Taking Chances free essay sample

My mother recently read me a commencement speech she had made at a local high school. She read it to me in hopes that shed satisfy my endless quest for answers answers to everything. Ever since I was very young, I have looked to my parents and grandparents, friends and teachers for guidance. Afraid to make a mistake, Ive asked countless times, What should I do? Maybe I was afraid to take my first baby step on my own, I dont remember. But somewhere along the line I decided I only wanted to do things the right way, even if it meant not doing something like trying out for a part in a play. I made doing it the right way my credo. What did I miss out on? Maybe I could have landed the part of Dorothy in a local community theatre. Maybe I could have sung a solo with my church choir. We will write a custom essay sample on Taking Chances or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Ill never know because I only wanted to do things the right way. Sometime during my high school years, I changed. Maybe it was when I decided to try for the lead in Beauty and the Beast. Never mind that I would have to memorize lines and choreographed movements, I would take the chance that I might fail. The day of tryouts I was still telling myself, If you dont think you can do it the right way, just dont go. My heart was pounding. My hands were shaking so badly when I got up to sing, that I wasnt certain the people holding the audition would hear me above the rattle of the paper. But something happened. When I opened my mouth to sing the sound was loud and powerful and sweet. I reached for the high notes that sometimes were beyond my reach and nailed them. With no crack in my voice. I was trying to do my best even though I might fail. But I didnt. That audition probably wasnt the end of the trap that kept me from trying but it was definitely an important part of the process. I guess I realize that if I want to live life to the fullest, I have to take chances. Now, I do take these chances, small ones, with baby steps, every day; voicing my opinions in class discussions or debates, reaching for a high note when I sing in chamber choir, performing in other school plays, riding my horse over a three-foot jump. Three years ago, I fractured two vertebrae when I fell from my horse going around a hunt course. That was a case of genuine risk that could be considered real failure. I couldnt carry a book bag or take gym for four months. It was both frightening and life limiting. I couldnt ride didnt want to ride for nearly a year. The injury meant I couldnt try out for basketball, which I loved. And, the day I tried out for track was the first day my doctor allowed me to do any physical activity at all. The coach didnt take me. I was, after all, out of shape. But I still liked to run, so I started running with my mom. No stop-watches. No cheering crowds. I hadnt made the track team, but I hadnt failed either. I have finally learned to push myself. Im taking a tough science course right now. Some of my friends are talking about auditing the class so a low grade wont affect their chances for college. Me, Im taking the chance that if I try hard enough, Ill get a good enough grade that it wont detract from my strong grades in other subjects. I have come to realize that even though my parents and grandparents, friends and teachers dont always have the answer to What should I do? they will be there to support me when I try. Life is learning to deal with Plan B, my mother stated in her speech. But I now know that the Plan As I make for my life will become easier and easier with every chance I take. Taking Chances free essay sample How do you feel about sharing secrets with someone youve technically never met? Or saying how your life Is to someone that you havent even seen face-to-face? What about Going to the extent of falling in love with that anonymous stranger? For some people, these things seem out of reach but actually, making a friend from the internet is quite thrilling. I know that most people would immediately say, Thats so dangerous, Id better not ever do that. , but actually, its not as scary as it may seem. How can I say this confidently?Just a year ago, I was as scared as a normal person But right now, after taking chances, I have made a few internet friends myself, and I dont regret one bit of it, in fact, its one of the best risks Vive taken. I wasnt planning to brag, I just dont want people to think that Im doing this without a bit of experience. We will write a custom essay sample on Taking Chances or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Its not even all that dangerous If you Just use your head, dont give out your address, dont share personal details Immediately, or dont give out your last name If you want if you dont feel comfortable yet, then just dont do it, its simple as that, use our head.If they pressure you, then stop talking to them, your mind knows where that leads to. Just think. Or better, if you cant stop yourself from being impulsive, then forget about it, stick to personal friends. The idea of earning a friend through the internet right now may seem distant but having one has its fair share of perks. For one, it feels great to actually say that you have a friend from another country, Just imagine having a friend from Japan or Korea for an instance. You learn the different lifestyle and culture of their country.Or even pick up a few words and phrases from their language. There might even be some cases where you have homework about their country or so, you can ask them a few questions for a bit of help. Second, If you meet someone older, around 18 and above, you can ask them for advice. Whether its because you dont know what to do about that certain person or because youre confused which is wrong or right. They can help you out a lot. Or even if youre feeling down, they can pick you right back up with a look on the brighter side.Third, if you go through forums, you can easily find people with your same interests and talking to them will never be awkward since you will always have a topic. Befriending people will be easier because youll never get awkward atmosphere with your common interests and through that, you will know each other better since one topic leads to another hundred topics, before you know it, its like both of you have been friends for a long time. The other reasons vary from what kind of friendship you have with that person. .. Web Its because they never let you be lonely, they literally make you laugh-out- loud through the internet, or because theres something about them that really you can never really see whats behind that other monitor But it can always be either good or bad. A kidnapper? A mental patient? What if theyre Just a teenager like you, Just looking for a friend? No one can ever know for sure. So the key in forging something as spontaneous as this is learning when to take the risk. When to make decisions and if that decision is for the best. Take the chance.

