Fundamental questions still remain regarding the detailed mechanisms of enzyme activity and its relationship to enzyme structure. The two most powerful tools that have been brought to bear on these questions in modern times are the continued development and use of biophysical probes of protein structure, and the application of molecular biological methods to enzymology. X-ray crystallography continues to be used routinely to solve the structures of enzymes and of enzyme—ligand complexes. In addition, new NMR methods and magnetization transfer methods make possible the assessment of the three-dimensional structures of small enzymes in solution, and the structure of ligands bound to enzymes, respectively.

The application of Laue diffraction with synchrotron radiation sources holds the promise of allowing scientists to determine the structures of reaction intermediates during enzyme turnover, hence to develop detailed pictures of the individual steps in enzyme catalysis. Other biophysical methods, such as optical (e.g., circular dichroism, UV—visible, fluorescence) and vibrational (e.g.,infrared, Raman) spectroscopies, have likewise been applied to questions of enzyme structure and reactivity in solution. Technical advances in many of these spectroscopic methods have made them extremely powerful and accessible tools for the enzymologist. Furthermore, the tools of molecular biology have allowed scientists to clone and express enzymes in foreign host organisms with great efficiency. Enzymes that had never before been isolated have been identified and characterized by molecular cloning. Overexpression of enzymes in prokaryotic hosts has allowed the purification and characterization of enzymes that are available only in minute amounts from their natural sources. This has been a tremendous advance for protein science in general.

The tools of molecular biology also allow investigators to manipulate the amino acid sequence of an enzyme at will. The use of site-directed mutagenesis (in which one amino acid residue is substituted for another) and deletional mutagenesis (in which sections of the polypeptide chain of a protein are eliminated) have allowed enzymologists to pinpoint the chemical groups that participate in ligand binding and in specific chemical steps during enzyme catalysis.

The study of enzymes remains of great importance to the scientific community and to society in general. We continue to utilize enzymes in many industrial applications. Moreover enzymes are still in use in their traditional roles in food and beverage manufacturing. In modern times, the role of enzymes in consumer products and in chemical manufacturing has expanded greatly. Enzymes are used today in such varied applications as stereospecific chemical synthesis, laundry detergents, and cleaning kits for contact lenses.

Perhaps one of the most exciting fields of modern enzymology is the application of enzyme inhibitors as drugs in human and veterinary medicine. Many of the drugs that are commonly used today function by inhibiting specific enzymes that are associated with the disease process. Aspirin, for example, one of the most widely used drugs in the world, elicits its antiinflammatory efficacy by acting as an inhibitor of the enzyme prostaglandin synthase. As illustrated in Table 1.1, enzymes take part in a wide range of human pathophysiologies, and many specific enzyme inhibitors have been
developed to combat their activities, thus acting as therapeutic agents. Several
of the inhibitors listed in Table 1.1 are the result of the combined use of biophysical methods for assessing enzyme structure and classical pharmacology
in what is commonly referred to as rational or structure-based drug design.
This approach uses the structural information obtained from x-ray crystallography
or NMR spectroscopy to determine the topology of the enzyme active site. Next, model building is performed to design molecules that would fit well into this active site pocket. These molecules are then synthesized and tested as inhibitors. Several iterations of this procedure often lead to extremely potent inhibitors of the target enzyme.

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Directed Enzyme Evolution: Screening and Selection Methods
(Methods in Molecular Biology)
From Humana Press


Click here for order

Product Description
Seasoned practitioners from many leading laboratories describe their best readily reproducible screening strategies for isolating useful clones. These techniques have been optimized for sensitivity, high throughput, and robustness, and are of proven utility for directed evolution purposes. The assays presented use a variety of techniques, including genetic complementation, microtiter plates, solid-phase screens with colorimetric substrates, and flow cytometric screens. An accompanying volume, Directed Evolution Library Creation: Methods and Protocols (ISBN 1-58829-285-1), describes readily reproducible methods for the creation of mutated DNA molecules and DNA libraries.

Copy for Both Volumes

Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods constitute an extraordinary collection of all the key methods used today for directed evolution research. Described in step-by-step detail to ensure robust experimental results, these methods will enable both newcomers and more experienced investigators to design and implement directed evolution strategies for the engineering of novel proteins. The first volume describes methods for the creation of mutated DNA molecules, or DNA libraries, encoding variants of desired proteins. The second volume describes methods for screening DNA libraries to isolate mutant proteins that exhibit a specified function.

