Hizballah Role In A Unified Middle East :: essays research papers fc

The Monroe Doctrine was developed because the United States and Britain were c whizz timerned over the possibility of European colonial expansion in the Americas. al-Jihad, or party of god, was developed along the same lines of separationism, because originally the Shiite Muslims began the organization as a revolt against Western influences and the Israelis occupation of Lebanon. The future of the Hizballah and the Islamic Resistance (the parties competitive wing) is unclear as well as the future of the entire fondness East. The Hizballah provide be forced to either adjust to the rest of the Middle East, or the organization get out have to be eradicated. In the event that the Middle East is unified and the Hizballah organization is able to adjust, the Hizballah will try and influence the rest of the area to take on a Monroe Doctrine approach to foreign affairs as well.The Islamic Resistance could abide a terrorist force even under a unified Middle East as long as the militia i s able to work with separate armed forces. In the past the Islamic Resistance has worked with the Irish Republic Army (IRA) in order to purchase arms, including surface-to-air missiles, rocket-propelled grenade launchers, machine guns, explosives, and detonators (Thomas 118). Although some(prenominal) of these groups have been known terrorist organizations (The IRA working for Sinn Fein), the birds of a feather policy was upheld because of mutual interests. Just like in the Monroe Doctrine the Hizballah worked with a once adversary, the IRA-a Western influence, in order to grapple with Israel over the colonization of the Lebanese land.Even under a Unified Middle East, the Hizballah party will continue to hold on to the belief that the Middle East is better off without any influence from the godless West. The party has already successfully conducted aims at the United States under the sponsorship of Iran and with the blessings of Syria (Jaber 21). In a Unified Middle East with two countries backing the party, the Hizballah is already steps ahead of other policy-making organizations. The principle of ridding Lebanon of the Israeli and Western influence will simply spread to ridding the Middle East of any outside influence. With this type of policy, an attack on one nation in the region will bring the other nations online to deal with and intervene if necessary to rid the area of outside influences.In order for the Hizballah party to build this modern Middle East doctrine, a strong political presence must be on hand to implement the policy.

Husbands Gender Ideology Essay -- Gender Roles, Women

In response to why women remain investing significantly more prison term in unpaid housework than men (see Shelton and John, 1996 Coltrane, 2000 for a thorough review) and specialised in types of housework, empirical work done by researchers in economics downplay focuses on relative resource approach that builds on Beckers model of exchange. Nevertheless, the economic approach is far from satisfactory in explaining why married women who ar financially independent perform more housework than their spouses. This brings the argument of gender ideology from the perspective of sociologists. The allocation of time among family members in the work that needs to be done, both in the market and in the folk, has important implications for the households consumption possibilities. Extending the benefits of labour peculiarity that documented in the standard economics textbook, Becker (1985, 1991) suggests that multiperson household often find it beneficial to specialise to some extent in the activities that they undertake, based on comparative advantage. A salient example of such intrahousehold specialisation is married men specialised in market work and married women in household production. This historically division of labour within households is arranged on the basis that women accumulate less human capital. Given womens relatively lower opportunity cost in work outside the home as compared to men, household members would arrange spouses labour in a manner that women should allocate more time to household labour and less to market work in order to yield a maximum utility for the family.However, women nowadays have acquired as much human capital as men be it in education, labour market experience, occupational attainme... ...economic dependence in housework performed between husband and married woman, Greenstein (2000) also found a U shaped pattern for women, in which breadwinner wives undertake a greater section of housework than their husbands and a reversed U shaped for economically dependent husbands. However, Greenstein emphasise the process of deviance neutralization instead of gender display in the division of housework. The author suggests that to neutralise a nonnormative provider role of women in the family, both husband and wife may restore to a traditional attitude to make up for gender deviance even if the relative resource approach suggests that the husband should share far more housework. With these theories and fact of the past as the backgorund, we turn our attention to the married couples in Kuching city to account for the asymmetrical distribution of household labour.

The Always Present Mother Essay -- essays research papers fc

The Always Present MotherThis is now bone of my bones and flesh of my flesh she shall be called woman, because she was taken out of man (Gen. 223). History has shown us that the Great Mother precedent has been with society since the beginning of time. Through stories, songs, poems and thoughts, man has always run aground the need for the Mother and the women that make this warning possible. Some be consider myths and legends, while others have been documented in history. Regardless of what they have done or thought to have done, they have made an impact on the way man foresees woman. I will discourse three women characters that play a persona in the develop archetype, and explain why these rolls are important to their culture. Demeter will be the first goddess in this examination on the mother archetype, followed by Isis. These women are man made stories, to try and help explain why certain things are the way they are. The last mother archetype I will discuss was a woman that is still worshipped today, and with the help of man made stories, she has become immortal. This woman is the Virgin Mary. Before this is discussed, I will explain what an archetype is and what traits and similarities one must have to become a Mother Archetype.The mother archetype is a term derived from a man by the build of Carl Gustav Jung. Jung was a predecessor of Sigmund Freud. According to Jungian psychology the archetypes of the collective unconscious are manifested in similar mythological motifs which are universal(Trachy and Hopkins 166). Jung defines a archetype in many ways.Archetypes appear in conscious as a universal and recurring image, pattern or motif representing a typical valet experience. Archetypal images come from the collective unconscious and are the basic concepts of religions, mythologies, legends and Artsthey emerge through dreams and visionsthey convey a sense of transpersonal power which transcend the swelled head. (Jung 46)The ego is the center of the consciousness and the base of the individuals experience of subjective identity. The mother archetype has definite qualities of her own, such as, life giving, wisdom, and nurturing traits. Not all the traits of the mother archetype are good. There is the evil side of the mother archetype as well, such as, darkness, witchery and death. All these aspect... ...left at the realization that they are thoughts. These goddess or mother archetypes are brought into our societies to teach man and woman the values in each other. To restore balance in ones lives. Demeter, she helped to explain grotesque occurrences in Greece as well as reveal the importance of womans traits to give life. Isis, she kept the Egyptian nation strong and united during her predominate by being everything at once. Mary, she has had mercy brought into the Christian beliefs against the sometimes stern male God. All three characters are important throughout history, and have managed to pass off their legends alive. Wo rks CitedEngelsman, Joan C. The Feminine Dimension of the Divine. Pennsylvania Westminster Press, 1997.Epiphanius, Heresies. In Mary In The Document of the Church. Palmer, P.F. ed. London Burns Oats, 1953.Holy Bible. New King James Version. capital of Tennessee Thomas Nelson, 1997.Hopkins, P., & Trachy, C.L. The Study of Story. North Carolina Hunter Textbooks Inc., 1996.Jung, C.G. Collected Works, Volume 9, Part1, Archetypes & the Collective Unconscious.London Routledge & Kegan Paul, 1968 (1933)

The involvement of the International Brigades in the Spanish Civil War :: European Europe History

The involvement of the outside(a) Brigades in the Spanish Civil WarOn the 18 July 1936, leading Generals of the Spanish Army led a revolt against the democratically elected Popular Front government of Spain. Within days the country was plunged into civil war with the Republicans flake the insurgent Nationalists for control of the country. The various democracies of the world turned their backs on Spains plight and even hindered the Republicans by supporting non-intervention in the conflict. However, many people came to help the Republic. Las Brigades Internacionales, the International Brigades, would eventually include almost 40,000 men and women from 53 different countries, from all around the world.The International Brigades began as an idea in July and August of 1936, but soon its formation became the main work of the Comintern (the body with the responsibility of fostering the world-wide spread of Communism). Each Communist party was instructed to raise volunteers who would be sent to Spain by call for or boat. Around 60% of the volunteers were Communists, but non-Communists were also welcomed. The first group of recruits came to Spain by train from Paris, and arrived at their base in Albacete, halfway between capital of Spain and Valencia, on the 14th of October. It was there that the 500 French, German and Polish recruits began training. The theme of the recruitment propaganda was based on the slogan that Spain should be The grave of European Fascism, and with this in mind volunteers continued to flow into Spain from France. One of the organisers of recruits in Paris was the future Marshal Tito - Joseph Broz. In Albacete the volunteers were organised into language groups and the base was put under the verify of Andre Marty. The Brigades were to be led by General Emilio Kleber and intensive training was to take place in the base before going to the front.The International Brigades baptism of fire came on the 8th of November 1936, when the XIth and XII th Brigades went to the Madrid front. They numbered about 3,500 men altogether, and were extremely important to the defence of Madrid. The fighting in Madrid eventually reached stalemate and the Brigades were transferred to other fronts. The XI, XIII and XV Brigades fought at the Brunete disgusting of July 1937, where losses were very high, and where Oliver Law, the Afro- American commander of the Lincoln Battalion was killed. The Brigades also played a major part in the Aragon offensive of August 1937, and were formally incorporated into the Republican Army around this time.

The Cartesian Doubt Experiment and Mathematics :: Mathematics Math Mathematical Papers

The Cartesian Doubt Experiment and MathematicsABSTRACT The view that Descartes c totallyed numerical propositions into doubt as he impugned all beliefs concerning common-sense ontology by assuming that all beliefs derive from perception seems to rest on the presupposition that the Cartesian problem of doubt concerning mathematics is an instance of the problem of doubt concerning man of substances. I argue that the problem is not whether I am counting actual objects or empty images, but whether I am counting what I count correctly. Considering Descartess early works, it is possible to see that for him, the proposition 2+3=5 and the argument I think, therefore I am, were equally evident. But Descartes does not found his epistemology upon the evidence of mathematical propositions. The doubt experiment does not seem to give positive results for mathematical operations. Consciousness of carrying out a mathematical proposition, however, unlike putting forth a result of an operation, is i mmune to doubt. Statements of consciousness of mathematical or logical operations are instances of I think and hence the argument I count, therefore I am is equivalent to I think, therefore I am. If impugning the veridicality of mathematical propositions could not pose a difficulty for Descartess epistemology which he thought to establish on consciousness of thinking alone, then he cannot be seen to debar the question. Discarding mathematical propositions themselves on the grounds that they are not immune to doubt evoked by a powerful agent does not generate a substantial problem for Descartes provided that he believes that he can justify them by appeal to Gods benevolence. The question whether Descartes impugned veridicality of mathematical propositions via the arguments of the First Meditation is of epistemologically significance for an inquiry into the genius of Descartes doubt experiment with a view to a plausible answer to this question may offer us clues to understand the na ture of Cartesian opening of justification and the nature of foundationalistic epistemology in general.The evil genius hypothesis introduced in the last paragraph of the First Meditation does not seem to call veridicality of mathematical propositions into question Descartes does not mention mathematical truths when he finalizes the setting of the doubt experiment. The text is ambiguous at this point and the reader is left ignorant whether dewy-eyed truths of arithmetics or geometry are held exempt from doubt evoked by the evil genius hypothesis. Does this final tool of the doubt experiment put emphasis on the dubitability of judgments of common-sense ontology based on sense perception alone?

