Proof reading Essay Example | Topics and Well Written Essays - 1000 words

Proof reading - Essay Example This is achieved by removing the device-specific hardware dependencies, which were previously part of almost any software development effort. Moreover, DirectX performance capabilities are similar or equal to those provided in DOS but allows hardware to be fully utilized. Multi-agent systems deal with the construction of complex systems involving multiple agents and their coordination. A multi-agent system (MAS) is a distributed computing system with autonomous interacting intelligent agents that coordinate their actions so as to achieve its goal(s) jointly or competitively. Agent technologies are now being applied to the development of large-scale commercial and industrial software systems. Such systems are complex, involving hundreds, perhaps thousands of agents and there is a pressing need for system modeling techniques that permit their complexity to be effectively managed, along with principled methodologies to guide the process of system design. Without adequate techniques to support the design process, such systems will not be sufficiently reliable, maintainable or extensible, will be difficult to comprehend, and their elements will not be re-usable. Kinetics calls into play a large number of concepts that are new to AI, for example, corporation, coordination and satisfaction. The difference between AI and kinetics is that with AI it is the individual that is intelligent, whereas with kinetics it is the organization that displays functionalities that can be characterized as intelligent. The main goal of the distributed system is the carrying out of tasks by making the best use of physically distributed resources (for example, memory, processor, data managers). On the other hand, the system of kinetics is much more open. The multi-agent system takes an overview and summary of the problem of interaction between individual entities, considering the distributed system as being merely one possible

Why Interracial Dating is still not accepted Essay Example for Free

Why Interracial Dating is still not accepted Essay The representatives of different races have always been founding shelter and new home for them on the U. S territory, but in the last hundred years the quantity of immigrants has increased. Thus, it is no wonder that nowadays dating and marriages between the representatives of different races have also become more often. The main racial groups that live on the U. S territory, White Americans, African Americans and Asian Americans nowadays enter mixed marriages much more often than forty or thirty years ago, as nowadays the impact of social norms that disapprove of interracial relationship has become much weaker. Thus, the Black vice White, and Asian vice White couples with kids can be seen in every American city. Nevertheless, while racial discrimination has diminished its influence in the marital aspect, new problems have appeared for the interracial couples, and new societal attitudes developed, which complicate the life of those, who decided to tie the knot with the representative of the race different than his or hers. The number of people who strongly disapprove of interracial dating has declined since the middle of the 20th century, but still there are citizens, who insist that marriages between the representatives of different races should be banned. They explain their viewpoint by the fear for the future of children born in these kinds of marriages, and by the imbalance in the social and cultural levels of people of different races. Nevertheless, it is not the main reason for the negative attitude towards interracial dating that still exists in the American society. The main cause of disturbance about interracial dating and marriages nowadays is statistical. The history of this phenomenon shows that with the outspread of this kind of marital relationships, the married/single ratios for the representatives of most races that live on the U. S territory has changed dramatically. The roots of this change lie in the lop-sided distribution of people in the interracial marriages. Statistics says that if we talk about marriages between the representatives of different races, than White vice Black and White vice Asian marriages are the most widespread. The problem is that in most White vice Black marriages black men marry white women, and in White vice Asian couples those are white men who have Asian wives. In the 1990 Census, 72 per cent of black-white couples consisted of a black husband and a white wife. In contrast, white-Asian pairs showed the reverse: 72 per cent consisted of a white husband and an Asian wife (Sailer, 1997). The 1992 Sex in America study of 3,432 people found that ten times more single white women than single white men reported that their most recent sex partner was black. In 1990, 1. 46 million Asian women were married, compared to only 1. 