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You browse ‘Enzyme Biocatalysis’ in category ‘Essay examples’.. 326 6th. 4. several Immobilization-Stabilization of Dehydrogenases………. 329 6. 5. 4 Jet Engineering……………………………. 330 6. 4. Production of Long-Chain Fat with Dehydrogenases 331 Recommendations…………………………………………….. 332 6th. 5 Utilization of Aldolases for Asymmetric Synthesis………………… 333? Josep L? pez-Sant? n, Gregorio Alvaro, and Pere Clap? s o? e 6th. 5. 1 Aldolases: Para? nitions and Classi? cation…………….. 334 6. 5. a couple of Preparation of Aldolase Biocatalysts……………….. 335 six. 5. three or more Reaction Performance: Medium Executive and Kinetics.. 339 six. 5. some Synthetic Applications…………………………. 346 six. 5. five Conclusions………………………………….. 352 References…………………………………………….. 352 6. 6 Application of Enzymatic Reactors intended for the Destruction of Very and Terribly Soluble Recalcitrant Compounds……………….. 355 o Juan M. Consigna, Gemma Eibes, Carmen T? pez, Meters. Teresa Moreira, and Gumersindo Feijoo 6. 6. 1 Potential Application of Oxidative Nutrients for Environmental Purposes………………………… 355 6. six. 2 Requirements for an Ef? cient Catalytic Cycle…………. 357 6. 6. three or more Enzymatic Aeroplano Con? gurations…………………. 358 6. 6. 4 Modeling of Enzymatic Reactors………………….. 364 6. 6. five Case Studies………………………………….. 365 6th. 6. 6 Conclusions and Perspectives…………………….. 374 References…………………………………………….. 375 Index…………………………………………………… 379 6. a few. 3 Foreword This book was written with all the purpose of offering a sound basis for the design of enzymatic reactions based on kinetic principles, nevertheless also to give an up to date vision in the potentials and limitations of biocatalysis, particularly with respect to recent applications in processes of organic and natural synthesis. The? rst? empieza chapters are structured as a book, going in the basic principles of enzyme framework and function to reactor design and style for homogeneous systems with soluble enzymes and heterogeneous systems with immobilized enzymes.

The last chapter of the book is split up into six sections that represent illustrative circumstance studies of biocatalytic processes of industrial significance or potential, written by experts in the respective? elds. We all sincerely hope that this guide will stand for an element inside the toolbox of graduate students in used biology and chemical and biochemical engineering and also of undergraduate students with formal training in organic chemistry, biochemistry, thermodynamics and chemical reaction kinetics. Beyond that, the publication pretends likewise to illustrate the potential of biocatalytic processes with case studies in the? ld of organic and natural synthesis, which we hope will be of interest for the instituto and professionals involved in R, D, My spouse and i. If some of our young readers ought to engage or persevere within their work in biocatalysis this will undoubtedly be our more valuable reward.? a Too much has been written about publishing. Nobel laureate Gabriel Garc? a Meters? rquez wrote one of its most inspired books by writing about writing (Living to Tell the Tale). There he had written “life can be not what one resided, but what a single remembers and exactly how one recalls it to be able to recount it. This barely applies to a scienti? book, but certainly highlights what is applicable to any book: its symbiosis with life. Authoring biocatalysis offers given me personally that privileged feeling, even more so because enzymes are genuinely the factors of your life. Biocatalysis is usually hardly separable from warring and publishing this book has become certainly more an ecstasy than an agony. A book is an object of love who better than good friends to build this. Eleven recognized professors and researchers have got contributed to this kind of endeavor with the knowledge, their particular commitment and their encouragement. Beyond our common language, My spouse and i share with all of them a view and a life-lasting friendship.

That is what is placed behind this book and made the construction a thrilling and satisfying experience. ix x Foreword Chapters 3 to 5 were drafted with the priceless collaboration of Claudia Altamirano and Lorena Wilson, two of my previous students, today my fellow workers, and my personal bosses My spouse and i am worried. Chapter 5 also included the experience of Jos? Manuel Guis? in, e a Roberto Fern? ndez-Lafuente and C? sar Mateo, all of them very good friends who have a electronic were kind enough to participate this project and improve the publication with their community known knowledge in heterogeneous biocatalysis. Section 6. is definitely the result of a cooperation sustained by a CYTED project that brought jointly Sonia Barberis, also a former graduate college student, now a prosperous professor and permanent collaborator and, over and above that, a dear friend, Fanny Guzm? in, a reputed scientist in the? eld of peptide a synthesis who is my partner, support and inspiration, and Josep L? pez, a well-known o man of science and professional but , above all, a friend at heart and a warm number. Section 6th. 3 was your result of a joint job with Gregorio Alvaro, a dedicated researcher who may have been a permanent collaborator with our group in addition to a very particular friend and kind host. Section 6. is the result of a collaboration, in a really challenging? eld of used biocatalysis, of Dr . Guisan’s group which we have a long-lasting educational connection and strong personal ties. Section 6. 5 represents an extremely challengo electronic ing job in which Josep L? ignorar and Gregorio Alvaro have joined Pere Clap? h, a dominant researcher in organic activity and a buddy through the years, to formulate an up to date review on a very attention grabbing? eld of enzyme biocatalysis. Finally, section 6. six is a collaboration of a special friend and outstanding instructor, Juan Consigna, and his exploration group that widens the scope of biocatalysis for the? ld of environmental executive adding a specific? avor to this? nal section. A substantial component to this book was written in Spain while doing a sabbatical inside the o Universitat Aut` noma de Barcelona, where I was warmly managed by the Chemical Engineering Office, as I likewise was during short stays at the Company of Catalysis and Petroleum Chemistry in Madrid with the Department of Chemical substance Engineering in the Universidad para Santiago para Compostela. My recognition for the persons within my institution, the Ponti? cia Universidad Feline? lica de Valpara? therefore , that recognized and motivated this project, particularly to o? the rector Prof.

