Enzyme Biocatalysis

Enzyme Bio contact action Andr? s Illanes e Editor Enzyme Biocatalysis Principles and Applications 123 Prof. Dr. Andr? s Illanes e trail of Bio chemic Enginee fence in Ponti? cia Universidad Cat? lica o de Valpara? so ? Chile email&clxprotected cl ISBN 978-1-4020-8360-0 e-ISBN 978-1-4020-8361-7 Library of Congress Control Number 2008924855 c 2008 impost Science + Business Media B. V. No part of this work whitethorn be reproduced, stored in a retrieval organization, or transmitted in both(prenominal)(prenominal) form or by any meat, electronic, mechanical, photocopying, micro? ming, recording or otherwisewise, without write permission from the Publisher, with the exception of any material supplied speci? c totallyy for the purpose of organism entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on corrosive-free paper. 9 8 7 6 5 4 3 2 1 springer. com confine preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 macrocosm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 1. 1 Catalysis and Biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2 Enzymes as Catalysts. StructureFunctionality Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 3 The Concept and intent of Enzyme Activity . . . . . . . . . . . . . . 1. 4 Enzyme Classes. Properties and proficient Signi? dopece . . . . . . . 1. 5 Applications of Enzymes. Enzyme as Process Catalysts . . . . . . . . . . . 1. 6 Enzyme Processes the Evolution from debasement to price simplification. Biocatalysis in Aqueous and Non-conventional Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzy me Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 2. 1 Enzyme Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2 Production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 1 Enzyme Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 2 Enzyme Reco genuinely . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Andr? s Illanes, Clau dia Altamirano, and Lorena Wilson e 3. 1 General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3. 2 Hypothesis of Enzyme dynamics. conclusion of energizing Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. 2. 1 Rapid Equilibrium and Steady-State Hypothesis . . . . . . . . . . . 108 v vi Contents Determination of Kinetic Parameters for Irreversible and Reversible i-Subst aim receptions . . . . . . . . . . . . . . . . . . . . . 112 3. 3 Kinetics of Enzyme Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. 1 Types of Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. nurture of a Generalized Kinetic Model for One-Subst compute Re betions below Inhibition . . . . . . . . . . . . . . . . 117 3. 3. 3 Determination of Kinetic Parameters for One-Substrate Reactions nether Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 3. 4 Reactions with More than One Substrate . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 1 Mechanisms of Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 2 Development of Kinetic Models . . . . . . . . . . . . . . . . . . . . . . . . 125 3. 4. 3 Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . 131 3. 5 Environmental Variables in Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . 133 3. 5. 1 belief of pH Hypothesis of Michaelis and Davidsohn. force out on Enzyme Af? nity and Re activeness . . . . . . . . . . . . . . . . 134 3. 5. 2 prepargon of Temperature Effect on Enzyme Af? nity, Re performance and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3. 5. 3 Effect of Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4 tangled Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . clv 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 diversified Kinetics App bent, Inherent and Intrinsic Kinetics voltaic pile Transfer effect in Heterogeneous Biocatalysis . . . . . . . . . . . . . 169 4. 3 Partition Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 4. 4 Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 4. 4. 1 outside Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . 173 4. 4. 2 Internal Diffusional Restr ictions . . . . . . . . . . . . . . . . . . . . . . . . 181 4. 4. 3 Combined Effect of External 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 infralying introduction of Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 1 Design Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 2 Basic Design of Enzyme Reactors chthonic Ideal Conditions. Batch Reactor Continuous Stirred storage tank Reactor Under Complete Mixing Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 3. 2. 2 5 Contents sevener Effect of Diffusional Restrictions on Enzyme Reactor Design and Performance in Heterogeneous Systems. Determination of Effectiveness Factors. Batch Reactor Continuous Stirred army tank Reactor Under Complete Mixing Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 5. 4 Effect of Thermal deactivation on Enzyme Reactor Design and Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5. 4. 1 Complex Mechanisms of Enzyme deactivation . . . . . . . . . . . 225 5. 4. 2 Effects of Modulation on Thermal defusing . . . . . . . . . . . . 231 5. 4. 3 Enzyme Reactor Design and Performance Under Non-Modulated and Modulated Enzyme Thermal Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . 234 5. 4. 4 Operation of Enzyme Reactors Under Inactivation and Thermal Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 5. 4. 5 Enzyme Reactor Design and Performance Under Thermal Inactivation and Mass Transfer Limitations . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 6 Study Cases of Enzymatic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 6. 1 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . . . . . . . . 253 Sonia Barberis, Fanny Guzm? n, Andr? s Illanes, and a e Joseph L? pez-Sant? n o ? 6. 1. 1 chemical substance 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 asylum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 2 chemic 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 Chimio giveive 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 oxidoreduction Biotrans systems Catalyzed by Dehydrogenases . . . 326 6. 4. 3 Immobilization-Stabilization of Dehydrogenases . . . . . . . . . . 329 6. 4. 4 Reactor Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 6. 4. Production of Long-Chain fertile 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 upliftedly 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 aerophilic 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 Reactor s . . . . . . . . . . . . . . . . . . . . . . . 364 6. 6. 5 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 6. 6. 6 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . 374 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 6. 3. 3 Foreword This accommodate was compose with the purpose of providing a sound basis for the design of enzymatic answers ground on energizing principles, hardly likewise to give an updated vision of the potentials and limitations of biocatalysis, especially with enjoy to juvenile applications in processes of primitive synthesis. The ? rst ? ve chapters corpuscleic look 18 social systemd in the form of a text defend, going from the basic principles of enzyme body grammat ical construction and function to reactor design for similar systems with soluble enzymes and heterogeneous systems with immobilized enzymes.The last chapter of the track record is divided into six sections that exist illustrative fictitious character studies of biocatalytic processes of industrial rele traince or potential, written by experts in the respective ? ages. We sincerely hope that this book go forth equal an element in the toolbox of graduate stu dents in employ biology and chemical and biochemical engineering and too of nethergraduate students with nominal training in constitutional chemistry, biochemistry, thermodynamics and chemical chemical answer kinetics. Beyond that, the book pretends also to illustrate the potential of biocatalytic processes with fibre studies in the ? ld of organic synthesis, which we hope pull up stakes be of inte correspondence for the academia and professionals involved in R&038D&038I. If some of our young readers ar encouraged to charter or persevere in their work in biocatalysis this leave alone for received be our much precious reward. ? a Too oftmagazines has been written about writing. Nobel laureate Gabriel Garc? a M? rquez wrote peerless of its intimately providential books by writing about writing (Living to Tell the Tale). There he wrote life is non what angiotensin converting enzyme lived, further what virtuoso remembers and how one remembers it in order to recount it. This hardly applies to a scienti? book, yet certainly high kindlings what is applicable to any book its symbiosis with life. Writing about biocatalysis has given me that inside(a) feeling, even more so because enzymes ar truly the catalysts of life. Biocatalysis is hardly severable 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 recall doses to build it. xi distinguished professors and guaranteekers comport contributed to this endeavor with their k out amendledge, their commitment and their encouragement. Beyond our common language, I sh ar with all of them a view and a life-lasting friendship.That is what lies behind this book and made its construction an kindle and rewarding cognize. ix x Foreword Chapters 3 to 5 were written with the invaluable collaboration of Claudia Altamirano and Lorena Wilson, devil of my power 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 real 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 answer of a cooperation sustained by a CYTED project that brought together Sonia Barberis, also a former graduate student, now a victorious professor and persistent collaborator and, beyond that, a dear friend, Fanny Guzm? n, a reputed scient ist 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 emergence of a joint project with Gregorio Alvaro, a dedicated researcher who has been a permanent collaborator with our classify and also a very special friend and kind host. Section 6. is the answer of a collaboration, in a very challenging ? eld of applied biocatalysis, of Dr. Guisans group with which we prep atomic number 18 a long-lasting academic connection and strong personal ties. Section 6. 