26 million Asian men. It is obvious that this distribution creates severe problems among African-American women and Asian males, who feel the scarcity of partners within their own race, and have little possibility to find them outside of it. The reason is that marriages between white man and African-American women, as well as between Asian males and white women are rare. African-American vice Asian marriages are even harder to found. Thus, every year the U. S society gets a considerable quantity of African-American women, and Asian-American men who are unable to find a partner. No wonder that African-American and Asian American communities arent silent about this problem. Numerous talk shows, books, and movies acquainted the American society with the problems African-American women have. The Afro-American activists created a feeling in most of the female representatives of this race that white women steal black man from Afro-American women. This attitude provokes hostility towards white women among the Afro-American ones, and also worsens the attitude towards interracial marriages in the whole society. Asian-American males are also downtrodden by the existing situation. They, in their turn, develop negative attitudes towards white men, who deprive them of the possibility to create families with Asian women. Some researchers propose that to solve this problem white males should be encouraged to marry Afro-American women, and white women be keener on having relationships with Asian males, but the other prove it would be ineffective for purely biological reasons. The thing is that, as the researchers proved, those are mostly hormonal reasons for which black males and Asian females are seen as the most attractive partners. The scientists found out that black men are on average more masculine than the white and Asian ones, while Asian females are the most feminine. In the same time, most men see Afro-American women as less feminine than white females, and Asian males, due to their build, are considered to be less masculine. In addition to the biological reasoning, these images are supported and promoted by the media stereotypes, where Afro-American males are depicted as hyper masculine, and Asian women – hyper feminine. Nature and society dictates that a person should choose a heterosexual partner with the most prominent features of the representative of the opposite gender, thus the Afro-American women and Asian males have problems finding partners, which, in its turn, creates strained attitudes towards interracial dating and marriage in the whole U. S society. The attitudes towards interracial dating and marriages have become much more tolerant in the past forty years, but the strain concerning this question still exists in the society. It is mostly caused by the fact that due to social and biological reasons Afro-American females, and Asian males experience problems trying to find a partner, as the considerable part of males and females who belong to their races prefer to have white partners. Works Cited 1. Sailer, S. Is love colorblind? public opinion about interracial marriage. National Review, 1997

Ecline and Fall and Sun also Rises :: Free Essay Writer

Ecline and Fall and Sun also Rises Though Ernest Hemingway's The Sun Also Rises, and Evelyn Waugh's Decline and Fall are written by two different authors, they share similar content and themes. In The Sun Also Rises, Brett desires Jake but cannot commit as a result of Jake's impotence. Similarly, in Decline and Fall, Margot cannot commit to Paul because of his time in jail. Both men seem to be infatuated with someone who does not share the same interests in their relationship. These relationships are hollow, showing no emotion and are only based on sex. In The Sun Also Rises, Brett claims she does not want to get involved with Jake, yet the underlying truth is more evident through her actions. While driving around the city, Brett and Jake have a deep discussion about their relationship. Brett says, "Don't touch me" (25). However, when Jake asks if she loves him, she responds, "Love you? I simply turn all to jelly when you touch me"(26). Their conversation becomes ironic when Brett says, "When I think of all the hell I've put chaps through. I'm paying for it now" (26). After hurting so many men, who she could not love, she is now being hurt by someone who could not love her. Later on in the novel, Brett visits Jake's apartment. After a conversation, Jake feels desperate and asks Brett. "couldn't we just live together?" (55). Brett responds by saying, " I don't think so. I'd just tromper...stand it" (55). Although it is quite evident that Brett wants to be with Jake, she still does not encourage the idea of living together. She openly admits that she will cheat on him if this was to ever happen. Jake's jealously is certainly displayed when Robert defends Brett. Jake tells Robert that Brett did not love any of the men she had married in the past. Robert tells Jake, "I didn't ask you to insult her" (39). By the end of their argument, one may feel a sense of pity for Robert and his childish behaviour. However, Jake feels more than pity for Robert; he feels a sense of anger and jealousy towards the man who is about to make a pass at the woman he loves. When Brett tells Jake that she is going to San Sebastian to get away from him, Jake abases himself and asks her if he can go too.

