The Economic Growth of Australia Samples for Students Myassignment

Question: Pick any Industry and Discuss the changes done or required in that Industry. Answer: Presentation For this exploration paper, I might want to pick the Australian agribusiness industry as an examination industry. The farming business contributes in the financial development of Australia. Alongside this, the Australian farming industry is one of the most refined and exceedingly specific endeavors on the planet. Additionally, in earlier years, bunches of changes have been happened in the horticulture business of Australia. These changes upgrade the efficiency as well as improve the economy and GDP of the country. The Australian government has built up various plans and techniques for the advancement of farming segment of the country. The Australian agribusiness industry is exceptionally perceived industry on the planet. It is a direct result of its fares top notch, protected and new items around the globe. The Australian government accentuation on the horticulture area due to this is the quick new division on the planet. What's more, this examination paper is useful to show the chan ges that include been occurred inside the business in recent decades. Besides, this exposition is invaluable to propose some significant changes that might be advantageous for the development just as improvement of the agribusiness business of Australia. Changes in the Australian Agriculture Industry The facts confirm that, the Australian agribusiness industry is a creating industry of the country. The governing bodies of nation give cautious thought on the turn of events and improvement of the cultivating industry. The guideline clarification for it is that, the horticulture business contributes in the money related just as monetary of the nation. It is a central bit of the economy of Australia. Furthermore, the cultivating division is the prime business in nation and common gatherings of Australia (Ferguson, 1998). Alongside this, the horticulture business of Australia contributes in the yearly GDP advancement pace of the nation. It contributes around 2.3% of creation in the GDP of the country. Likewise, the improvement of the Australian cultivating industry generally depends on upon the changes that are made by the organization of nation. For instance, CAP strategy (Common Agricultural Policy) is seen as a vital change in this industry. Under this methodology, the governing body extended the typical size of Australian farms to overhaul the proficiency just as benefit of nation. Alongside this, with the help of this game plan, agriculturists or creators end up discernibly prepared to guarantee their own farms (Marsh MacAulay, 2002). As a result, about, 95 percent properties are family-asserted in Australia. They work and manage the estates themselves. They don't spend too much money on the properties of others. Additionally, the family-had estates extended the age level of farms. In like manner, Australian has transformed into the prime exporter of cultivating items on the planet. In like manner, it tends to be estimated an imperative change in the horticulture business of Australia. In a similar way, the legislature of country caused the business to convey arranged. This is another enormous change in the cultivation fragment of Australia. Besides, due to this change, the business was benefitted at the period of deregulation. The business stays a key supporter of charge pay of Australia. The toll obtaining is a basic supporter of the financial turn of events and progression of nation (Vitalis, 2007). This thing will improve the dependence of various countries on the Australian market. Due to this dependence, the Australia's farmers will get appropriate estimations of their things. The charge organized structure of the business upgrades the lifestyles of Australia's farmers inside the nation. Moreover, the organization of nation made changes in the working environment of the business. The farmers got another economic situation to upgrade their effectiveness and to battle in the market suitably. Likewise, the diminishing of sponsorships is an important change that has occurred inside the business. The Australia government diminished the sponsorships by 95% just to reduce the dependence of people on the lawmaking body of nation. Additionally, with the help of this, the organization needs to make the Australian's planners increasingly prosperous (Edwards, 2003). This change has reduced the variations in help those were proficient to engage insufficiencies in resource utilize across over estates. The change has enormous impact on the money related and plausible headway of the nation. Aside from this, the Australian cultivation industry has pushed toward turning out to be market to Asian countries. It is an immediate aftereffect of its offers high bore and fresh things