Alfonso Muga, and instructors Atilio Bustos and Graciela Mu? ounces n Last but not least, my greatest appreciation to the persons for Springer: Marie Johnson, Meran Owen, Tanja van Gaans and Padmaja Sudhakher, who had been always delicate, diligent and encouraging. Dear audience, the wisdom about the merchandise is yours, but beyond the item there is a procedure whose splendor I hope to have been able to transmit. I actually count on the indulgence with language that, despite the effort of our publisher, may still reveal each of our condition of nonnative English speakers. Andr? h Illanes e Valpara? therefore , May 15, 2008? Part 1 Launch Andr? s Illanes electronic. 1 Catalysis and Biocatalysis Many chemical reactions can occur spontaneously, others need to be catalyzed to carry on at a signi? cannot rate. Factors are elements that reduce the magnitude from the energy hurdle required to always be overcame to get a substance to get converted chemically into another. Thermodynamically, the magnitude with this energy hurdle can be quickly expressed with regards to the free-energy change. Since depicted in Fig. 1 ) 1, catalysts reduce the value of this hurdle by virtue of it is interaction with all the substrate to form an triggered transition sophisticated that gives the product and frees the catalyst.

The catalyst is not used or modified during the response so , in principle, it can be used inde? nitely to convert the base into product, in practice, yet , this is restricted to the stability of the catalyst, that may be, its ability to retain its active composition through period at the conditions of reaction. Biochemical reactions, this is, the chemical reactions that comprise the metabolism coming from all living cells, need to be catalyzed to proceed at the speed required to maintain life. This kind of life factors are the enzymes. Each one of the biochemical reactions from the cell metabolic process requires to be catalyzed by one particular speci? chemical. Enzymes are protein substances that have evolved to perform ef? ciently under the mild circumstances required to preserve the functionality and integrity of the biological systems. Enzymes can be viewed as then since catalysts that have been optimized through evolution to perform their physical task where all varieties of life hinge. No wonder how come enzymes are equipped for performing a wide range of chemical reactions, many of which extremely complex to do by substance synthesis. Not necessarily presumptuous to state that any kind of chemical reaction currently described could have an enzyme able to catalyze it.

Actually the feasible primary constructions of an enzyme protein made up of n protein residues is 20n so that for a alternatively small healthy proteins molecule containing 100 amino acid residues, you will discover 20100 or 10130 possible School of Biochemical Architectural, Ponti? cia Universidad Kitten? lica sobre Valpara? so , Avenida País e do mundo o? 2147, Valpara? so , Chile. Telephone: 56-32-273642, fax: 56-32-273803, email: [email, protected] cl? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Research + Organization Media W. V. 08 1 two Trasition Condition A. Illanes Catalyzed Path Uncatalyzed Route

Free Energy Ea Ea’ Reactans? G Products Reaction Improvement Fig. 1 . 1 System of catalysis. Ea and Ea will be the energies of activation with the uncatalyzed and catalyzed response.? G is a free energy modify of the effect amino acid sequences, which is a fabulous number, higher even than the number of elements in the whole universe. To get the proper enzyme for a certain reaction is then a matter of search and this is certainly challenging and exciting in the event that one knows that a small fraction of most living varieties have been previously isolated.

It is even more promising when one particular considers the possibility of obtaining GENETICS pools in the environment lacking to know the organism from which it comes then expressed that into a ideal host organism (Nield et al. 2002), and the opportunities of genetic remodeling of structural family genes by site-directed mutagenesis (Abi? n ain al. 2004). a Enzymes have been the natural way tailored to conduct under physical conditions. Yet , biocatalysis identifies the use of enzymes as method catalysts underneath arti? cial conditions (in vitro), so that a major problem in biocatalysis is to enhance these hysiological catalysts in to process factors able to carry out under the generally tough effect conditions of your industrial method. Enzyme catalysts (biocatalysts), as any catalyst, act by minimizing the energy hurdle of the biochemical reactions, without having to be altered as a result of the reaction they promote. Yet , enzymes display quite specific properties when compared with chemical factors, most of these real estate are a outcome of their sophisticated molecular composition and will be analyzed in section 1 . 2 .