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Product Details
Amazon Sales Rank: #869739 in Books
Published on: 2003-05-16
Original language: English
Number of items: 1
Binding: Hardcover
370 pages

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Editorial Reviews
Review
"...covers a considerable number of protocols for a broad range of enzymes...very useful..." - ChemBioChem

covers a considerable number of protocols for a broad range of enzymes very useful ChemBioChem

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INTRODUCTION
Alcohol-based fuels have been important energy sources since the 1800s. As early as 1894, France and Germany were using ethanol in internal combustion engines. Henry Ford was quoted in 1925 as saying that ethanol was the fuel of the future [1]. He was not the only supporter of ethanol in the early 20th century. Alexander Graham Bell was a promoter of ethanol, because the decreased emission to burning ethanol [2]. Thomas Edison also backed the idea of industrial uses for farm products and supported Henry Ford’s campaign for ethanol [3]. Over the years and across the world, alcohol-based fuels have seen short-term increases in use depending on the current strategic or economic situation at that time in the country of interest. For instance, the United States saw a resurgence in ethanol fuel during the oil crisis of the 1970s [4]. Alcohols have been used as fuels in three main ways: as a fuel for a combustion engine (replacing gasoline), as a fuel additive to achieve octane boosting (or antiknock) effects similar to the petroleum-based additives and metallic additives like tetraethyllead, and as a fuel for direct conversion of chemical energy into electrical energy in a fuel cell.

Alcohols are of the oxygenate family. They are hydrocarbons with hydroxyl functional groups. The oxygen of the hydroxyl group contributes to combustion. The four most simplistic alcoholic fuels are methanol, ethanol, propanol, and butanol. More complex alcohols can be used as fuels; however, they have not shown to be commercially viable. Alcohol fuels are currently used both in combustion engines and fuel cells, but the chemistry occurring in both systems is the same. In theory, alcohol fuels in engines and fuel cells are oxidized to form carbon dioxide and water. In reality, incomplete oxidation is an issue and causes many toxic by-products including carbon monoxide, aldehydes, carboxylates, and even ketones. The generic reaction for complete alcohol oxidation in either a combustion engines or a fuel cell is



It is important to note this reaction occurs in a single chamber in a combustion engine to convert chemical energy to mechanical energy and heat, while in a fuel cell, this reaction occurs in two separate chambers (an anode chamber where the
alcohol is oxidized to carbon dioxide and a cathode chamber where oxygen is
reduced to water.)

ETHANOL
Ethanol (also known as ethyl alcohol) is the most common of alcohols. It is the form of alcohol that is in alcoholic beverages and is easily produced from corn, sugar, or fruits through fermentation of carbohydrates. Its chemical structure is
CH3CH2OH. It is less toxic than methanol. The LD50 for oral consumption by a rat is 7060 mg/kg [5]. The LD50 for inhalation by a rat is 20,000 ppm for 10 hours [6]. The NIOSH recommended exposure limit is 1000 ppm for 10 hours [7]. Ethanol is available in a pure form and a denatured form. Denatured ethanol contains a small concentration of poisonous substance (frequently methanol) to prevent people from drinking it. Ethanol is a colorless liquid with a melting point of –144°C and a boiling point of 78°C. It is less dense than water with a density of 0.789 g/ml and soluble at all concentrations in water. Ethanol is frequently used to form blended gasoline fuels in concentrations between 10–85%. More recently, it has been investigated as a fuel for direct ethanol fuel cells (DEFC)and biofuel cells. Ethanol was deemed the “fuel of the future” by Henry Ford and has continued to be the most popular alcoholic fuel for several reasons: (1)it is produced from renewable agricultural products (corn, sugar, molasses, etc.) rather than nonrenewable petroleum products, (2) it is less toxic than the other alcohol fuels, and (3) the incomplete oxidation by-products of ethanol oxidation (acetic acid (vinegar) and acetaldehyde) are less toxic than the incomplete oxidation by-products of other alcohol oxidation.

BUTANOL
Butanol is the most complex of the alcohol-based fuels. It is a four-carbon alcohol
with a structure of CH3CH2CH2CH2OH. Butanol is more toxic than either methanol or ethanol. The LD50 for oral consumption of butanol by a rat is 790 mg/kg. The LD50
for skin adsorption of butanol by a rabbit is 3400 mg/kg. The boiling point of butanol is 118°C and the melting point is –89°C. The density of butanol is 0.81 g/mL, so it is more dense than the other two alcohols, but less dense than water. Butanol is commonly used as a solvent, but is also a candidate for use as a fuel. Butanol can be made from either petroleum or fermentation of agricultural products. Originally, butanol was manufactured from agricultural products in a fermentation process referred to as ABE, because it produced Acetone-Butanol and Ethanol. Currently, most butanol is produced from petroleum, which causes butanol to cost more than ethanol, even though it has some favorable physical properties compared to ethanol. It has a higher energy content than ethanol. The vapor pressure of butanol is 0.33 psi, which is almost an order of magnitude less than ethanol (2.0 psi) and less than both methanol (4.6 psi) and gasoline (4.5 psi). This decrease in vapor pressure means that there are less problems with evaporation of butanol than the other fuels, which makes it safer and more environmentally friendly than the other fuels. Butanol has been proposed as a replacement for ethanol in blended fuels, but it is currently more costly than ethanol. Butanol has also been proposed for use in a direct butanol fuel cell, but the efficiency of the fuel cell is poor because incomplete oxidation products easily passivate the platinum catalyst in a traditional fuel cell.