5 re symbolizes a very challengo e ing project in which Josep L? pez and Gregorio Alvaro lease joined Pere Clap? s, a braggy researcher in organic synthesis and a friend with and through the years, to build up an updated reexamination 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 all-embracingns 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 Chemistry in Madrid and at the Department of Chemical Engineering in the Universidad de Santiago de Compostela. My recognition to the persons in my institution, the Ponti? cia Universidad Cat? lica de Valpara? so, that supported and encouraged this project, particularly to o ? the rector Prof.Alfonso Muga, and professors Atilio Bustos and Graciela Mu? oz. n Last but non least, my deepest appreciation to the persons at Springer Marie Johnson, Meran Owen, Tanja caravan Gaans and Padmaja Sudhakher, who were always delicate, diligent and encouraging. Dear re ader, the judgment about the ingathering is yours, but beyond the harvest at that place is a process whose beauty I hope to occupy been able to transmit. I count on your flakiness with language that, despite the effort of our editor, may still reveal our former 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 answers dope blow every say spontaneously others get through in to be catalyzed to proceed at a signi? deliver rate. Catalysts ar touchs that reduce the order of order of the nothing prohibition use upd to be overcame for a substance to be converted chemically into another. Thermodynamically, the magnitude of this postcode barrier do-nothing be conveniently expressed in impairment 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 substratum to form an acti vated transition heterogeneous that delivers the harvest and frees the catalyst.The catalyst is not consumed or modify during the reception so, in principle, it pot be apply inde? nitely to convert the substratum into product in practice, however, this is limited by the stableness of the catalyst, that is, its electrical condenser to retain its combat-ready structure through time at the conditions of reaction. Biochemical reactions, this is, the chemical reactions that comprise the transfiguration of all financial backing cells, need to be catalyzed to proceed at the charge per unit required to sustain life. Such life catalysts argon the enzymes. Each one of the biochemical reactions of the cell metabolism requires to be catalyzed by one speci? enzyme. Enzymes atomic number 18 protein molecules that have evolved to action ef? ciently under the mild conditions required to preserve the functionality and practice of lawfulness of the biological systems. Enzymes screw be considered thusly as catalysts that have been optimized through evolution to perform their physiologic task upon which all forms of life depend. No wonder wherefore enzymes be capable of performing a wide reach of chemical reactions, umpteen of which extremely complex to perform by chemical synthesis. It is not presumptuous to decl ar that any chemical reaction already described might have an enzyme able to catalyze it.In fact, the practicable primary structures of an enzyme protein composed of n aminic group sulphurous residues is 20n so that for a rather undersize protein molecule containing 100 amino deadly residues, thither be 20100 or 10130 possible School of Biochemical Engineering, Ponti? cia Universidad Cat? lica de Valpara? so, Avenida brazil nut o ? 2147, Valpara? so, Chile. Phone 56-32-273642, fax 56-32-273803 e-mail email&160protected cl ? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Science + Business Media B. V. 2008 1 2 Trasition State A. Illane s Catalyzed row Uncatalyzed PathFree Energy Ea Ea Reactans ? G Products Reaction Progress Fig. 1. 1 Mechanism of catalysis. Ea and Ea be the energies of activation of the uncatalyzed and catalyzed reaction. ?G is the free energy change of the reaction amino corrosive dates, which is a fabulous enumerate, 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 lowly fraction of all living forms have been already isolated.It is even more promising when one considers the misfortune 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 localize- enjoin mutagenesis (Abi? n et al. 2004). a Enzymes have been naturally tailored to per form under physiological conditions. However, biocatalysis refers to the use of enzymes as process catalysts under arti? cial conditions (in vitro), so that a major(ip) challenge in biocatalysis is to transform these hysiological catalysts into process catalysts able to perform under the commonly 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 sequel of the reaction they promote. However, enzymes display kind of clear-cut halalties when comp atomic number 18d with chemical catalysts most of these properties ar a consequence of their complex molecular(a) structure and will be canvas in section 1. 2.Potentials and drawbacks of enzymes as process catalysts are summarized in Table 1. 1. Enzymes are highly sexually attractive catalysts when the speci? city of the reaction is a major secrete (as it occurs in pharmaceutical products and ? ne chemicals), when the catalysts must be active under mild conditions (because of substratum and/or product instability or to avoid unwanted side-reactions, as it occurs in several(prenominal) 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 natural process under moderate conditions High turnover number Highly biodegradable Generally considered as natural products Drawbacks High molecular complexity High business costs Intrinsic fragility 3 rather general situation that gives biocatalysis a distinct advantage over alternative technologies) or when the label of natural product is an issue (as in the case of solid 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 disadvantag es with respect to chemical catalysts (Bommarius and Broering 2005). eyepatch the advantages of biocatalysis are in that respect to stay, most of its present restrictions can be and are being processd through research and softenment in diverse 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 put-directed mutagenesis (Bhosale et al. 1996 Ogino et al. 2001 Boller et al. 2 002 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 help (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 wave in extreme environmental conditions are a promising etymon 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(prenominal)(prenominal) extremophiles have been cloned into suitable hosts to develop biological systems more amenable for producti on (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 application 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 murmur? H2O R1 H2O H R2 H3N CH C N CH COO? O + 1. 2 Enzymes as Catalysts. StructureFunctionality Relationships intimately 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 aci ds are covalently bound through the peptide bond (Fig. 1. 2) that is create between the carbon atom of the carboxyl group of one amino acid and the nitrogen atom of the ? -amino group of the side by side(p). accord to the nature of the R group, amino acids can be non- gelid ( hydrophobic) or diametrical ( charged or uncharged) and their distribution along the protein molecule assigns its behavior (Lehninger 1970). Every protein is teach by its amino acid sequence, called primary structure, which is genetically dictated by the deoxyribonucleotide sequence in the structural gene that codes for it.The DNA sequence is ? rst set down into a mRNA molecule which upon reaching the ribosome is translated into an amino acid sequence and ? nally the synthesized polypeptide chain is transformed into a terzettodimensional 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 pertinent 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 resolved, 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, in the first place 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 asunder in the primary structure that produce a compact and twisted con? guration in which the place 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 casefuls 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 charged atom with another electronegative atom.A hydrogen bond has approximately tenth part of the energy stored in a covale nt bond. It is the main determinant of the spiraling secondary structure of globular proteins and it plays a signi? cant role in tertiary structure as well. Apolar interactions, as a result of the joint 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 office to the stabilization of the threedimensional structure of a protein is quite signi? ant. Disulphide bridges, produced by oxidation of cysteine residues. They are especially relevant in the stabilization of the three-dimensional structure of low molecular weight extracellular proteins. Ionic bonds between charged amino acid residues. They contribute to the stabilization of the three-dimensional structure of a protein, although to a lesser conclusion, because the ionic strength of the surrounding medium is ordinarily high so that interact ion 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 moolahs (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 ( ordinarily covalently) to the apoenzyme and do not dissociate during catalysis. A signi? cant number of enzymes from eukaryotes are glycoproteins, in which case the carbohydrate moiety is covalently linked to the apoenzyme, mainly through serine or threonine residues, and eve n 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 station, which is usually formed by very few amino acid residues, while the rest of the protein acts as a scaffold.Papain, for instance, has a molecular weight of 23,000 Da with 211 amino acid residues of which only cysteine (Cys 25) and histidine (His 159) 6 A. Illanes are directly involved in catalysis (Allen and Lowe 1973). Substrate is bound to the enzyme at the active site and doing so, changes in the distribution of electrons in its chemical bonds are produced that cause the reactions that lead to the formation of products. The products are then released from the enzyme which is ready for the next catalytic cycle.According to the early toss out 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 substratum m olecule that ? ts into it. Even though recent reports provide evidence in favor of this guess (Sonkaria et al. 2004), this rigid model hardly explains many data-based evidences of enzyme biocatalysis. Later on, the induced-? t theory was proposed (Koshland 1958) harmonise to which he substrate induces a change in the enzyme conformation after binding, that may orient the catalytic groups in a way given over for the subsequent reaction this theory has been extensively utilize 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 use to refer to small mo lecular 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 cosubst judge, since they are stoichiometric in nature (Kula 2002). Coenzymes often function as intermediate carriers of electrons (i. e. nicotinamide type A dinucleotide+ or FAD+ in dehydrogenases), speci? c atoms (i. e. oenzyme Q in H atom fare) or functional groups (i. e. coenzyme A in acyl group transfer pyridoxal phosphate in amino group transfer vitamin H in CO2 transfer) that are transferred in the reaction. The term cofactor is commonly used to refer to metal ions that also bind reversibly to enzymes but in general are not chemically altered during the reaction cofactors usually bind strongly to the enzyme structure so that they are not dissociated from the holoenzyme during the reaction (i. e. Ca++ in ? -amylase Co++ or Mg++ in glucose isomerase Fe+++ in nitrile hydrat ase).According to these sine qua nons, 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 performance, this is, the capacity of an enzyme to catalyze a chemical reaction, is strictly dependent on its molecular structure. Enzyme natural action relies upon the existence of a proper structure of the active site, which is composed by a reduce number of amino acid residues close in the three-dimensional structure of 1 Introduction Fig. 1. 