Memorable Lucille Ball Quotes

Memorable Lucille Ball Quotes Lucille Ball began her career in musical comedy, became a success in radio comedy, starred in several movie comedies, and achieved her greatest popular success with her TV show, I Love Lucy, first airing in 1951 and running until 1957. She also starred in The Lucy Show (1962-68) and Heres Lucy (1968-74). Lucille Ball and Desi Arnaz, who produced I Love Lucy together as well as starred in the show, were married from 1940 to 1960. Lucille Ball managed Desilu Productions from 1962 to 1967 and Lucille Ball Productions from 1967 to 1989. Selected Lucille Ball Quotations I never thought I was funny. I dont think funny. Im not funny. What I am is brave. Ability is of little account without opportunity. The secret of staying young is to live honestly, eat slowly, and lie about your age. If you want something done, ask a busy person to do it. The more things you do, the more you can do. Luck? I dont know anything about luck. Ive never banked on it, and Im afraid of people who do. Luck to me is something else: Hard work - Â  and realizing what is opportunity and what isnt. One of the things I learned the hard way was that it doesnt pay to get discouraged. Keeping busy and making optimism a way of life can restore your faith in yourself. I think knowing what you cannot do is more important than knowing what you can do. In fact, thats good taste. I would rather regret the things that I have done than the things that I have not. In life, all good things come hard, but wisdom is the hardest to come by. I have an everyday religion that works for me. Love yourself first, and everything else falls into line. You really have to love yourself to get anything done in this world. Once in his life, every man is entitled to fall madly in love with a gorgeous redhead. My God, Im outliving my henna. Womens lib?...It doesnt interest me one bit. Ive been so liberated it hurts. Politics should be the part-time profession of every citizen who would protect the rights and privileges of free people and who would preserve what is good and fruitful in our national heritage. Its a helluva start, being able to recognize what makes you happy. Not everything that is faced can be changed, but nothing can be changed until it is faced. I regret the passing of the studio system. I was very appreciative of it because I had no talent. What could I do? I couldnt dance. I couldnt sing. I could talk. Heaven, no. I was shy for several years in my early days in Hollywood until I figured out that no one really gave a damn if I was shy or not, and I got over my shyness. You see much more of your children once they leave home. Use a make-up table with everything close at hand and don’t rush; otherwise you’ll look like a patchwork quilt. A man who correctly guesses a womans age may be smart, but hes not very bright. What we did on [i[I Love Lucy was not slapstick. I worked with the Three Stooges years ago, and they were masters of slapstick, so I know what slapstick is. The best thing I learned from working with the Stooges was when to duck! Its true. Your timing has to be right so that you dont get hurt in the scene. The Stooges were always teaching people on the set how to duck. You spell Bob Hope C-L-A-S-S. I dont do T A very well because I havent got much of either. Quote collection assembled by Jone Johnson Lewis.

