics of the Baroque epoch showed long, narrow naves that w ere replaced by a broader, or on occasion with round signifiers. It displayed dramatic usage of visible radiation that could be either strong light-and-shade contrast ( known aschiaroscuro) effects ; or they used unvarying lighting by agencies of several Windowss. Another characteristic was deluxe usage of decorations ( puttos made of wood ( frequently gilded ) , plaster or stucco, marble or fake coating ) , they used large-scale ceiling frescoes and Baroques external facade is frequently characterized by a dramatic cardinal projection, nevertheless the inside is frequently no more than a shell for picture and sculpture which was seen in the late Baroque period. Baroque features besides include illusive effects like trompe l'oeil and the blending of picture and architecture and in other states such as Bavaria, Czech, Polish, and Ukrainian the Baroque manner contained, pear domes that were are omnipresent.FeaturesThe chief features for the Baroque epoch were energy, great sums of ten seness and a sense of motion from the edifices. Its picture, sculpture and architecture evolved from idiosyncrasy and broke off from the regulations of modern-day architecture, they demanded freedom to program, design and adorn their edifices with what they wanted. Columns had twisted shafts which were placed in forepart of pilasters surrounded by valances and covered with curving and broken pediments. They contained â€Å"over the top† and frequently unsuitable inside informations with carven ornament. Insides had gilded sculptures frequently in awkward airss ; the architecture was noted for its curving lines. Many of Baroques add-ons were finished in bronze, marble, gold and Ag. Baroque had a dynamic expression and experience to its design ; it was a utile categorization for insulating the inclinations and merchandises of stylistic alteration. It was seen as broad, superb, theatrical, passionate, animal, enraptured, deluxe, excessive, various and ace. It was an age of enlar gement following on an age of find, its enlargement led to still farther find about architectural design and ornament. Section A: Insides S.Maria Della Salute ( 1631-1682 ) The inside of S.Maria Della Salute is a really good illustration of Baroque Architecture and design. It displays the Baroque kernel in a manner but is non wholly over ornamented nor does it incorporate any unsuitable inside informations. Marble is chiefly used in the columns and the base appears to be gilded in bronze. Sculptures are carved from marble and stand high in the unit of ammunition of the dome. An communion table can be seen and one might presume it is besides gilded in bronze. It is an graphics in a manner but it is non â€Å"over the top† in any manner which can be seen from this position point. S.Pietro ( 1656 – 1667 ) The S.Pietro is another great illustration of Baroque architecture and design. In this exposure we can see the bronze communion table that stands merely in forepart of the apsis, we can see pictures that are decorated with gold lodgers, marble columns and high walls, the domes contain gilded ceilings. Walls are decorated with sculptures in free standing places in the walls besides made of marble. Light is given through high standing Windowss and the chief dome from above through a sky visible radiation. Versailles ( 1660 – 1685 ) The Palace of Versailles is the most good known piece of Baroque Architecture and design known to day of the month. It defines what the Baroque period was approximately. It was excessive, animal, dynamic, passionate ( pictures ) , various and deluxe. Decorations were non silver but pure gold. Soft cloths, bright colorss and beautiful sculptures decorated the insides of the Palace. In the Hall of Mirrors as seen in figure 8 sculptures themselves were cast in gold high Windowss offered light and glass pendants hung from a ceiling decorated in picture and gilded gold lodgers. In the Queens Chambers as seen in figure 7 one can state that non even the sleeping rooms were unbroken simple. Gold was besides used and to an extent it was excessively used. The focal point of 1s oculus is led to the gold ornament and non the architectural values of the edifice. The outside as seen in figure 6 one can see that the balcony railings were gilded in gilded ornament as the Sun makes this easy to see. Marble is besides used on the facade of the edifice but it is chiefly decorated in gold. Rich vivacious colorss can be seen throughout the castle as seen in figure 7 of the Queens Chambers. Section B: Interior Design Although the Baroque epoch contributed to the great edifices we see today, one can non bury about the insides. Although extravagantly decorated the insides are really good designed to suit certain facets. Architects need to believe like that in order to obtain a good sense of what works and what does non. I believe interior design does really suggest about it along the lines. We design what we think the client would wish and so acquire an thought if the client likes it of non, if non we merely do a few alterations to acquire a better feel and a better position of what they want. It is the same now as it is so. Interior interior decorators design the infinite in which the client will be in every twenty-four hours. It requires a great trade of penetration and a cognition about a individual, one might see reading your client by speaking to them, when you do that you can acquire better thoughts. Simple designs frequently make better designs in these yearss nevertheless in the past if it was large, broad and contained munificent ornaments it was classified as good.DecisionSo from looking at the Baroque architecture one can state that it played a great function in the design of edifices today. Many people who obtain an over and above wage sometimes have one or two suites that give recollection to the Baroque manner. It was surely munificent, dynamic and â€Å"over the top† ornaments were decidedly a spot much for today’s modern society.BibliographyTansey, R.G & A ; Kleiner, F.S. 1954. Chapter 24.Gardener’s Art Through The Ages: 10th edition. United States of America: Ted Buchhloz. Pages 816 – 904.Gardener, L. Chapter 10.Gardener’s Art Through The Ages: 4th edition.United States of America: G.Bell and Son Ltd, London. Page 397 onwards.Honour, H & A ; Fleming, J. 1982. Separate 3: Chapter 13.A World History of Art.United Kingdom: RB Macmillion. Page 426 onwards.Wikipedia. 2008.Baroque.[ online ] . [ Accessed August 20, 2008 ] . Availa ble from universe broad web: & lt ; hypertext transfer protocol: //www.wikipedia.org & gt ;Versailles.[ online ] . [ Accessed August 20, 2008 ] . Available from universe broad web: & lt ; hypertext transfer protocol: //www.bc.edu/bc_org/arp/cas/fnart/arch/versailles.html & gt ;History of Baroque Architecture.[ online ] . [ Accessed August 20, 2008 ] . Available from universe broad web: & lt ; hypertext transfer protocol: //web.kyoto-inet.or.jp/org/orion/eng/hst/baroque.html & gt ;Baroque Age.[ online ] . [ Accessed August 20, 2008 ] . Available from universe broad web: & lt ; hypertext transfer protocol: //library.thinkquest.org/16545/data/low/baroque.html & gt ;