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+ ,

Who Is to Be Blame for Macbeth’s Downfall

Macbeth is a famous tragic play written by William Shakespeare, a well-known English poet and an Elizabethan playwright, in the 1500’s. Macbeth tells about the downfall of a war hero who chose an evil path to achieve his ambition. Therefore, Macbeth is responsible for his own downfall. Although some may argue that he was influenced by the witches and his wife, Lady Macbeth, however it is his choice to act upon those suggestions, which he eventually did. So, Macbeth is totally responsible for his own downfall.Throughout the play, the witches have played a very significant role of influencing Macbeth to think and act evilly. Initially, Macbeth was a good man, a good soldier who is fiercely loyal to King Duncan and to his country, Scotland. In fact, he is a valiant warrior against who fought courageously against the Norwegian forces, where he was praised as â€Å"brave†, â€Å"noble† and a â€Å"peerless kinsman†. But the witches, through prophecy, plant a se ed in his mind that appeal to Macbeth’s superstitions and ambition to be king, â€Å"All hail Macbeth, hail to thee, Thane of Glamis†¦ Thane of Cawdor†¦ that shalt be king hereafter†.These prophecies then provoked evil thoughts inside Macbeth’s mind. After Macbeth was told by Ross that Duncan had praised him with the title â€Å"Thane of Cawdor†, visions of murdering Duncan began to appear in his mind, â€Å"my thought, whose murder yet is but fantastical, shakes so my single state of man that function is smothered in surmise, and nothing is, but what is not†. Other horror thoughts includes, â€Å"this is a step on which I must fall down, or else o’erleap, for in my way it lies†.After Duncan announced that his son, â€Å"Prince of Cumberland†, will succeed him to throne, Macbeth realizes that he either have to get rid of Malcolm or give up in his ambition. The witches don’t tell Macbeth what to do; but it was M acbeth himself who thinks of murder. The three witches’ intention is only to disrupt the natural order and through Macbeth, this is accomplished. From the above, it is clear that the witches are only responsible for encouraging Macbeth’s ambition and confidence but is not responsible for the killings.It is Macbeth who has killed Duncan, Banquo and Macduff’s family; therefore, he is responsible for the killings and also for his own downfall. Besides the three witches, Lady Macbeth is another major influence on Macbeth. She is manipulative and encourages Macbeth to achieve his ambition. She is portrayed as a strong, fiercely determined woman who, unlike her husband, shows no doubt of hesitation about killing Duncan. She acts quickly seeking to take advantage of the opportunity to kill Duncan, â€Å"O never shall sun that morrow see†, when Duncan decides to stay overnight at their castle.She also called upon â€Å"the spirits that tend on mortal thoughts to unsex me here†, so that she could be evil enough to commit the murder. When Macbeth preferred to be king â€Å"without my stir†, she attacked his manhood, saying, â€Å"When you broke this enterprise to me then you were a man and should you do this then you would be so much more the man†. Even though Lady Macbeth appears to be the evil mastermind behind the murdering, but in the end, it is also partly because of Macbeth’s own ambition to be king that encourages Macbeth to go into Duncan’s chamber and murders him.Besides Duncan, Macbeth has also murdered other people such as the grooms, Macduff’s family and also his best friend, Banquo. Due to that, he was later referred as a â€Å"tyrant†, a â€Å"butcher†, and a terrifying ruler of Scotland. His downfall is a result of his misuse of power, and Lady Macbeth is no longer involved. Therefore, only Macbeth himself shall be blamed for his own downfall. Even though the witches and L ady Macbeth certainly play an integral part in influencing Macbeth, but the choice is ultimately his. He could have ignored the â€Å"hags'† prophecy, like Banquo does.He did not have to share his dark desires with his wife, either. Again, it is his ambition that provokes him to do those evil deeds, â€Å"I have no spur to prick the sides of my intent but only vaulting ambition which o’erleaps itself and falls on th’other†. Once he is bent on becoming king, Macbeth became evil and ruthless, as he is willing to kill anyone in his way, even including women, children, and his friends and countrymen. Were it not for this ambition in Macbeth's character, he would have been happy with his position of thane and never sought the throne.In the end, he has no one to blame but himself. Conclusively, it is pretty clear to the readers that Macbeth has brought his own downfall to himself. It is his lust for crown that has consumed him. Although some may argue that the witches and his wife, Lady Macbeth have influenced him at some stage, but in the end, it is Macbeth who made the decisions to kill ruthlessly, as he could have ignored those suggestions. Therefore, Macbeth should solely hold full responsible for his own downfall and not the witches or Lady Macbeth.