Compare and Contrast two Counterinsurgency campaigns Research Paper

Compare and Contrast two Counterinsurgency campaigns - Research Paper Example They used various tactics to win the support of the people; both policies of attraction and chastisement. After taking over, the Americans, set their bases in Philippines and colonized it. The Americans used the policies of attraction and chastisement to woo the support of the locals. This paper is going to highlight the application of policies of attraction and chastisement in both Cuba and Attraction. The policy of attraction was an appealing message, while the policy of chastisement was using of force. It will show how Americans were trying to bring change in a short time. Spanish were failing the economies of both Cuba and Philippines and this paper will highlight the reason Americans removed Spanish authorities in these two countries. Spain was in the war with Cuba. They had invaded the territory of the South American nation. Americans used their press called the yellow papers to spread the news of the war all over the world (Company, 1999). American policy makers felt the investments, which Americans had invested in Cuba would face destruction. Many people lost their lives. The European countries put in place the Monroe Doctrine that prohibited any form of war, whether internally or externally. European countries felt they had to react and stop the war too. There was pressure from all quarters towards Spain, so that they stopped the war. The American government sent a warship to Cuba as a warning to Spain as they wanted a lasting peace. The ship sent to deliver the warning was bombed (Archives, 2014). American press sent out rumors that the Spanish troops destroyed the ship. In reality, Cuban rebels were responsible for the ambush. This triggered American congress to declare war against the Spanish forces in Cub a (Blumenfeld, 2003). US wanted the Spanish to withdraw its troops immediately. America declared war. It was the first time for almost eighty years since US was in the war against a European country.

Comparison of 3-Dimensional Radiotherapy with IMRT for nasopharyngeal Dissertation

Comparison of 3-Dimensional Radiotherapy with IMRT for nasopharyngeal cancer - Dissertation Example The treatment modality therefore, revolves around successful and complete removal of the tumour mass without damage to the vital organs around the tumour (Tham and Lu, 2010). Most of the critical analysis presented in this research is based on findings that have been carried out on patients of Eastern Asian origin. This is because nasopharyngeal carcinomas are prevalent in this population group. On the same note, some of the most significant research results and tabulations have been obtained from studies carried out in these geographical areas. One of the most significant facts about NPC is that these are diagnosed in later stages of cancer (Afqir, Ismaili and Errihani, 2009, pp 3). The close proximity of different regions leads to many problems after treatment is carried out. Adverse effects to the pituatory, thyroid and hypothalamic glands are commonly seen post treatment (Stevens et al, 1998). Soft tissue fibrosis leading to trismus and necrosis of the eye structures is also comm only seen (Stevens et al, 1998). Surgical treatment options are somewhat reserved due to the complex location of the tumour. Studies researching the efficacy of surgical treatment have shown higher tissue destruction, and increased chances in recurrence and metastasis, which are reflected in high mortality rates, with five year survival rates falling in the range of 33-57% only (Leu and Lee, 2009, pp 103). A similar study assessed the effects of salvage surgery on patients with recurrent nasopharyngeal carcinoma after radiation therapy. The study selected 38 patients with recurrence in NPC who underwent surgery (Chang et al, 2004, pp 499). The results showed a 3 year survival rate and local control rate of 69 and 72.8% respectively. Local control rates at the intracranial and skull base levels were 83.3%, and the overall morbidity rates were below 14% (Chang et al, 2004, pp 500). Morbidity rates with radiotherapy treatment alone fall between 40 To 50%, whereas combined radio and che motherapy provide survival rates of 55 to 80% respectively (Paulino and Louis, 2010). However, this study has very limited number of participants, making it difficult to state that the results are significant. However, when viewed in comparison to many other studies on the efficacy of combined radiotherapy, the results bear some significance. Another important consideration is that surgery can still become an option in cancers which are of larger size, in order to help reduce the severity and duration of the chemoradiation therapy. Very large cancers can be removed with the help of fine surgical instruments, after which pockets of tumour can be removed through radiation and chemotherapy. Surgical treatments however, have been largely replaced by the newer and more sophisticated technology of irradiation. However, chemotherapy is another major contender for treatments of NPC. Many drugs have been introduced, but so far, trials and researches have gathered evidence for cisplatin and 5 -florucil respectively. Trials of use of cisplatin combined with radiotherapy for NPC have shown an overall survival rate of 76% at two to three years, which is comparatively better than therapy from any one of the methods alone. Another trial on 130 patients showed a 46% rate of survival with combined chemotherapy compared to 25% of it when treatment was done with radiation alone at five years (Siewert, Salama and Vokes, 2006, pp 168 and 169). Other drugs that are being used or considered for combined