The Cartesian Doubt Experiment and Mathematics :: Mathematics Math Mathematical Papers

The Cartesian Doubt Experiment and MathematicsABSTRACT The view that Descartes called mathematical propositions into doubt as he impugned all beliefs concerning common-sense ontology by assuming that all beliefs derive from perception reckons to rest on the presupposition that the Cartesian worry of doubt concerning mathematics is an instance of the problem of doubt concerning existence of substances. I argue that the problem is not whether I am counting actual objects or empty images, but whether I am counting what I count correctly. Considering Descartess early works, it is possible to see that for him, the proposition 2+3=5 and the argument I think, therefore I am, were equally evident. But Descartes does not build his epistemology upon the evidence of mathematical propositions. The doubt experiment does not seem to give positive results for mathematical operations. Consciousness of carrying out a mathematical proposition, however, unlike pose forth a result of an operation, i s immune to doubt. Statements of consciousness of mathematical or logical operations are instances of I think and hence the argument I count, therefore I am is tantamount(predicate) to I think, therefore I am. If impugning the veridicality of mathematical propositions could not pose a difficulty for Descartess epistemology which he thought to establish on consciousness of thinking alone, then he cannot be seen to avoid the question. Discarding mathematical propositions themselves on the grounds that they are not immune to doubt evoked by a powerful agent does not generate a substantial problem for Descartes provided that he believes that he can justify them by appeal to Gods benevolence. The question whether Descartes impugned veridicality of mathematical propositions via the arguments of the First Meditation is of epistemologically significance for an inquiry into the constitution of Descartes doubt experiment with a view to a plausible answer to this question may offer us clues to understand the nature of Cartesian theory of justification and the nature of foundationalistic epistemology in general.The evil genius hypothesis introduced in the last paragraph of the First Meditation does not seem to call veridicality of mathematical propositions into question Descartes does not mention mathematical truths when he finalizes the setting of the doubt experiment. The text is ambiguous at this point and the reader is leave ignorant whether simple truths of arithmetics or geometry are held exempt from doubt evoked by the evil genius hypothesis. Does this final tool of the doubt experiment repose emphasis on the dubitability of judgments of common-sense ontology based on sense perception alone?