Free sample - Review Of Hitlers Germany. translation missing

Review Of Hitlers Germany. Review Of Hitler's GermanyIn April 1933, during the early months after the Nazis ascended to power in Germany, a law which commonly came to be termed as the Aryanan Paragraph came into effect. It outlawed any person of Jewish descent from government employment. This was the first piece of legislature to be effected in a then heightening assault on Jews led by the Third Reich Hitler and evidently expressed in his toxic rhetoric and ideological imperatives. This placed German Churches at a focal point: They either had to resist these attacks on Jews or dismiss all Jewish preachers and employees so as to preserve their subsidies. Most of the churches publicly or silently fell in line with Hitler’s demands. These in effect became the onset of the world’s bloodiest World War II and the context of Roderick Stackelberg’s book on Hitler’s Germany: Origins, Interpretations and Legacies which provide an interesting read and meets its chief objective of introducing a ny reader to the history and the atrocities committed in the Nazi Germany. The book extends from the abortive 1923 Beer Hall Putsch to the World War II and the aftermath in the 1940’s. This therefore gives Stackelberg’s novel a wide coverage while ensuring the reader is totally engrossed in the narrative as the story unfolds. Stackelberg , a humanities professor at Gonzaga University in Spokane, he cogently sets out to argue out that the Nazi Regime was supported and maintained through a mass consensus by the majority of the German citizens rather than the implied coercion by most authors. He is hence in agreement with Daniel Goldhagen and his views as phrased in his narrative, Hitler's Willing Executioners of which he has recognized and praised. He points out that Germans expressed conviction and expediency in their support and collaboration with the Nazi regime. He endeavors to balance ‘intentionalist’ versus ‘functionalist’ approaches to th e Holocaust committed against Jews so as to amply show the Nazi’s adherence to the fatal eugenic belief of exterminating all those deemed to be "life unworthy of life". This resulted in the death of two-thirds of the Jews in Europe at the time. Stackelberg successfully combines dramatic writing with a dispassionate analysis so as to aptly provide a rich historical context the barbaric behavior and actions of the Third Reich by boldly depicting a pre-history of Nazism such as the absolutist rule put forward by his predecessor Otto Van Bismarck, the 19th-century nationalist propagandistsand the Free Corps hooligan squads who not only crushed the 1919 Spartacist revolt but also murdered Rosa Luxemburg. He further covers the Nuremberg trials, the German denazification and the modern-day resurgence of militant neo-Nazi extremists. Although the work presented herein has already been documented in other books, he manages to author an interesting and engrossing superb read on the Naz i Germany history. The book first provides a detailed coverage of the roots of fascist ideologies, its constituency and the conditions that facilitated its growth in Germany. It then reflects on the key problems facing German unity which Stackelberg clearly and comprehensively covers as absolutism and particularism. This serves as a basis as to why the German Empire changed from a democratic state to social imperialism and finally landed on the path to war. Stackelberg clinically examines the Germanic ideology that was instituted into the masses by the political class so as to influence support. He finds that the politicians managed to drive the cause for nationalism towards fanatism while coupling this with vulgarized idealism and anti-Semitism.   Stackelberg has also provided a rich context for German’s history and involvement in the First World War and the resultant crisis in imperial Germany under Bismarck. He goes on further to examine the Weimar Republic through a well-documented study a nd the weakness of liberal democracy in Germany. This led to the consequent fall of the Weimar republic and the rise of Nazism further facilitated by the Great Depression. The Nazis managed to consolidate power in the 1933-1934 under the Third Reich Hitler whose governance in the 1933-1939 period has been fully analyzed under the aspects of politics, society and culture hence providing a rich and diverse read. Further, Stackelberg manages to depict hideous details of the persecution of the Jews and the Holocaust in this period. The origins of the Second World War, its spread from a European to a global war and its ensuing transformation from triumph to defeat in 1942-1945 have been elaborately covered while providing ample contextual information that leaves a clear imaginative image in the readers’ minds. Finally, the book evaluates the aftermath of the war and Germany’s National Socialism. The last chapter examines Hitler’s place in history and memory and the v ital lessons learnt from the ordeal. In the introduction, Stackelberg clarifies why he wrote the book despite a myriad number of historical books in the market dealing with a similar subject matter. He feels there is a need to write a book that not only covers the Nazi regime but also the 19th century background and the aftermath. Despite the book’s title, only seven out of sixteen chapters are dedicated to the Nazi regime. It provides a rich and