Save the Tiger Essay Example for Free

Save the Tiger Essay This article needs additional citations for verification. Please help improve this article by adding citations to reliable sources. Unsourced material may be challenged and removed. (October 2009) Save the Tiger is a 1973 film about moral conflict in contemporary America. It stars Jack Lemmon, Jack Gilford, Laurie Heineman, Thayer David, Lara Parker and Liv Lindeland. The film was directed by John G. Avildsen. The screenplay was adapted by Steve Shagan from his novel of the same title (the first book by the author of The Formula and other thrillers, and generally regarded to be his most successful novel by literary standards). Lemmon won an Academy Award for his role as Harry Stoner, an executive at a Los Angeles apparel company on the edge of ruin. Throughout the film, Stoner struggles with the complexity of modern life versus the simplicity of his youth. He longs for the days when pitchers wound up, jazz filled the air, and the flag was more than a pattern to put on a pants pocket. He wrestles with the guilt of surviving the war and yet losing touch with the ideals for which his friends died. To Harry Stoner, the world has given up on integrity, and threatens to destroy anyone who clings to it. He is caught between watching everything he has worked for evaporate, or becoming another grain of sand in the erosion of the values he once held so dear. Plot A bleak story that depicts an outwardly successful man questioning the value of the material prosperity he is desperately trying to maintain, it follows the uncertain path of Harry Stoner, the real tiger (Jack Lemmon), an executive at an apparel company close to ruin. With no legal way to keep the company from going under, Stoner considers torching his warehouse for the insurance settlement. Meanwhile, he drinks, laments the state of the world, and tries his best to keep the business rolling as usual. This last task is complicated when a client has a heart attack in the arms of a prostitute provided by Stoner. With nerves still shaky, Stoner takes the stage at the premiere of his companys new line, only to be overcome by war memories. He ends the day spontaneously deciding to go home with a young, free-spirited hitchhiker, whose ignorance of his generation underscores his isolation from the world around him. At the end of the film, Stoner agrees to the warehouse getting torched and then walks by a Little League game and attempts to act as pitcher to the children. One child shouts out, You cant play with us, Mister! , leaving Stoner yet again isolated from another part of society. Production and reception The movie was written by Steve Shagan and directed by John G. Avildsen. Lemmon was determined to make the movie, despite its limited commercial prospects, and so he waived his usual salary and worked for scale. The movie was filmed in sequence after three weeks of rehearsal in Los Angeles. There is also a novel version of Save the Tiger, by Shagan: the title comes from a campaign to save tigers from extinction to which Stoner contributes. The movie failed financially at the box office, but critics and viewers who saw it liked the Oscar-winning performance of Jack Lemmon as Stoner. Why should we save tigers? At the turn of the 20th century, according to estimates, India probably had many thousand tigers in the wild. In 2002, based on a census using the pug mark technique, this number was 3,642. As per the monitoring exercise by Wildlife Institute of India in association with National Tiger Conservation Authority (NTCA), Government of India, using camera traps, in 2008 we were left with only 1,411 tigers. This number is so small that they will be gone soon if we don’t wake up to the crisis. The tiger is not just a charismatic species or just another wild animal living in some far away forest. The tiger is a unique animal which plays a pivotal role in the health and diversity of an ecosystem. It is a top predator which is at the apex of the food chain and keeps the population of wild ungulates in check, thereby maintaining the balance between prey herbivores and the vegetation upon which they feed. Therefore, the presence of tigers in the forest is an indicator of the well being of the ecosystem. The extinction of this top predator is an indication that its ecosystem is not sufficiently protected, and neither would it exist for long thereafter. If the tigers go extinct, the entire system would collapse. For e. . when the Dodos went extinct in Mauritius, one species of Acacia tree stopped regenerating completely. So when a species goes extinct, it leaves behind a scar, which affects the entire ecosystem. Another reason why we need to save the tiger is that our forests are water catchment areas. Therefore, it’s not just about saving a beautiful animal. It is about making sure that we live a little longer as the forests are known to provide ecological services like cl ean air, water, pollination, temperature regulation etc.