PROPANOL
Although propanols are three carbon alcohols with the general formula C3H8O, they are rarely used as fuels. Isopropanol (also called rubbing alcohol) is frequently
used as a disinfectant and considered to be a better disinfectant than ethanol, but it is rarely used as a fuel. It is a colorless liquid like the other alcohols and is flammable. It has a pungent odor that is noticeable at concentrations as low as 3 ppm. Isopropanol is also used as an industrial solvent and as a gasoline additive for dealing with problems of water or ice in fuel lines. It has a freezing point of –89°C and a boiling point of 83°C. Isopropanol is typically produced from propene from decomposed petroleum, but can also be produced from fermentation of sugars. Isopropanol is commonly used for chemical synthesis or as a solvent, so almost 2M tons are produced worldwide.

CONCLUSIONS
In today’s fuel market, methanol and ethanol are the only commercially viable fuels. Both methanol and ethanol have been blended with gasoline, but ethanol is the current choice for gasoline blends. Methanol has found its place in the market as an additive for biodiesel and as a fuel for direct methanol fuel cells, which are being studied as an alternative for rechargeable batteries in small electronic devices. Currently, butanol is too expensive to compete with ethanol in the blended fuel market, but researchers are working on methods to decrease cost and efficiency of production to allow for butanol blends, because the vapor pressure difference has environmental advantages. Governmental initiatives should ensure an increased use of alcohol-based fuels in automobiles and other energy conversion devices.

From:
Alcoholic Fuels
Shelley Minteer
Saint Louis University
Missouri

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Enzyme Assays: A Practical Approach

(The Practical Approach Series, 257)

From Oxford University Press, USA




Product Description
Enzyme assays are among the most frequently performed procedures in biochemistry and are routinely used to estimate the amount of enzyme present in a cell or tissue, to follow the purification of an enzyme, or to determine the kinetic parameters of a system. The range of techniques used to measure the rate of an enzyme-catalysed reaction is limited only by the nature of the chemical change and the ingenuity of the investigator. This book describes the design and execution of enzyme assays, covering both general principles and specific chapters. Building upon the highly popular first edition, this book combines revised or rewritten chapters with entirely new contributions. Topics include include experimental protocols covering photometric, radiometric, HPLC, and electrochemical assays, along with methods for determining enzyme assays after gel electrophoresis. The theory underlying each method is outlined, together with a description of the instrumentation, sensitivity and sources of error. Also included are chapters on the principles of enzyme assay and kinetic studies; techniques for enzyme extraction; high- throughout screening; statistical analysis of enzyme kinetic data; and the determination of active site concentration. This second edition of Enzyme Assays will be valuable not only to biochemists, but to researchers in all areas of the life sciences.

Product Details
Amazon Sales Rank: #671394 in Books
Published on: 2002-06-20
Original language: English
Number of items: 1
Binding: Paperback
302 pages

Editorial Reviews
Review Provides a very detailed and useful discussion of practical issues in kinetic analysis, illustrates with relevant examples, and provides primary references to the biochemical literature, which are up-to-date ... It is clearly written and illustrated, and will appeal to professional biochemists interested in enzymes. Natural Product Reports
Review 'Although the approach is practical, underlying theory is explained for each technique and the book will be useful to both laboratory researchers and advanced students.' Aslib Book Guide, Vol. 57, No. 8, August 1992
About the Author Robert Eisenthal is in the Department of Biology and Biochemistry, University of Bath, UK. Michael Danson is in the Department of Biology and Biochemistry, University of Bath, UK.

Customer Reviews
Enzyme Assays Explained
In an age of computerized and automated asay systems, it is possible to gather data without understanding the chemistry or biochemistry involved. This may be suitable for lab techs who work for commercial companies, but it is not a good idea for serious scientists. This book explains how assays work, what can go wrong and what the limitations are. It also reviews the types of instrumentation commonly used in assays. It is an excellent resource for laboratory instructors and graduate students.
A good guide to the principles of enzyme assay development. Enzyme Assays: A Practical Approach is a good guide to the principles of assay development. It contains excellent chapters on the theoretical development of kinetic expressions and the statistical treatment of results. Different assays are illustrated (photometric, radiometric, chromatographic, and electochemical) in their respective chapters that include important considerations for assay development/use with each system. A chapter on buffers and protein determination is also included. Although the book provides some examples (in useful detail) of each kind of assay it is NOT a comprehensive overview. The chapters on electrochemical assays and the techniques for enzyme extraction are incomplete due only to the age of the volume (>5 years old). This book would be an excellent volume for those interested in enzyme assay development and would be suitable for a first year graduate chemistry/biochemistry course.