essential understanding of the Hitler-led Nazi regime. This was a decision he reached at after having taught the subject matter for over twenty years. Stackelberg feels that the book approaches the Nazi regime under a two dimension: He provides an accurate and complete account of Nazi rule and goes further to provide an interpretive framework that endeavors to explore the reasons as to the extraordinary occurrence in German history. The book provides a clear guideline to the reader whereas incorporating the complex and vast complexities of historical causation as experie nced by the contemporary figures that lived in that turbulent and violent era. In creating a rich analysis and reconstruction of the Nazi regime in 1933 to 1945, the author places the period in a larger context which enables him to ably provide a sufficient background of the regime while ensuring various critical arguments are brought forward. First, Stackelberg feels that history is inseparable from its interpretative analysis. No author, in Stackelberg’s view should present the bare facts of a historical occurrence without endeavoring to provide a parallel interpretive theory as to why the historical phenomenon took place. Historical books and journals have always depicted the Nazi era under a barbaric and destructive scope and it is almost viewed entirely as the world’s greatest battle of evil versus good. This approach is rather heightened by the atrocities committed such as the irrational racial obsessions and the Holocaust with an aim to wipe out all Jews. Any other approach, such as a metaphysical approach, would definitely not successfully account for the success and popularity of Nazism in Germany. However, rather than approach the Nazi era under a moral and evil conception as multiple authors’ have, Stackelberg endeavors to define the rise of the Nazi regime under a political analysis. Stacke lberg feels it is essential to establish why the Germans at the time felt that Nazism was a reconstructive force in the quest for National Socialism that would utterly propel them into a superpower state. He critically notes in a catchy headline that history is past politics, hence, even the atrocities committed under the anti-Semitism derive must have a cognitive understanding. Unlike facts which if in dispute can easily be ratified among historians, an analysis of the reasons as to why German Nazism was widely popular can only be perceived under the analysts own political and societal values. These are highly diverse among historians and are therefore bound to bring forth a degree of controversy. In a review of egalitarian governments, Stackelberg depicts how left-wing movements can easily gain popularity through â€Å"championing for emancipation from oppressive governments whereas the right-wing lobbyists defend traditional and hierarchical governments.† The left extremists can effortlessly apply authority in the running of governments so as to create egalitarian societies as depicted by the 21st century communist governments. The conservatives in the right wing endeavor to create liberal societies through curtailing government power and promotion of individual freedom. In this book, Stackelberg addresses this contemporary left-right spectacle in their respective egalitarian perspectives by a case scenario of American politics. American conservatives have been documented as in opposition of powerful governments bringing them closer to the left’s camp but with absolutely diverse goals which are the key to any government. In their campaign against the powerful g overnment and their defense for laissez-faire, American conservatives have been found to depict similar traits to those of 19th century conservatives in continental Europe and the Nazis’ fascism. In America, the highly liberal society either leans on personal freedom or social equality. The leftists rather lean on social equality while the right conservatives opt for freedom. Stackelberg further provides a distinction between moderates and extremists in which extremists are rather authoritative, prejudiced and inclined towards violence, deception and collectivism across the left-right extremists. They are intolerant of any opposition or deviations from the ideal entailed by freedom and seek to forcefully impose these ideals on individuals. Communism bordered on the left while fascism was composed of right extremist. However, most authors, with the exception of the well-sourced Stackelberg’s book, feel that communism and fascism are inherently related which a critical e valuation in Hitler’s Germany depicts that they are fundamental opposites. While communism mainly appealed to workers who owned minimal properties due to its enhancement of a greater degree of equality, fascism mainly appealed to the middle class and propertied workers who felt that they would in essence lose from the implementation of egalitarian principles. Therefore, proponents of each group were arch enemies since communism maltreated the higher and mighty classes while fascism greatly victimized the â€Å"lower races† and poor classes of humanity. In exploring the causative force behind the Fascism variant Nazism, Stackelberg examines counter-revolutionary concepts in contrast to revolutionary concepts in his apt and wide description of the Hitler Regime. He feels that in contrast to neo-conservatism in the United States, fascism in Germany is much