Strengths and weaknesses of terrorism research

Strengths and weaknesses of terrorism research Terrorism can be termed an ever evolving dynamic, widely disputed complex phenomenon that finds its roots in psycho-social and political realms. According to Lacquer (1999)  [1]  and Gordon (2004)  [2]  every instance or act of terrorism is inherently different and possess distinctive characteristics similar to biometrics. As stated by Gordon (2010) futile attempts have been made to form terrorism typologies according to terrorists methods of operations, regions of the world, organization and ideologies  [3]  . Based on these assertions it is evident that terrorism as a research field is unclear and still in it normative stages and as such lends itself to structured, synergized future development. Subsequent to the attacks of September 11, 2001 terrorism research experienced a massive influx of scholarly, semi-academic and popular writings from scholars, law enforcement personnel (both retired and active), and journalists depicting various academic, historic, religious, cultural, ethnic and social perspectives all wanting to postulate on the so called new phenomenon of modern terrorism. This apparent thrust according to Jackson (2007)  [4]  has led to the subject matter of Terrorism being transformed into a standalone field of study with its own dedicated journals, research centers, leading scholars and experts, research funding opportunities, conferences and university programmes. Further, Professor Andrew Silke, Director of terrorism studies at the University of East London in an interview with The Guardian, a UK newspaper dated 3rd July 2007, headline The rise and rise of Terrorism Studies has claimed that if current trends continue, more than 90 percent of all terrorism studies literature will have been published post 9/11, 2001, and that a new book on terrorism is published every six hours in the English language.  [5]   Also, in a study on Terrorism and knowledge growth done by Dr. Avishag Gordon, Senior Information Expert in the Computer Science Library at the Technion-Israel Institute of Technology, in 2004, using publishing databases, it was found that prior to September 2001, terrorism publications had grown over 234 percent between the period 1988-2001  [6]  but post September 2001, there was an explosion of such a proportion that Dr Richard Jackson; senior lecturer in international politics at Manchester University believes that scholarly papers in the discipline have increased by 300% since then.  [7]   Cognizant of the above terrorism research environment and the continued pace of the publication of work on terrorism, this paper will provide a critical examination of Terrorism Research using current literature in order to elucidate the distinguishing aspects, deficiencies and limitations and conclude by providing ideas/ suggestions on the way forward. During the conduct of the analysis this paper will use secondary data to draw attention to the evolution of the unrelenting pace of voluminous outputs purporting to be Terrorism Research. The approaches to the conduct of terrorism research, the challenges associated with the field, the comparison to other fields of discipline and the perception of the driving force behind Terrorism Research will all be examined. The Definition Dilemma Before any incision into Terrorism Research can occur and in order to establish a basis for any arguments for the development of this essay, the issue of the failure to develop a universally acceptable definition must be discussed. The definition of Terrorism is crucial and the most important foundation upon which to build because it ultimately determines the way in which this and any Research on Terrorism should be conducted. To date there is no universal definitions of terrorism accepted by scholars, experts, journalists or theorists. So, rather than revisit the seemingly never ending debate on the definition of terrorism, the paper will adopt Bruce Hoffmans, one of the worlds leading analysts on terrorism definition on terrorism. Hoffman (2005a)  [8]  defines terrorism as: Ineluctably political in aims and motives; Violent-or, equally important, threatens violence; Designed to have far-reaching psychological repercussions beyond the immediate victim or target; Conducted either by an organization with an identifiable chain of command or conspiratorial cell structure (whose members wear no uniform or identifying insignia) or by individuals or a small collection of individuals directly influenced, motivated, or inspired by the ideological aims or example of some existent terrorist movement and/or its leaders; and Perpetrated by a sub-national group or non-state entity. This definition was chosen because it comes from an authoritative source; it is encompassing and gives the widest possible consideration to all actors and all forms of terrorism. The definition elucidates the need to use power to coerce individuals to conformity; it also conveys the violent nature and attendant resonating fear inducing component of the strategy achieved through death and destruction; moreover, it puts the end state of the strategy into context with the aims and motives of terrorists by specifying the strong political nature. Finally the definition combines all the above inferences into a statement whereby the principal deduction can be that its expression is quite simply without doubt terrorism. However making this definition operative in any debate is anything but easy. A major problem was that terrorism almost always has a pejorative connotation and