Potentials and drawbacks of enzymes as procedure catalysts will be summarized in Table 1 ) 1 . Nutrients are highly desired catalysts when the speci? city of the reaction is actually a major issue (as it arises in pharmaceutical products and? nenni chemicals), if the catalysts must be active underneath mild conditions (because of substrate and product instability or to prevent unwanted side-reactions, as it arises in several reactions of organic and natural synthesis), the moment environmental constraints are exacting (which is currently a 1 Advantages Table 1 ) 1 Advantages and Drawbacks of Nutrients as Factors Advantages Large speci? ity High activity under moderate conditions High turnover quantity Highly biodegradable Generally viewed as natural goods Drawbacks High molecular complexity High development costs Inbuilt fragility a few rather basic situation which gives biocatalysis a definite advantage more than alternative technologies) or when the label of natural system is an issue (as in the case of meals and aesthetic applications) (Benkovic and Ballesteros 1997, Wegman et ‘s. 2001). Nevertheless , enzymes happen to be complex molecular structures which have been intrinsically momentaneo and expensive to produce, that happen to be de? ite disadvantages regarding chemical catalysts (Bommarius and Broering 2005). While the features of biocatalysis is there to stay, the majority of its present restrictions could be and are getting solved through research and development in various areas. Actually enzyme leveling under method conditions is known as a major issue in biocatalysis and lots of strategies have already been developed (Illanes 1999) including? 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 approach. 2001, Mateo et approach. 2005, a Kim ain al. 06\, Wilson et al. 006), crystallization (H? ring and Schreier 99, Roy a and Abraham 2006), aggregation (Cao et al. 2003, Mateo ain al. 2005, Schoevaart et al. 2004, Illanes ou al. 2006) and the modern techniques of protein executive (Chen 2001, Declerck et al. the year 2003, Sylvestre et al. 06\, Leisola and Turunen 2007), namely site-directed mutagenesis (Bhosale et ‘s. 1996, Ogino et ‘s. 2001, Boller et ‘s. 2002, van den Burg and Eijsink 2002, Adamczak and Hari Krishna 2004, Bardy ain al. 2005, Morley and Kazlauskas 2005), directed advancement by conjunction mutagenesis (Arnold 2001, Brakmann and Johnsson 2002, Alexeeva et ‘s. 003, Boersma et ‘s. 2007) and gene-shuf? e based on polymerase assisted (Stemmer 1994, Zhao et ‘s. 1998, Shibuya et al. 2000, Kaur and Sharma 2006) and, more recently, ligase assisted recombination (Chodorge ainsi que al. 2005). Screening intended for intrinsically stable enzymes is usually a dominant area of research in biocatalysis. Extremophiles, that is certainly, organisms able to survive and thrive in extreme environmental conditions can be a promising resource for highly stable enzymes and exploration on all those organisms is incredibly active at the moment (Adams and Kelly 1998, Davis 1998, Demirjian ainsi que al. 001, van family room Burg the year 2003, Bommarius and Riebel 2005, Gomes and Steiner 2004). Genes by such extremophiles have been cloned into appropriate hosts to build up biological systems more amenable for development (Halld? rsd? ttir ainsi que al. 98, o um Haki and Rakshit the year 2003, Zeikus ou al. 2004). Enzymes will be by no means great process catalysts, but their incredibly high speci? city and activity below moderate conditions are dominant characteristics that are to be increasingly appreciated by several production areas, among that this pharmaceutical and? ne-chemical industry (Schmid et al. 001, Thomas ou al. 2002, Zhao et al. 2002, Bruggink et al. 2003) have included with the more classic sectors of food (Hultin 1983) and detergents (Maurer 2004). 5 Fig. 1 ) 2 System of peptide bond development between two adjacent? -amino acids R1 + H3N CH C OH Um A. Illanes H R2 + H N CH COO? INGESTING WATER R1 WATER H R2 H3N CH C In CH COO? O + 1 . 2 Enzymes while Catalysts. Structure”Functionality Relationships The majority of the characteristics of enzymes because catalysts get from their molecular structure. Nutrients are protein composed by a number of amino acid residues that range from 95 to several hundreds.

These proteins are covalently bound through the peptide bond (Fig. 1 ) 2) that may be formed between carbon atom of the carboxyl group of one amino acid plus the nitrogen atom of the? -amino group of the subsequent. According to the character of the 3rd there’s r group, amino acids can be non-polar (hydrophobic) or polar (charged or uncharged) and their distribution along the proteins molecule determines its patterns (Lehninger 1970). Every healthy proteins is trained by the amino acid collection, called primary structure, which is genetically dependant on the deoxyribonucleotide sequence in the structural gene that unique codes for it.

The DNA collection is? rst transcribed to a mRNA molecule which after reaching the ribosome is translated into an amino acid collection and? nally the produced polypeptide sequence is transformed into a threedimensional structure, called native framework, which is normally the one endowed with biological efficiency. This modification may include many post-translational reactions, some of which is often rather relevant for its functionality, like proteolytic tits, as it occurs, for instance, with Escherichia coli penicillin acylase (Schumacher ainsi que al. 986) and glycosylation, as it takes place for several eukaryotic enzymes (Longo et approach. 1995). The three-dimensional framework of a proteins is then genetically determined, yet environmentally trained, since the molecule will interact with the surrounding medium. This is specifically relevant to get biocatalysis, where the enzyme works in a channel quite different from the one in which will it was synthesized than can alter its native functional framework. Secondary 3d structure is a result of connections of protein residues proximate in the main structure, generally by hydrogen bonding of the amide groupings, for the ase of globular protein, like digestive enzymes, these communications dictate a predominantly ribbon-like coiled que incluye? guration known as? -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 garbled con? guration in which the area is abundant with polar alanine 1 Launch 5 residues, while the inner part can be abundant in hydrophobic amino acid elements. This tertiary structure is essential for the biological efficiency of the protein.

Some aminoacids have a quaternary three-dimensional structure, which is common in regulatory healthy proteins, that is the response to the discussion of different polypeptide chains constituting subunits which could display similar or several functions within a protein complicated (Dixon and Webb lates 1970s, Creighton 1993). The main types of relationships responsible for the three-dimensional framework of aminoacids are (Haschemeyer and Haschemeyer 1973): ¢ Hydrogen provides, resulting from the interaction of a proton linked to an electronegative atom with another electronegative atom.