Taking a Final Exam Essay Example | Topics and Well Written Essays - 250 words

Taking a Final Exam - Essay Example This will not result in a high grade because information needs to be taken in gradually. If you have revised all your study material leading up to the exam, then it is best not to do any study on the day of the exam because your mind needs to be relaxed and at ease. Carrying on from this idea, part of your preparation is getting enough rest the night before a crucial exam. The likelihood is that tiredness will cause you to perform below your capabilities. A rested mind increases the chances of doing well because you will be able to think clearly when answering each question. Furthermore, you can better prepare for a final exam by giving yourself short quizzes to check if you know the material. This can be best done with one of your classmates—you can even quiz each other. These questions should simple questions from your textbook. In conclusion, exam preparation is a key part of performing well on a final exam. If you fail to prepare properly for a final exam, then you should expect to do poorly. On the other hand, if you study all your material, rest your mind, and quiz yourself, the chances of scoring highly will increase dramatically. The more of these tips that can be done means it is more likely that you will attain a good

MUSIC in Britain Essay Example | Topics and Well Written Essays - 1250 words

MUSIC in Britain - Essay Example They have a fusion of different genres of music: sacred, secular, popular and new age music. Each of the Great Nations that originally made up the Great Britain-Ireland, Whales, England, and Scotland- maintained a unique instrumentation and music forms. British music was largely impacted by continental developments. British composers contributed a lot to significant music movements in the Great Britain. Such movements included the polyphony that later gave birth to national as well as international classical. Over the ages British musicians created distinctive musical forms such as carol, antiphons, the rota, and Countenance Angloise. Church music predominant in the 16th century was largely influenced by the Protestant Reformation. Thus, the songs and music at this time were themed around worship, national music and belief. Following the church music was the Baroque, largely viewed by critiques as a culmination of masques, lute ayres, and English magrigals during the Renaissance Period (Baggett et al. 1996, p24). The Baroque music was developed in the 17th century. By contrast, court music was more integrated into the larger Great Britain. It is important to mention that the Baroque music was largely associated by the British Isles. Baroque music was predominant between the medieval time and the Renaissance. It became more formalized and fully accepted orchestral classical music in the mid 18th century. The music was differentiated with intricate musical ornamentation, variation in musical notes, novel instrumentation; and new techniques of playing instruments as the ascent of musical forms such as opera. Although traditionally used in the dawn of the 17th century, the full impact of Baroque music was not felt until mid 17th century; the effects were delayed due to native reluctance in incorporating the music, wars between the Great Nations, as well as cultural and religious differences. With

Leadership styles Research Paper Example | Topics and Well Written Essays - 1750 words