thus falls in the same category of words such as tyranny and genocide, unlike such relatively neutral terms such as war and revolution that can be used to convey the same act. One can aspire to objective and dispassionate research, but one cannot be neutral about terrorism any more than one can be neutral about slavery and genocide. Thus, defining terrorism became an effort not only to delineate a subject area but also to maintain its illegitimacy. Even the most clinical inquiry was laden with values and therefore political issues. The very study of terrorism implied to some a political decision and or objective. Qualitative, case-study research method has dominated the terrorism topic for many years. Since the number of first hand observations in the greater part of this work is really small, researchers have been cautious to delineate terrorism to fit the cases under examination. The undersized quantity of observations, regrettably, often disallows unreliable dubious parts of the definition. In one country, for instance, hostility against the military might take place, but in the second country it might not. In an assessment of the first country, one could vary the definition beyond civilian targets to military targets. In an assessment of the second country, one could not adjust the definition to investigate the implications of unreliable degrees from minimal to maximal definitions (Lesser, 1999). XYZ In current research on terrorism in the political science writings, there is plenty of room to tailor the definition of terrorism to identify with its consequences. Specifically, there is no need to decide on one particular definition of terrorism; multiple definitions can be allowed and then the effects can be empirically sorted out. Empirical analysis might generate two measures of terrorism: one with civilians as the target and the second with both civilians and the military at peace time as the target. Moreover, empirical analysis may demonstrate whether results are alike or diverse dependent on the measure. And any results would have implications for future theoretical and empirical research (American Association for the Advancement of Science, 2004). EXAMINATION OF THE STRENGTHS AND WEAKNESSES OF TERRORISM RESEARCH Terrorism research has been noted to be somewhat self regulating, though the critiques and reviews of the fields achievements and failures extend over the past two decades. Some of the most important reviews  [9]  include key theorists, experts and analysts in the field. The highlighted strengths and weaknesses are examined below: Weaknesses Poor Concepts, Theories and Methods Terrorism research has been criticized for its less than rigorous theories and concepts primarily due to the absence of a definition of terrorism  [10]  . This conundrum may never ever be resolved however evidence suggests that the current approach by most luminaries in the field seems to be one of sidestepping the definitional issue in favor of variance of term for its use according to the circumstances. This approach reeks of interference and points to external motivations according to purposes. The weakness that this approach portrays is reflected in limitation in the research and studies of terrorism. Another criticism levied in Silke (2004)  [11]  that highlights poor research methods and procedures is the over reliance on interviews and secondary data as opposed to the outputs of primary research. Though there are benefits to be derived from these methodologies, the obvious limitations override them. If evidence that supports the use of other methodologies were present this would bolster the claim for terrorism research to be an independent discipline with its own theoretical framework. Sadly support for an eclectic approach to methodologies used is absent and thus this lack of complementarity exposes the gaps in terrorism research. Another shortcoming in terrorism research as postulated by Richard Jackson (2007)  [12]  is that the outputs tend towards ahistoricity and acontextuality. This view as espoused by Jackson suggests that much of recent terrorism research ignores historical data pre September 2001 and virtually do not take into account experiences of other countries. Most modern researchers and experts tend to view terrorism tabular rasa post September 2001 and consequently refer to terrorist activities as modern terrorism. This misnomer can easily be dispelled as terrorism existed as early as 1880. Further there remains a view that terrorism research is acontextual primarily because researchers do not look at terrorist activity within the context from which they emerged rather terrorist activity is viewed and analyzed to develop trend and pattern analysis from which extrapolation can occur. Another related flaw as espoused by Jackson (2007)  [13]  is that since the events of September 2001 terrorism research tends towards exceptionalizing the experiences of the United States and Al Qaeda. Another expert Louise Richardson (2006)  [14]  described this tendency as American Exceptionalism, the sense that America is different from (and implicitly superior to) the rest of the world. These comments suggests that terrorism researchers had delved in the field without even considering any previous relationship thereby creating a myopia linked to activities post September 2001. In Silke (2004)  [15]  Research on Terrorism, Frederick Schulze notes that Schmid and Jongman (1988) identifies that though a lot has been written about Terrorism, it is not empirically based and lacks substance. In fact Schmid and Jongman note