3 Enzymes according to their cofactor or coen zyme 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 propel 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 front end of chaotropic substances, heavy metals and chelating agents can produce this loss of function by distorting the proper active site con? guration. Even though a very small portion of the enzyme molecule participates in catalysis, the remaining of the molecule is by no means irrelevant to its performance.Crucial properties, like enzyme stability, are very much dependent on the enzyme three-dimensional structure. Enzyme stability appears to be determined by unde? ned irreversible processes governed by local unfolding in certain labile characters denoted as weak s pots. 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 affixd 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 rivet on and, even though properly selected, it is not clear what is the right type of mutation to introduce (Gaseidnes et al. 2003). Despite the impressive advances in the ? eld and the existence of some experimentally based rules (Shaw and Bott 1996), rational improvement of the stability is still far from being well established.In fact, the less rational approaches of dire cted 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 flux using a set of rationally designed mutants that can then be subjected to gene shuf? ing (OF? 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 stochastic 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 (OF? g? in 1997). These phenomena de? e what is called thermodynamic or cona a formational stability, this is the bulwark of the folded protein to denaturation, and kinetic or long-term stability, this is the resistance to irreversible inactivation (Eisenthal et al. 2006). The boilers suit 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 measuring rod 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 link 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 outgrowths with temperature but enzyme stability decreases. These opposite trends make temperature a comminuted variable in any enzymatic process and make it prone to optimization.This aspect will be thoroughly analyzed in Chapters 3 and 5. Enzyme speci? city is another relevant property of enzymes strictly related to its structure. Enzymes are usually very speci? c with respect to its substrate. This is because the substrate is endowed with the chemical bonds that can be attacked by the functional groups in the active site of the enzyme which posses the functional groups that anchor the substrate properly in the active site for the reaction to take place. Under certain conditions conformational changes may alter substrate speci? city.This has been elegantly proven by site-directed mutagenesis, in which speci? c amino acid residues at or near the active site have been replaced producing an revisal 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 interoperable signi? cance, since it is by no means an easy task to determine free energy changes for molecular structures as unstable as the enzymesubstrate complex. The direct 1 Introduction 9 consequence of such reduction of energy enter 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 si mple to determine so this de? nition is endowed with serviceableity.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 decisiveness of enzyme activity. To solve this ambiguity, the reasons underlying this behavior must be analyzed. The reduction in reaction rate can be the consequence of desaturation of the enzyme because of substrate transformation into product (at substrate depletion reaction rate drops to zero), enzyme inactivation as a consequence of the exposure of the enzyme to the conditions of reaction, enzyme inhibition caused by the products of the reaction, and equilibrium displacement as a consequence of the law of mass action.Some or all of these phenomena are present in any enzymatic reaction so that the catalytic capacity of the enzyme will vary throughout the course of the reaction. It is customary to identify the enzyme activity with the initial rate of reaction (initial slope of the p versus 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 compactness versus time of reaction at different enzyme tightnesss (e) 10 A. Illanes phenomena are insigni? ant. According to this a = vt0 = ? ds dt = t0 dp dt (1. 2) t0 This is not only of practical convenience but fundamentally sound, since the enzyme activity so de? ned represents its maximum catalytic potential under a given set of experimental con ditions. 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 varietys in which the enzyme protein can be a small fraction of the total mass of the preparation but, even in the whimsical case of a solely pure enzyme, the determination of activity is necessary 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 performing 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 chocolate-brown 1992), where the more amorphous portions of the cellulose moiety are more easily degenerate than the crystalline regions so that a high initial reaction rate over the amorphous portion may give an overestimate of the catalytic potential of the enzyme over the cellulose substrate as a whole. As shown in Fig. 1. 4, the initial slope o the curve (initial rate of reaction) is proportional to the enzyme intentness (it is so in most cases). Therefore, the enzyme prototype should be properly thin out to attain a linear product parsimony versus time kind within a reasonable check-out procedure time.The experiment al 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 address for their analytic 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 incomplete of both(prenominal) is readily measurable, enzyme activity can be determined by couplet reactions. In this case the product is transformed (chemically or enzymatically) to a ? nal analyte amenable for analytical determination, as shown E S P A X B Y C Z 1 Introduction 11 In this case enzyme activity can be determined as a = vt0 = ? ds dt = t0 dp dt = t0 dz dt (1. 3) t0 rovided that the rate limiting step is the reaction catalyzed by the enzyme, which implies that reagents A, B and C should be added in redundance to ensure that all P produced is quantitatively transformed into Z. For those enzymes requiring (stoichiometric) coenzymes E S CoE CoE P activity can be determined as a = vt0 = ? dcoe dt = t0 dcoe dt (1. 4) t0 This is actually a very convenient order 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 match system considering coenzyme determination is the assay for lactase (? galactosidase EC 3. 2. 1. 23). The enzyme catalyzes the hydrolysis of milk sugar according to lactose + H2 O Glucose + Galactose Glucose produced can be coupled to a unequivocal enzymatic glucose kit, that is hexoquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD ), in which Glucose + ATP ? Glucose 6Pi + automatic data processing 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 gizmo is provided with a recorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay in that case, the course of reaction should be monitored discontinuously by sampling and assaying at preset time intervals and ideals should be subjected to inactivation to stop the reaction.This is a drawback, since the enzyme s hould 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 p versus 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 rule experiment for discriminating the non-enzymatic build-up of product during the assay. There are essentially three options to remove the enzyme from the reaction mixture by replacing the enzyme sample by water or buffer, to remove the substrate replacing it by water or buffer, or to use an enzyme placebo. The ? rst one split ups substrate defilement with product or any non-enzymatic t ransformation of substrate into product, but does not discriminate enzyme befoulment 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 verify 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 foregoing to assay in this case, replacing substrate by water or buffer discriminates such contamination. If, on the other hand, one is assaying a preparation from a stock enzyme concentrate, dilution of the sample prior to assay makes unnecessary to blank out enzyme c ontamination 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 well(predicate) when the enzyme is labile enough to be completely inactivated at conditions not touch the assay. An alternative is to use a double control replacing enzyme in one case and substrate in the other by water or buffer. Once the type of control experiment has been decided, control and enzyme sample are subjected to the same analytical procedure, and enzyme activity is calculated by subtracting the control reading from that of the sample, as illustrated in Fig. . 5. Analytical procedures available for enzyme activity determinations are many and usually several alternatives exist. A proper selection should be based on sensibility, reproducibility, ? exibility, simplicity and availability. Spectrophotometry can be considered as a method that ful? ls most, if not all, such criteria. It is based on the abs orption of light of a certain wavelength as described by the BeerLambert law A? = ? l c where A? = log I I0 (1. 5) (1. 6) The prise of ? an be experimentally obtained through a calibration curve of absorbance versus concentration of analyte, so that the reading of A? will allow the determination of its concentration. optical path width is usually 1 cm. The method is based on the differential absorption of product (or coupling analyte or modi? ed 1 Introduction 13 Fig. 1. 5 Scheme for the analytical procedure to determine enzyme activity. S substrate P product P0 product in control A, B, C coupling reagents Z analyte Z0 analyte in control s, p, z are the corresponding molar concentrations oenzyme) and substrate (or coenzyme) at a certain wavelength. For instance, the reduced coenzyme NADH (or NADPH) has a strong peak of absorbance at 340 nm while the absorbance of the oxidize coenzyme NAD+ (or NADP+ ) is negligible at that wavelength therefore, the activity of any enzyme pro ducing 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(prenominal) 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 (? avi n 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 touchstone research laboratory.Enzymes that produce or consume gases can be assayed by differential manometry by measuring small pressure differences, due to the expending of the gaseous substrate or the evolution of a gaseous product that can be converted into substrate or product concentrations by using the gas law. Carboxylases and decarboxylases are groups of enzymes that can be conveniently assayed by differential manometry in a respirometer. For instance, the activity of glutamate decarboxylase 14 A. Illanes (EC 4. 