to everybody. Moreover, the use of the moved advancements is a critical change done in the business (Australia, 2014). Along these lines, it very well may be acknowledged that, there are heaps of changes have gott en done with the agriculture business of the nation. These progressions contributed into the cultivating productivity advancement of Australia. Aside from this, as per me, the council should roll out a couple of improvements for the future turn of events and improvement of the business. To the exclusion of everything else, the lawmaking body should give force loosening up to the farmers. It is an immediate aftereffect of the farmers of nations gets low expenses of their things. The extended obligation will reduce the effectiveness and lifestyle of them. Also, the organization should guide getting ready ventures to show progressively creative strategies for developing (Connell, 2016). The governing body should make suitable assistance procedures for agriculturists; along these lines, they can demand the assistance if they required. These movements would be helpful to upgrade the advancement of the business. End On the reason of the above assessment, one may state that, the farming business of Australia is quick creating industry on the planet. The business is passage arranged and it conveys incredible things to various nations. Nearby this, it is moreover watched that, the Australian government generally emphasis on the agribusiness zone to redesign the lifestyle of the agriculturists. Also, it is examined that, loads of dynamic and dynamic changes have done inside the cultivating division of Australia. These progressions contributed in the effectiveness advancement of the cultivating (farming) industry of Australia. References Australia, D. (2014). Australian Dairy Industry. Dairy Australia. Recovered From: https://www.australiandairyfarmers.com.au/PDF/Dairy-industry-reaction to-agvet-substance change proposals.pdf Connell, J. (2016). Country Change in Australia: Population, Economy, Environment. New York: Routledge. Edwards, G. (2003). The tale of deregulation in the dairy business. Australian Journal of Agricultural and Resource Economics, 47(1), 75-98. Ferguson, J. (1998). Farming: A contextual analysis in mechanical relations change. Australian Journal of Agricultural and Resource Economics, 42(3), 321-332. Bog, S. P., MacAulay, T. G. (2002). Land change and the improvement of business farming in Vietnam: approach and issues. Agribusiness Review, 10(1), 1-20. Vitalis, V. (2007). Horticultural endowment change and its suggestions for reasonable turn of events: the New Zealand experience. Natural Sciences, 4(1), 21-40.

African Americans and the Prison System Essay Example for Free

African Americans and the Prison System Essay I. The History of Oppression and African Americans? K? K? K? K? K? K? K? K.. III. The enduring impacts of bondage: consistent mistreatment? K? K? K? K? K? K? K a. The lost feeling of culture and social pride: Feeling of inadequacy b. No monetary establishment c. Unleveled playing field IV. Looking after persecution? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K. Section 2 : THE NEW AGE SLAVERY: The Prison System I. The Prison Institution? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K II. Race and the Prison System? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K.. III. The enduring mistreatment? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K.. IV. The impacts of persecution? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K a. Downfall of the Black family b. Lost political voice V. Arrangements? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K. VI. Shutting? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K? K I. Presentation In the book the Mugging of Black America, Earl Ofari Hutchinson transfers a fascinating encounter by a journalist. The columnist, who burned through over two hours watching speculates walk before Washington, D. C. Unrivaled Court Judge Morton Berg, noticed that everything except one of these subjects was Black. He expressed, ?  §There is an odd air about the quick evening? Xan climate like that of British Africa in frontier times? Xas the parade of worn out, grieved, glaring, poor blacks confess or not liable to charges of medication ownership, tranquilize dispersion, ambush, outfitted burglary, robbery, breaking in, extortion and illegal conflagration. As per Hutchinson, the correspondent saw in excess of a court scene; he saw the inheritance of bondage. This paper will endeavor develop Hutchinson?  ¦s hypothesis. It will do as such by first depicting bondage and its enduring effect then it will endeavor to show how the present criminal equity framework mirrors subjection. Section 1: Slavery I. The History of Oppression and African Americans The historical backdrop of the mistreatment as it identifies with African Americans started in 1619. It was for the current year wherein a Dutch boat carried the principal slaves from Africa to North America. Following this appearance of twenty Africans in Virginia, white European-Americans made the foundation of subjugation. Bondage spread so rapidly that by 1860 the first twenty slaves transformed into almost 4,000,000. Before all else the legitimate status of these Africans was indistinct. This missing definition made an absence of sureness which took into consideration a few captives to turn out to be free following quite a while of administration. This just kept going quickly. During the 1660s, nonetheless, the states started authorizing laws that characterized and managed slaves and the organization of servitude. One of the most significant of these was the arrangement that dark slaves, and the offspring of slave ladies, would serve forever. These ?  §breeding laws were only the start. Before long, subjugation in the United States was administered by an assortment of laws created from the 1660s to the 1860s. Despite the fact that each slave state had its own slave code and case law, it became all inclusive that subjugation was a changeless condition. Notwithstanding subjugation being a lasting condition, slaves were additionally, under these laws, thought about property. Slaves, being property, couldn't claim property or be involved with an agreement. Since marriage is a type of an agreement, slave relationships had no legitimate standing. Most codes additionally had segments controlling free blacks. Under these codes blacks who were not slaves were as yet liable to controls on their developments and work. These laws served as a physical confinement, yet an ideological one moreover. Notwithstanding giving slave proprietors and white individuals control over slaves and sometimes free blacks, the laws likewise allowed slaveholders and white-Europeans an elusive wellspring of intensity. Socially, the organization of subjugation permitted white slave proprietors to accept they had physical control, yet physical and mental predominance over the slaves. With just a couple of exemptions, all slaves were Africans. This reality set the name of inadequacy on dark skin. The real establishment of subjugation as it identifies with ace and slave kept going up in till the Civil war. The American Civil War was battled, to a limited extent, over bondage. During the war, President Abraham Lincoln gave the Emancipation Proclamation, which ?  §freed all slaves. This apparently, brought the finish of servitude all through the United States, yet shockingly left an enduring impression. Starting here on subjugation took on another structure as previous slaves being related with the mark of inadequacy. II. The enduring impacts of bondage: persistent persecution Slavery is characterized by Webster?  ¦s word reference as ?  §The condition of being heavily influenced by someone else . Aalthough the genuine physical control and brutality probably finished after the liberation decree, The elusive hypothesis of matchless quality got from the foundation of servitude brought about many enduring impacts. These impacts all by themselves are a type of power, a structure subjugation. a. The lost feeling of culture and social pride: Feeling of inadequacy Slave drivers put forth incredible attempts to take out African culture. For example Africans were beaten in the event that they were discovered communicating in their local dialects or completing local customs . Along these lines, they couldn't adequately pass the dialects, stories and conventions on to their kids. This constrained concealment brought about the loss of verbal records and a rich inheritance of history. Its a well known fact that there is pride in culture. Removing the way of life removes the pride and the inspiration and results in sentiments of uselessness. b. no financial establishment Slave drivers not just endeavored to deny the Africans of there culture and pride, yet they effectively looted them monetarily. Slaves had to work without pay for a considerable length of time while cushioning the pockets of the slave proprietors. This shortfall of financial aspects brought about a failure to build up a monetary establishment in the United States. c. Unleveled playing field Along with the hardship of money related assets, another huge factor concerning the province of African Americans is captured improvement. Slaves were denied of chances to learn and turn out to be increasingly serious in numerous territories of society. Dark individuals were not permitted to peruse or figure out how to peruse, so they couldn't exploit composed content. All these enduring impacts set blacks in a seriously distraught state when bondage was nullified, driven a financial structure where white individuals by and large held the most noteworthy positions and Black individuals for the most part held the least positions. III. Keeping up persecution In request to keep up this financial structure, there consistently is by all accounts another type of abuse set up to keep up ?  §slavery. As though the above impeding impacts of bondage were insufficient, the White southerners were on edge to keep up more straightforward force and command over individuals with dark skin, in spite of there order as ?  §free. The White southerners chose to, once more, utilize the law all together take shape there hypothesis of mediocrity and keep dark individuals at the most minimal positions. In 1865, southerners made Black Codes, which filled in as an approach to control and restrain the opportunity of ex-slaves. These noteworthy Codes controlled practically all parts of life, and disallowed African Americans from practically all the opportunities that had been won during the Civil War. The codes, which were explicitly supremacist and abusive, were in the long run suspended in June 1866, during the ? §reconstruction time. During this timeframe in America and notwithstanding opposition, African-Americans were gradually turning out to be a piece of this countries consideration. By 1868, the fourteenth Amendment to the Constitution affirmed the hotly anticipated citizenship for Blacks in America. By 1870, the fifteenth Amendment was added to the Constitution which made it illicit to deny the option to cast a ballot dependent on race. The Reconstruction period, albeit fleeting, demonstrated the main genuine endeavors of comprehensive opportunity for African-Americans since the abrogation of bondage. Additions were occurring: Citizenship, Voting, Education, and Politics. Be that as it may, the basic want to have control over those in dark skin never died down. Much the same as the dark Codes, this craving to command again showed itself in another structure, Jim Crow Laws. These laws advanced separation and the refusal of equivalent assurance by law. Much the same as the codes, they also were in the end abrogated. Much the same as the Codes, Jim Crow laws, the craving of our general public to smother those in dark skin will before long take another structure. Today that structure is the Criminal Justice System. Section 2 The New Age Slavery: The Prison System I. The Prison Institution Prisons are large in the United States. During the previous 20 years, the United States encountered a monstrous increment in detainment. The jail populace expanded fourfold, from 330,000 out of 1980 to about 1. 4 million of every 1999, and the imprisonment rate expanded from around 140 to around 476 for every 100,000 occupant populaces. Today there are in excess of 2,000,000 Americans in a correctional facility. However, significantly more alarming is the way that more than one-portion of these imprisoned Americans have dark skin. Albeit dark Americans just make up about 12% of the US populace, they represent all the more then 30% everything being equal, 44% all things considered and 40% of detainees waiting for capital punishment. II. Race and the Prison System These undeniable variations in the criminal equity framework can be credited to a wide range of things extending from racial profiling to the absence of chance and poor training, yet most criminal equity spectators accept that these incongruities have risen up out of the hidden presumptions established in bondage. The supposition that slaves were second rate has continued to today. At present this hypothesis of inadequacy and want to keep up mistreatment impacts one of the

DSM-5 Criteria for Diagnosing Panic Disorder

DSM-5 Criteria for Diagnosing Panic Disorder Panic Disorder Diagnosis Print DSM-5 Criteria for Diagnosing Panic Disorder By Sheryl Ankrom linkedin Sheryl Ankrom is a clinical professional counselor and nationally certified clinical mental health counselor specializing in anxiety disorders. Learn about our editorial policy Sheryl Ankrom Medically reviewed by Medically reviewed by Steven Gans, MD on November 07, 2019 Steven Gans, MD is board-certified in psychiatry and is an active supervisor, teacher, and mentor at Massachusetts General Hospital. Learn about our Medical Review Board Steven Gans, MD on November 07, 2019 Sturti / istock More in Panic Disorder Diagnosis Symptoms Treatment Coping Related Conditions In This Article Table of Contents Expand What Is DSM-5? DSM-5/Panic Disorder Defining Panic Attacks Agoraphobia Professional Diagnosis View All Back To Top Panic disorder is classified as an anxiety disorder in DSM-5. According to the guidelines, in order to be diagnosed with a panic disorder, you must experience unexpected panic attacks on a regular basis. What else does DSM-5 say about a  panic disorder? How does the way its diagnosed in DSM-5 compare to the previous edition of the manual? Among the updates are clarification on the types of panic attacks and how