related to the traditional continental Europe conservatism though it has some anti-conservative features. Multiple radical methods were adopted into German Nazism from the practices of its arch foe, communism such as mass mobilization techniques, violence and propaganda. This was a highly critical countermeasure identified by Stackelberg in which the Nazis used the tactics employed by the left against the left. Counter-revolutionary concepts however did not characterize the left-right distinction as much as the core goal of preventing equality much agitated for by the left by a vehement denial of its existence through the structure governing various races and their coexistence. In t his book therefore, Stackelberg finds it crucial that most historians have neglected the fact that these ruthless and radical measures were put in place to counter socialism by purported National Socialists through the eradication of the significant proportion of production contributed by private property. Stackelberg therefore feels that the term socialist has been misused since the party was not true to the doctrines it purported to support and further. Hence, he feels that the Nazis were counter-revolutionary since it endeavored to curb developments in the transformation of the property sector while upholding the Puritist nature of the fascist regime. This book also makes a very interesting and engrossing read since it answers the contentious question on the relationship between fascism and Nazism and their relation to other political movements of the past centuries. It further answers the crucial questions on how Nazism managed to ascend to power in such a civilized, industrialized and urbanized context. In a well-analyzed and well-sourced background study, Stackelberg examines the rise in popularity of Nazism by interpreting it basing heavily on the Sonderweg thesis. This is categorically analyzed in chapter 2 whereby the variation in the development of democracy in Germany was remarkably different from other European nations. Most authors have neglected to write a detailed account of the pre-Hitler administrations which would otherwise provide vital historical clues to the rise of Nazism. This is utterly reflected in the book. However, Stackelberg expresses caution that a study to chiefly analyze pre-Nazi Germany in the 19th ce ntury as a pure preliminary stage to the Nazi regime and its aftermath would not only be a narrow-minded approach but also historically inadequate and unjustifiable. Although Stackelberg feels that greater and much more vital events such as Russian Bolshevik Revolution, the defeat in the First World War and the conflict of political interests in the Weimar Republic provide a crucial basis for evaluation of Nazism, the lack of the development of democracy can partially be attributed to Nazism. Finally, Stackelberg furthers debate as to whether Nazism is a modernizing or anti-modern debate. At the time, Germany’s economy was at its peak but the inability of political liberalization and democracy to keep pace with the advances in technology led to a flaw in its development hence depicting a rejection of modernity. This point of analysis as put forward by Stackelberg is further supported by the Sonderweg thesis whereby major evidence of anti-modernity such as â€Å"blood and soil† ideology that depicts a German-only agrarian culture under threat of urbanization and the resultant industrialization. This was in effect promoting capitalism whereby the Jews were viewed as the major beneficiaries at the expense of the former chief producers, the Mittelstand. However, Stackelberg also evaluates Nazism as a facilitator of modernity through the implementation of advanced technology in the military during World War II. Though this is highly complemented by pioneer studie s in space technology, the rejection of Jewry physics in the development of nuclear weapons further served as a major factor in deterring modernism. Stackelberg has used a wide variety of sources that span from The German Empire, ideologies, the First World War, the Weimar Republic and its collapse, the Nazi consolidation of power, the society, culture and politics during Hitler’s rule, the Holocaust and the anti-Semitism, the Second World War, the Aftermath and the modernity debate. For instance, it is crucial to note on Stackelberg’s reference to Taylor’s famous and controversial book, The origins of the Second World War in which he strongly criticizes The failure of the British to conclusively put in place a pact with the then Soviet Union so as to put an end to the war. The sources used herein in this book are highly relevant and serve in meeting the objective of the book. It gives this work a high credential. Further, Stackelberg has written the sources in a well-organized and presentable manner depicting that the book was written after a conducting a research for a period of twenty years during which he was teaching a similar course. This therefore enables Stackelberg to write an objective, chronological account and a must-read book that not only expands on Hitler’s Germany, but also on the 19th century pre-Nazi period and the 20th Century post-war period and the aftermath. This serves to give the book an edge over other historical books written at the time. (Stackelberg, 1999) Reference Stackelberg, R. (1999). Hitler's Germany: origins, interpretations, legacies. Routledge Press.