Enzyme Biocatalysis

Enzyme Bio contact action Andr? s Illanes e Editor Enzyme Biocatalysis Principles and Applications 123 Prof. Dr. Andr? s Illanes e School of Biochemical techno put downy Ponti? cia Universidad Cat? lica o de Valpara? so ? Chile emailprotected cl ISBN 978-1-4020-8360-0 e-ISBN 978-1-4020-8361-7 Library of Congress Control Number 2008924855 c 2008 Springer Science + Business Media B. V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in every form or by any means, electronic, mechanical, photocopying, micro? ming, recording or otherwise, without write permission from the Publisher, with the exception of any material supp lied speci? c entirelyy for the purpose of universe entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid- openhanded paper. 9 8 7 6 5 4 3 2 1 springer. com Contents Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 1. 1 contact action and Biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2 Enzymes as Catalysts. StructureFunctionality Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 3 The Concept and finale of Enzyme Activity . . . . . . . . . . . . . . 1. 4 Enzyme Classes. Properties and Technological Signi? pukece . . . . . . . 1. 5 Applications of Enzymes. Enzyme as p wretched Catalysts . . . . . . . . . . . 1. 6 Enzyme Processes the Evolution from Degradation to deduction. Biocatalysis in Aqueous and Non-conventional Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme output signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 2. 1 Enzyme Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2 mathematical production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 1 Enzyme discount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 2 Enzyme reco truly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 3 Enzyme Puri? cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 4 Enzyme Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 8 16 19 31 39 57 57 60 61 65 74 84 89 2 3 Homogeneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Andr? s Illanes, Claudia Altamirano, and Lo rena Wilson e 3. 1 General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3. 2 Hypothesis of Enzyme Kinetics. De end pointination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. 2. 1 Rapid Equilibrium and Steady-State Hypothesis . . . . . . . . . . . 108 v vi Contents finis of Kinetic Parameters for Irreversible and Reversible One- subst ordain responses . . . . . . . . . . . . . . . . . . . . . 112 3. 3 Kinetics of Enzyme forbidding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. 1 Types of Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. Development of a Generalized Kinetic Model for One-Substrate Reactions low Inhibition . . . . . . . . . . . . . . . . 117 3. 3. 3 Determination of Kinetic Parameters for One-Substrate Reactions Under Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . long hundred 3. 4 Reactions with More than One Substrate . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 1 Mechanisms of Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 2 Development of Kinetic Models . . . . . . . . . . . . . . . . . . . . . . . . 125 3. 4. 3 Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . 131 3. 5 environmental Variables in Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . 133 3. 5. 1 Effect of pH Hypothesis of Michaelis and Davidsohn. Effect on Enzyme Af? nity and Re legal action . . . . . . . . . . . . . . . . 134 3. 5. 2 Effect of Temperature Effect on Enzyme Af? nity, Reactivity and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3. 5. 3 Effect of Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4 Hetero geneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Andr? s Illanes, Roberto Fern? ndez-Lafuente, Jos? M. Guis? n, e a e a and Lorena Wilson 4. 1 Enzyme Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 4. 1. 1 Methods of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 4. 1. 2 Evaluation of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . 166 4. 2 Heterogeneous Kinetics Appargonnt, Inherent and Intrinsic Kinetics Mass tilt Effects in Heterogeneous Biocatalysis . . . . . . . . . . . . . 169 4. 3 Partition Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 4. 4 Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 4. 4. 1 immaterial Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . 173 4. 4. 2 Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . 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 Basic Design of Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 1 Design Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 2 Basic Design of Enzyme Reactors Under Ideal Conditions. caboodle Reactor Continuous Stirred Tank Reactor Under Complete Mixing Continuous Packed-Bed Reactor Under Plug fly the coop Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 3. 2. 2 5 Contents vii Effect of Diffusional Restrictions on Enzyme Reactor Design and death penalty in Heterogeneous Systems. Determination of Effectiveness Factors. Batch Reactor Continuous Stirred Tank Reactor Under Complete Mixing Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 5. 4 Effect of thermic Inactivation on Enzyme Reactor Design and Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5. 4. 1 Complex Mechanisms of Enzyme Inactivation . . . . . . . . . . . 225 5. 4. 2 Effects of Modulation on Thermal Inactivation . . . . . . . . . . . . 231 5. 4. 3 Enzyme Reactor Design and Performance Under Non-Modulated and Modulated Enzyme Thermal Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . 234 5. 4. 4 Operation of Enzyme Reactors Under Inactivation and Thermal optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 5. 4. 5 Enzyme Reactor Design and Performance Under Thermal Inactivation and Mass Transfer Limitations . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 6 Study Cases of enzymatic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 6. 1 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . . . . . . . . 253 Sonia Barberis, Fanny Guzm? n, Andr? s Illanes, and a e Joseph L? pez-Sant? n o ? 6. 1. 1 Chemical Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . . 254 6. 1. 2 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . 257 6. 1. 3 Enzymatic Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . 258 6. 1. 4 Process Considerations for the Synthesis of Peptides . . . . . . . 263 6. 1. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 6. 2 Synthesis of ? -Lactam Antibiotics with Penicillin Acylases . . . . . . . 273 Andr? s Illanes and Lorena Wilson e 6. 2. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 2 Chemical Versus Enzymatic Synthesis of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 3 Strategies of Enzymatic Synthesis . . . . . . . . . . . . . . . . . . . . . . 276 6. 2. 4 Penicillin Acylase Biocatalysts . . . . . . . . . . . . . . . . . . . . . . . . . 277 6. 2. 5 Synthesis of ? -Lactam Antibiotics in Homogeneous and Heterogeneous Aqueous and Organic Media . . . . . . . . . . . . . . 279 6. 2. 6 Model of Reactor Performance for the Production of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . 282 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 6. 3 Chimioselective Esteri? cation of Wood Sterols with Lipases . . . . . . . 292 ? Gregorio Alvaro and Andr? Illanes e 6. 3. 1 Sources and Production of Lipases . . . . . . . . . . . . . . . . . . . . . . 293 6. 3. 2 Structure and Functionality of Lipases . . . . . . . . . . . . . . . . . . . 296 5. 3 viii Contents Improvement of Lipases by Medium and Biocatalyst Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 6. 3. 4 Applications of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 6. 3. 5 Development of a Process for the Selective Transesteri? cation of the Stanol Fraction of Wood Sterols with Immobilized Lipases . . . . . . . . . . . . . . . . . . . . . . 308 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 6. 4 Oxidoreductases as Powerful Biocatalysts for Green Chemistry . . . . 323 Jos? M. Guis? n, Roberto Fern? ndez-Lafuente, Lorena Wilson, and e a a C? sar Mateo e 6. 4. 1 Mild and Selective Oxidations Catalyzed by Oxidases . . . . . . 324 6. 4. 2 Redox Bio changes Catalyzed by De henryases . . . 326 6. 4. 3 Immobilization-Stabilization of Dehydrogenases . . . . . . . . . . 329 6. 4. 4 Reactor Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 6. 4. Production of Long-Chain Fatty Acids with Dehydrogenases 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 6. 5 Use of Aldolases for Asymmetric Synthesis . . . . . . . . . . . . . . . . . . . . . 333 ? Josep L? pez-Sant? n, Gregorio Alvaro, and Pere applaud? 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 Kinetic s . . 339 6. 5. 4 Synthetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 6. 5. 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 6. 6 Application of Enzymatic Reactors for the Degradation of Highly and Poorly Soluble fidgety Compounds . . . . . . . . . . . . . . . . . . . . 355 o Juan M. Lema, Gemma Eibes, Carmen L? pez, M. Teresa Moreira, and Gumersindo Feijoo 6. 6. 1 Potential Application of Oxidative Enzymes for Environmental Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 6. 6. 2 Requirements for an Ef? cient Catalytic Cycle . . . . . . . . . . . . . 357 6. 6. 3 Enzymatic Reactor Con? gurations . . . . . . . . . . . . . . . . . . . . . . 358 6. 6. 4 Modeling of Enzymatic Reactors . . . . . . . . . . . . . . . . . . . . . . . 364 6. 6. 5 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 6. 6. 6 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . 374 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 6. 3. 3 Foreword This harbour was written with the purpose of providing a sound basis for the creation of enzymatic reactions establish on energising principles, besides also to give an updated vision of the strengths and limitations of biocatalysis, especially with respect to recent applications in processes of organic tax deduction. The ? rst ? ve chapters atomic number 18 structure in the form of a textbook, going from the basic principles of enzyme structure and function to reactor design for homogeneous systems with soluble enzymes and heterogeneou s systems with immobilized enzymes.The last chapter of the book is dissever into half dozen sections that represent illustrative casing studies of biocatalytic processes of industrial rele wagon traince or potential, written by experts in the respective ? elds. We sincerely wish that this book allow for represent an element in the toolbox of graduate students in applied biology and chemical and biochemical engineering and also of lowgraduate students with statuesque training in organic chemistry, biochemistry, thermodynamics and chemical reaction kinetics. Beyond that, the book pretends also to illustrate the potential of biocatalytic processes with eccentric person studies in the ? ld of organic synthesis, which we hope pull up stakes be of interest for the academia and professionals involved in R&D&I. If some of our young readers atomic number 18 encouraged to engage or continue in their work in biocatalysis this will certainly be our more precious reward. ? a Too much has been written to the highest degree writing. Nobel laureate Gabriel Garc? a M? rquez wrote angiotensin converting enzyme of its nigh inspired books by writing about writing (Living to Tell the Tale). There he wrote life is not what one lived, but what one remembers and how one remembers it in order to recount it. This s natestily applies to a scienti? book, but certainly highlights what is applicable to any book its symbiosis with life. Writing about biocatalysis has given me that privileged feeling, even more so because enzymes ar truly the catalysts of life. Biocatalysis is hardly separable from my life and writing this book has been certainly more an transfer than an agony. A book is an object of love so who better than boosters to build it. Eleven distinguished professors and re lookupers bring on contributed to this endeavor with their k this instantledge, their commitment and their encouragement. Beyond our common language, I share with all of them a view and a life -lasting friendship.That is what lies behind this book and made its construction an exciting and rewarding experience. ix x Foreword Chapters 3 to 5 were written with the invaluable collaboration of Claudia Altamirano and Lorena Wilson, two of my former students, now my colleagues, and my bosses I am afraid. Chapter 4 also included the experience of Jos? Manuel Guis? n, e a Roberto Fern? ndez-Lafuente and C? sar Mateo, all of them very good friends who a e were kind enough to join this project and enrich the book with their world known expertise in heterogeneous biocatalysis. Section 6. is the end of a cooperation sustained by a CYTED project that brought together Sonia Barberis, also a former graduate student, now a successful professor and permanent collaborator and, beyond that, a dear friend, Fanny Guzm? n, a reputed scientist in the ? eld of peptide a synthesis who is my partner, stand up and inspiration, and Josep L? pez, a substantially-known o scientist and engineer but, above all, a friend at heart and a warm host. Section 6. 