A hydrogen relationship has around one-tenth in the energy kept in a covalent bond. Is it doesn’t main determinant of the helical secondary framework of globular proteins and it plays a signi? cant function in tertiary structure as well. ¢ Apolar interactions, due to the common repulsion from the hydrophobic protein residues by a polar solvent, like normal water. It is a alternatively weak conversation that does not symbolize a proper chemical bond (approximation between atoms exceed the van welcher Waals radius), however , the contribution towards the stabilization with the threedimensional structure of a protein is quite signi? ant. ¢ Disulphide connections, produced by oxidation of cysteine residues. They can be especially relevant in the leveling of the 3d structure of low molecular weight extracellular proteins. ¢ Ionic a genuine between charged amino acid elements. They contribute to the stabilization with the three-dimensional composition of a proteins, although to a lesser degree, because the ionic strength with the surrounding medium is usually high so that interaction is created preferentially between amino acid residues and ions in the method. Other weak type relationships, like vehicle der Waals forces, in whose contribution to three-dimensional composition is not considered signi? cant. Proteins can be conjugated, this is, linked to other substances (prosthetic groups). In the case of digestive enzymes which are conjugated proteins (holoenzymes), catalysis always occur in the protein area of the chemical (apoenzyme). Prosthetic groups could possibly be organic macromolecules, like carbohydrates (in the situation of glycoproteins), lipids (in the case of lipoproteins) and nucleic acids (in the truth of nucleoproteins), or straightforward inorganic agencies, like material ions.

Prosthetic groups happen to be tightly certain (usually covalently) to the apoenzyme and do not dissociate during catalysis. A signi? cant quantity of enzymes by eukaryotes happen to be glycoproteins, whereby the carbohydrate moiety can be covalently linked to the apoenzyme, largely through serine or threonine residues, and even though the carbs does not participate in catalysis it confers relevant properties towards the enzyme. Catalysis takes place in a small portion of the enzyme named the energetic site, which is usually produced by hardly any amino acid residues, while the remaining portion of the protein provides for a scaffold.

Papain, for instance, has a molecular pounds of twenty three, 000 De uma with 211 amino acid residues of which just cysteine (Cys 25) and histidine (His 159) six A. Illanes are straight involved in catalysis (Allen and Lowe 1973). Substrate is likely to the enzyme at the active site and doing so, modifications in our distribution of electrons in the chemical bonds are produced that cause the reactions that lead to the formation of products. These products are then simply released from the enzyme which is ready for the next catalytic cycle.

According to the early on lock and key version proposed by Emil Fischer in 1894, the active site includes a unique geometric shape that may be complementary towards the geometric shape of the substrate molecule that? ts into it. Even though recent surveys provide evidence in favor of this theory (Sonkaria et al. 2004), this kind of rigid model hardly clarifies many trial and error evidences of enzyme biocatalysis. Later on, the induced-? capital t theory was proposed (Koshland 1958) relating to which he substrate induces a change in the enzyme conformation after joining, that may orient the catalytic groups in a way prone pertaining to the subsequent effect, this theory has been substantially used to make clear enzyme catalysis (Youseff ou al. 2003). Based on the transition-state theory, enzyme catalysis has been discussed according to the hypothesis of chemical transition condition complementariness, which will considers the prefc erential binding with the transition express rather than the substrate or merchandise (Benkovi? and Hammes-Schiffer 2003).

Many, however, not all, enzymes require little molecules to do as catalysts. These molecules are called coenzymes or cofactors. The word coenzyme can be used to refer to small molecular weight organic and natural molecules that associate reversibly to the enzyme and are certainly not part of its structure, coenzymes bound to digestive enzymes actually take part in the reaction and, therefore , will be sometime called cosubstrates, since they are stoichiometric in nature (Kula 2002). Coenzymes often function as intermediate service providers of bad particals (i. electronic. NAD+ or perhaps FAD+ in dehydrogenases), speci? c atoms (i. elizabeth. oenzyme Queen in L atom transfer) or useful groups (i. e. coenzyme A in acyl group transfer, pyridoxal phosphate in amino group transfer, vitamin h in LASER transfer) which have been transferred in the reaction. The term cofactor is usually used to make reference to metal ions that as well bind reversibly to nutrients but in standard are not chemically altered throughout the reaction, cofactors usually combine strongly for the enzyme structure so that they are certainly not dissociated through 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 organizations as represented in Fig. 1 . three or more: (i) those that do not need of an further molecule to do biocatalysis, (ii) those that require cofactors that remain unaltered and securely bound to the enzyme performing in a catalytic fashion, and (iii) all those requiring coenzymes that are chemically modi? ed and dissociated during catalysis, performing within a stoichiometric style. The requirement of cofactors or coenzymes to perform biocatalysis has outstanding technological implications, as will be analyzed in section 1 ) 4.

Enzyme activity, this can be, the capacity associated with an enzyme to catalyze a chemical reaction, is usually strictly dependent upon its molecular structure. Chemical activity depends upon the presence of a proper structure of the energetic site, which is composed with a reduced number of amino acid residues close inside the three-dimensional composition of 1 Launch Fig. 1 . 3 Nutrients according for their cofactor or coenzyme requirements. 1: zero requirement, 2: cofactor needing, 3: coenzyme requiring H 1 7 P Elizabeth E CoE 2 S i9000 E-CoE P E CoE 3 Elizabeth CoE’ Electronic P S i9000 E-CoE the protein nevertheless usually much apart in the primary structure.

Therefore , any kind of agent that promotes healthy proteins unfolding is going to move a part the elements constituting the active site and will then simply reduce or destroy it is biological activity. Adverse circumstances of temperature, pH or perhaps solvent and the presence of chaotropic chemicals, heavy metals and chelating agents will produce this decrease of function simply by distorting the proper active web page con? guration. Even though a really small area of the enzyme molecule participates in catalysis, the remaining with the molecule through no means irrelevant to its performance.