because of the lack of rigorous research based literature; the works produced are narrative, overly descriptive, derivative, derogatory and prescriptive rather than analytical. These identified flaws adequately tell a story of the quality, validity and reliability of the current research. Consequently the focus of the current terrorism research is limited to the sexiest topics while gaps in the literature remain unexplored. Terrorism by its very nature is interdisciplinary as asserted by Joshua Sinai in Silke (2004) yet researchers have not collaborated on much integrated work worldwide. Furthermore, interdisciplinarity and synergies amongst fields are crucial to the development and growth of a research field. Collaborative efforts bring varying perspective together that develop innovative approaches to research agendas. Moreover interdisciplinarity enhances and creates alternate pathways to achieving solutions that are sometimes elusive. Ranstorp (2006)  [16]  stated that In essence interdisciplinary focus and innovation will remain absolutely vital in efforts to develop a critical knowledge base in future terrorism research. It is obvious that for terrorism research to be able to create an expansive valid knowledge base that scientific collaboration across fields must occur. Further in the quest to be recognised as an independent field terrorism research must leverage existing knowledge pools to its advantage. According to Gordon (2010)  [17]  , for the terrorism research field to be considered mature it must go through the development stages variable that includes collaboration as a foundation principle. Yet it is apparent that in the quest for recognition that individualistic behaviours have subsumed the common sense approach of using knowledge bases and methods that exist within other disciplines. One of the harshest criticisms levied against the field is that research priorities, projects topics and perspectives are motivated by a problem solving approach funded by governments. Consequently the general view held is that research produced on behalf of sovereign nations is tainted and state centric because of the obvious relationships. This espoused view cast doubts on the outcome of sponsored work and questions the ability of researchers to remain independent. Andrew Silke (2004)  [18]  has concluded that much terrorism research is driven by policy concerns and is limited to addressing government agendas. This view can at times be myopic as the effects of terrorism will always be a national issue that must be addressed by government. The consequences of any institutional financial political relationship are the risk of ascribed influence peddling. However it can be argued that such a relationship is necessary to advance any field of research. It is believed that researcher s must understand that they should operate within the accepted codes of ethics and conduct and must remain independent lest their credibility and integrity become irreparably damaged. XYZ XYZ Finally, the adhesive that should hold the terrorism research field together is the unity of focus and the concentration of effort among its luminaries. Sadly all indications are that there is a disparate approach funneled by the advancement of personal agendas. Accordingly the leadership needed to close the obvious gaps, to cross fertilize, to synergize and integrate with other existing fields remains absent while the crab in a barrel syndrome pervades. A suggestion is for the creation of an association similar to that of the medical profession with the mandatory accreditation of individuals. This approach is seen as a viable option to guide, assess and focus the work to be conducted in the terrorism research field. Strengths Inputs, developments and effectiveness Terrorism researchers for years have been exploring the root causes of the phenomenon in an attempt to negate the effects of the physical and psychological violence on the wider society. According to Sinai in Silke (2004)  [19]  researchers have through the social sciences using accepted theories and methodologies systematically identified, itemized and correlated root causes ranging from general to the specific, including those at the individual, group, societal and governmental levels. This assertion has provided support that researchers have to a comforting degree been able to understand the origins and the structural theories of terrorism thereby assuring the completion of the early developmental stages of the field of terrorism research. Though some early works have been conducted and methodologies, theories and models for understanding the phenomenon of terrorism have been proffered, additional focus on contributory apparatus and processes in which additional aspects and ci rcumstances further act as motivators for terrorist activities are yet to be explored. As well terrorism research has not yet fully embraced and leveraged existing technologies to assist with computational and mapping challenges. Terrorism research has been able to enhance the tracking of day to day terrorist activities with the advent of chronologies electronic databases such as the Memorial institute for the Prevention of Terrorism (MIPT). This advancement has greatly boosted the collection of terrorist activities globally. The examination of the compiled data is significant to the furthering of longitudinal studies, trend analyses, geographical stamping, establishing relationship among groups, mapping strategies employed, determining intensification or deceleration in activities, shaping effectiveness of countermeasures