agoraphobia is associated with panic disorder. What Is DSM-5? The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) by the American Psychiatric Association (APA) is the system used in the United States to diagnose mental health disorders. The DSM contains diagnostic criteria used by mental health professionals to classify and describe every mental illness. The 2013 release of DSM-5 is the first significant update since 1994. In this edition, many changes were made and this includes some updates to the diagnosis of panic disorder. This system is not without controversy. Many disorders have overlapping symptoms. Some professionals question the validity of this type of  classification system, while others feel there is a great deal of subjectivity in its application.?? Despite these reservations, a diagnosis is often necessary for treatment, research, and insurance reimbursement. Many professionals feel that this system is far better than no system at all. How DSM-5 Diagnoses a Panic Disorder The  diagnostic criteria for panic disorder  are  defined in the DSM-5. It is an anxiety disorder  based primarily on the occurrence of panic attacks, which are recurrent and often unexpected.?? In addition, at least one attack is followed by one month or more of the person fearing that they will have more attacks. This causes them to change their behavior, which  often includes avoiding situations that might induce an attack.?? Its important to note that a panic disorder diagnosis must rule out other potential causes for the panic attack or what feels like one.?? The attacks are not due to the direct physiological effects of a substance (such as  drug use or a medication) or a general medical condition.The attacks are not better accounted for by another  mental disorder. These may include a  social phobia  or another  specific phobia, obsessive-compulsive disorder,  post-traumatic stress disorder,  or  separation anxiety disorder How to Tell If Panicky Symptoms Are a Sign of Disorder Defining Panic Attacks Since panic attacks  are key to a panic disorder diagnosis, they are well defined and rather specific. This is where the updates in DSM-5 are significant. The previous version classified panic attacks into three categories:  situationally bound/cued, situationally predisposed, or unexpected/uncued. DSM-5 simplifies it into two very clear categories: expected and unexpected panic attacks.?? Expected panic attacks are those associated with a specific fear like that of flying. Unexpected panic attacks have no apparent trigger or cue and may appear to occur out of the blue. According to DSM-5, a panic attack is characterized by four or more of the following symptoms:?? Palpitations, pounding heart, or accelerated heart rateSweatingTrembling or shakingSensations of shortness of breath or smotheringA feeling of chokingChest pain or discomfortNausea or abdominal distressFeeling dizzy, unsteady, lightheaded, or faintFeelings of unreality (derealization) or being detached from oneself (depersonalization)Fear of losing control or going crazyFear of dyingNumbness or tingling sensations (paresthesias)Chills or hot flushes The presence of fewer than four of the above symptoms may be considered a limited-symptom panic attack. Agoraphobia Now Stands Alone From Panic Disorder In previous versions of DSM, agoraphobia  was associated with panic disorder. With the updates of DSM-5, it is now a separate and codable diagnosis. This is one of the biggest differences in the updates. Within the update to agoraphobia, DSM-5 notes that a person must experience intense fear or anxiety in a minimum of two situations. These include being out in public, open spaces, and in crowds, essentially anywhere in which youre outside of the home. It also notes that avoidance behaviors must be exhibited. These are a result of the fear of being in situations that may induce panic attacks or anxiety in which help may not be available or that its difficult to flee. Only a Professional Can Diagnose Panic Disorder It is important to know that the symptoms of panic disorder may mimic many other anxiety disorders and/or medical conditions. Only your doctor or mental health professional can diagnose panic disorder. Panic Disorder Discussion Guide Get our printable guide to help you ask the right questions at your next doctors appointment. Download PDF When seeking professional help in order to evaluate your symptoms and potentially reach a diagnosis, remember that honesty is key. You may even see one therapist and decide youd like to see a different one instead. Remember to do what you are comfortable with. Although it may feel difficult at first to discuss your feelings, remember that your doctor is there to help you and that speaking openly about your condition is the first step toward managing your symptoms in a healthy way. What Is the Biological Cause of Panic Disorder?