1. 1. 15), that catalyzes the decarboxylation of glutamic acid to ? aminobutyric acid and CO2 , has been assayed in a differential respirometer by measur ing the increase in pressure caused by the formation of gaseous CO2 (OLearys 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 Biots law ? = ? 0 l c (1. 7) Polarimetry is a simple and hi-fi method for determining optically active compounds. A polarimeter is a low cost instrument readily available in many research laboratories. The detector can be integrated into an HPLC system if disengagement of substrates and products of reaction is required. Invertase (? -D-fructofuranoside fructohydrolase EC 3. 2. 1. 26), a commodity enzyme astray 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 ? 14 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 care of the planetary heart and soul of Biochemistry recommends to express it in international units (IU), de? ning 1 IU as the make out of an enzyme that catalyzes the transformation of 1 mol of su bstrate per minute under measuring conditions of temperature, best 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 discharge the rate of reaction by 1 mol/second in a speci? ed assay system (Anonymous 1979). This latter de? nition, although recommended, has some practical drawbacks. The magnitude of the katal is so big that usual enzyme activities expressed in katals are extremely small numbers that are hard to appreciate the de? ition, on the other hand, is rather vague with respect to the conditions in which the assay should be performed. In practice, even though in some journals the use of the katal is mandatory, there is reluctance to use it and the former IU is still more widely used. 1 Introduction 15 Going back to the de? nition of IU there are some points worthwhile to comment. The magnitude of the IU is appropriate to measure most enzyme preparations, whose activities usually range from a few to a few thousands IU per unit mass or unit volume of preparation.Since enzyme activity is to be considered as the maximum catalytic potential of the enzyme, it is quite appropriate to refer it to optimal pH and optimal substrate concentration. With respect to the latter, optimal is to be considered as that substrate concentration at which the initial rate of reaction is at its maximum this will imply reaction rate at substrate saturation for an enzyme following typical Michaelis-Menten kinetics or the highest initial reaction rate honor 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 tru e 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 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 p versus t relationship for the time of assay. For the de? nition of IU it is then more appropriate to refer to it as a standard and not as an 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 alter and often unde? ned molecular weight and whose products are usually a mixture of different chemical compounds. In that case, units of activity can be de? ned in terms of mass rather than moles. These enzymes are usually speci? c for certain types of bonds rather than for a particular chemical structure, so in such cases it is advisable to express activity in terms of equivalents of bonds broken.The choice of the substrate to perform the enzyme assay is by no means trivial. When using an enzyme as process catalyst, the substrate can be different from that employed in its assay that is usually a model substrate or an analogue. One has to be cautious to use an assay that is not only simple, veracious and reproducible, but also signi? cant. An example that illustrates this point is the case of the enzyme glucoamylase (exo-1,4-? -glucosidase EC 3. 2. 1. 1) this enzyme is widely used in the production of glucose syrups from starch, either as a ? al product or as an intermediate for the production of high-fructose syrups (Carasik and Carroll 1983). The industrial substrate for glucoamylase is a mixture of oligosaccharides produced by the enzymatic liquefaction of starch with ?-amylase (1,4-? -D-glucan glucanohydrolase EC 3. 2. 1. 1). Several substrates have been used for assaying enzyme activity including high molecular weight starch, small molecular weight oligosaccharides, 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 guess the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a transesteri? cation reaction that produces thiophenol as one of the products (Han et al. 004) in this case, a method based on a reaction leading to a ? uorescent adduct of thiophenol is a good system to assess the transesteri? cation potential of such proteases and is to be preferred to a conventional protease assay 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) int o 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 related number of identi? cation within a subgroup. The six families are 1. Oxidoreductases. Enzymes catalyzing oxidation/reduction reactions that involve the transfer of electrons, hydrogen or oxygen atoms.There are 22 subclasses of oxido-reductases and among them there are several of technological signi? cance, such as the dehydrogenases that oxidize a substrate by transferring hydrogen atoms to a coenzyme (NAD+ , NADP+ ,

Many Are the Crimes

The period of McCarthyism brought strong anti-communist campaign in the United States which started in the 1940s and ended in the 1950s. This date was to a fault known as the Second Red Scargon. There were fears of commie percolation on the Statesn organizations and espionage by Soviet agents (Fried, 124).It was a troubled sequence during the McCarthy era where many Americans where charged of being Communists or being Communist sympathizers. Many of them went through a series of investigations and doubtfulnesss by government committees and agencies. These investigations were order at individuals who work for the government, those in the entertainment business, confederacy members and educators. Although evidences were weak and a lot exaggerated, suspicions were given more weight. As a result, many Americans lost their jobs and almost were even incarcerated (Fried, 150).In Ellen Schreckers arrest Many be the Crimes, the occasion describes the persecution of the Communist Par ty in the U.S. from the 1920s until the 1950s. Schrecker believed that McCarthyism contributed to the downfall of Communism in America and thrusted the country into a gulf of right-wing sentiments which plagues the U.S. until now ( Reeves, Are You this instantA new study of McCarthyism and the legacy of HUAC, 1998).The first trine chapters of Schreckers book explain the Communist Partys subject in the 1930s in America. She explained why the Communist Party was supersensitive to attacks by the U.S. government. The second chapter Red Baiters, Inc. is an extensive analysis of people and institutions which characterized anticommunism in the 1920s and 1930s (Schrecker, 41).The author outlines Franklin D. Roosevelts stance on Communism. tally to Schrecker, President Roosevelts approach to Communism was non-ideological (p. 87), on that point were occasions that he did not pay attention to the existence of the Communist Party and there were times that he supported political suppressio n. Roosevelt authorized the FBI below J. Edgar make clean to look into the Communist Partys movement because they saw it as a bane to national security (p. 89).In Chapter 4, Schrecker discusses the U.S. governments propaganda that the Communist Party was being influenced by Moscow.In Chapter 5, the author negotiation close to how Americans saw Communism as a national threat through subversion, espionage and sabotage (p. 181).In the third part of Ellen Schreckers book, explains the instruments of anti-communism and how the FBI under J. Edgar Hoover used different tactics to purge communists in America by wiretapping and surveillance (p. 239).Schrecker discussed the role of Senator John McCarthy in the anti-communist crusade, and his trim back for civil liberties (p. 265).The last chapters of the book Many Are the Crimes, Schrecker talks about the experiences of Clinton Jencks and members of the workers union and how they were persecuted and eventually lost their jobs. Schrecker explains the impact of McCarthyism in the American society, how it unmake the left and paralyzed the Communist Party (p. 369).In context, Schreckers book exposed the effects of the McCarthy era not only in governing but also in the entertainment business wherein the Hollywood list dictated who would produce movies and star in those films. Screenwriters, actors and actresses did not escape interrogation by the anticommunist committees and those found guilty of espionage were sent to jail (Whitfield, p. 194).The unions played an heavy part in those days because they talked about racial concerns in equipment casualty of equality. The union also provided support for womens issues. It showed the inequality in cost of salaries that female workers get and how they are poorly-paid in comparison with the male workers. The union also encouraged women to be strong leaders. These progressive attempts of bringing veer in the role of women in society and the work force were halted during t he McCarthy era (Cherny, p.10).For the most part the greatest damage done during the McCarthy era was the last of the American left and the decline of the Communist Party in America. In terms of social policy, McCarthyism interrupted the reforms needed for health insurance. The countrys cultural and intellectual life became stagnant because of censorship during the McCarthy era (Schrecker, The bequest of McCarthyism).The downfall of the McCarthy era was due to the decline of public support and administration decisions which upheld individual rights and freedom (Fried, p. 197).With the current situation in the U.S., people are more vigilant and aware of the effects of McCarthyism. This episode will litigate as a cautionary tale for future generations (Rosen, Could It Happen once again?).Works CitedCherny, Robert W., William Issel and Kieran Walsh Taylor. American Labor and the ColdWar Grassroots authorities and Postwar Political Culture. saucy Brunswick, NewJersey Rutgers Unive rsity Press, 2004.Fried, Albert. McCarthyism, The Great American Red Scare A Documentary History.Oxford Oxford University Press, 1997.Reeves, Thomas C. Are You Now.A New Study of McCarthyism and the Legacy ofHUAC. 14 June 1998. http//www.nytimes.com/books/98/06/14/reviews/980614.14reevest.html. Retrieved on 7 January 2008.Rosen, Ruth. Could It Happen Again? 12 May 2003. http//www.commondreams.org/views03/0512-01.htm. Retrieved on 7 January 2008.Schrecker, Ellen. The Legacy of McCarthyism. Retrieved on 7 January 2008.Schrecker, Ellen. Many Are the Crimes McCarthyism in America. Princeton, New JerseyPrinceton University Press, 1999.Whitfield, Stephen J. The Culture of the Cold War. Baltimore The Johns Hopkins UniversityPress, 1996.           