3 was the result of a joint project with Gregorio Alvaro, a dedicated researcher who has been a permanent collaborator with our group and also a very special friend and kind host. Section 6. is the result of a collaboration, in a very challenging ? eld of applied biocatalysis, of Dr. Guisans group with which we have a long-wearing academic connection and pie-eyed personal ties. Section 6. 5 represents a very challengo e ing project in which Josep L? pez and Gregorio Alvaro have joined Pere Clap? s, a prominent researcher in organic synthesis and a friend through the years, to build up an updated review on a very provocative ? eld of enzyme biocatalysis. Finally, section 6. 6 is a collaboration of a dear friend and outstanding teacher, Juan Lema, and his research group that widens the scope of biocatalysis to the ? ld of environmental engineering adding a particular ? avor to this ? nal chapter. A substantial part of this bo ok was written in Spain while doing a sabbatical in the o Universitat Aut noma de Barcelona, where I was warm hosted by the Chemical Engineering Department, as I also was during short stays at the Institute of Catalysis and Petroleum Chemistry in capital of Spain and at the Department of Chemical Engineering in the Universidad de Santiago de Compostela. My recognition to the persons in my institution, the Ponti? cia Universidad Cat? lica de Valpara? so, that supported and encouraged this project, particularly to o ? the rector Prof.Alfonso Muga, and professors Atilio Bustos and Graciela Mu? oz. n Last but not least, my deepest appreciation to the persons at Springer Marie Johnson, Meran Owen, Tanja van Gaans and Padmaja Sudhakher, who were always delicate, diligent and encouraging. Dear reader, the judicial decision about the product is yours, but beyond the product at that coif is a process whose beauty I hope to have been able to transmit. I count on your indulgence with langu age that, scorn the effort of our editor, may still reveal our condition of non- inseparable English speakers. Andr? s Illanes e Valpara? so, May 15, 2008 ? Chapter 1 Introduction Andr? s Illanes e . 1 Catalysis and Biocatalysis Many chemical reactions can occur spontaneously others require to be catalyzed to proceed at a signi? camber rate. Catalysts are molecules that reduce the order of magnitude of the dynamism barrier required to be overcame for a substance to be converted chemically into another. Thermodynamically, the magnitude of this energy barrier can be conveniently uttered in terms of the free-energy change. As depicted in Fig. 1. 1, catalysts reduce the magnitude of this barrier by virtue of its impelion with the substratum to form an activated enactment complex that delivers the product and frees the catalyst.The catalyst is not consumed or altered during the reaction so, in principle, it can be apply inde? nitely to convert the substrate into product in practi ce, however, this is limited by the stability of the catalyst, that is, its cognitive content to retain its vigorous structure through time at the conditions of reaction. Biochemical reactions, this is, the chemical reactions that comprise the metabolism of all living booths, need to be catalyzed to proceed at the pace required to sustain life. Such life catalysts are the enzymes. Each one of the biochemical reactions of the cell metabolism requires to be catalyzed by one speci? enzyme. Enzymes are protein molecules that have evolved to perform ef? ciently under the mild conditions required to preserve the functionality and integrity of the biological systems. Enzymes can be considered then as catalysts that have been optimized through evolution to perform their physiological task upon which all forms of life depend. No wonder why enzymes are fitted of perform a wide shop of chemical reactions, umteen of which extremely complex to perform by chemical synthesis. It is not pres umptuous to state that any chemical reaction al ca-ca described might have an enzyme able to catalyze it.In fact, the possible primary structures of an enzyme protein composed of n amino acid residues is 20n so that for a rather micro protein molecule containing 100 amino acid residues, there are 20100 or 10130 possible School of Biochemical Engineering, Ponti? cia Universidad Cat? lica de Valpara? so, Avenida brazil nut o ? 2147, Valpara? so, Chile. Phone 56-32-273642, fax 56-32-273803 e-mail emailprotected cl ? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Science + Business Media B. V. 2008 1 2 Trasition State A. Illanes Catalyzed Path Uncatalyzed PathFree Energy Ea Ea Reactans ? G Products Reaction Progress Fig. 1. 1 Mechanism of catalysis. Ea and Ea are the energies of activation of the uncatalyzed and catalyzed reaction. ?G is the free energy change of the reaction amino acid chronological successions, which is a mythological egress, higher even than the number of mole cules in the whole universe. To get the right enzyme for a certain chemical reaction is then a matter of search and this is certainly challenging and exciting if one realizes that a very small divide of all living forms have been already isolated.It is even more promising when one considers the possibility of obtaining DNA pools from the environment without requiring to know the organism from which it comes and then denotative it into a suitable host organism (Nield et al. 2002), and the opportunities of genetic remodeling of structural genes by site-directed mutagenesis (Abi? n et al. 2004). a Enzymes have been naturally tailored to perform under physiological conditions. However, biocatalysis refers to the use of enzymes as process catalysts under arti? cial conditions (in vitro), so that a study challenge in biocatalysis is to translate 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 consequence of the reaction they promote. However, enzymes display quite distinct properties when compared with chemical catalysts most of these properties are a consequence of their complex molecular structure and will be analyzed in section 1. 2.Potentials and drawbacks of enzymes as process catalysts are summarized in Table 1. 1. Enzymes are highly desirable catalysts when the speci? city of the reaction is a major issue (as it occurs in pharmaceutical products and ? ne chemicals), when the catalysts must be active under mild conditions (because of substrate and/or product instability or to avoid unwanted side-reactions, as it occurs in some(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 sp eci? ity High activity under moderate conditions High up treated number Highly biodegradable Generally considered as natural products Drawbacks High molecular complexity High production greets 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 issue of food and cosmetic applications) (Benkovic and Ballesteros 1997 Wegman et al. 2001). However, enzymes are complex molecular structures that are intrinsically labile and costly to produce, which are de? ite disadvantages with respect to chemical catalysts (Bommarius and Broering 2005). While the advantages of biocatalysis are there to stay, most of its present restrictions can be and are being solved through research and development in different areas. In fact, enzyme stabilization under process conditions is a major issue in biocatalysis and several strategies have been developed (Illanes 1999) that inc lude ? chemical modi? cation (Roig and Kennedy 1992 Ozturk et al. 2002 Mislovi? ov? c a et al. 2006), immobilization to solid matrices (Abi? n et al. 2001 Mateo et al. 2005 a Kim et al. 2006 Wilson et al. 006), crystallization (H? ring and Schreier 1999 Roy a and Abraham 2006), aggregation (Cao et al. 2003 Mateo et al. 2004 Schoevaart et al. 2004 Illanes et al. 2006) and the modern techniques of protein engineering (Chen 2001 Declerck et al. 2003 Sylvestre et al. 2006 Leisola and Turunen 2007), namely site-directed mutagenesis (Bhosale et al. 1996 Ogino et al. 2001 Boller et al. 2002 van den Burg and Eijsink 2002 Adamczak and Hari Krishna 2004 Bardy et al. 2005 Morley and Kazlauskas 2005), directed evolution by tandem mutagenesis (Arnold 2001 Brakmann and Johnsson 2002 Alexeeva et al. 003 Boersma et al. 2007) and gene-shuf? ing ground on polymerase assisted (Stemmer 1994 Zhao et al. 1998 Shibuya et al. 2000 Kaur and Sharma 2006) and, more recently, ligase assisted recombination (Ch odorge et al. 2005). Screening for intrinsically stable enzymes is also a prominent area of research in biocatalysis. Extremophiles, that is, organisms able to bring through and thrive in extreme environmental conditions are a promising source for highly stable enzymes and research on those organisms is very active at present (Adams and Kelly 1998 Davis 1998 Demirjian et al. 001 van den Burg 2003 Bommarius and Riebel 2004 Gomes and Steiner 2004). Genes from much(prenominal) extremophiles have been cloned into suitable hosts to develop biological systems more amenable for production (Halld? rsd? ttir et al. 1998 o o Haki and Rakshit 2003 Zeikus et al. 2004). Enzymes are by no means ideal process catalysts, but their extremely high speci? city and activity under moderate conditions are prominent characteristics that are being increasingly appreciated by different production sectors, among which the pharmaceutical and ? ne-chemical industry (Schmid et al. 001 Thomas et al. 2002 Zhao et al. 2002 Bruggink et al. 2003) have added to the more traditional sectors of food (Hultin 1983) and detergents (Maurer 2004). 4 Fig. 1. 2 stratagem of peptide bond formation between two adjacent ? -amino acids R1 + H3N CH C OH O A. Illanes H R2 + H N CH COO? H2O R1 H2O H R2 H3N CH C N CH COO? O + 1. 2 Enzymes as Catalysts. StructureFunctionality Relationships Most of the characteristics of enzymes as catalysts derive from their molecular structure. Enzymes are proteins composed by a number of amino acid residues that range from 100 to several hundreds.These amino acids are covalently bound through the peptide bond (Fig. 1. 2) that is formed between the carbon atom of the carboxyl group group of one amino acid and the nitrogen atom of the ? -amino group of the following. According to the nature of the R group, amino acids can be non- diametral (hydrophobic) or polar (charged or uncharged) and their distribution along the protein molecule confines its behavior (Lehninger 1970). Every protein is conditioned by its amino acid sequence, called primary structure, which is genetically obstinate by the deoxyribonucleotide sequence in the structural gene that codes for it.The DNA sequence is ? rst transcribed into a mRNA molecule which upon reaching the ribosome is translated into an amino acid sequence and ? nally the synthesized polypeptide chain is transform into a threedimensional structure, called native structure, which is the one endowed with biological functionality. This transformation may include several post-translational reactions, some of which can be quite relevant for its functionality, similar 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-d structure of a protein is then genetically determined, but environmentally conditioned, since the molecule will interact with the surrounding medi um. 