The Temperance Movement Essay - 2243 Words

In the early parts of the 20th Century, Canada experimented with banning alcohol consumption. There were some exceptions to this, but most of Canada’s Provincial governments issued some sort of prohibitory laws. The exception being Quà ©bec who only prohibited hard liquor, meaning that they allowed the production and consumption of beverages, such as, beer. This drive towards prohibition started during the mid-19th Century. It all started during the Temperance Movement, when proponents voluntarily abstained from alcohol. This abstention was due to alcohol’s, perceived, moral downfalls. However, slowly, the various provinces reversed their restrictions on alcohol and moved from prohibition to system of coordination. There were several†¦show more content†¦They thought that ridding the world of alcohol was necessary for their saviors return. They used the Temperance Movement as a means to achieve their ultimate goal—social salvation. The Temperance Move ment was not just about abstaining, or riding the world from alcohol, but it was all about religion. In fact, the movement had a positive correlation with religion; which, meaning that when religious fever increases so does the popularity of the Temperance Movement . In essence, the temperance was a religious, moral, crusade to prepare society for the second coming of the one true savior—Jesus Christ. Therefore, this shows that the Temperance Movement was a religious movement as well. The movement went further than just trying to cleanse society from the grips of alcohol. It centered on the health and well-being of the family unit. The movement consisted of various different organizations. Two examples of numerous temperance groups are The Bands of Hope of the Sons of Temperance (BHST) and the Women’s Christian Temperance Union (WCTU). The BHST for instance â€Å"encouraged children to abstain from liquor, tobacco, and bad language.† This shows that the Temperance Movement wanted to go beyond alcohol reform. It could be argued that all fore mentioned things are immoral, thus they must be abstained from. The reformers also tried to change, or create, a new definition of what it is to be a â€Å"real man.† The proponents of temperance said that â€Å"real men† doShow MoreRelatedThe Events Of Temperance Movement1332 Words   |  6 Pages Part 1: The Event; Temperance Movement â€Å"Second Great Awakening was not focused simply on promotion individual conversions; it was also intended to reform human society, which was said by Lyman Beecher a champion of evangelic Christian revivalism† (Tindall and Shi 508). The United States, which was known for a nation of separation and church and state was swept with religious revivals during 1790 to 1830s known as the Second Great Awakening. From the Second Great Awakening in 1842, the UnitedRead MoreAmerican Temperance Movement Essay1770 Words   |  8 Pagesto control alcohol consumption, or advocate temperance, has been a goal of humanity throughout countless periods of history. Many countries have had organized temperance movements, including Australia, Canada, Britain, Denmark, Poland, and of course, the United States. The American temperance movement was the most widespread reform movement of the 19th century, culminating in laws that completely banned the sale of all alcoholic beverages. The movemen t progressed from its humble local roots to nationwideRead MoreAmerican Temperance Movement Essay1815 Words   |  8 Pagesto control alcohol consumption, or advocate temperance, has been a goal of humanity throughout countless periods of history. Many countries have had organized temperance movements, including Australia, Canada, Britain, Denmark, Poland, and of course, the United States. The American temperance movement was the most widespread reform movement of the 19th century, culminating in laws that completely banned the sale of all alcoholic beverages. The movement progressed from its humble local roots to nationwideRead MoreThe Temperance Movement Of Antebellum America708 Words   |  3 PagesAntebellum Temperance The Temperance Movement in Antebellum America was one of the largest moral reforms of in 1800s. Several members of the community fought for the prohibition of alcohol, rather than just limiting the about being consumed. However, â€Å"many farmers argued that the society and its desire to eradicate King Alcohol—as temperance advocates often termed alcoholic beverages—were a scheme to deprive the people of their liberty. Starting with main in the 1851, twelve states and territoriesRead MoreWomen s Christian Temperance Movement Essay1385 Words   |  6 PagesThe recently formed Women s Christian Temperance Movement (WCTU) took up the campaign for the vote in 1885. The movement was strongly linked to church and had the motto ‘For God, Home and Humanity.’ The WCTU had previously been involved in a temperance movement and this was one of the main reasons they decided to campaign for the vote. According to Wood â€Å"social climate had the greatest effect on mobilising women into a combined effort to rid themselves of laws that discriminated against them. DrunkennessRead MoreThe Temperance Movement Of The Early 1900 S1934 Words   |  8 Pagesand feel no pain. But this destroyed families because they were almost constantly drunk. They would sometimes lose their only job because of the drinking. So, a lot of religious groups and many women started the temperance movement. There have been many people who have supported temperance in the past and it dates all the way back to when the Bible was written. Eventually the 18th Amendment was passed on January 26th 1919. This amendment was known as the â€Å"Noble Experiment†. This turned out to be aRead MoreThe Temperance Movement Essay1374 Words   |  6 PagesTemperance Movement What was the purpose of the Temperance Movement and Prohibition on alcohol? The Temperance Movement was an anti-alcohol movement. The Temperance Movement took place back in the early 20th century. The Christian abolitionists who fought slavery also prayed to the same God to end the scourge of alcohol. The purpose of the Temperance Movement was to try to abolish alcohol in the early 1900’s. â€Å"’We Sang Rock of Ages‘: Frances Willard Battles Alcohol in the late 19th Century† (Willard)Read MoreNASCAR and the Temperance Movement Essay603 Words   |  3 Pagesis the largest sanctioning body of stock car racing in the United States. NASCAR headquarters are located in Daytona Beach Florida. The temperance movement is what started the prohibition. The temperance movement of the 19th century was a movement that tried to moderate the consumption of alcohol and they pressed for complete absence of alcohol. The movement was mostly followed by women. Well in 1920 the prohibition was passed. Which made it illegal to sell, produce, import, or transport alcoholRead MoreProhibition During The Era Of 1920-1933 Essay814 Words   |  4 Pages During the era of 1920-1933 could best be characterized as the path to a sober nation. This time of history consisted of the eighteenth amendment which was passed on October 28th 1919; Volstead Act. The Volstead act was created to carry out the movement of prohibition. In addition, Prohibition is the legal prohibiting of the manufacture and sale of alcoholic drinks for common consumption according to dictionary.com. Furthermore, this amendment concurs that alcohol beverages could not be made, transportedRead MoreProhibition and the Effect on America756 Words   |  3 Pagesteenagers often turn to the underage consumption of alcohol to make them seem â€Å"cool,† or as a form of rebellion against parental and governmental authorities. The temperance movement acted as a predecessor to the prohibition of alcohol in the 1920’s. The temperance movement relied heavily on the efforts put forth by the Women’s Christian Temperance Union (WCTU) and the Anti-Saloon League. The WCTU lead thousands of women united against alcohol to make great pushes against alcoholism; they introduced an

Cliques And Its Effect On Society - 1341 Words

It has always been curious to me as to why cliques begin forming in adolescence and then become so important to us for the rest of our lives. Cliques make many parents, including myself, feel uncomfortable when thinking about our own children. This is because this word has a connotation of creating a separation between the â€Å"cool kids† and the â€Å"losers.† As a parent I would like to think that children are always kind and understanding with each other, but whether I like it or not, cliques begin forming early on and unfortunately for some, lead to feelings of being ostracized. This leads me to question what drives the formation of cliques and how early on in our lives do these driving forces begin the process. Friendships form based on the tendency to associate with individuals who we share many similarities with. Cliques begin forming as children when we choose friends who are similar to us. Friends then influence each other and result in them becoming more alike as the friendships develop over time. Studies have also shown that cliques are beginning to form earlier with the emergence of daycares and children being socialized at younger ages (NICHD, 2006). In my personal experience working in a daycare, it was evident that kids began grouping themselves based on age and genders but mutual interests would begin to form which solidified the friendships. It would make sense then that this behavior would then transfer over into elementary school and progress to become moreShow MoreRelatedThe, Freaks, And The Non Elites Essay1416 Words   |  6 Pages Geeks, Freaks, and the Rest of the Non-Elites Who are the geeks, freaks, and the non-elites? In every high school there is social stratification and social cliques. Social stratification is the division of society according to rank, class, or caste [See Figure 1.]. Orientation toward peers and immersion in friendships are defining features of adolescence . â€Å"Through friendships, adolescents learn about what others are doing, anticipate accepted and expected behaviors, figure out how to presentRead More The Effect of Cliques on High School Students Essay examples1563 Words   |  7 PagesThe Effect of Cliques on High School Students Most college freshman can still vividly remember their high school days. These days included ruling the school as seniors, or running from the seniors as lowly ninth graders. These days included having lunch with friends, and gossiping in the hallways between classes. Whatever was done, it was usually done with a friend or a group of friends. Most of these groups can be considered cliques. Cliques are groups where there is some kind of commonRead MoreTeen Delinquency And Its Effects On Society963 Words   |  4 PagesIn modern society juvenile delinquency has been an issue engaged in by minors. More and more adolescents are committing delinquent crimes. Delinquency can be seen as the direct result of negative affective states. Gangs are a group of youths who collectively engage in delinquent behavior. Peer groups can provide a lot of social and emotional support for anti-social activities. Peer relations, in all cultures have been linked to adolescent behavior choices including substance abuse and delinquencyRead MoreEssay on Adolescent Development in Mean Girls1402 Words   |  6 Pagesschool system. During her first week of school Cady becomes friends with two people in one of her classes named Janis and Damien, who unbeknownst to her are a part of the outcast crowd. They convince her to befriend â€Å"The Plastics† who are a well-known clique of girls, beca use it would be fun to see what their lives are like on the other side that people don’t see. She goes along with this plan and quickly her life begins to change. 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Enzyme Biocatalysis Free Essays