Leadership styles - Research Paper Example Their ability to perform the aforementioned things relies on the leadership style adopted. Thus, at any time the leader or the manager should employ these styles. An important issue arises and this will be subject in this paper, how are the managers capable of altering their leadership styles (as described in situational leadership theory) to communicate and motivate the employees. There are four leadership styles (the selling or coaching style, the participating or supporting style, the telling or directing style, and the delegating style) and each style will be discussed in detail and applied in the case of General Electric. Specifically, the paper will explore the leadership style of Jeff Immelt, the Chief Executive Officer (CEO) of General Electric. General Electric is among many companies that have excelled in their leadership styles. The leadership style employed has been used as a benchmark for most organizations and companies. It is noted that the four leadership styles are c rucial for the success or failure of a company. As seen in General Electric case, a leader or a manager can apply all the four leadership styles to steer the organization. However, there is a tendency to use one leadership style more than the others are. General Electric In 1876, in Menlo Park, New Jersey, Thomas Alva Edison opened a laboratory where he could discover the prospects of the dynamo and other electrical tools or devices that he had realized in the exposition. By the year 1890, Edison launched the Edison General Electric Company by merging his various businesses. At the same time, a competitor appeared, the Thomson-Houston Company. Thomson-Houston Company became the principal electrical company through a series of unions or mergers led by Charles A. Coffin (General Electric Company, 2012). As the businesses grew, it became very difficult for either of the company to generate complete electrical installations depending entirely on their own technologies and patents. The t wo companies merged in the year 1892, and the new organization was called the General Electric Company. A number of Edison’s initial business offerings are still a portion of General Electric (GE) today and they include transportation, power transmission, medical equipment, industrial products, and lighting. The initial GE Appliances electric fans were generated at the Ft. Wayne electric works in the early 1890s. Full line of cooking and heating devices was first produced in 1907(General Electric Company, 2012). The GE Aircraft Engines started its operations in 1917 when the United States government started its search for a company that could produce the first airplane booster. Edison’s experiments with plastic filaments for the light bulbs started in 1893 and this led to the creation of the GE Plastics department in 1930. The General Electric leaders over the years have created a diverse portfolio of prominent businesses;a series of dominant company-wide initiatives t hat reduces cost and drives growth; Controllership and financial strength that permit it capitalize on openings through several cycles; and a collection of common values which permit it to face every environment with confidence(General Electric Company, 2012). Analysis The Hersey-Blanchard Situational Leadership Model illustrates the relation between the willingness of the followers and leadership style based on relationship and task behaviors of leaders.

Impacts of Keystone Holdings, LLCs Merger with Compagnie de Saint-Gob Assignment

Impacts of Keystone Holdings, LLCs Merger with Compagnie de Saint-Gobain - Assignment Example The Keystone Holdings tried to acquire the Advanced Ceramics Business of Saint- Gobain and thereby eliminate the competitor from the market for alumina wear tile. Government tries to ensure competition in the market and thereby maximum choice and minimum price to the customers. My goal in this essay is to portrait the impact of the acquisition on society and firms. Keystone is the holding company of CoorsTek, Inc. (CoorsTek), which is a leading technical ceramics manufacturer, supplying ceramics based products for use in defense, medical, automotive, semiconductor, and power generation applications, among others. Keystone is headquartered in Golden, Colorado with facilities in North America, Europe, and Asia. Keystone manufactures and sells alumina wear tile for use in high wear applications at its facilities in Golden, Colorado. Saint-Gobain is a highly diversified, multinational company, headquartered in Courbevoie, France. The Advanced Ceramics Business includes ceramic components such as hot surface igniters, electro-ceramic parts for household appliances, ceramic balls for high-performance bearings, automobile water pump seals, special components for the semiconductor industry, agricultural spray nozzles, and other dense alumina components, such as alumina wear tile. Saint-Gobain manufactures and sells alumina wear tile out of its Latrobe, Pennsylvania facility. Oligopoly is a market organization in which there are only a few sellers of a product. So the actions of each seller affect other sellers also. Mergers and acquisitions are mainly a part of the oligopolistic market. The alumina wear tile market in North America can be considered as an oligopolistic market as there are very few firms in the industry. As the alumina wear tile market is an oligopolistic market, any action that the Advanced Ceramics Business takes has an impact on other competitors like Keystone Holdings. If Advanced Ceramics reduces the price of their product, the other players in the market are forced to reduce their prices also.