75 Persuasive Essay Topic Ideas

75 Persuasive Essay Topic Ideas The persuasive essay is one type of writing that you will likely come across in your academic career. A persuasive essay, if youre unfamiliar, is one in which you have to make an argument. You need to choose a side and prove why youre correct by using hard evidence and convincing language. The idea is that you want to convince the reader that your argument is the right one, so youll definitely want to pick a topic that youre passionate about and something that youll get excited about researching and writing. This exercise is designed so that you can clearly articulate your opinion and understand why its important to have evidence to back up your claim.Your teacher or instructor will probably have specific guidelines on what your essay should entail, but you might have a little bit of free reign on what kinds of topics you can explore and argue about in your essay. With so many things to argue about and for, it might be a little overwhelming to come up with a topic on your own. When y ou feel like youre stuck on brainstorming ideas, take a look at the following list of 75 persuasive essay topics. You may find something you can use, or something you can adapt for the specific guidelines of your paper. Happy writing!Educational persuasive essay topicsThere are so many things that can be discussed when it comes to education. In our country (and globally), there are many different opinions on how education should be handled and what tactics teachers or academic administrators should use. Here are a few topics on education (which could be expanded or changed to fit your teachers guidelines) that might be of interest to you.Should soda be offered in school cafeterias?Should schools teach abstinence-only education?Why should schools teach financial literacy?Do all students need to go to college?Should students take a gap year after high school?Do all students need to learn a foreign language?Is online or homeschool an effective way to learn?Should standardized tests det ermine whether or not you go on to another grade level?Should all students be required to participate in the arts?Should a college education be free?Should high school journalists be protected under the First Amendment?Some universities just have pass/fail grades instead of letter grades. How do you feel about this?Should teachers/professors be unbiased in the classroom?Should you still learn cursive in elementary school? What are the disadvantages/ advantages?Many college campuses have speakers come in occasionally. These speakers can range in political opinion and some can be controversial. Should you let speakers come to schools that have controversial rhetoric or ideas to uphold free speech?Political persuasive essay topicsThey say that you should never talk about politics or religion because its not polite. But in a persuasive essay, that rule is completely extinguished. Politics and religion are hotbed subjects for a reason- because so many people have radically different idea s of how a society and a country should operate. What side of these political persuasive topics are you on? Take a stab at one of these and the paper will likely fly out onto the keyboard.Should protesters be allowed to block traffic? Do they pose a threat to public safety?Why should you vote?Should same-sex marriage be legal?What is your opinion on protecting religious liberties?What is your opinion on separating church and state?Why has the country become so divided politically over the past few years? Can it be fixed?Many industries (like coal and manufacturing) are tough to find a job in and many Americans are out of work. How should we solve this problem?Should citizens under 18 be able to vote?Should a National Voter ID law be passed to avoid voter fraud?What does the phrase fake news mean?Local newspapers are dwindling. What should be done, if anything, about this problem?Should local municipalities do more to combat global warming? If so, how?How should we reduce the threat of terrorism in the United States?Females have traditionally lower participation in politics. Why do you think that is?Some people say that the top 1% of earners dont pay enough taxes. How do you feel about this?Will a huge wall on the southern border with Mexico solve the United States immigration problem?How should we solve the United States immigration problem?The voter turnout for the 2016 presidential election was less than 60%, which is much lower than in other democratic societies. Why do you think this is and what can be done about it (or should anything be done about it)?Millennials are graduating college with a lot of student loan debt. What should be done to avoid a debt crisis?Many say that minimum wage jobs are low skill and the workers in them shouldnt be compensated more for their work, but others claim that a minimum wage job isnt enough money to live off of. Which side do you land on?What do you think of celebrities who are vocal about environmental issues but who f requently fly on private, and not commercial, jets?Crime and legal persuasive essay topicsCrime in any society is an unfortunate