Crucial houses, like enzyme stability, are extremely much dependent upon the chemical three-dimensional framework. Enzyme stability appears to be based on unde? ned irreversible procedures governed by local unfolding in certain momentaneo regions denoted as weakened spots. These types of regions at risk of unfolding are definitely the determinants of enzyme steadiness and are usually located in or close to the area of the proteins molecule, which is why the surface structure of the enzyme is so necessary for its catalytic stability (Eijsink et ‘s. 2004). These regions have been completely the target of site-speci? c mutations for increasing stability.

Though substantially studied, rational engineering from the enzyme molecule for improved stability has been a very complex task. Typically, these fragile spots aren’t easy to identify so it is unclear to what location of the necessary protein molecule will need to one always be focused on and, even though effectively selected, not necessarily clear what is the right form of mutation to introduce (Gaseidnes et ing. 2003). In spite of the impressive developments in the? eld and the living of several experimentally primarily based rules (Shaw and Bott 1996), rational improvement in the stability continues to be far from being well-established.

In fact , the less realistic approaches of directed progression using error-prone PCR and gene shuf? ing have been completely more successful in obtaining more stable mutant enzymes (Kaur and Sharma 2006). Both equally strategies can combine by using a set of detailed designed mutants that can then be subjected to gene shuf? ent (O’F? g? in 2003). a a A perfectly organised native chemical expressing their biological activity can suffer the loss by unfolding of their tertiary structure to a random polypeptide string in which the amino acids located in the active site are no longer lined up closely enough to perform it is catalytic function.

This sensation is known as denaturation and it may be invertable if the denaturing in? uence is taken off since simply no chemical changes 8 A. Illanes have occurred in the proteins molecule. The enzyme molecule can also be put through chemical changes that generate irreversible decrease of activity. This kind of phenomenon is definitely termed inactivation and usually happens following unfolding, since a great unfolded protein is more prone to proteolysis, decrease of an essential cofactor and assimilation (O’F? g? in 1997). These tendency de? e what is called thermodynamic or perhaps cona a formational stableness, this is the amount of resistance of the folded away protein to denaturation, and kinetic or long-term stability, this is the resistance to irreversible inactivation (Eisenthal ainsi que al. 2006). The overall technique of enzyme inactivation can then be displayed by: N <>U? >I where N represents the native active conformation, U the unfolded conformation and I the irreversibly inactivated enzyme (Klibanov 1983, Bommarius and Broering 2005). The? rst step can be de? ned by the equilibrium constant of unfolding (K), while the second is de? ed in terms of the rate constant for irreversible inactivation (k). Stability is not related to activity and in many cases they have opposite trends. It has been suggested that there is a trade-off between stability and activity based on the fact that stability is clearly related to molecular stiffening while conformational? exibility is bene? cial for catalysis. This can be clearly appreciated when studying enzyme thermal inactivation: enzyme activity increases with temperature but enzyme stability decreases. These opposite trends make temperature a critical variable in any enzymatic process and make it prone to optimization.

This aspect will be thoroughly analyzed in Chapters 3 and 5. Enzyme speci? city is another relevant property of enzymes strictly related to its structure. Enzymes are usually very speci? c with respect to its substrate. This is because the substrate is endowed with the chemical bonds that can be attacked by the functional groups in the active site of the enzyme which posses the functional groups that anchor the substrate properly in the active site for the reaction to take place. Under certain conditions conformational changes may alter substrate speci? city.

This has been elegantly proven by site-directed mutagenesis, in which speci? c amino acid residues at or near the active site have been replaced producing an alteration of substrate speci? city (Colby et al. 1998, diSioudi et al. 1999, Parales et al. 2000), and also by chemical modi? cation (Kirk Wright and Viola 2001). K k 1. 3 The Concept and Determination of Enzyme Activity As already mentioned, enzymes act as catalysts by virtue of reducing the magnitude of the barrier that represents the energy of activation required for the formation of a transient active complex that leads to product formation (see Fig. . 1). This thermodynamic de? nition of enzyme activity, although rigorous, is of little practical signi? cance, since it is by no means an easy task to determine free energy changes for molecular structures as unstable as the enzyme”substrate complex. The direct 1 Introduction 9 consequence of such reduction of energy input for the reaction to proceed is the increase in reaction rate, which can be considered as a kinetic de? nition of enzyme activity. Rates of chemical reactions are usually simple to determine so this de? nition is endowed with practicality.

Biochemical reactions usually proceed at very low rates in the absence of catalysts so that the magnitude of the reaction rate is a direct and straightforward procedure for assessing the activity of an enzyme. Therefore, for the reaction of conversion of a substrate (S) into a product (P) under the catalytic action of an enzyme (E): S? >P v=? ds dp = dt dt (1. 1) E If the course of the reaction is followed, a curve like the one depicted in Fig 1. 4 will be obtained. This means that the reaction rate (slope of the p vs t curve) will decrease as the reaction proceeds.

Then, the use of Eq. 1. 1 is ambiguous if used for the determination of enzyme activity. To solve this ambiguity, the reasons underlying this behavior must be analyzed. The reduction in reaction rate can be the consequence of desaturation of the enzyme because of substrate transformation into product (at substrate depletion reaction rate drops to zero), enzyme inactivation as a consequence of the exposure of the enzyme to the conditions of reaction, enzyme inhibition caused by the products of the reaction, and equilibrium displacement as a consequence of the law of mass action.