Sexual Assault of Children and Youth and Other Sexual Offenses Essay

Sexual Assault of Children and Youth and Other Sexual Offenses - Essay Example Sexual assault is not common only in women. Today, wives, children, grandmothers, mothers, and even boys and men face sexual assault. The belief is that co-workers, classmates, neighbors, or a stranger would commit a sexual assault, but it is also possible that a family member or friend, or relative to sexually assault someone. "Sexual assault of children is a crime that our society abhors in the abstract, but tolerates in reality" (Burgess and Groth 15). Incest is one of the forms of sexual assault of children. Incest is when there is sexual activity between close relatives, which is illegal or taboo in the society. Sexual abuse of children can include trickery, sexual contact by force, bribery where there is a difference in age, power, size, or knowledge. Sexual abuse in a particular child could be once or multiple incident over a long period of time. It is found that abuse is usually committed by a person who knows the child. The abuse committed on a child can increase over time, especially if the abuser is a part of the family or a close relative. Prostitution, exhibitionism, and pornography are some ways in which children are abused other than the sexual assaults mentioned above. When people think of a child molester, they imagine an ugly old man taking children away by giving them some candy. No one pictures their neighbor, mom, dad, or uncle. This misconception has been dismissed. A child molester can come from all walks of life and all social and economic groups. They can be rich, poor, male, female, religious or non-religious, from any race or culture. Children can be molested by strangers or people they don't know and by people they know, like friends, relatives, or caregivers. Though majority of the people found guilty of sexual offence to children are men, women also molest children. Child molesters can be divided into two groups based on their behavioral patterns. A small percentage of child molesters who have a life long attraction to children, these people do not get attracted to adults. Majority of child molesters have adult partners and do not have an exclusive attraction towards children, also, they do not molest children multiple times. Sometim es an adult molests a child of the same sex, however, they need not be homosexual. Though it is believed that child molestation occurs when children are picked up from the street and molested forcibly, in the majority of child molestation cases, adults seduce children through delicate persuasion and intimidation and are usually known to the child. Child molesters who do not know their victims use methods like force, tricks, or bribery, or pretend to be friendly, to attract their victim. A child molester has many different characteristics. The child molester is usually married, prefers the company of children than adults, are often homosexuals and sometime bisexuals as well, they often wish to work for programs involving children, feel the want to have an emotional relationship with children, sometimes collect photographs of their victims and also collect child-adult pornography and child erotica, may be alcoholic, or a drug addict, may offer to take children out for walks or picnics, and many more such traits are examples of characteristics of child molesters. There are various definitions of the term pedophilia or paedophillia found in law enforcement, the

The Practical Benefits of Personal Skills Essay

The Practical Benefits of Personal Skills - Essay Example The four skills that I have chosen are coordination, active learning, active listening, and critical thinking. Possession of these life skills is said to be very beneficial and eases difficult conditions, both in terms of personal matters and in terms of those that deal with the workplace. Indeed, when faced with life's problems, which are natural however, one should possess the basic life skills so that he may be armed in defeating all life's foes. My chosen skills are beneficial to a person in a variety of ways that one can imagine. Its importance is seen in the tendency of a suicidal patient to end his life, which is a certain manifestation of hardship in dealing with a current problem. This condition only mirrors the truth that this person has not equipped himself effectively enough with life's basic skills. The reason may perhaps be because he is not aware that these are important skills that must be developed and carried on in life, or it may be that he did not have the opportunity to develop them in the first place, the environment he is in is not one conducive enough for such development, or he simply did not have the knowledge of how important they are to his own advantage. Below are the four chosen basic skills and their benefits: Coordinatio Coordination Skills Coordination is adjusting actions in relation to others' action.1 It implies one's ability to adapt to situations and the people around it. Getting something through whatever means possible is contrary to the development of this basic skill, since one has to consider others - their ideas, feelings, and impacts of an undertaking or a decision - and unite them with one's ideas in making things work out the most possible and desirable way. Adjusting one's actions with those of others in a pursuit to achieve an individual or a group goal is the best way of dealing with differences.2 The benefits that this basic skill extends to an individual are massive. The results are manifested in a healthy work environment, a give-and-take relationship, and new and renewed friendships. People will also appreciate one's consideration and ability of involving them in his worldview, a rare capacity that must be developed, especially in the workplace. This mere appreciation is a benefit in itself, for it leads to the extension of one's sphere of influence and linkage building, necessary in making certain goals achievable and with ease. Coordination skill is one that I have acquired recently and I am reaping various positive outcomes as a result. Developing and sustaining new friendships is one benefit that I directly experience. Coordination has made me develop my receptivity as a person, a trait, which I think is important especially that people always want to have their ideas and insights be considered and being repulsive to them will scare them just the same. It does not mea n however, that this receptivity is just simply allowing people get their way. Rather, it is more of digesting their insights, weighting them with mine, considering them if they are attuned to my values and ethics, and informing people the reasons why I do not consider them in case I