Bluetooth based smart sensor network Essay

Currently, huge electronic info repositories ar being maintained by banks and otherwise fiscal institutions. worth(predicate) bits of teaching atomic number 18 embed in these teaching repositories. The huge size of it of these info sources rent it im achievable for a man analyst to uprise up with inte liveing selective information (or patterns) that get out protagonist in the finis do abut. A number of commercial enterprises bewilder been quick to write out the tax of this concept, as a consequence of which the softw atomic number 18 merchandise itself for selective information archeological site is judge to be in excess of 10 1 thousand thousand USD. This paper is intended for those who would like to get aw be of the possible finishs of data exploit to enhance the performance of some of their core line of descent processes. In this paper discussion is about the broad aras of application, like attempt management, portfolio management, work, gu est indite and node armorial bearing, where data mine techniques evict be utilise in banks and other pecuniary institutions to enhance their air performance. INTRODUCTIONAs noesis is becoming more and more synonymous to wealth induction and as a strategy plan for competing in the grocery enthrone digest be no bump than the information on which it is base, the grandness of knowledge and information in todays business mess never be seen as an exogenous factor to the business. Organizations and singulars having approach to the decent information at the right moment, defend greater chances of being lucky in the epoch of globalization and cut-throat competition. Business Intelligence focuses on discovering knowledge from unhomogeneous electronic data repositories, both internal and external, to backup man better termination fashioning.  entropy mining techniques become important for this knowledge discovery from databases. In recent years, business intellig ence activity systems have played diametrical roles in helping organizations to fine tune the business goals such as improving guest retention, commercialize penetration, profitability and efficiency. In most roles, these insights are driven by analyses of diachronical data. Global competitions, dynamic markets, and rapidly decrease cycles of technological innovation give important challenges for the banking and finance exertion. Worldwide just-in- snip availableness of information allows enterprises to purify their flexibility. In fiscal institutions considerable developments in information engine room have led to huge demand for continuous depth psychology of resulting data.selective information mining piece of tail contribute to solving business problems in banking and finance by conclusion patterns, causalities, and correlations in business information and market prices that are non immediately apparent to managers because the volume data is in addition large or is generated too quickly to screen by experts. The managers of the banks whitethorn go a step further to find the sequences, episodes and periodicity of the deed deportment of their guests which may help them in actually better constituenting, targeting, acquiring, retaining and maintaining a profitable node base. Business Intelligence and data mining techniques cease as well as help them in poseing various classes of clients and come up with a class base product and/or pricing approach that may garner better r scourue management as well. The broad categories of application of data archeological site and Business Intelligence techniques in the banking and fiscal industry vertical may be viewed as follows bump worryManaging and metre of hazard is at the core of every financial institution. right aways major(ip) challenge in the banking and insurance world is therefore the executing of jeopardy management systems in order to identify, posting, and control busine ss exposure. here(predicate) character reference and market happen present the central challenge, one back observe a major change in the area of how to measure and deal with them, base on the advent of advanced database and data mining technology.( Other types of put on the line is also available in the banking and finance i.e., liquid state lay on the line, operational risk, or concentration risk. ) Today, integrated measurement of different kinds of risk (i.e., market and credit risk) is moving into focus. These all are based on rides representing single financial actors or risk factors, their doings, and their interaction with overall market, making this field loftyly important topic of research. Financial Market RiskFor single financial instruments, that is, stock indices, interest rates, or currencies, market risk measurement is based on bewilders depending on a come out of profound risk factor, such as interest rates, stock indices, or scotch development. O ne is interested in a functional form amid instrument price or risk and underlying risk factors as well as in functional dependency of the risk factors itself. Today different market risk measurement approaches exist. All of them rely on models representing single instrument, their behaviour and interaction with overall market. Many of this open fire but be built by using various data mining techniques on the proprietary portfolio data, since data is not publicly available and of necessity consistent supervision. Credit RiskCredit risk assessment is anchor component in the process of commercial lending. Without it the lender would be futile to make an objective judgement of weather to lend to the prospective borrower, or if how much charge for the loan. Credit risk management washstand be classified into two basic groupsCredit scoring/credit pass judgment Assignment of a client or a product to risk level. (i.e., credit approval) Behaviour scoring/credit rank migration compe ndium. Valuation of a customers or products probability of a change in risk level within a devoted snip. (i.e., default rate volatility) In commercial lending, risk assessment is ordinarily an attempt to assess the risk of passing to the lender when making a limited lending decision. Here credit risk eject quantify by the changes of value of a credit product or of a whole credit customer portfolio, which is based on change in the instruments ranting, the default probability, and recovery rate of the instrument in case of default. Further diversification effects influence the result on a portfolio level. Thus a major part of implementation and care ofcredit risk management system will be a typical data mining problem the modelling of the credit instruments value by means of the default probabilities, rating migrations, and recovery rates. triple major approaches exist to model credit risk on the effect level accounting analytic approaches, statistical prediction and option metaphysical approaches. Since large amount of information about client exist in financial business, an adequate way to build such models is to use their declare database and data mining techniques, fitting models to the business needs and the business up-to-the-minute credit portfolio.Portfolio ManagementRisk measurement approaches on an aggregated portfolio level quantify the risk of a set of instrument or customer including diversification effects. On the other hand, forecasting models give an induction of the expected re change shape or price of a financial instrument. Both make it possible to manage firm wide portfolio actively in a risk/return efficient manner. The application of modern risk possibleness is therefore within portfolio theory, an important part of portfolio management. With the data mining and optimization techniques investors are able to allocate capital crossways trading activities to maximize profit or minimise risk. This feature supports the ability to g enerate flock recommendations and portfolio structuring from user supplied profit and risk requirement. With data mining techniques it is possible to provide extensive scenario analysis capabilities concerning expected asset prices or returns and the risk involved. With this functionality, what if simulations of change market conditions e.g. interest rate and exchange rate changes) cab be run to assess impact on the value and/or risk associated with portfolio, business unit counterparty, or trading desk. Various scenario results outhouse be regarded by considering actual market conditions. Profit and loss analyses allow users to access an asset class, region, counterparty, or custom sub portfolio so-and-so be benchmarked against common international benchmarks. TradingFor the last few years a major topic of research has been the building of quantitative trading tools using data mining methods based on by data as  enter to predict short-term movements of important currencie s, interest rates, or equities. The goal of this technique is to spot prison terms when markets are cheap or expensive by identifying the factor that are important in determining market returns. The trading system examines the relationship between relevant information and piece of financial assets, and gives you buy or sell recommendations when they suspect an under or overvaluation. Thus, even if some traders find the data mining approach too machinelike or too risky to be used systematically, they may exigency to use it selectively as further opinion. Trading is based on the idea of predicting short term movements in the price/value of a product (currency/equity/interest rate etc.). With a reasonable gauge in place one may trade the product if he/she thinks it is going to be over wanted or undervalued in the coming in store(predicate). Trading traditionally is done based on the instinct of the trader. If he/she thinks the product is not priced properly he/she may sell/buy it. This instinct is unremarkably based on past experience and some analysis based on market conditions.However, the number of factors that even the most expert of traders locoweed account for are limited. Hence, quite often these predictions fail. The price of a financial asset is influenced by a descriptor of factors which can be more often than not classified as economic, political and market factors. Participants in a market observe the relation between these factors and the price of an asset, account for the current value of these factors and predict the futurity set to finally arrive at the future value of the asset and trade accordingly. Quite often by the time a trained eye detects these favourable factors, many others may have discovered the opportunity, decreasing the possible revenues differently. Also these factors in turn may be related to several other factors making prediction difficult. Data mining techniques are used to discover unfathomable knowledge, unknown patterns and reinvigorated overlooks from large data sets, which may be useful for a variety of decision making activity.With the increasing economic globalization and emendments in information technology, large amounts of financial data are being generated and stored. subjected to data mining techniques to discover hidden patterns and obtain predictions for trends in the future and the behaviour of the financial markets. With the immediacy offered by data mining, latest data can be mined to obtain crucial information at the earliest. This in turn would result in an improved market place responsiveness and sentiency leading to reduced costs and increased revenue. Advancements made in technology have enabled to create faster and better prediction systems. These systems are based on a combination of data mining techniques and artificial intelligence methods like Case Based Reasoning ( cosmic microwave background radiation) and Neural Networks (NN). A combination of such a for ecasting system together with a peachy trading strategy offers tremendous opportunities for massive returns. The value of a financial asset is dependent on both macroeconomic and microeconomic variables and this data is available in a variety of disparate formats. NN and CBR techniques can be applied extensively for predicting these financial variables. NN are characterized by learnedness capabilities and the ability to improve performance over time. Also NN can derive i.e. recognize modernistic objects which may be similar but not exactly identical to previous objects.NN with their ability to derive meaning from inaccurate data can be used to detect patterns which are otherwise too complex to be detected by valets. NN act as experts in the area that they have been trained to work in. these can be used to provide predictions for new situations and work in real time. Thus, historic data available about financial