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. alternate three-dimensional structure is the result of interactions of amino acid residues proximate in the primary structure, mainly by hydrogen bonding of the amide groups for the ase of globular proteins, like enzymes, these interactions set up a predominantly ribbon-like coiled con? guration termed ? -helix. Tertiary three-dimensional structure is the result of interactions of amino acid residues located apart in the primary structure that produce a compact and twisted con? guration in which the surface is rich in polar amino acid 1 Introduction 5 residues, while the inner part is abundant in hydrophobic amino acid residues. This tertiary structure is essential for the biological functionality of the protein. few proteins have a tetrad three-dimensional structure, which is common in regul atory proteins, that is the result of the interaction of different polypeptide chains constituting subunits that can display identical or different functions at heart a protein complex (Dixon and Webb 1979 Creighton 1993). The main types of interactions responsible for the three-dimensional structure of proteins are (Haschemeyer and Haschemeyer 1973) Hydrogen bonds, resulting from the interaction of a proton linked to an electronegative atom with another electronegative atom.A hydrogen bond has approximately one-tenth of the energy stored in a covalent bond. It is the main determinant of the helical secondary structure of globular proteins and it plays a signi? cant role in tertiary structure as well. Apolar interactions, as a result of the mutual repulsion of the hydrophobic amino acid residues by a polar solvent, like water. It is a rather sluttish interaction that does not represent a proper chemical bond (approximation between atoms exceed the van der Waals radius) however, its contribution to the stabilization of the threedimensional structure of a protein is quite signi? ant. Disulphide bridges, produced by oxidation of cysteine residues. They are especially relevant in the stabilization of the three-dimensional structure of low molecular weight extracellular proteins. Ionic bonds between charged amino acid residues. They contribute to the stabilization of the three-dimensional structure of a protein, although to a lesser extent, because the ionic strength of the surrounding medium is accustomedly high so that interaction is produced favourentially 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 dispense of the en zyme (apoenzyme). Prosthetic groups may be organic macromolecules, like shekelss (in the case of glycoproteins), lipids (in the case of lipoproteins) and nucleic acids (in the case of nucleoproteins), or guileless inorganic entities, like metal ions.Prosthetic groups are tightly bound (usually covalently) to the apoenzyme and do not dissociate during catalysis. A signi? cant number of enzymes from eukaryotes are glycoproteins, in which case the carbohydrate moiety is covalently linked to the apoenzyme, mainly through serine or threonine residues, and even though the carbohydrate does not participate in catalysis it confers relevant properties to the enzyme. Catalysis income tax returns place in a small portion of the enzyme called the active site, which is usually formed by very few amino acid residues, while the rest of the protein acts as a scaffold.Papain, for instance, has a molecular weight of 23,000 Da with 211 amino acid residues of which only cysteine (Cys 25) and histidi ne (His 159) 6 A. Illanes are directly involved in catalysis (Allen and Lowe 1973). Substrate is bound to the enzyme at the active site and doing so, changes in the distribution of electrons in its chemical bonds are produced that cause the reactions that lead to the formation of products. The products are then released from the enzyme which is ready for the next catalytic cycle.According to the early lock and key model proposed by Emil Fischer in 1894, the active site has a unique geometric shape that is complemental to the geometric shape of the substrate molecule that ? ts into it. Even though recent reports provide evidence in favor of this theory (Sonkaria et al. 2004), this rigid model hardly explains many experimental evidences of enzyme biocatalysis. Later on, the induced-? t theory was proposed (Koshland 1958) according to which he substrate induces a change in the enzyme conformation after binding, that may level the catalytic groups in a way prone for the subsequent rea ction this theory has been extensively used to explain enzyme catalysis (Youseff et al. 2003). Based on the transition-state theory, enzyme catalysis has been explained according to the hypothesis of enzyme transition state complementariness, which considers the prefc erential binding of the transition state rather than the substrate or product (Benkovi? and Hammes-Schiffer 2003).Many, but not all, enzymes require small molecules to perform as catalysts. These molecules are termed coenzymes or cofactors. The term coenzyme is used to refer to small molecular weight organic molecules that associate reversibly to the enzyme and are not part of its structure coenzymes bound to enzymes actually take part in the reaction and, therefore, are sometime called cosubst order, since they are stoichiometric in nature (Kula 2002). Coenzymes oftentimes function as intermediate carriers of electrons (i. e. NAD+ or FAD+ in dehydrogenases), speci? c atoms (i. e. oenzyme Q in H atom transfer) or func tional groups (i. e. coenzyme A in acyl group transfer pyridoxal phosphate in amino group transfer biotin in carbonic acid gas transfer) that are transferred in the reaction. The term cofactor is commonly used to refer to metal ions that also bind reversibly to enzymes but in general are not chemically altered during the reaction cofactors usually bind strongly to the enzyme structure so that they are not dissociated from the holoenzyme during the reaction (i. e. Ca++ in ? -amylase Co++ or Mg++ in glucose isomerase Fe+++ in nitrile hydratase).According to these requirements, enzymes can be classi? ed in three groups as depicted in Fig. 1. 3 (i) those that do not require of an special 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 req uirement of cofactors or coenzymes to perform biocatalysis has profound technological implications, as will be analyzed in section 1. 4.Enzyme activity, this is, the capacity of an enzyme to catalyze a chemical reaction, is strictly dependent on its molecular structure. Enzyme activity relies upon the existence of a proper structure of the active site, which is composed by a reduced number of amino acid residues close in the three-dimensional structure of 1 Introduction Fig. 1. 3 Enzymes according to their cofactor or coenzyme requirements. 1 no requirement 2 cofactor requiring 3 coenzyme requiring S 1 7 P E E CoE 2 S E-CoE P E CoE 3 E CoE E P S E-CoE the protein but usually far apart in the primary structure.Therefore, any agent that promotes protein florescence will move apart the residues constituting the active site and will then reduce or destroy its biological activity. Adverse conditions of temperature, pH or solvent and the presence of chaotropic substances, surd metals an d 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 blossoming in certain labile regions denoted as weak spots. These regions prone to unfolding are the determinants of enzyme stability and are usually located in or close to the surface of the protein molecule, which explains why the surface structure of the enzyme is so important for its catalytic stability (Eijsink et al. 2004). These regions have been the target of site-speci? c mutations for increasing stability.Though extensively studied, rational engineering of the enzyme molecule for increased stability has been a v ery 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 cerebrate 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 found rules (Shaw and Bott 1996), rational improvement of the stability is still far from being well established.In fact, the less rational approaches of directed evolution using error-prone PCR and gene shuf? ing have been more successful in obtaining more stable mutant enzymes (Kaur and Sharma 2006). Both strategies can combine using a set of rationally designed mutants that can then be subjected to gene shuf? ing (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 random polypeptide chain in which the amino acids loc ated 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 take away 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 resistance of the folded protein to denaturation, and kinetic or long-term stability, this is the resistance to irreversible inactivation (Eisenthal et al. 2006). The overall process of enzyme inactivation can then be represented by N U ? I where N represents the native active conformation, U the unfolded conforma tion and I the irreversibly inactivated enzyme (Klibanov 1983 Bommarius and Broering 2005). The ? rst step can be de? ned by the equilibrium constant of unfolding (K), while the second is de? ed in terms of the rate constant for irreversible inactivation (k). Stability is not related to activity and in many cases they have opposite trends. It has been suggested that there is a trade-off between stability and activity based on the fact that stability is clearly related to molecular stiffening while conformational ? exibility is bene? cial for catalysis. This can be clearly appreciated when studying enzyme thermal inactivation enzyme activity increases with temperature but enzyme stability decreases. These opposite trends make temperature a critical variable in any enzymatic process and make it prone to optimization.This aspect will be thoroughly analyzed in Chapters 3 and 5. Enzyme speci? city is another relevant property of enzymes strictly related to its structure. Enzymes are usua lly 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 burn down the active site have been replaced producing an alteration of substrate speci? city (Colby et al. 1998 diSioudi et al. 1999 Parales et al. 2000), and also by chemical modi? cation (Kirk Wright and Viola 2001). K k 1. 3 The Concept and Determination of Enzyme Activity As already mentioned, enzymes act as catalysts by virtue of reducing the magnitude of the barrier that represents the energy of activation required for the formation of a transient active complex that leads to product f ormation (see Fig. . 1). This thermodynamic de? nition of enzyme activity, although rigorous, is of little practical signi? cance, since it is by no means an easy task to determine free energy changes for molecular structures as unstable as the enzymesubstrate complex. The direct 1 Introduction 9 consequence of such diminution of energy infix 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 undecomposable to determine so this de? nition is endowed with practicality.Biochemical reactions usually proceed at very low rates in the absence of catalysts so that the magnitude of the reaction rate is a direct and straightforward procedure for assessing the activity of an enzyme. Therefore, for the reaction of conversion of a substrate (S) into a product (P) under the catalytic action of an enzyme (E) S ? P v=? ds dp = dt dt (1. 1) E If the course of the reaction is f ollowed, 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 end 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 uprightness 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 reacti on (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 concentration versus time of reaction at different enzyme concentrations (e) 10 A. Illanes phenomena are insigni? ant. According to this a = vt0 = ? ds dt = t0 dp dt (1. 2) t0 This is not only of practical convenience but fundamentally sound, since the enzyme activity so de? ned represents its maximum catalytic potential under a given set of experimental conditions. To what extent is this catalytic potential going to be expressed in a given situation is a different matter and will have to be assessed by modulating it according to the phenomena that cause its reduction. All such phenomena are amenable to quanti? ation as will be presented in Chapter 3, so that the function of this maximum catalytic potential is fundamental for any study regarding enzyme kinetics. Enzymes should be quanti? ed in terms of its cataly tic potential rather than its mass, since enzyme preparations are rather impure mixtures in which the enzyme protein can be a small fraction of the total mass of the preparation but, even in the unusual case of a completely pure enzyme, the determination of activity is unavoidable since what matters for evaluating the enzyme performance is its catalytic potential and not its mass.Within the context of enzyme kinetics, reaction rates are always considered then as initial rates. It has to be pointed out, however, that there are situations in which the determination of initial reaction rates is a poor predictor of enzyme performance, as it occurs in the determination of degrading enzymes acting on heterogeneous polymeric substrates. This is the case of cellulase (actually an enzyme complex of different activities) (Montenecourt and Eveleigh 1977 Illanes et al. 988 Fowler and Brown 1992), where the more amorphous portions of the cellulose moiety are more easily degraded than the crystal line regions so that a high initial reaction rate over the amorphous portion may give an overestimate of the catalytic potential of the enzyme over the cellulose substrate as a whole. As shown in Fig. 1. 4, the initial slope o the curve (initial rate of reaction) is proportional to the enzyme concentration (it is so in most cases). Therefore, the enzyme sample should be properly reduce to attain a linear product concentration versus time relationship within a reasonable assay time.The experimental determination of enzyme activity is based on the measurement of initial reaction rates. Substrate depletion or product build-up can be used for the evaluation of enzyme activity according to Eq. 1. 