inevitability. Why does crime happen and what should be done about it? These are just a few of the things to explore in these crime/legal persuasive essay topics.What should we do about a city with a high crime rate like Chicago?Should guns be allowed on college campuses?Should gun laws be more restrictive?Do we have a right to privacy?Trends have shown that many recent terrorists have been convicted or accused of domestic violence. What should be done and how do you feel about this?Should we have the death penalty? If so, when should it be used?Many prisoners are incarcerated for minor drug charges (such as possession of drugs or drug paraphernalia). Should we try to rehabilitate these prisoners or should they serve their full sentences?Colorado has legalized marijuana for recreational purposes. What is your opinion of this?Do you think marijuana is a gat eway drug which leads some users to harder drugs?Can criminals be rehabilitated?Many prisoners who enter the system are likely to have a high recurrence of criminal activity. What can be done to solve this?Many people are starting to use drones for recreational activity. Should there be restrictions on where and how you can use your personal drone?Self-driving cars are expected to become increasingly used on city roads. If a self-driving car gets into an accident, whose fault is it? The engineers?Health persuasive essay topicsHealth is something that we all have to worry about. Whether its our own health or the health of a loved one, there are many things to think about and research on. Whats your opinion on the healthcare system in our country? Should we treat drug addiction like a disease? How should we handle end-of-life care? Try out one of these essay topics to research and gain insight on some of the biggest challenges and questions that our society faces when it comes to heal th.Opioid addiction is at an all-time high in states like Ohio. What should we do to combat this?Should healthcare be universal?How do you feel about paternity leave?Should women get guaranteed maternity leave?The state of California requires that you display nutrition facts about menu items in restaurants. Should all states do this?Should fast food be sin taxed like cigarettes are?There is an effort to repeal and replace the Affordable Care Act. Should we do this or not? If we should, what improvements can be made to a replacement act?Many soldiers are coming back from warfare with Post Traumatic Stress Disorder. What should we do to help them?Many Americans are overweight. What has caused this health crisis and what can be done about it?Should vitamins and supplements be more tightly regulated?Should health insurance companies provide more financial incentives for subscribers to work out and eat more healthfully?Womens and gender persuasive essay topicsAre there inherent differenc es between men and women or is that just a societal myth? Women have gained a lot more rights over the last 100 years in America, but some say they still have a long way to go before they achieve equal rights. How do you feel about this and other womens and gender issues? Explore the following fascinating topics.Women have what is known as the second shift (meaning that as soon as they get home from work they have additional responsibilities that require their attention immediately). What do you think about this concept and should anything be done about it?There are many womens rights and minority rights advocates. Should there be mens rights advocacy groups? What about Caucasian advocacy groups?Some people say that gender is a socially constructed norm. What do you think?Women who participate in body building competitions are trying to build the ideal figure, which some claim is an outdated, sexist idea. But some argue that building muscles is considered a sport and a traditionally masculine idea. Which side do you agree with?Some people think that beauty pageants are outdated and anti-feminist and shouldnt be televised anymore. How do you feel?New wave feminism is the idea that feminism can encompass many different ideas of what it is to be a feminist. Its the idea that you can have choices (whether thats staying at home with children or trying to be a CEO). How do you feel about new wave feminism?Miscellaneous persuasive essay topicsOf course, there are more categories of essay topics than what are listed above. Here are some additional essay topics if you havent found one yet that captures your interest.Does social media improve or hurt our society?Is it important or frivolous to travel the world?Many Americans watch a lot of reality TV shows. Why do you think this is?With many people reading digital copies of books, are libraries necessary anymore?Should anything be done to curb the rise in offensive lyrics in music?Should pregnant women be allowed to par k in handicapped parking spots?Recent studies have shown that pets improve the mental and the physical health of their owners. Should pet-related expenses be tax-deductible?What do you think about net neutrality?With the rise in selfies and Instagram photo filtering apps, do you think we have become a more self-obsessed society?