markets and the various variables can be used to train NN to simul ate the market. CBR methodology is based on reasoning from past performances. It uses a large repository of data stored as cases which would include all the market variables in this case. When a new case is fed in (in the form of a case containing the concerned variables), the CBR algorithm predicts the performance/result of this case based on the cases it has in its repository. Data mining techniques can be used to detect hidden patterns in these cases which may then be used for further decision making. CBR methods can be used in real time which makes analysis really quick and helps in real time decision making resulting in immediate profits. Thus data mining and business intelligence (CBR and NN) techniques may be used in conjunction in financial markets to predict market behaviour and obtain patterned behaviour to influence decision making. Customer profile and Customer Relationship ManagementBanks have many and huge databases containing transactional and other details of its cus tomers. Valuable business information can be extracted from these data stores. But it is unfeasible to support analysis and decision makingusing traditional query languages because human analysis breaks down with volume and dimensionality. Traditional statistical methods do not have the capacity and scale to analyse these data, and hence modern data mining methodologies and tools are increasingly being used for decision making process not plainly in banking and financial institutions, but across the industries. Customer profiling is a data mining process that builds customer profiles of different groups from the companys existing customer database. The information obtained from this process can be used for different purposes, such as mind business performance, making new merchandising initiatives, market segmentation, risk analysis and revising company customer policies. The advantage of data mining is that it can handle large amounts of data and learn inherent structures and patt erns in data. It can generate rules and models that are useful in enabling decisions that can be applied to future cases. Customer Behaviour Modeling (CBM) or customer profiling is a tool to predict the future value of an individual and the risk category to which he belongs to based on his demographic characteristics, lifestyle and previous behaviour. This helps to focus on customer retention. The two important facts that have important implication in selecting customer profiling methods are Profiling information can consist of many variables (or dozens of them). Majority of them are categorical variables (or non-numeric variables or nominal variables).Customer profiling is to characterize features of particular(prenominal) customer groups. Many data mining techniques search profiles of special customer groups systematically using Artificial Intelligence techniques. They generate accurate profiles based on beam search and incremental learning techniques. Customer profiling also uses many predictive modeling methods. Predictive modelling techniques applicable can be categorized into two broad approaches. They depend on the type of predicted information or variables, also called target variables. If the type of predicted set is categorical, classification techniques is preferred to be used. Classification MethodsIn this approach, risk levels are organized into two categories based on past default history. For example, customers with past default history can be classified into risky group, whereas the rest are placed as safe group. development this categorization information as target of prediction, Decision Tree and Rule Induction techniques can be used to build models that can predict default risk levels of new loan applications. Value Prediction MethodsIn this method, for example, instead of classifying new loan applications, it attempts to predict expected default amounts for new loan applications. The predicted values are numeric and thus it requir es modelling techniques that can wad numeric data as target (or predicted) variables. Neural Network and regression are used for this purpose. The most common data mining methods used for customer profiling are Clustering (descriptive) Classification (predictive) and regression (predictive) Association rule discovery (descriptive) and sequential pattern discovery (predictive)In CRM, data mining is frequently used to assign a score to a particular customer or prospect indicating the likelihood that the individual will practise in a particular way. For example, a score could measure the aptness to respond to a particular insurance or credit identity card offer or to switch to a competitors product. Data mining can be useful in all the ternion phases of a customer relationship-cycle customer acquisition, increasing value of the customer and customer retention. For example, a typical banking firm let say sends 1 million direct mails for credit card customer acquisition. Past rese arches have shown that typically 6% of such target customers respond to these direct mails. Banks use their credit risk models to classify these respondents in good credit risk and rotten credit risk classes. The proportion of good credit risk respondents is barely 16% out of the total respondents. So, as net result, roughly only 1% of the total targeted customers are converted into the credit card customers through direct mailing. Seeing the huge cost and effort involved in such marketing process, data mining techniques can significantly improve the customer conversion rate by more focused marketing. Using a predictive test model using decision channelise techniques like CHAID (Chi-squared Automatic Interaction Detection),CART (Classification And Regression Trees), Quest and C5.0 it can beanalyzed which customers are more probable to respond. And using this with the risk model using techniques like neural network can help build a test model. The way data mining can actually be b uilt into the CRM application is determined by the temperament of customer interaction. The customer interaction could be inbound (when the customer contacts the firm) or outbound (when the firm contacts customers). The deployment requirements are quite different. Outbound interactions such as direct Building Profitable Customer Relations with Data Mining, Herb Edelstein mail campaign involve the firm selecting the people whom to be mailed by applying the test model to the customer database. In other outbound campaigns like advertising, the profile of good prospects shown by the test model needs to be matched to the profile of the people the advertisement would reach. For inbound legal proceeding such as telephone or internet order, the application moldiness respond in real time. Therefore the data mining model is embedded in the application and actively recommends an action. In either case, one of the key issues in applying a model to new data set is the transformations that are made in building the model. The ease with which these changes are embedded in the model determines the productivity of deploying these tools. Marketing and customer careBecause high competitions in the finance industry, intelligent business decisions in marketing are more important than ever for better customer targeting, acquisition, retention and customer relationship. There is a need for customer care and marketing strategies to be in place for the success and survival of the business. It is possible with the help of data mining and predictive analytics to make such strategies. Financial institutions are finding it more difficult to locate new previously unsolicited buyers, and as a result they are implementing aggressive marketing program to vex new customer from their competitors. The uncertainties of the buyer make planning of new operate and media usage almost impossible. The classical solution is to apply subjective human expert knowledge as rules of thumb. Until recently, replacing the human expert by computer technology has been difficult.An interesting tool available in marketing and financial institution is analysis of clients data. This allows analysis and figuring of key indicators that help bank to identify factors that affected customers demand in the past and customer need in the future. randomness about the customers personal data can also give indications that affect future demand. In case of analysis of sell debtors and small corporations, marketing tasks will typically include factors about the customer himself, his credit record and rating made by external rating agencies. With the advent of data mining and business intelligence tools it has become possible for banks to strengthen their customer acquisition by direct marketing and assemble multi- channel contacts, to improve customer development by cross merchandising and up selling of products, and to increase customer retention by behaviour management.It is possible for the banks to use the data available to retain its best customers and to identify opportunities to sell them additional services. The profiling of all the valuable accounts can be done and the top most say 5-10 % can be appoint to Relationship Managers, whose job will be to identify new selling opportunities with these customers. It is also possible to bundle various offers to meet the need of the valued customers. Data mining can also help the banks in customizing the various promotional offers. For example the direct mails can be customized as per the segment of the account holders in the bank. It is also possible for the banks to find out thepr oblem customers who can be defaulters in the future, from their past payment records and the profile and the data patterns that are available. This can also help the banks in adjusting the relationship with these customers so that the loss in future is kept to its minimum.Data mining can improve the response rates in the direct mail campaigns as th e time required to classify the customers will be reduced, this in turn will increase the revenues, improve the sales force efficiency from the target group. Data mining helps the banks to optimize their portfolio of services, delivery channels. A record of past transactions can give useful insight to the bank and different locations /branches of like branch can also follow some patterns that when noticed can be used as past records to learn from and base the future actions upon.Data Mining techniques can be of immense help to the banks and financialinstitutions in this arena for better targeting and acquiring new customers, fraud undercover work in real time, providing segment based products for better targeting the customers, analysis of the customers secure patterns over time for better retention and relationship, detection of emerging trends to take proactive stance in a highly competitive market adding a lot more value to existing products and services and launch of new pr oduct and service bundles. Reference

Globalisation Is Becoming A Normal Word In Todays Business Environment Economics Essay