Enzyme Assays: A Practical Approach is a good guide to the principles of assay development. It contains excellent chapters on the theoretical development of kinetic expressions and the statistical treatment of results. Different assays are illustrated (photometric, radiometric, chromatographic, and electochemical) in their respective chapters that include important considerations for assay development/use with each system. A chapter on buffers and protein determination is also included. Although the book provides some examples (in useful detail) of each kind of assay it is NOT a comprehensive overview. The chapters on electrochemical assays and the techniques for enzyme extraction are incomplete due only to the age of the volume (>5 years old). This book would be an excellent volume for those interested in enzyme assay development and would be suitable for a first year graduate chemistry/biochemistry course.

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The oldest known reference to the commercial use of enzymes comes from a description of wine making in the Codex of Hammurabi (ancient Babylon,circa 2100 ..). The use of microorganisms as enzyme sources for fermentation was widespread among ancient people. References to these processes can be found in writings not only from Babylon but also from the early civilizations of Rome, Greece, Egypt, China, India. Ancient texts also contain a number of references to the related process of vinegar production, which is based on the
enzymatic conversion of alcohol to acetic acid. Vinegar, it appears, was a common staple of ancient life, being used not only for food storage and preparation but also for medicinal purposes.

Dairy products were another important food source in ancient societies. Because in those days fresh milk could not be stored for any reasonable length of time, the conversion of milk to cheese became a vital part of food production, making it possible for the farmer to bring his product to distant markets in an acceptable form. Cheese is prepared by curdling milk via the action of any of a number of enzymes. The substances most commonly used
for this purpose in ancient times were ficin, obtained as an extract from fig trees, and rennin, as rennet, an extract of the lining of the fourth stomach of a multiple-stomach animal, such as a cow. A reference to the enzymatic activity of ficin can, in fact, be found in Homer’s classic, the Iliad:

As the juice of the fig tree curdles milk, and thickens it in a moment though it be liquid, even so instantly did Paee¨ on cure fierce Mars.

The philosopher Aristotle likewise wrote several times about the process of milk curdling and offered the following hypothesis for the action of rennet:

Rennet is a sort of milk; it is formed in the stomach of young animals while still being suckled. Rennet is thus milk which contains fire, which comes from the heat of the animal while the milk is undergoing concoction.

Another food staple throughout the ages is bread. The leavening of bread by yeast, which results from the enzymatic production of carbon dioxide, was well known and widely used in ancient times. The importance of this process to ancient society can hardly be overstated.

Meat tenderizing is another enzyme-based process that has been used since antiquity. Inhabitants of many Pacific islands have known for centuries that the juice of the papaya fruit will soften even the toughest meats. The active enzyme in this plant extract is a protease known as papain, which is used even today in commercial meat tenderizers. When the British Navy began exploring the Pacific islands in the 1700s, they encountered the use of the papaya fruit as a meat tenderizer and as a treatment for ringworm. Reports of these native uses of the papaya sparked a great deal of interest in eighteenth-century Europe, and may, in part, have led to some of the more systematic studies of digestive enzymes that ensued soon after.

From: Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
Robert A. Copeland

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Life depends on a well-orchestrated series of chemical reactions. Many of these reactions, however, proceed too slowly on their own to sustain life. Hence nature has designed catalysts, which we now refer to as enzymes, to greatly accelerate the rates of these chemical reactions. The catalytic power of enzymes facilitates life processes in essentially all life-forms from viruses to man. Many enzymes retain their catalytic potential after extraction from the living organism, and it did not take long for mankind to recognize and exploit the catalytic power of enzyme for commercial purposes. In fact, the earliest known references to enzymes are from ancient texts dealing with the manufacture of cheeses, breads, and alcoholic beverages, and for the tenderizing of meats.

Today enzymes continue to play key roles in many food and beverage manufacturing processes and are ingredients in numerous consumer products, such as laundry detergents (which dissolve protein-based stains with the help of proteolytic enzymes). Enzymes are also of fundamental interest in the health sciences, since many disease processes can be linked to the aberrant activities of one or a few enzymes. Hence, much of modern pharmaceutical research is based on the search for potent and specific inhibitors of these enzymes.

The study of enzymes and the action of enzymes has thus fascinated scientists since the dawn of history, not only to satisfy erudite interest but also because of the utility of such knowledge for many practical needs of society. This brief chapter sets the stage for our studies of these remarkable catalysts by providing a historic background of the development of enzymology as a science. We shall see that while enzymes are today the focus of basic academic research, much of the early history of enzymology is linked to the practical application of enzyme activity in industry.

From: Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.
Robert A. Copeland

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