Some or all of these phenomena are present in any enzymatic reaction so that the catalytic capacity of the enzyme will vary throughout the course of the reaction. It is customary to identify the enzyme activity with the initial rate of reaction (initial slope of the “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 = vt>0 =? ds dt = t>0 dp dt (1. 2) t>0 This is not only of practical convenience but fundamentally sound, since the enzyme activity so de? ned represents its maximum catalytic potential under a given set of experimental conditions. To what extent is this catalytic potential going to be expressed in a given situation is a different matter and will have to be assessed by modulating it according to the phenomena that cause its reduction. All such phenomena are amenable to quanti? ation as will be presented in Chapter 3, so that the determination of this maximum catalytic potential is fundamental for any study regarding enzyme kinetics. Enzymes should be quanti? ed in terms of its catalytic potential rather than its mass, since enzyme preparations are rather impure mixtures in which the enzyme protein can be a small fraction of the total mass of the preparation, but, even in the unusual case of a completely pure enzyme, the determination of activity is unavoidable since what matters for evaluating the enzyme performance is its catalytic potential and not its mass.

Within the context of enzyme kinetics, reaction rates are always considered then as initial rates. It has to be pointed out, however, that there are situations in which the determination of initial reaction rates is a poor predictor of enzyme performance, as it occurs in the determination of degrading enzymes acting on heterogeneous polymeric substrates. This is the case of cellulase (actually an enzyme complex of different activities) (Montenecourt and Eveleigh 1977, Illanes et al. 988, Fowler and Brown 1992), where the more amorphous portions of the cellulose moiety are more easily degraded than the crystalline regions so that a high initial reaction rate over the amorphous portion may give an overestimate of the catalytic potential of the enzyme over the cellulose substrate as a whole. As shown in Fig. 1. 4, the initial slope o the curve (initial rate of reaction) is proportional to the enzyme concentration (it is so in most cases). Therefore, the enzyme sample should be properly diluted to attain a linear product concentration versus time relationship within a reasonable assay time.

The experimental determination of enzyme activity is based on the measurement of initial reaction rates. Substrate depletion or product build-up can be used for the evaluation of enzyme activity according to Eq. 1. 2. If the stoichiometry of the reaction is de? ned and well known, one or the other can be used and the choice will depend on the easiness and readiness for their analytical determination. If this is indifferent, one should prefer to measure according to product build-up since in this case one will be determining signi? ant differences between small magnitudes, while in the case of substrate depletion one will be measuring small differences between large magnitudes, which implies more error. If neither of both is readily measurable, enzyme activity can be determined by coupling reactions. In this case the product is transformed (chemically or enzymatically) to a? nal analyte amenable for analytical determination, as shown: E S P A X B Y C Z 1 Introduction 11 In this case enzyme activity can be determined as: a = vt>0 =? ds dt = t>0 dp dt = t>0 dz dt (1. 3) t>0 rovided that the rate limiting step is the reaction catalyzed by the enzyme, which implies that reagents A, B and C should be added in excess to ensure that all P produced is quantitatively transformed into Z. For those enzymes requiring (stoichiometric) coenzymes: E S CoE CoE P activity can be determined as: a = vt>0 =? dcoe dt = t>0 dcoe dt (1. 4) t>0 This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i. e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase (? galactosidase, EC 3. 2. 1. 23). The enzyme catalyzes the hydrolysis of lactose according to: Lactose + H2 O >Glucose + Galactose Glucose produced can be coupled to a classical enzymatic glucose kit, that is: hexoquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD), in which: Glucose + ATP? >Glucose 6Pi + ADP Glucose 6Pi + NADP+??>6PiGluconate + NADPH where the first rate of NADPH (easily measured within a spectrophotometer, discover ahead) can be then stoichiometrically correlated to the initial price of lactose hydrolysis, so long as the additional enzymes, Hx and G6PD, and co-substrates are added in excess.

Enzyme activity can be discovered by a continuous or unsuccessive[obs3], broken, interrupted assay. If the analytical system is provided with a recorder that register the course of reaction, the initial level could be very easily determined from your initial incline of the merchandise (or substrate, or coupled analyte, or perhaps coenzyme) attention versus period curve. It is far from always conceivable or easy to set up a consistent assay, in that case, the span of reaction should be monitored discontinuously by sample and assaying at predetermined time intervals and trials should be exposed to inactivation to avoid the reaction.

This is certainly a disadvantage, since the enzyme should be quickly, completely and irreversibly inactivated by revealing it to harsh conditions that can interfere with the G6PD Hx doze A. Illanes analytical process. Data items should describe a thready “p versus “t romance within the time interval pertaining to assay to ensure that the initial charge is being measured, if not, enzyme test should be diluted accordingly. Assay time ought to be short enough to make the effect of the products around the reaction price negligible and also to produce a negligibly reduction in substrate concentration. A major issue in enzyme activity perseverance is the para? ition of your control research for dainty the nonenzymatic build-up of product during the assay. You will find essentially three options: to take out the enzyme from the effect mixture by replacing the enzyme test by normal water or barrier, to remove the substrate replacing it by water or perhaps buffer, or use an chemical placebo. The? rst a single discriminates base contamination with product or any type of non-enzymatic transformation of base into merchandise, but would not discriminate enzyme contamination with substrate or perhaps product, the second one functions exactly the contrary, the third one can in rinciple discriminate both equally enzyme and substrate contaminants with merchandise, but the mistake in this case is a risk of not having inactivated the enzyme totally. The control of choice depends upon what situation. For example, when you are producing a great extracellular chemical by fermentation, enzyme test is likely to be polluted with substrate and or product (that may be constituents from the culture medium or goods of metabolism) and may end up being signi? ant, since the test probably contains a low enzyme protein focus so that it is not diluted prior to assay, in this case, exchanging substrate simply by water or buffer discriminates such contaminants. If, alternatively, one is assaying a prep from an investment enzyme put emphasis, dilution with the sample ahead of assay makes unnecessary to blank away enzyme contamination, replacing the enzyme by water or perhaps buffer may discriminate substrate contamination that is in this case even more relevant.