Families Essay Example | Topics and Well Written Essays - 500 words - 3

Families - Essay Example While men are often looked at as the fundamental breadwinners for the family, it becomes quite difficult for some to provide their children with the time and association they require. Children require contact and interaction. Most men complain that they lack association with their father simply because their fathers did not provide for them when they needed them. Men tend to reserve more time for their children than what they had been given by their fathers. Most fathers tend to feel protective about their children besides letting them out and explore their world on their own. This is so because they are aware of all the threats their children might encounter because they did face them themselves. Many fathers in the present age have lived a much free and leveraged childhood in terms of freedom as compared to what their children are allowed. Childhood in the past was enriched with an outdoor taste while today, outdoor games have been replaced with technical indoor activities, both related to school and otherwise. Today’s fathers are more protective towards their children. Many fathers tend to provide their children with sufficient leverage so that they can decide their future as per their interests and aptitude. Many children might even consider this a neglect on the part of the parents, but it is a fact that in most cases, parents hardly have time to help their child with his/her studies. However, parents do all to ensure that the child gets the finest education available. I think the authors have been quite rational in their realization of the four traits of fatherhood. Good fatherhood is an integration of provision, protection, endowment and emotional closeness. These are four factors many men miss about their relationship with their fathers. Emotional closeness is the most fundamental trait of fatherhood that decides the level of intimacy a father would develop with his children. Fathers need to provide their children with time,

Choice one of the topic Essay Example | Topics and Well Written Essays - 2000 words

Choice one of the topic - Essay Example Generally, quality can be managed by clearly defining quality characteristics, considering how it can be measured, setting quality standards and lastly, monitoring and improving standards (Kelemen, 2002, p.29). Some of the problems in managing quality of service include measurements because it is very difficult to measure something that is not tangible. Another problem is setting or meeting the required standards since it is not easy to meet diverse expectations of different customers. In addition, monitoring and improving quality service is also a challenge because it is extremely difficult to monitor something that cannot be observed. Some scholars equate the concept service quality to satisfaction (Hernon and Whitman 2000, p.14) claiming that service quality is concerned with satisfaction and meeting individuals’ expectations. Agreeably, service managers face different challenges as opposed to production managers and some of these challenges include answering customer call, satisfying customers by providing timely service, ensuring quality service, and time factors among others. This paper presents a discussion on quality, challenges that service managers face as opposed to product managers especially when trying to manage quality. Feigenbaum not only focuses on manufacturing but he also covers various departments which contribute to the quality of the product and services that an organizations offer to satisfy customer’s needs (Knowles, n.d, p.10). Managing quality is one big challenge to many managers in an organization. Product quality covers features, performance and defects among others whereas service quality incorporates time delivery and knowledge of delivery personnel among others. Product features is one way in which quality can be measured in that customers focus on the features of the product as the most important factor in meeting their expectations (Juran, 1989, p.19). Product quality and service quality differ in

Othello and Race Essay Example for Free

Othello and Race Essay In Shakespeare’s literary creation â€Å"Othello,† the protagonist, Othello, is a high ranking black soldier in a community of white people. Though he had gained his high marks by sheer perseverance and dedication, as it is in the old times, there are the people’s doubts. He was, after all, still a black man. Shakespeare, through this tragedy had shown the stereotypes of our modern times; that being white presents superiority or a sense of control over people of a different race, specifically those of a darker color. Shakespeare, however, did not mean to degrade or discriminate but rather, he seemed to point out the common mistakes of people that ultimately caused racism. Othello’s race and basically his skin color had played a major factor in the story’s main theme, progress and twists. The story mainly revolve on the struggle of a black man and how he found his happiness, but was cut short by other people’s jealousy and deceit. In his fight for love, the fact that he was black was used against him. It was presented to the woman’s father in a sense that generalizes black people as untrustworthy. It was also the main reason Iago used to point out that his wife Desdemona betrayed him for the love of another man, a man who happens to be of pure white ancestry.