2. If the stoichiometry of the reaction is de? ned and well known, one or the other can be used and the choice will depend on the easiness and readiness for their analytical determination. If this is indifferent, one should prefer to measure according to product build-up since in this case one will be determining signi? ant differences between small magnitudes, while in the case of substrate depletion one will be measuring small differences between large magnitudes, which implies more error. If neither of both is readily measurable, enzyme activity can be determined by coupling reactions. In this case the product is transformed (chemically or enzymatically) to a ? nal analyte amenable for analytical determination, as shown E S P A X B Y C Z 1 Introduction 11 In this case enzyme activity can be determined as a = vt0 = ? ds dt = t0 dp dt = t0 dz dt (1. 3) t0 rovided that the rate limiting step is the reaction catalyzed by the enzyme, which implies that reagents A, B and C should be added in excess to attend that all P produced is quantitatively transformed into Z. For those enzymes requiring (stoichiometric) coenzymes E S CoE CoE P activity can be determined as a = vt0 = ? dcoe dt = t0 dcoe dt (1. 4) t0 This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i. e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase (? galactosidase EC 3. 2. 1. 23). The enzyme catalyzes the hydrolysis of lactose according to Lactose + H2 O Glucose + Galactose Glucose produced can be coupled to a classical enzymatic glucose kit, that is hexoquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD), in which Glucose + ATP ? Glucose 6Pi + ADP Glucose 6Pi + NADP+ ? ? ? ? 6PiGluconate + NADPH where the initial rate of NADPH (easily measured in a spectrophotometer see ahead) can be then stoichiometrically correlated to the initial rate of lactose hydrolysis, provided that the auxiliary enzymes, Hx and G6PD, and co-substrates are added in excess.Enzyme activity can be determined by a continuous or discontinuous assay. If the analytical device is provided with a recorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay in that case, the course of reaction should be monitored discontinuously by sampling and assaying at predetermined time intervals and samples should be subjected to inactivation to stop the reaction.This is a drawback, since the enzyme should be rapidly, completely and irreversibly inactivated by subjecting it to harsh conditions that can interfere with the G6PD Hx 12 A. Illanes analytical procedure. Data points should describe a linear 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 control experiment for discriminating the non-enzymatic build-up of product during the assay. There are essentially three options to remove the enzyme from the reaction mixture by successor 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 withdraws substrate contamination with product or any non-enzymatic transformation of substrate into product, but does not discriminate enzyme contamination with substrate or product the second one acts exactly the opposite the third one can in rinciple discriminate both enzyme and substrate contamination with product, but the pitfall in this case is the risk of not having inactivated the enzyme completely. The control of choice depends on the situation. For instance, when one is producing an extracellular enzyme by fermentation, enzyme sample is in all likelihood to be contaminated with subs trate and or product (that can be constituents of the culture medium or products of metabolism) and may be signi? ant, since the sample probably has a low enzyme protein concentration so that it is not diluted prior to assay in this case, replacing substrate by water or buffer discriminates such contamination. If, on the other hand, one is assaying a preparation from a stock enzyme concentrate, dilution of the sample prior to assay makes unnecessary to blank out enzyme contamination replacing the enzyme by water or buffer can discriminate substrate contamination that is in this case more relevant.The use of an enzyme placebo as control is best(predicate) when the enzyme is labile enough to be completely inactivated at conditions not affecting the assay. An alternative is to use a double control replacing enzyme in one case and substrate in the other by water or buffer. Once the type of control experiment has been decided, control and enzyme sample are subjected to the resembling a nalytical procedure, and enzyme activity is calculated by subtracting the control reading from that of the sample, as illustrated in Fig. . 5. Analytical procedures available for enzyme activity determinations are many and usually several alternatives exist. A proper selection should be based on sensibility, reproducibility, ? exibility, simplicity and availability. Spectrophotometry can be considered as a method that ful? ls most, if not all, such criteria. It is based on the absorption of light of a certain wavelength as described by the BeerLambert law A? = ? l c where A? = log I I0 (1. 5) (1. 6) The value of ? an be experimentally obtained through a calibration curve of absorbance versus concentration of analyte, so that the reading of A? will allow the determination of its concentration. Optical path width is usually 1 cm. The method is based on the differential absorption of product (or coupling analyte or modi? ed 1 Introduction 13 Fig. 1. 5 Scheme for the analytical proced ure to determine enzyme activity. S substrate P product P0 product in control A, B, C coupling reagents Z analyte Z0 analyte in control s, p, z are the corresponding molar concentrations oenzyme) and substrate (or coenzyme) at a certain wavelength. For instance, the reduced coenzyme NADH (or NADPH) has a strong peak of absorbance at 340 nm while the absorbance of the oxidized coenzyme NAD+ (or NADP+ ) is negligible at that wavelength therefore, the activity of any enzyme producing or consuming NADH (or NADPH) can be determined by measuring the increase or decline of absorbance at 340 nm in a spectrophotometer. The assay is sensitive, reproducible and simple and equipment is available in any research laboratory.If both substrate and product absorb signi? cantly at a certain wavelength, coupling the detector to an appropriate high performance transparent chromatography (HPLC) column can solve this interference by separating those peaks by differential retardation of the analytes in the column. HPLC systems are increasingly common in research laboratories, so this is a very convenient and ? exible way for assaying enzyme activities. Several other analytical procedures are available for enzyme activity determination.Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (? avin adenine dinucleotide) and FMN (? avin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less ? exible and the equipment not so common in a standard research laboratory.Enzymes that produce or consume gases can be assayed by differential manometry by measuring small pressure differences, due to the consumption of the gaseous substrate or the evo lution of a gaseous product that can be converted into substrate or product concentrations by using the gas law. Carboxylases and decarboxylases are groups of enzymes that can be conveniently assayed by differential manometry in a respirometer. For instance, the activity of glutamate decarboxylase 14 A. Illanes (EC 4. 1. 1. 15), that catalyzes the decarboxylation of glutamic acid to ? aminobutyric acid and CO2 , has been assayed in a differential respirometer by measuring the increase in pressure caused by the formation of gaseous CO2 (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 sim ple and accurate method for determining optically active compounds. A polarimeter is a low cost instrument readily available in many research laboratories. The detector can be integrated into an HPLC system if separation of substrates and products of reaction is required. Invertase (? -D-fructofuranoside fructohydrolase EC 3. 2. 1. 26), a commodity enzyme wide 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 b een determined by measuring the reduction in viscosity of the corresponding olymer solutions. A comprehensive review on methods for assaying enzyme activity has been recently published (Bisswanger 2004). Enzyme activity is expressed in units of activity. The Enzyme Commission of the foreign Union of Biochemistry recommends to express it in international units (IU), de? ning 1 IU as the amount of an enzyme that catalyzes the transformation of 1 mol of substrate per minute under standard conditions of temperature, optimum pH, and best substrate concentration (International Union of Biochemistry).Later on, in 1972, the Commission on Biochemical Nomenclature recommended that, in order to adhere to SI units, reaction rates should be expressed in moles per second and the katal was proposed as the new unit of enzyme activity, de? ning it as the catalytic activity that will raise the rate of reaction by 1 mol/second in a speci? ed assay system (Anonymous 1979). This latter de? nition, alt hough 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 substrat e concentration at which the initial rate of reaction is at its maximum this will imply reaction rate at substrate saturation for an enzyme following typical Michaelis-Menten kinetics or the highest initial reaction rate value in the case of inhibition at high substrate concentrations (see Chapter 3).With respect to pH, it is straightforward to determine the value at which the initial rate of reaction is at its maximum. This value will be the true operational optimum in most cases, since that pH will lie within the region of maximum stability. However, the opposite holds for temperature where enzymes are usually quite unstable at the temperatures in which higher initial reaction rates are obtained actually the concept of optimum temperature, as the one that maximizes initial reaction rate, is quite misleading since that value usually re? cts nothing more than the deflexion of the linear p versus t relationship for the time of assay. For the de? nition of IU it is then more appropri ate 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 varying and often unde? ned molecular weight and whose products are usually a mixture of different chemical compounds. In that case, units of activity can be de? ned in terms of mass rather than moles. These enzymes are usually speci? c for certain types of bonds rather than for a particular chemic al structure, so in such cases it is advisable to express activity in terms of equivalents of bonds broken.The choice of the substrate to perform the enzyme assay is by no means trivial. When using an enzyme as process catalyst, the substrate can be different from that employed in its assay that is usually a model substrate or an analogue. One has to be cautious to use an assay that is not only simple, accurate and reproducible, but also signi? cant. An example that illustrates this point is the case of the enzyme glucoamylase (exo-1,4-? -glucosidase EC 3. 2. 1. 1) this enzyme is widely used in the production of glucose syrups from starch, either as a ? al product or as an intermediate for the production of high-fructose syrups (Carasik and Carroll 1983). The industrial substrate for glucoamylase is a mixture of oligosaccharides produced by the enzymatic liquefaction of starch with ?-amylase (1,4-? -D-glucan glucanohydrolase EC 3. 2. 1. 1). Several substrates have been used for assa ying enzyme activity including high molecular weight starch, small molecular weight oligosaccharides, malt sugar and maltose synthetic analogues (Barton et al. 1972 Sabin and Wasserman 16 A. Illanes 1987 Goto et al. 1998). None of them probably re? cts properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a transesteri? cation reaction that produces thiophenol as one of the products (Han et al. 004) in this case, a method based on a reaction leading to a ? uorescent pull of thiophenol is a good system to assess the tran sesteri? 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) into six families, based on the type of chemical reaction catalyzed.A four digit number is assigned to each enzyme by the Enzyme Commission (EC) of the IUBMB the ? rst one denotes the family, the second denotes the subclass within a family and is related to the type of chemical group upon which it acts, the third denotes a subgroup within a subclass and is related to the particular chemical groups involved in the reaction and the forth is the correlative number of identi? cation within a subgroup. The six families are 1. Oxidoreductases. Enz ymes 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+ ,