The use of an chemical placebo because control is advisable if the enzyme is usually labile enough to be completely inactivated at conditions not really affecting the assay. Another solution is to use a double control replacing enzyme in one case and base in the various other by drinking water or barrier. Once the form of control try things out has been determined, control and enzyme test are put through the same deductive procedure, and enzyme activity is worked out by subtracting the control reading from that of the test, as illustrated in Fig.. 5. Conditional procedures readily available for enzyme activity determinations are many and usually many alternatives exist. A proper assortment should be based upon sensibility, reproducibility,? exibility, simpleness and supply. Spectrophotometry can be viewed as a technique that complete? ls the majority of, if only a few, such requirements. It is based upon the consumption of light of a certain wavelength since described by Beer”Lambert rules: A? =? m c where: A? = journal I I0 (1. 5) (1. 6) The value of? an be experimentally obtained through a calibration contour of absorbance versus attention of analyte, so that the browsing of A? enables the determination of its concentration. Optic path breadth is usually you cm. The strategy is based on the differential consumption of product (or joining analyte or modi? impotence 1 Intro 13 Fig. 1 . your five Scheme for the conditional procedure to ascertain enzyme activity. S: base, P: item, P0: item in control, A, B, C: coupling reactants, Z: analyte, Z0: analyte in control, t, p, unces are the matching molar concentrations oenzyme) and substrate (or coenzyme) at a certain wavelength. For instance, the reduced coenzyme NADH (or NADPH) includes a strong optimum of absorbance at 340 nm while the absorbance of the oxidized coenzyme NAD+ (or NADP+ ) is minimal at that wavelength, therefore , the activity of any kind of enzyme creating or consuming NADH (or NADPH) can be discovered by measuring the increase or perhaps decline of absorbance at 340 nm in a spectrophotometer. The assay is very sensitive, reproducible and and equipment is available in any research clinical.

If both equally substrate and product absorb signi? cantly at a certain wavelength, coupling the metal detector to an appropriate high performance water chromatography (HPLC) column can solve this kind of interference by separating those peaks simply by differential reifungsverzögerung of the analytes in the line. HPLC devices are more and more common in research laboratories, so this is an extremely convenient and? exible way for assaying enzyme activities. Several other analytical types of procedures are available for enzyme activity dedication.

Fluorescence, this is the ability of certain substances to absorb light at a specific wavelength and emit it at an additional, is a house than can be used for enzymatic analysis. NADH, but likewise FAD (? avin adenine dinucleotide) and FMN (? avin mononucleotide) have this property that can be used for those enzyme needing that substances as coenzymes (Eschenbrenner ain al. 1995). This method stocks some of the very good properties of spectrophotometry and can also be incorporated into an HPLC system, nonetheless it is less? exible and the tools not so common in a standard research lab.

Enzymes that produce or consume fumes can be assayed by differential box manometry by simply measuring tiny pressure distinctions, due to the intake of the gaseous substrate or perhaps the evolution of your gaseous merchandise that can be converted into substrate or product concentrations by using the gas law. Carboxylases and decarboxylases are categories of enzymes that could 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 CARBON DIOXIDE, has been assayed in a gear respirometer simply by measuring the increase in pressure caused by the formation of gaseous CO2 (O’Learys and Brummund 1974). Digestive enzymes catalyzing reactions involving optically active ingredients can be assayed by polarimetry. A compound is considered to be optically active in the event polarized light is rotated when passing through it. The magnitude of optical rotation is determined by the molecular structure and focus of the optically active element which has a unique speci? rotation, as para? ned in Biot’s law:? =? 0 m c (1. 7) Polarimetry is a simple and correct method for determining optically energetic compounds. A polarimeter can be described as low cost instrument readily available in many research labs. The metal detector can be integrated into an HPLC system in the event separation of substrates and products of reaction is required. Invertase (? -D-fructofuranoside fructohydrolase, EC 3. 2 . 1 ) 26), a commodity enzyme widely used in the food market, can be quickly assayed by polarimetry (Chen et approach. 2000), since the speci? optical rotation in the substrate (sucrose) differs from that of the items (fructose additionally glucose). Several depolymerizing digestive enzymes can be ideally assayed by viscometry. The hydrolytic actions over a polymeric substrate can make a signi? cant reduction in kinematic viscosity that can be correlated towards the enzyme activity. Polygalacturonase activity in pectinase preparations (Gusakov et ing. 2002) and endo? 1″4 glucanase activity in cellulose preparations (Canevascini and Gattlen 1981, Illanes and Schaffeld 1983) had been determined by computing the decrease in viscosity of the corresponding olymer solutions. An extensive review in methods for assaying enzyme activity has been lately published (Bisswanger 2004). Enzyme activity is expressed in units of activity. The Enzyme Commission of the Intercontinental Union of Biochemistry recommends to express it in intercontinental units (IU), de? ning 1 IU as the number of an enzyme that catalyzes the alteration of 1 mol of base per minute beneath standard conditions of temp, optimal ph level, and optimal substrate focus (International Union of Biochemistry).