Outline On Galileo Galilei

Outline On Galileo Galilei Thesis statement main argument The astronomer Galileo Galilei contributed to the field of astronomy majorly by observing the sky with a telescope he had built, observations which resulted in his discovery of many astronomical phenomena further proving that the Earth was not the center of the solar system. Statement of purpose (scope of the essay): Following a brief biography of Galileo Galilei, this paper will determine the state of the scientific knowledge prior to Galileos astronomical discoveries, explain what Galileos contribution to astronomy was, and discuss how his findings subsequently changed humanitys conception of the universe. Body Topic sentence of paragraph 1: Besides being known as a very influential astronomer, Galileo Galilei was also known for being an Italian scientist and philosopher. Birth date: February 15, 1564; Place of birth: Pisa, Italy. (Galileo Biography, 2013) Death date: January 8, 1642; Place of death: Arcetri, Italy. When he died, he was blind and very ill, and was under house arrest for heresy. (Galileo Biography, 2013) Galileo first started in a monastery school because he wanted to become a monk, but he eventually left the monastery and attended the University of Pisa to study medicine, like his father wished. However, he never completed his medicine degree and instead found an interest in mathematics and philosophy. (Bellis, 2013; Galileo Galilei, 2013) Galileo taught for three years at the University of Pisa, but transferred to the University of Padua when his three-year contract at Pisa ended. (Bellis, 2013) In 1609, Galileo heard rumours of a spyglass having been created by a Dutch spectacle-maker. Galilei decided to create his own spyglass, later renamed a telescope, and eventually made it more powerful than the Dutch spyglass. One night, he pointed his telescope towards the sky and his astronomical discoveries began then. (Bellis, 2013) Topic sentence of paragraph 2: Prior to Galileos astronomical contribution, the Catholic Church and the Bible were the principal sources of explanation for most of the phenomena that occurred on Earth and in space. In that time period, the geocentric model, suggested by Claudius Ptolemy at the beginning of the 2nd century A.D., argued that the Earth was in the center of the solar system and that the other planets and the Sun revolved around it. This model was widely accepted and encouraged by the Catholic Church. (Mochà ©, 2009; Redd, 2013) However, a more recent model had been brought forward by Nicholaus Copernicus, in 1543. This model was called the heliocentric model and declared that the Earth was not in the center of the solar system, but rather that this place was occupied by the Sun and that all the planets, including the Earth, rotated around the Sun. (Mochà ©, 2009; Redd, 2013) Galileo Galilei supported the Copernican theory (Galileo Biography, 2013), but this theory was considered against the teachings of the Church. As a result, Copernicus writings were banned by the Church. (Machamer, 2009; Mochà ©, 2009) Topic sentence for paragraph 3: Galileo Galilei made more than one contribution to the field of astronomy by observing the sky with his telescope, but his major discoveries were the first moons of Jupiter and the phases of Venus. His two major discoveries provided proof that the heliocentric model, introduced by Copernicus, was truly the one that was representative of the solar system. (Mochà ©, 2009; Weisstein, 2007) The moons of Jupiter (*the names of those moons will be included in the final essay*) that Galileo observed rejected the geocentric models argument against the Copernican theory. This argument stated that if the Sun was the center of the solar system, Earth would lose its moon because it circulated around the Sun; Earth could only keep its moon if it was in the center. However, with the moons of Jupiter (later named the Galilean moons in honour of Galileo) rotating around Jupiter, the scientist community could only face the fact that a planet could keep moons, even though it was not in the center of the solar system. (Mochà ©, 2009; Galileo Biography, 2013) The phases of Venus further encouraged the heliocentric model. The phases of Venus indicated that Venus must circle the Sun for its phases to be visible from Earth, just like the phases of the moon were. In the geocentric model, Venus would show no phases and would always be a crescent shape because the Sun would not be in the center of its orbit. (Mochà ©, 2009; Galileo Biography, 2013) Topic sentence of paragraph 4: Although Galileos observations and discoveries were not first accepted by the religious community, evidence of Galileos findings started to circulate and the Church was eventually forced to admit that Galileo had been right. (Galileo Biography, 2013; Bellis, 2013) Galileo had already published multiple books (*the names and dates will be included in the final essay*) prior to being charged of heresy by the Church and placed under house arrest. (Galileo Biography, 2013; Bellis, 2013) While being under house arrest, Galileo continued to write and publish books (Galileo Biography, 2013; Bellis, 2013), although he was becoming blind from having stared too much at the Sun with his telescope for another of his astronomical discoveries. (Our solar system, 2011) In 1758, the Church was forced to face the truth and lifted the ban on most of the books that supported the Copernican theory and the heliocentric model. In 1835, it abandoned its opposition against this model completely. (Galileo Biography, 2013) In the 20th century, some popes acknowledged the revolutionary work done by Galileo. In 1992, Pope John Paul II publicly apologized and showed regret on how the case of Galileo had been delt with. (Galileo Biography, 2013; Bellis, 2013) Conclusion Restatement of thesis statement: Galileo Galilei significantly contributed to astronomy primarily by observing the sky with a telescope, which resulted in his discovery of many astronomical phenomena proving that the Earth was not the center of the solar system. Summary of main points: Prior to Galileos findings, the Church believed in the geocentric model, introduced by Claudius Ptolemy. However, Galileos discovery of the moons of Jupiter and the phases of Venus instead supported the heliocentric model, suggested by Nicolaus Copernicus. Galileos contribution took a long time to be recognized, but it allowed for a better understanding of the way the solar system functions. Other discoveries based on the astronomers contribution: Galileos use of a telescope to observe the sky also allowed him to observe the Suns dark patches known as sunspots, part of the star cloud of the Milky Way, the rings of Saturn that he identified as ears, and the Moons crater-covered surface. (Mochà ©, 2009) There is also recent evidence that Galileo may have discovered Neptune nearly two centuries before it was official found by satellites and modern telescopes. (Redd, 2013) APA References Bellis, M. (2013). Galileo Galilei. About.com Inventors. Retrieved April 13, 2013, from http://inventors.about.com/od/gstartinventors/a/Galileo_Galilei.htm Famous Astronomers and Astrophysicists (2012). Retrieved April 13, 2013, from http://cnr2.kent.edu/~manley/astronomers.html Galileo Biography. (2013). Biography.com. Retrieved April 13, 2013, from http://www.biography.com/people/galileo-9305220 Galileo Galilei (1564-1642). (2013). BBC History. Retrieved April 13, 2013, from http://www.bbc.co.uk/history/historic_figures/galilei_galileo.shtml Machamer, P. (2009). Galileo Galilei. Stanford Encyclopaedia of Philosophy. Retrieved April 13, 2013, from http://plato.stanford.edu/entries/galileo/ Mochà ©, D. L. (2009). Astronomy: A self-teaching guide (7th edition). [ebrary version]. Retrieved from http://site.ebrary.com/lib/champlaincollege/docDetail.action?docID=10342867 Our solar system: Galileos observations of the Moon, Jupiter, Venus and the Sun. (2011, February 10). Solar System Exploration NASA. Retrieved April 13, 2013, from http://solarsystem.nasa.gov/scitech/display.cfm?ST_ID=2259 Redd, N. T. (2013). Galileo Galilei: Biography, inventions other facts. Space.com. Retrieved April 13, 2013, from http://www.space.com/15589-galileo-galilei.html Weisstein, E. W. (2007). Galileo Galilei (1564-1642). Scienceworld.wolfram.com. Retrieved April 13, 2013, from http://scienceworld.wolfram.com/biography/Galileo.html *** Most of these sources are preliminary sources (i.e.: websites). For the final essay, I will find books or other academic sources to replace them, particularly for Galileos biography.***