Memorandum Mandatory Use of Turkish Language Essay

This Memorandum aims to provide brief information on (i) provisions and restrictions imposed by the right on Mandatory Use of Turkish Language by Economic Enterprises numbered 805 and dated 10 April 1926 (the rightfulness no(prenominal) 805) on Turkish and immaterial enterprises (ii) consequences of non-compliance with the Law No. 805 and (iii) application of the dear faith principle in the light of the precedents of the Turkish Supreme salute of Appeals (the Court of Appeals).A. SCOPE OF THE jurisprudence zero(prenominal) 805The Law No. 805 is applicable to every(prenominal) Turkish and foreign enterprises with respect to transactions listed in words 1 and 2 of the Law No. 805 however, application of the Law No. 805 differs depending on the nationality of the enterprise.1. Turksh EnterprsesIn accordance with Article 1 of the Law No. 805, Any type of companies and enterprises which have Turkish nationality shall use Turkish phrase in all kinds of transactions, agreements, correspondences, accounts and books within Turkey. According to Article 1 of the Law No.805, each company and enterprise a the Turkish nationality is obliged to conserve any kind of transaction, records and books and execute all agreements and make all communications with each other in Turkish run-in within the territory of Turkey. Accordingly, the single exclusion for the obligation of the use of Turkish lecture in transactions and communications of Turkish companies and enterprises is the case where such transactions and communications are punish/performed outside the territory of Turkey.In that respect, it is clear that an agreement to be penalize between two or more Turkish companies should be prepared and write in Turkish language, or if it is p colligatered to be kill in a foreign language, to be accompanied with a Turkish version which shall attain in the event of discrepancy. There are several(prenominal) court decisions in this respect. In one of its judgments, the Court of Appeals has upheld that the parties, in the text of the agreement may refer to non-Turkish (international) term and/ or clauses, only if (i) these terms cannot be replaced by a counterpart in Turkish and (ii) the parties are obliged to include such terms in the agreement.However, it has also underlined by the Court of Appeals that this rule is valid only for terms and expressions either with no counterpart in Turkish or which cannot be written in Turkish. Similarly, in a later decision, the Court of Appeals has ruled that if the parties are both Turkish then all agreements must be executed in Turkish although international terms and expressions may be incorporated into a Turkish agreement as a special condition. In this decision, the Court of Appeals has not accepted the drill of a maturity clause which was drafted in English language in an agreement executed in Turkish language since such maturity clause could easily be drafted in Turkish language as well.2. Foregn ente rprsesIn accordance with Article 2 of the Law No. 805, This obligation applies to foreign companies and enterprises only in their communications, transactions and relations with the Turkish institutions and persons and for the documents, books and records which must be submitted to governmental offices and government officers.Pursuant to Turkish legislation, a foreign enterprise is a duly incorporated legal entity that has its registered office outside of Turkey. For foreign enterprises, as tell above, the requirement of Turkish language usage is limited only to (i) transactions, correspondences and communications with persons and legal entities subject to Turkish laws and (ii) documents, books and records which will be presented to Turkish authorities.In contrast with Article 1 of Law No. 805, Article 2 of the Law No. 805 has a narrower a scope of obligation to use Turkish language. The wording agreement is willingly not stated in Article 2 on purpose. In other words, the Law No. 805 does not specifically refer to agreements under the provision concerning foreign entities. This inattention indicates that the Law No. 805 allows the execution of an agreement between a foreign entity and a Turkish entity in a foreign language.There are several court decisions with respect to Article 2 of the Law No. 805. In a decision by the 11th Circuit of the Court of Appeals, the Court of Appeals has upheld that all accounts and books have to be in Turkish language, when submitted to the relevant Turkish authorities. Otherwise, they do not have any validity before Turkish courts. The Court of Appeals has gain to a similar conclusion summarizing that Turkish enterprises may enter into agreements with foreign enterprises in any language since Article 2 of the Law No. 805 specifically lists instances where foreign enterprises are prohibited from using a foreign language and this list does not include agreements.Accordingly, the Court of Appeals has concluded that agreements m ay be executed in any foreign language, where one of the parties is a foreign enterprise and the other is Turkish. According to this decision, an agreement executed in a foreign language between a foreign company and a Turkish company is valid and binding on the parties pursuant to and under Turkish laws. However, under Article 4 of the Law No.805, the parties are compelled to use Turkish language in all correspondences with respect to the agreement, although the agreement is allowed to be drafted in a foreign language.B. CONSEQUENCES OF NON-COMPLIANCE WITH THE LAW NO. 805Pursuant to Article 7 of the Law No. 805, any party violating the related provisions is imposed to a judicial fine of at least 100 days. The Turkish Penal Code numbered 5237 has regulated the judicial fine provisions and accordingly the judicial fine for one day shall be determined between TL 20 TL 100 depending on (i) severity of violation (ii) specific and economic conditions of persons and (iii) discretion of j udges.C. GOOD FAITH principlePursuant to Article 2 of the Turkish Civil Code, raising a good faith get is possible regarding the conflicts in an agreement. While deciding on the validity of the good faith avers, the determination shall be made on a case-to-case basis. In terms of disputes regarding non-compliance with the Law No. 805, there is not a definite provision for the application of the good faith principle and we have not come across any judgment of the Court of Appeals regarding the good faith claims has raised with respect to the application of the Law No. 805. However, with respect to the general provisions regarding the good faith claims, by analogy, the following solution may present to conflicts on the mandatory use of Turkish language in agreements.In the event that the parties have carried out their performances to the fullest extent, considering that the agreement is valid, the agreement, itself, should be considered valid, as well. The Court of Appeals, in one of its judgments, has stated that if parties have carried out their obligations under a contract for a long time, it would be a violation of the good faith principles to claim that the related contract is invalid. Similarly, the Assembly of Civil Chambers of the Court of Appeals has ruled in a former decision that the right to raise a good faith claim is restricted when the party raising the claim is acting against the good faith principle.Although the aforementioned judgment does not directly refer to a dispute under the Law No. 805, the following conclusion could be drawn when one or both of the parties is awaring (or shall be aware) of the mandatory use of Turkish language requirement and the aware party (or parties) disregards this rule on purpose, they should not be able to rely on a good faith claim regarding the language of the agreement.