Later on, 39 years ago, the Percentage on Biochemical Nomenclature advised that, in order to adhere to SI units, effect rates should be expressed in moles every second as well as the katal was proposed since the new product of chemical activity, para? ning it as the catalytic activity that will enhance the rate of reaction by 1 mol/second in a speci? ed assay system (Anonymous 1979). This latter para? nition, although recommended, has some practical disadvantages. The magnitude of the katal is so big that usual enzyme actions expressed in katals are incredibly small figures that are hard to appreciate, the de? ition, on the other hand, is quite vague with respect to the conditions in which the assay should be performed. In practice, even though in certain journals the katal is mandatory, there may be reluctance to work with it and the former IU is still extensively used. 1 Introduction 15 Going back to the de? nition of IU there are some points worthwhile to comment. The magnitude from the IU is acceptable to measure most chemical preparations, in whose activities usually range from a few to a few countless numbers IU per unit mass or product volume of prep.

Since enzyme activity is usually to be considered as the maximum catalytic potential of the chemical, it is quite appropriate to refer it to optimum pH and optimal base concentration. According to latter, ideal is to be considered as that substrate concentration when the initial charge of reaction is at its maximum, this will likely imply effect rate for substrate vividness for an enzyme subsequent typical Michaelis-Menten kinetics or maybe the highest primary reaction level value in the case of inhibition for high substrate concentrations (see Chapter 3).

With respect to ph level, it is simple to determine the value at which the first rate of reaction is in its optimum. This value will be the true operational ideal in most cases, since that pH will lie within the region of optimum stability. However , the opposite keeps for temperature where digestive enzymes are usually quite unstable in the temperatures through which higher initial reaction prices are attained, actually the concept of “optimum temperatures, as the one that maximizes preliminary reaction charge, is quite deceiving since that value generally re? cts nothing more than the departure in the linear “p versus “t relationship intended for the time of assay. Intended for the sobre? nition of IU it is then more appropriate to refer to it as being a “standard and never as a great “optimal temperature. Actually, it is rather dif? conspiracy to sobre? ne the right temperature to assay chemical activity. Most probably that value will differ from the one at which the enzymatic process will probably be conducted, it is best then to acquire a mathematical manifestation for the result of temperatures on the primary rate of reaction to be able to transform the units of activity in line with the temperature of operation (Illanes et ing. 000). It is not always possible to express chemical 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 include a varying and often unde? ned molecular weight and whose goods are usually a blend of different chemical compounds. In that case, devices of activity can be sobre? ned in terms of mass instead of moles. These kinds of enzymes are often speci? c for certain types of provides rather than for your chemical composition, so in such cases it is advisable to share activity in terms of equivalents of bonds damaged.

The choice of the substrate to accomplish the enzyme assay through no means trivial. Whenever using an chemical as procedure catalyst, the substrate could be different from that employed in it is assay that is usually a model substrate or perhaps an advertising agency. One has being cautious to work with an assay that is not just simple, accurate and reproducible, but also signi? cannot. An example that illustrates this time is the case of the chemical glucoamylase (exo-1, 4-? -glucosidase, EC three or more. 2 . 1 ) 1): this enzyme can be widely used within the manufacturing of glucose syrups from starch, either as a? al item or since an more advanced for the availability of high-fructose syrups (Carasik and Carroll 1983). The industrial substrate to get glucoamylase can be described as mixture of oligosaccharides produced by the enzymatic liquefaction of starch with? -amylase (1, 4-? -D-glucan glucanohydrolase, EC several. 2 . 1 ) 1). Several substrates had been used for assaying enzyme activity including excessive molecular weight starch, small molecular weight oligosaccharides, maltose and maltose synthetic équivalents (Barton ou al. 72, Sabin and Wasserman 18 A. Illanes 1987, Goto et ‘s. 1998). Probably none of them probably re? cts properly the enzyme activity over the genuine substrate, therefore it will be a couple of judgment and experience to pick the most essential assay with regards to the actual make use of the enzyme. Hydrolases are currently assayed regarding their hydrolytic activities, yet , the increasing use of hydrolases to perform reactions of activity in nonaqueous media make this type of assay not quite adequate to evaluate the synthetic potential of this kind of enzymes. As an example, the protease subtilisin has become used being a catalyst for a transesteri? cation reaction that produces thiophenol as one of the goods (Han ou al. 004), in this case, a way based on a reaction leading to a? uorescent adduct of thiophenol is a good system to assess the transesteri? cation potential of such proteases and is to be preferred into a conventional protease assay depending on the hydrolysis of a proteins (Gupta ou al. 99, Priolo ou al. 2000) or a unit peptide (Klein et ‘s. 1989). 1 ) 4 Enzyme Classes. Houses and Scientific Signi? cance Enzymes will be classi? impotence according to the suggestions of the Nombre Committee in the International Union of Biochemistry and biology and Molecular Biology (IUBMB) (Anonymous 1984) into six families, based on the type of reaction catalyzed.

A four digit number is usually assigned to each enzyme by the Enzyme Commission rate (EC) with the IUBMB: the? rst one denotes the family, the other denotes the subclass within a family and is related to the type of substance group upon which it acts, the 3rd denotes a subgroup within a subclass and is related to the particular chemical organizations involved in the effect and the on is the correlative number of identi? cation within a subgroup. The six people are: 1 . Oxidoreductases. Nutrients catalyzing oxidation/reduction reactions that involve the transfer of electrons, hydrogen or fresh air atoms.

There are 22 subclasses of oxido-reductases and one of them there are several of technological signi? cance, including the dehydrogenases that oxidize a substrate simply by transferring hydrogen atoms to a coenzyme (NAD+, NADP+