string(56) " Redox Biotransformations Catalyzed by Dehydrogenases \." 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 aillanes@ucv. We will write a custom essay sample on Enzyme Biocatalysis or any similar topic only for you Order Now 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 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 Substrate . . . . . . . . . . . . . . . . . . . . . . . . 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 External and Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Enzy me 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 Enzyme 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 and 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 Chimioselective 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 L ipases . . . . . . . . . . . . . . . . . . . 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 . You read "Enzyme Biocatalysis" in category "Essay examples" . . 326 6. 4. 3 I mmobilization-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 represent 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 RDI. 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 Wilson, 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 an d 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. Section 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 whil e 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 Chemistry 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 spontaneously; 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 con veniently 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 catalyzed 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 c apable 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 20100 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: aillanes@ucv. cl ? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Science + Business Media B. V. 2008 1 2 Trasition State A. Illanes Catalyzed Path Uncatalyzed Path Free 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 ch emical 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 label of natural product is an issue (as in the case of food and cosmetic app lications) (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 Catalysts. Structure–Functionality Relationships Most of the characteristic s 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 and ? 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 struct ure. Secondary three-dimensional structure is the result of interactions of amino acid residues proximate in the primary structure, mainly 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 proteins. †¢ Ionic bonds between charged amino acid residues. They contribute to the stabilization of the three-dimensional structure of a protein, although to a lesser exten t, 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 covalently) 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 released 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; cofactors 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 structure 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 much 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 the ? 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 place. 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 consequence 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 behavior 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? ant. According to this: a = vt0 = ? ds dt = t0 dp dt (1. 2) t0 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 unavoidable 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 proportional 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 determined 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 = vt0 = ? ds dt = t0 dp dt = t0 dz dt (1. 3) t0 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 = vt0 = ? dcoe dt = t0 dcoe dt (1. 4) t0 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 reaction 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 reaction 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 case, replacing substrate by water or buffer discriminates such contamination. If, on the other hand, one is assaying a preparation from a stock enzyme concentrate, dilu tion 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 alternatives 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 coenzyme) 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 manometry 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 deter mined 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 HPLC 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 act ivity. 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 hard 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-Menten 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 pr ocess 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 broken. 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 oligosaccharides, mal tose 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 assay bas ed 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, hydrogen 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+ , How to cite Enzyme Biocatalysis, Essay examples