Advantages and Disadvantages of ICT in the Social World

Advantages and Disadvantages of ICT in the Social World ADVANTAGES AND DISADVANTAGES OF DIFFERENT TYPES OF CURRENT INFOMATION TECHNOLOGY IN RELATION TO COMMUNICATION. Introduction. Information Technology has rapidly changed the way how people communicate in the last two decades. Just consider the concept of IT communications on the society it is apparent that it has been changed the way how it affects business and social and personal lives. People can communicate now either by telephonic connection to talk or send messages, or over internet link to nearly anywhere in the world. Also order a huge number of items from any place on planet to do the business. This report will explain advantages and disadvantages of ICT (Information Communication Technology) in the business world and social live in society. Advantages. The new electronic independence re-creates the world in the image of a global village. Mc Luhan, M. 1964, Understanding Media: The Extensions of Man Published by McGraw-Hill. Marshall McLuhan speaks about Global village in 1960s and his quote became reality in todays living. There are advantages using Information Communication Technology: Communication Speed/time and money can be saved because it is much quicker to send/share information around. Communication is more efficient to contact either business partners or friends and family members all over the world. ICT expands availability for communications. Social network sites and Social media such as Skype allows making video-conference calls with immediate response. Messages can be send to numerous people/companies across distances. Lives have been affected by ICT in most positive ways bringing families together across the world. Cost effectiveness Numerous offers from telecommunication companies and smart phones therefore making far cheaper than in the past. For business ICT saves incredible amount of money on business flights and accommodations. Only few years ago there was no way to send free message through to the phone, but now people uses social network for free communication e.g. Viber, Skype, Facebook. Saving time and money for petrol as people can go shopping from home through online shopping. Greater availability Websites are open for communication in every minute of the year. This means that a business can be open anytime anywhere giving a customer the capability to make purchase from different sites and different countries. Bridging the cultural gap Greater access to the ICT has helped to build the bridges between different cultures giving them opportunity to exchange views and ideas. Also, educate both sides of communication bridge thereby increasing awareness and reducing prejudice. Creation of jobs The best advantage of ICT has been a creation of new and interesting jobs in IT sectors. Computer programmers, web designers etc. have great employment opportunities created through the advancement of technology. Education There is new opportunity for further education to improve qualification in so many economic sectors. A degree can be completed online from persons home. It is possible to hold a job and still do degree. Disadvantages. Lack of Security/Privacy Though IT may have changed and more convenient, it also brought along privacy and security issues. From email hacking, phone signal interception etc. people are worried that personal information may become public knowledge. IT keeps changing almost every day which means that the individuals must be up to date in IT to secure their jobs. There is also risk factors with the systems computer viruses, malware, spam, Trojans etc., attacking. Unemployment While IT may have streamlined the business process, it has created job redundancies and subcontracting. Using the computers instead of human resources employers save huge amount of money but employees are losing their jobs as not needed anymore. Social media The network pages are open to everyone including teenagers and young childrens which can affect their mental and physical health by watching and playing violent games. They became addicted to the phones, iPod, gaming consoles forgetting about outside activities and communication in the society. Cyber bulling It is so easy now bullying and threatening others in social network pages that this has become much easier for internet users all over the world. They dont realize what the consequences are to those reading/hearing unpleasant comments. In the recent past there have been so many investigation cases regarding cyber bullying with lethal consequences. Avoid hurting someones feelings by emails or other forms of electronic communication; Respect other peoples online rights; Avoid insulting someone; If someone insults you be calm; Avoid crashing discussion groups or fora; Respect the privacy of other people online; Be responsible online. Ref.http://www.garda.ie/Documents/ Reliance on technology People dont bother to read, calculate or write without computers anymore in same time losing abilities of hand writing (why write if can use spell-checker), calculate without calculator even for minor addition, reading books (why read if there so much information in internet). Conclusion The abovementioned has explained numerous advantages and disadvantages that are increasing as the technology improves. People must be cautious with how and who they give in the personal information to. The list can be endless. What happens in the future; will advantages outweigh disadvantages? Who will win in this battle, computers or human beings, and how much will be lost in this battle remains to be seen. Bibliography Tutor notes http://cyberbullying.org/ Mc Luhan, M. 1964, Understanding Media: The Extensions of Man Published by McGraw-Hill. http://www.garda.ie/ http://bookboon.com/