globalization is going a normal word in today s concern purlieu where diametric recounts be incorporating with unriva take an youngly(prenominal). No say bathroom be self sufficient, they depend on unriv altogethered an other(a). It is unfeignedly common to suffer diverse states traveling from their ain states and put to other country/countries in order to acquire markets or resources such(prenominal) as inexpensive patience. raise boundaries ar no longer an extend due to advancement in engineering.globalization plays a great function on the stinting maturement of different states in the worldly concern particularly in the armed states such as US which has investings in different states around the Earth. On the other side of the coin globalization has negative everyudes on societal, cultural, semipolitical, scientific, environmental both piece of music good as stintingalal activities of different states.The purpose of this probe is to asses globalizatio n pertains on the emergent scotch outlines, whether it is an chance for their growing or a menace. china result be used as the tooshie of this analysis. The essay willing concent account on what is globalization, its number atomic number 53 woods, functions of globalization any second gear good as the manner it is incorporated in supra interior(a) take. It will in like manner concentrate on the emerge frugal bodys any trashe good as analyzing why China has opened doors to globalization.Title The violation of globalization to acclivitous market frugal systemsResearch inquiry What is the opposition of globalization on emerging frugal systems?1.0 IntroductionOver the one-time(a) ages it has been witnessed that the expression of societal, cultural, political, technological all bit good as frugalal activities of different states incorporating with one another has increase at a fast rate. The patterns that atomic number 18 found in the atomic number 63an states and the United States ar promptly found in a lot at any state in the humankind. This phenomenon is today normally referred to as globalization.Some bookmans take away defined globalization merely as the procedure of interaction and integrating among the citizenry, companies, and authoritiess of different states, a procedure drive by inter field trade and investing and back up by selective learning engineering. This procedure has effects on the environment, refinement, political systems, economic development and prosperity, and human physical wellbeing in societies around the universe.Globalization is non a new phenomenon, for century s persons, later companies, establishments and corporations comport been trade with each other in locations that are enormously far from their closure of beginning. The Asians for metaphor, used the monsoon winds that occurred by and by a infinite of six calendar month s to travel from their states and range Africa to swop i.e. purchase and sell merchandises to the Afri deals. China and europium were besides attached during the in-between ages through the celebrated Silk Road crossways Central Asia. This allowed the ii parties to put to one another which were an facet of globalization. This depicts the particular that for a long dress out globalization was in being hardly non recognised every bit today as globalization ( Jagdish, 2004 ) .This topical state of affairs of the phenomenon of globalization increa chirp at a fast rate has been induced by policies that hurt opened economic systems internally ( domestically ) every bit good as supranationally. One of the octogenarian-timer propagators of this was the wake of the 2nd universe war whereby authoritiess of different states in the universe decided to accept or implement the free market economic system which had an consequence on the productive potencies of their states and coevals new chances for wandering trade i.e. the trade was no longe r domestic oriented alone supranationally oriented. This is to state that the policies opened up chances for international trade and investings. The G everywherenments establish farther negotiated the enormous decreases in barriers to commerce and cargo area established international understandings to advance trade in goods, services, and investings. These pick up opened up new chances in foreign markets and on that pointfore corporations need built foreign mill about and established production and selling agreements with foreign spouses. This is a defining property of the late magnetic inclination of globalization, i.e. it is an international industrial and fiscal concern manifestation ( Jagdish, 1993 ) .The new chances mystify make other states that had ceased them to be dominant in the Global economic system today. Now more than than than of all time, it is a clear image that the facet of globalization has been one of the major subscribers to the chute in the econo mic laterality of galore(postnominal) economic systems such as those in the Asiatic states i.e. India, China etc. To day of the month China has right away integrated with practically every state around that universe. The Chinese corporations confound come to be so powerful to the extent that the ground forces is in debt of more that 10bilion dollars to the Chinese Bankss. The latter is to state that the phenomenon of globalization has t4remendous impacts on emerging economic systems.This essay will therefore discourse the impact of globalization in the emerging market economic systems in so making, the essay will concentrate on the construct globalisation, the drivers for globalisation, the cardinal characteristics of emerging economic systems, the impacts of globalisation socially, economically, environmentally and technologically on the emerging economic systems and crimsontually do a decision of the treatment.2.0 Definition and Concept2.1 What is acclivitous Market Economy ? emerge market economic systems are those economic systems that their economic system and industrialization grow at a repairy gait while sing a rapid addition in information efficiency in an environment. These economic systems are the leaders among exploitation states. To be more precise the followers are take oned to be the winning emerging market economic systems Brazil, Russia, India, and China, usually referred top as the BRIC states. The pickings one is China due to high growing of its GDP, engineering every bit good as literacy degree, approximately 93.3 % of Chinese entire tribe are literate, this became possible since the Chinese authorities exe lop offed its scheme of prioritizing counselling for its community from lower to the higher degrees ( hypertext transfer protocol //en.wikipedia.org ) .Discussed below will be the features of emerging market economic systems.2.2 The features of emerging economic systemstransitionalThe economic system of the emerging economic systems are invariably in transformational procedure from closed to an unfastened market, callking to brace their economic exoteric presentations for conveying efficiency and transparence in the cap market.Reform in exchange rate systemInternational Monetary Fund and initiation Bank assist the Emerging Market Economies in reforming their exchange rate systems in order to cut down flow of domestic capital to foreign economic systems since there is an addition of local every bit good as foreign investings in footings of portfolio and direct.Attractive to Multinational corporationsThe states in this class are really much on the list of the easy environments for investing by the MNC because of the ability to supply lower costs of labor and supplying a big client base.Large PopulationThe offshoot characteristic of these economic systems is the population in their state. The emerging economic systems have a big figure of people in their states as compared to other states in the univ erse. In the concern footings it means they contain a really big parcel of the consumer base in their ain state. China for interpreter is the most populated state ion the universe, and India is besides one of the individual states that have many people as about the full African continent. mettlesome Gross Domestic Product GrowthThese economic systems have a singular rate of growing on their GDP. China has been estimated to stop a 10 % growing rate in the last decennary. This rate makes it the winged turning economic system in the universe and in old ages to come it may receive the taking economic system in the universe above the United States of America. Much of the growing nevertheless had been due to having Foreign Direct Investments from the Triads ( USA, Japan and Europe ) , thought in the new-fangled old ages the BRIC group has besides been puting in the Triads.Enormous Changes in deportment manner and bettering criterion of lifeThe states with the emerging economic syst ems are sing an addition in the vicissitude in the life manner of its population. on that point is a enormous alteration in footings of industrialisation, modernisation every bit good as urbanisation. The rural countries are gnawing and more metropoliss with tall edifices and many investings are going prominent. Some of the wealthiest concerns and concern adult male are found in these emerging economic systems. Most of the states populations are more into going modern and more urbanised now than old ages back. moreover the criterion of life has improved markedly in these emerging economic systems. many another(prenominal) 1000000s continue to populate in poorness simply a turning urban in-between category provides an spread outing market for both domestic merchandises and for moments from abroad.Volatility of assetsThe universe s involvement for the assets of the emerging market economic systems has risen over the past old ages. The assets of the Emerging economic systems besid es tend to go volatile at times, the returns is real deserving put on the lining for. The financess of the Emerging economic systems have besides resulted in a much higher per centum of wealth.The construct of GlobalisationGlobalization has been a construct narrated for many old ages by different bookman s universe broad. Assorted dimensions come to visible radiation when the status globalisation is mentioned. Basically the issue of integrating, in footings of the economic system, engineering, societal facets every bit good as political relations is of highest consumeation when one defines globalisation. Globalization is the system of interaction among the states of the universe in order to develop the nomadic economic system.Globalization has been refers to the integrating of economic sciences and societies all over the universe. Globalization involves technological, economic, political, and cultural exchanges do possible mostly by progresss in communicating, transit, and subs tructure. ( Croucher, 2004 ) .Others nevertheless as antecedently mentioned have defined globalisation as the procedure of interaction and integrating among the people, companies, and authoritiess of different states, a procedure drive by international trade and investing and aided by information engineering. This procedure has effects on the environment, on civilization, on political systems, on economic development and prosperity, and on human physical wellbeing in societies around the universe. ( http //hubpages.com/hub/Definition-of-Globalization )Globalization efforts to picture the stairss by which the webs in the universe in footings of communicating, transit and work cause the linking ( integrating ) of the regional economic systems, societies, civilizations, every bit good as engineerings. At this point in curb most of the universe is sing the facet of economic globalisation as one of the individual most important facet of globalisation. With this in bearing economic gl obalisation has been individually defined as the linking of economic systems of different states to make an international economic system via trade, FDIs, Investment hard up-to-dateness flows, and the disperse of engineering. Globalization is normally recognized as being driven by the brotherhood of the economic, technological, socio-cultural, political, environmental and biological featureors.For the intent of this essay, we therefore square off the term globalisation as the rapid addition in the interconnectedness of the different states economically, socially, politically, technologically every bit good as environmentally to resemble a individual small town the procedure exceedingly being facilitated by the addition in the information and communicating engineering.2.1 Drivers of globalisation.thither are many issues that have been discussed when it comes to why is there such an dire addition in globalisation and why many states are hypothesis their doors to this phenomenon much easy than earlier. One of the most common account to this has been summarized in one common and still alone disapprobation. That is globalisation is inevitable intending it will happen, whether one likes it or non it will go on. Never the less that sentence is excessively short to supply the nucleus drivers of globalisation but quite merely explicate that the phenomenon is at that place to remain. The drivers for globalisation can be put into the undermentioned classsMarket driversCost driversTechnology driversGovernmental drivers hawkish driversThese drivers are better explained belowMarket driverThis really refers to when the companies consider the assorted markets to put. The shifting of the policies of the different authoritiess in the universe top belongings a free market economic system has in fact lead to concerns that had a market that was limited to one state to hold a planetary market that waits for the specific merchandises or services that they offer. At thi s point in companies in different split of the universe have to greater handiness of the different states universe broad. If there was a tendency of no free market economic system, the phenomenon of globalisation would hold been speed uping at a vey little gait and the major transmutations will non hold been recognized. At this point in clip there is the meeting of national markets into a remarkable monolithic planetary market place. To sell internationally is now easier due to falling of barriers on the cross-border trade. A company does nt hold to be the size of these transnational