Priority Sector Lending in India

Definition and more details5 precedency sector A expect5 Priority Sector level Financial Reforms Effect6 Effect of reforms on anteriority sector lending6 Priority Sector Specific sector guidelines8 Agriculture8 Small(a) enterprises8 Weaker section9 Other sectors9 Priority Sector ease up status10 Participating Entities Targets to be met10 Participating Entities How much is achieved11 Public Sector marges11 Private Sector money boxs11 Foreign entrusts12 Participating Entities penalties in case of failure in achieving the target12 Priority Sector Advantages12Priority Sector Major Issues13 Strategies Ahead13 Exhibits15 References18 INTRODUCTION Priority sector bank lending was mainly started by the government to reach the unbanked areas through regular banks which were work on that time not much giveing to go to rude and undeveloped areas. It was one most important tool in our financial polity to fetter banks to increase their loanable customers. Before independence, b anks were mostly semiprivately owned and they used to lend only to the sectors in which they were assured of returns.According to the reports from 1940s, 79% of bank finances were make procurable to industry and commerce. Of that amount too, around 32% went to large industries of jute, cotton and sugar mills. When looking at the less rosy picture, the advances to agriculture sector stood a paltry 4%. Post independence, according to rbi survey of 1954, in 1951-52, of all citation disbursal by reference book agencies to cultivators, only 7. 3 % was from institutional reliance agencies. Of this diminished contribution, the part of banks was only 0. 9%. Rest was given by government and cooperative agencies.From this statistics, it is clear that the rest of the deferred payment was availed by the cultivators from non-institutional quotation agencies. When the spare-time activity rates aird by these agencies was checked, they were found to be usuriously high with professional moneylenders charging 41. 9% interest rate while agricultural moneylenders charged 23. 9% interest rate which was 5-6 times more than the normal bank rate. It shows that if a farmer is getting loan at this interest rate, chances are more that he will never be able to repay it fully and fall in the vicious circle of loans.By getting work capital at such high interest rates, it was equally difficult to breakeven. So, agriculture and small and medium enterprises were in deep need for credit at easy terms. PRIORITY arena DEFINITION AND MORE DETAILS Priority sector and its coverage area kept changing all through these years, mostly due to economic and policy- devising pressures. Although its definition arse be divided in two parts i. e. pre-reform and post reform period.Pre reform period definition It included agriculture, Small outstrip industry (including setting up of industrial estates), small road and water transport operators, small business, retail trade, professional and se lf employed persons, state sponsored organizations for SC/STs, nurtureal loans granted to individuals by banks chthonic schemes, Credit schemes for weaker sections and refinance by sponsor banks to Regional Rural strands. About the post reform definition we will talk later in details when dealing in the section about precession sector guidelines. PRIORITY SECTOR A NEED world support and employment generation According to the definition of antecedency sector it covers about 70% of Indias population by rough estimates. So, by making it mandatory for the banks to lend to priority sector, government is actually trying to cover a big part of population. Priority sector mostly includes agriculture and assort sector which employs largest estimate of people in our country. Freedom from non-institutional credit The priority sector cut out by government was mostly the one which was ahead victorious loans from non-institutional sources and was always indebted because of usurious rat es of interest.By creating priority sector lending, it was tried to make institutional credit available to a bigger section, at affordable interest rates. Willingness of banks Most of the banks were not willing to lend to this sector because of the risk have-to doe with here as strong as more paperwork take to lend smaller loans to large number of people. They were happy lending to urban sector which was more reliable and trustworthy. They preferred lending to industry, commerce, trade and securities as their tralatitious loanees and who were supposed to default less. Location of banks blasphemes were earlier situated mostly in urban area where the business was and so, it was geographical recordically also difficult for them to lend to rural and backward areas where there was no banking mesh earlier. It was difficult to know about the credit history of borrower and the po ten-spottial ability of loaned to repay the loan as swell as potential of the project for which loan was to be given. So, they were skeptical about loaning to those sectors. Institutional credit By allowing priority sector credit to flow, RBI and government actually allowed large amount of institutional credit to flow in this area.So, as it became mandatory for the banks to complete certain target for priority sector, they started meddling for viable projects and loaners who can successfully repay the loan. For this to happen branches were opened in rural areas and people were encouraged to take loan from banks. Many people availed loan under priority sector lending and got involved in successful enterprises. PRIORITY SECTOR FINANCIAL REFORMS EFFECT After financial sector reforms, priority sector lending underwent lots of change.As earlier, it was only focused towards weaker and rural section of society but afterwards it included many new sectors as well as the definition of earlier sectors was widened to include more areas in them Priority sector targets are Table 1 Priority Sector T argets to be achieved by Banks Before 1991 After 1991 Total priority sector credit 40% of net bank credit 40% of net bank credit rustic credit 18% of net bank credit 18% of net bank credit Weaker section credit 10% of net bank credit 10% of net bank credit Export credit 12% of net bank credit for foreign banks SSI credit 10% of net credit for foreign banksSource Reserve Bank of India Banking norms EFFECT OF REFORMS ON PRIORITY SECTOR LENDING A chorological sequence of changes in priority sector lending policy is given below which show how the definition of priority sector has changed in all these years 1. 1992-1993 In the light of reforms, and many new industries coming up in all sectors, government and RBI decided to help out industry with credit facilities and asked banks to fulfill demand of small scale industries upto Rs. 100 hundred cat valium limit for setting institutional framework to rejuvenate potentially viable small scale industry units. . 1993-1994 The overall targe t of net bank credit to be given for priority sector remained unchanged but the direct and indirect target for lending to agricultural sector was clubbed together to make a sub target of 18% for agricultural lending. But, in this system also, the indirect lending was not supposed to extend one-fourth of the total sub target. loaning higher up this in indirect lending, was not to be considered in priority sector lending. At least 40% of total credit was supposed to go to small scale and khadi and small town industries within limit of Rs. 5 lakh.Foreign banks were asked to revise their priority sector advance target from 10% to 32%. deuce more sectors were included in that i. e. advances to small-scale industries and export sector were made with each being 10%. 3. 1995-1996 In case of any shortfall in PSL (agricultural sector), banks were required to bring in to Rural Infrastructure Development Fund (RIDF), which was set up under NABARD, the level best of which was 1. 5 % of banks net credit. shortage in case of opposite areas, they were required to provide Rs. 1000 crores for financing in Khadi and Village Industries Commision (KVIC).All the refinances which was done to RRBs by the banks was now to be considered under priority sector lending. 4. 1996-1997 In this year Union Budget provided Rs. 2500 crore for RIDF fund. Export credit target increased from 10% to 12% in this year. Credit go to priority sector increased this year very much. From the last year numbers, it increased from 30. 37% of net bank credit to 32. 4%. 5. 1997-1998 The scope of priority sector lending was increased for road and water transport operators, with number of eligible vehicles increasing from not more than six to not more than ten.The credit limit for housing in rural and urban areas also increased upto Rs. 5 lakh. 6. 1998-1999 In this year, the interest rate subsidy for loan in PSL was taken away on the argument that now priority sector lending is also commercially viable for banks. Banks were also given the option to assign the PSL shortfall by lending to NABARD/SIDBI, so the restriction of not lending to profitable sector was slowly being taken away. 7. 1999-00 Banks were asked to lend to NBFCs and MFIs under priority sector, to enable them to lend to rural and weaker section.INSTITUTIONAL AND NON-INSTITUTIONAL CREDIT IN INDIA Before independence, the credit which was available to farmers was just non-institutional credit or in other words private money lenders. But, after independence, government took major steps to uproot this problem which was eating up the unequal population and was hampering with the countrys economic growth. In 1951, institutional credit accounted to 92. 7% of the total credit availed (Refer Graph-1) where as all these reforms positively impacted the credit scenario in India making the Non-institutional credit accounted to be 38. % in the year 2002. Graph 1 Trend of Institutional and Non-institutional credit in India PRIORITY SECTOR SPECIFIC SECTOR GUIDELINES AGRICULTURE 1. Direct finance pay given to individual farmers (including SHG & JLG) for agricultural and allied activities are included under this sector. This includes short-term loans for raising crops, advances upto 10 lakh against pledge of agricultural produce for level best 12 months period, working capital and term loans, for purchase of land, to indebted distressed farmers, for pre and post harvest activities.Loans given to partnerships, corporate and institutions for agricultural activities, and upto 1 crore for most of the activities mentioned above also come under direct finance. 2. Indirect finance It covers vast range i. e. corporate, Primary agricultural Credit societies, Farmers service societies, Large sized Adivasi Multi social occasion Societies, cooperative societies, and for the construction of warehouse, agricultural input dealers, arthias, NCDC, NBFCs, NGOs, MFIs, RRBs and overdraft upto 25000 for no-frills account in rural and semi-urban areas. SMALL ENTERPRISES 1. Direct finance a.For manufacturing enterprises, for small enterprises the upper cap for taking loans is less than 5 crores, while for micro enterprises it is upto 25 lakh only. b. For service enterprises, for small enterprises it is upto 2 lakh, while for micro enterprises it is only 10 lakh. c. For khadi and village industries it is upto 60% of small enterprise segment. 2. Indirect finance a. It is made available for the person involved in marketing activities of artisans, village and cottage industries. b. Under this Loans made by NABARD, SIDBI and commercial banks to NBFCs and cooperatives involved in this sector also come.WEAKER SECTION In weaker section, small and marginal farmers with less than 5 acres land holding, landless labourers, artisans, village and cootage industries, beneficiaries of SGSY, SC, ST, DRI, SJSRY, SLRS, self help groups, distressed poor, minority communities etc are included. They are given loans under priority s ector loans. OTHER SECTORS Retail trade Retailers involved in essential commodities, consumer co-operative stores, private retail traders, upto the limit of Rs. 20 lakh. Micro-credit For poor indebted borrower of non-institutional credit, it is given against collateral or group security.The upper limit for it is upto Rs. 50000 per borrower. press out sponsored organization It is for scheduled castes/tribes for extending credit for purchase of input or for marketing of output. Education Within India the maximum cap for education loan granted is 10 lakh, while outside India it is 20 lakh. It is applicable for individuals as well as NBFCs. Housing a. For purchase and construction of houses, the maximum loan allowed is 20 lakh. b. For repair of houses, the maximum loan allowed is 1 lakh in rural India and 2 lakh in urban areas. c.For government agencies for construction of dwelling units, or for slum dwellers, upto a maximum of Rs. 5 lakh is allowed. PRIORITY SECTOR PRESENT STATUS f ighting(a) ENTITIES TARGETS TO BE MET The Reserve Bank of India from time to time has issued a number of guidelines/instructions/directives to banks in lending credit to Priority sector. In priority sector diverse banks that are involved are- public and private sector bank under domestic banks and foreign banks. There are separate targets to be met for all the banks which are set by the RBI.RBI issues a master circular containing all the guidelines for incorporation of priority sector lending. If the targets are not met, then various penalties are to be borne by them. The targets set for the domestic and foreign banks working in India are already mentioned before in Table-1. The total advances that a domestic bank has to offer for the priority sector is 40% where as for foreign banks working in India is 32 %. These advances are further bifurcated into the advances provided to agricultural sector, small scale industries (SSI), export credit and weaker sections.However, domestic bank s dont have to contribute to SSI and foreign banks dont have to contribute to agricultural advances and weaker sections. Over the years, the advances provided to this sector are increasing in gross value and some other sectors like education, housing, retail trade which were not the part of this sector previously were also included. The trend observed during the last three years is explained in the graph provided below. In the year 2006, the advances offered by the public sector banks were Rs. 409. 745 thousand crores where as private sector provided Rs. 06. 556 thousand crores. Then in year 2008, these advances increased to Rs. 605. 965 thousand crores and Rs. 165. 225 thousand crores by public and private sector bank respectively. This marked a growth rate of 48% in public sector and 53. 5 % in private sector.Source Reserve Bank of India- Trend and Progress of Indian Banking 2008-09 The share of various sectors i. e. agriculture, SSI, education, housing have also registered a ch ange as shown in the figure given below. The share of advances provided to agriculture sector is more or less same where as the dvances provided to SSI has been replaced by small enterprises, housing and education where housing accounted for 30% of the advances and education accounted for 25% of the advances. Source Reserve Bank of India- Trend and Progress of Indian Banking 2008-09 The rationale of including these sectors was to provide the holistic phylogeny to the poor people. It was understood that its not just the credit requirement which has to be fulfilled but also the education which would ensure the socio-economic development of the society. In all, those sectors which can impact large section of populations are to be a part of priority sector.But, how efficiently are banks able to achieve these set targets is still questionable. combat-ready ENTITIES HOW MUCH IS ACHIEVED PUBLIC SECTOR BANKS Exhibit-1 shows the targets achieved by public sector bank. The public sector ba nks were able to meet the target of 40% till 2005-06 but in 2007 they fell short by 0. 7%. There were 28 banks in total, out of which- seven banks failed to achieve the target (Allahabad Bank, Oriental Bank of Commerce, Syndicate Bank, IDBI Ltd. , introduce Bank of India, State Bank of Mysore and State Bank of Patiala).However, only 8 banks were able to meet target of agricultural lending and only 7 for weaker sections. secluded SECTOR BANKS Exhibit-2 shows the targets achieved by private banks in lending to the priority sector. Out of 26 private sector banks, four banks (Bank of Rajasthan Ltd. , Centurian Bank of Punjab Ltd. , Jammu and Kashmir Bank Ltd. and Karnataka Bank Ltd. ) didnt achieve the target as stipulated for the priority sector lending. However, only three banks were successful in meeting agricultural credit target and no bank met the target for weaker sections. FOREIGN BANKSExhibit-3 shows the targets achieved by foreign banks in lending to the priority sector. Out of 29 foreign banks working in India five banks (Abu Dhabi Commercial Bank, Bank of Tokyo-Mitsubishi, Citi Bank, HSBC Ltd. and Mizuho Corporate Bank) did not achieve the target. However, only Seven banks (Bank of Nova Scotia, Bank of Tokyo-Mitsubishi, Citi Bank, HSBC Ltd. , JP Morgan Chase Bank, Mizuho Corporate Bank and Shinhan Bank) were not able to achieve SSI target and three banks (American take out Bank, Bank International Indonesia and Mizuho Corporate Bank) were not able to achieve the export credit target.The banks which failed to achieve the target have to pay the penalties decided by the RBI. PARTICIPATING ENTITIES PENALTIES IN CASE OF FAILURE IN ACHIEVING THE TARGET DOMESTIC BANKS Domestic banks which fail to achieve the target have to contribute to Rural Infrastructure development Fund (RIDF) established with NABARD or funds with other financial institutions, as condition by RBI by giving them one months notice. The particulars of this fund are decided in the beginni ng of financial year. Interest rate and period of deposit are also to be decided by RBI.FOREIGN BANKS Foreign banks which fail to achieve the target have to contribute to Small Industries Development Bank of India (SIDBI) or funds with other financial institutions, as specified by RBI . The particulars of this fund are decided in the beginning of financial year. Interest rate and period of deposit are also to be decided by RBI. Non-achievement of meeting the priority sector targets are considered while granting regulatory approvals for various purposes. PRIORITY SECTOR ADVANTAGES 1.Financial Inclusion It provided credit availability for small-marginal farmers, and to those sections which were previously deprived of taking any credit from the institutions. 2. Previously because of high default rate amongst the weaker sections,the institutions were reluctant to give credit to those people which forces the farmers or the weaker people to go to the money-lenders who charged them high r ate of interests (varying between 10% to 50%). Mandatory lending to priority sector has eradicated this problem and ensured advances by the institutions. 3.Poverty Alleviation If the timely credit is provided to small households, they can give more inputs to their produces which will result in better productivity. In effect agricultural GDP grows, which helps in upliftment of both the base and secondary sector which are dependent on small scale industries and agriculture, directly or indirectly. It generates more employment, hence, resulting in poverty alleviation. 4. Social Inclusion Poorer sections previously were deprived of participating in various community activities. The rise in their livelihood has given them a strong support to participate in various social activities.PRIORITY SECTOR MAJOR ISSUES 1. High Non-performing assets Since borrowers are not able to repay the loan on time, have created a fear in the banks and get them to make slow disbursement of loans. 2. Qua ntitative targets Since, the stringent targets has been set by RBI, this has resulted in lowering the quality of delivering targets. 3. regime interference Due to the regional Government intervention, the more influential people get the loan, and the poorer still get ignored. So, rich gets more richer. 4. Transaction cost Handling disbursement of huge quantity of small loans requires more time and labor. 5.Low absorption of credits -This occurs due to lack of capital infrastructure in agriculture and other small scale industries. 6. Low advantageousness -Low rate of interest charged from the borrowers makes this sector vulnerable. STRATEGIES AHEAD 1. Initiatives by Government a) Recovery of Non-Performing Assets Establishing Debt-recovery tribunals this will act as a mediator between the bank and borrower and will help bank in better recovery from the borrowers. Internal audit before sanctioning of loan should be done. b)Strengthen the cooperative bank network to increase credi t advances to the farmers. c)Link crop-insurance with loan amount.This mitigates the risk for Lender and borrower. d)Promote group lending to people group lending develops a collective responsibility amongst the borrowers which decreases the default rate. e)Government need to promote rigorous extension activities for promoting modern agricultural techniques for increasing production. f)Strict actions needs to be taken against the banks for not meeting the priority sector criteria. 2. Initiatives by Bank a)Banks should increase the term and delay the installments under term loan in case the borrowers are not able to repay in time. b)They should not charge compound interest on the loan amount.In a nutshell, Government need to strengthen backward and forward linkage both to provide inputs, increase productivity and develop markets. EXHIBITS Exhibit 1 Target achieved by Public Sector banks Exhibit 2 Target achieved by Private Banks Exhibit 3 Target achieved by foreign banksREFERENCES P riority Sector lending information (2010). Retrieved on August 4, 2010 from-http//www. rbi. org. in/scripts/FAQView. aspx? Id=8Trends, issues and strategies (2010). Retrieved on Aug 5, 2010 from-http//www. academicjournals. org/jat/PDF/Pdf2009/December/Uppal. pdfPlanning Commission reports on labour and employment (2010). Retrieved on Aug 5, 2010 from-http//books. google. co. in/books? id=qOOmWsfqfe4C&pg=PA96&lpg=PA96&dq=priority+sector+lending+appraisal&source=bl&ots=HZTEbRCSVo&sig=QtcebyqWJ5xWqkZ_TMdmPzCp4-4&hl=en&ei=KbFaTLK7DISXrAe9u52-DA&sa=X&oi=book_result&ct=result&resnum=9&ved=0CEsQ6AEwCAv=onepage&q&f=falseAll India Debt and Investment Surveys (2002). Retrieved on August 6 ,2010 from- http//www. rbi. org. in/scripts/BS_SpeechesView. aspx? Id=298Trend and Progress of Indian Banking 2008-09 (2009). Retrieved on August 6, 2010 from- http//www. rbi. org. in/scripts/AnnualPublications. aspx? head=Trend%20and%20Progress%20of%20Banking%20in%20India