The Impact Of Information Technology On Work Organisations Essay

The Impact Of Information Technology On Work Organisations The impact of information technology has significant effects on the structure, management and functioning of most organisations. It demands new patterns of work organisation and effects individual jobs, the formation and structure of groups, the nature of supervision and managerial roles. In the case of new office technology it allows the potential for staff at clerical/operator level to carry out a wider range of functions and to check their own work. The result is a change in the traditional supervisory function and a demand for fewer supervisors. IT has prompted a growing movement towards more automated procedures of work. There is a movement away from large scale, centralized organization to smaller working units. Processes of communication are increasingly limited to computer systems with the rapid transmission of information and immediate access to their national or international offices. Changes wrought by IT means that individuals may work more on their own, from their personal work stations or even from their own homes, or work more with machines than with other people. One person may be capable of carrying out a wider range of activities. There are changes in the nature of supervision and the traditional heirachal structure of jobs and responsibilities. Therefore the introduction of IT undoubtedly transforms significantly the nature of work and employment conditions for staff. The ma...

My Personal Goals :: essays research papers

My Personal Goals The personal goal  Ã‚  Ã‚  Ã‚  Ã‚  s I want to achieve as a student at University of Phoenix is to receive a college degree and make my mother proud. I know once that has been achieved, my future prospects are limitless. I am a product of a parent who grew up on welfare, but not only obtained her Bachelor’s degree, but went back to school two more times to obtain her Doctorate of Education. Her mother sacrificed plenty to send her to college and my mother always knew the power of education.   Ã‚  Ã‚  Ã‚  Ã‚  As my sister and I were growing up we would always listen to my mother tell stories about how she had to walk about 10 miles to and from school, but she was never discouraged. She would also tell us about having to go to the cotton fields on some days to work and then go to school. My mother worked at a very early age and saved for college any time she had extra money to spare. With the money my mother saved for college, the college fund my grandmother set up, and a partial scholarship, my mother was able to attend Prairie View A&M University. She decided to go into nursing, because she enjoyed helping people. After 4 very hard years, my mother graduated magna cum laude from college. She told us that was the best day of her and my grandmother’s life. My grandmother told her she was so proud of her.   Ã‚  Ã‚  Ã‚  Ã‚  Eight months after my mother’s graduation, my grandmother passed away. My mother was devastated, but she knew she had to go on because it was what her mother would want. One year later, my mother got married and moved to Salt Lake City, Utah where she found a job as a pediatric nurse at the Children’s Hospital. She worked at the hospital about a year and then decided to go back to school to receive her Master’s degree. Her husband was not happy with her decision to do such a thing, and was not supportive of her at all. At that time my mother realized she had made the best choice to go back to school because it was apparent her marriage would not last.   Ã‚  Ã‚  Ã‚  Ã‚  My mother graduated sum cum laude from University of Utah and was divorced shortly thereafter. The relationship became so strained while she was in school, by the time she graduated; she and her husband had grown far apart.

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+ ,