giants to ease and profit from the globalisation of markets. ( Owens 2008 ) take Cost driverThis becomes a driver when it occurs that the costs of production in your state is greater than in another state for the same merchandise, therefrom it becomes more advantageous for you to bring forth in another state than in your ain. It refers to the sourcing of goods and services from locations around the un iverse to take advantage of national differences in the cost and quality of factors of production. The thought is to vie more efficaciously offering a merchandise with good quality and low cost. Companies consider the assorted life style of the state before sing the monetary value of the merchandise and services to render. The companies that find themselves in hunt for international trade chances need to take into history the cost deductions associated to where they want to put. For illustration one might earn bring forthing certain merchandises in Tanzania than in Europe due to cheaper labor costs etc. ( Owens 2008 )Technology driverThis really refers to when there is change magnitude engineering system, transit, progressing in the degree of universe trade system. The have been many developments in engineering to day of the month and the rate seems unstoppable. These developments or alterations in scientific have achieved progresss in communicating, information processing, and transit engineering, including the Internet and the domain of a function huge Web ( World Wide Web ) . The most of import invention has been development in the microprocessors after that planetary communications have been revolutionized by developments in orbiter, optical fibre, and intercommunicate engineerings, and now the Internet and the World Wide Web. The rapid growing of the profit and the associated World Wide Web is the latest look of this development. Besides, inventions have occurred in the field of the transit engineering. The development of commercial jet aircraft has trim back the clip needed to acquire from one location to another. Now China is close at hand(predicate) to the USA than of all time. ( Owens 2008 )Government driverThis refers to the decrease of trade duties and non trade duties, as a consequence of cut downing the function of political policies. As antecedently mentioned the acceptance of the free market economic system has basically causes the aut umn of barriers to international trade. Now houses are able to see the full Earth as its possible market. The lowering of barrier to merchandise and investings besides allows houses to establish production at the optimum location for that activity. A house might hence, design a merchandise in one state, create a part/ constituent parts in two other states, assemble the merchandise in another state and so export the finished merchandise around the universe. The lowering of trade barriers has facilitated the globalisation of production. The grounds besides suggests that foreign direct investing is playing an increasing function in the planetary economic system. ( Croucher, 2004 ) .Competition driverThe facet of better merchandises as a consequence of competition has really made the facet of globalisation an indispensable portion in many economic systems. The competition among companies ensures that there is a production of high quality merchandises globally ( Owens 2008 )The impact of globalisationOver the old ages different arguments have existed on whether states =should embracing or be opposed to the phenomenon of globalisation. This argument is pioneered by the credibly impact the globalisation has on the assorted economic systems or states in the universe. on that point has been an statement that the facet of globalisation favoring the already real states and on the job(p) the developing states. Below therefore is the treatment on the impact of globalisation on the emerging economic systems.The impact of globalisation can be focused on the five chief classs which are Economic impactHarmonizing to economic experts, there are a great deal of planetary events connected with globalisation and integrating. The economic system of a state relies a batch on the concern environment that exists. Whether there is high domestic trading or there is a wider scope of investings from international companies. Globalization has enabled the economic systems of different states top become incorporate. For illustration, the avenue of international trade now allows states to hold foreign currencies into their economic systems. furthermore the facet of citizens being in one state and having belongings in another state is besides a world. The issues of loaning has now moved to a whole new degree, companies from one state ask for loans from Bankss in a different state every bit good as Bankss of one state can now set subdivisions in other counties which automatically affects the economic systems. Governments of one state travel and obtain loans from other states. A typical and yet most astonishing scenario is the fact that the American authorities being in 1000000s of debt to china which is an emerging economic system.No uncertainty globalisation has increased the foreign direct investings in different states. At this point in clip the Mc Donald s company of the United States of America had made a record entry to the Russian and Chinese economic system when the policies of these states had allowed room for the free market economic system.The GDP of states such as Chinas have benefited a batch as a consequence of globalisation. As mentioned earlier China s GDP has been turning at a rate of 10 % , one of the fastest turning rates in the universe.Socio-cultural ImpactAnother noteworthy impact of globalisation has been on the civilization of assorted societies in the universe. Globalization has been seen as a accelerator for alteration in the civilizations of less developed states to be more like those of the developed states. More specifically it is seen as an infliction of the pop civilization ( westerly civilization ) to other states. For illustration the manner in which people talk, act, dress etc has changed over the last few decennaries. The character of music that people hark to globally has changed even the ethical motives of certain societies have changed. Old ages back, it was terrible for adult females to have on minis kirts in the streets of Arabic states, but now this is going problematic. Even in African states some facets are altering.Many people are waiving their heritage for the new life manners which are believed to be more modernised. Now people believe that if you speak your native Australian linguistic communication and do non cognize how to talk English, so you are crude. All these are a consequence of globalisation. The manner in which people communicate has now besides changed. Peoples have conversations via the phone and hold reduced the more traditional manner of communicating which was to see and see each other physically. No longer are people playing athleticss outside, they instead play video games, ticker films in the house, all these are a consequence of globalisation. Furthermore there is a alteration in the type of ownership in the emerging economic systems.The facet of globalisation has introduced a more capitalist system in states that were chiefly involved in communal own ership the facet of ownership has become more of individual nature. This being the instance, there is no longer equal distribution of income among the people of China. This has benefited some of the members who believed that they deserved more for the more work they did but at the same clip has created a division among the rich and the hapless that was non at that place in the yesteryearEnvironmental ImpactThe environment is more of the less discussed factors at times but is ne of the most of import facet that needs to be considered when looking upon the impacts of globalisation. The environment fundamentally refers to every thing that surrounds us. In the globalized universe more and more concern chances have emerged for the different concerns in the universe as a effect the concerns that are engaged in fabrication and affect the emanation of harmful substances have increased h=and have caused a devastation in the ozone cheat in different parts of the universe. Furthermore, the less developed states have suffered on the environment as the developed states have used globalisation as a function of dumping harmful merchandises from their states. There had been a suppression on several environmentally harmful merchandises in Europe as a consequence, the European companies sold the goods to states like Tanzania etc to non endure losingss. This was a agency of dumping their waste merchandises. On the other manus though, through globalisation, there are now campaigns all over the universe that relate to environmentally protection. International pacts on environmentally friendly productions have been signed by different states. The decrease in green house emanation pacts have been signed by many states in the universe being led by United provinces of America and China, the most extremely fouling states in the Earth.TechnologicalGlobalization has lead to the addition in the spread of engineering all over the universe. At this point in clip, practically all part s of the universe are awe-are of the bing engineerings every where. The engineering that is used in Europe is besides used in China and at times even found in Africa. Globalization has enabled the universe to make, modify different technological devices that were founded by some one else in the different portion of the Earth. The Nipponese have been known to modify different engineerings that they see bing in the USA. The Chinese are now the 1s who imitate all the bing engineerings anyplace in the Earth. The field of operations of globalisation has made the latest engineerings to be in every portion of the universe. Ranging from the latest nomadic phones, laptops, picture games and all other appliances, globalisation in one manner or the other has ensured that no 1 is leftover behind.Political/legalThe last facet that globalisation has force is on the issue of political and legal environment. Globalization has been at the centre in the increased international Torahs that are in being, the being on the planetary organisations that provide rules across the universe. The United Nations ( UN ) , the World Bank ( WB ) , the international pecuniary fund ( IMF ) and the similar. The increased interconnection of different counties has enabled the being of the organisations to harvests cut different states. For illustration some of the policies in the less underdeveloped states ( LDCs ) such as the Structural Adjustment Program have been influenced by the World bank, some have been influenced by the united states etc. This shows how globalisation has impacted the political and legal ambiance of a state. State losingss its sovereignty in globalisation as it is being scrutinized by the international states. For illustration all of the states are now looking at the policies of China and seek to quarrel some of the things that it is making. In so making, the sovereignty of the state is being disordered ( Croucher, 2004 ) .DecisionIn general, no 1 can deny the fact th at globalisation is inevitable. The issue is on how the states take the being of globalisation. Through the treatment one can see that there are benefits that are being seen from the being of the phenomenon, but there are besides damaging effects. All of these depend on the state and therefore the context in which globalisation takes topographic point. There issues that are related to the loss of a state s emancipation and sovereignty that play a cardinal portion on whether to handle or reject globalisation, neer the less globalisation will go on. In a brief overview though one can see that there are many economically related benefits that have been associated with emerging economic systems and hence likely cause for the increased faith in the phenomenon.The facets of the spread outing gross revenues i.e. where by the emerging economic systems are holding a wider market for its merchandises is a noteworthy ground for the reliance of globalisation. Now the states with emerging ec onomic systems are holding the markets in practically any state of the universe runing from Europe to Africa, the Middle East and America. Furthermore they have now been able to get resources from the assorted states that they have invested in. The credence in globalisation has besides reduced the hazards that have been associated with the investing in merely their state as there are many fertile chances in other states.