in different geographical location and can generally be useful in assisting with prediction and the impact on societies, be it physical, social, economical or psychological. The downside to heavy reliance on a tool such as this is the increased probability that underestimation and wrongful predictions due to the use of arbitrary criteria when inputting data. This can lead to problems associ ated with the garbage in, garbage out theory. Furthermore the dearth of knowledge generated by current terrorism research has been instrumental in assisting governments in crafting counter terrorism strategies and policies while providing the foundation for the development of emergency management, law enforcement, security and defence agencies doctrine. More specifically, at the tactical level researchers have provided practitioners with useful information on profiles, character traits, and patterns of behaviors that has allowed law enforcement, security and defence personnel to be able to detect, deter and disable attacks. Moreover terrorism research has assisted government with developing approaches to address, neutralize and manage the effects of the phenomenon of terrorism. Critique The way forward The opportunities that are created by the current disarray in the field of terrorism studies are immeasurable. The gaps in existing literature and the lack of focus and unity provide fertile ground for budding researchers to sow intellectual hybrids for the harvest of a plural solution to a universal problem. The time to adopt a more conciliatory approach that creates synergy with other established fields is now or risks the chance of disappearing into ignominy. The prudence of this approach is a greater understanding and the ability to better inform all stakeholders in the interdiction and the management of the effects of terrorism. The thought of being the pioneer for the further development of the broader theoretical framework must continue to be an interesting prospect. The need to interrogate the core concepts of the field in order to provide satisfactory definitions and theoretical formulations must be seen as alluring. Opportunities for the alignment of methodology and the structuring of the discipline into topic areas, the apportioning of noted gaps to scholarship must be vigorously pursued as this structured approach will create an environment that generates funding for additional terrorism research. The upgrade in software technologies to better able researchers to understand, predict and forecast activities beckons on the horizons but the instigator is urgently needed. A serious examination of the political and strategic roots of terrorism is also essential if current tendencies towards acontextuality and ahistoricism are to be effectively countered. The establishment of new terrorism research journals as part of an attempt to foster a reflective and critical approach to the field is needed for encouraging the identification and exploitation of original information sources. The need for focus and expansion beyond the state-centric orientation of contemporary research is particularly urgent to change the perception of puppetry and biases. CONCLUSION If the benchmark for the acceptance of whether terrorism research field has attained maturity is the voluminous contributions by scholars, experts, theorists and analysts then one can opine that the intended status has been achieved. However, when a comprehensive analysis is conducted to provide insight into a difficult subject area, it is apparent that the field of terrorism research is dichotomous and fragmented. Terrorism research is yet to be considered a complete field primarily because of key issues such as definition, the absence of a theoretical framework, a general lack of focus, variance with interdisciplinarity and the absence of a focused research agenda. Coupled with the stated gaps and the inability to replicate and prove research studies, terrorism research as a field continues to be an elusive endeavor. Moreover, it is apparent that terrorism research has not been allowed to evolve through its developmental stages as other fields (the field was not allowed to creep before it learned to walk). Based on the events of September 2001, the research field appeared to have been given an injection akin to Somatotropin a forbidden synthetic human growth stimulant that has forced its maturity. This premature development which is without a solid foundation and littered with potential dangers and pitfalls is attempting to force its way into becoming an established research field, without first paying its dues. Consequently the environment has had a proliferation of works purporting to be legitimate discourse. Further the statistics from Gordon (2004) attest to the fact that the field has seen the most single author contributions than any other field of research. This must be worrisome as the interpretations can only suggest the appearance of some cultist fad which will eventually wither. The concerns at this time must be what will be the trigger to turn around this annoying trend? The answers lie with the experts, scholars, analysts and researchers and the ability to come together and re-focus a field that is critical to the continued existence of the global population. The responsibilities associated with terrorism research and understanding of the importance to the decrease of the fear of terrorism to the world must be the dilutive to the greed and egotistical aura that permeate the field today.

Enzyme Biocatalysis

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