Biocatalysis and Biodegration: Microbial Transformation of Organic Compounds
Authors: Lawrence P. Wackett1, C. Douglas Hershberger2Category: Applied and Industrial Microbiology; Environmental Microbiology
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Designed for use as a textbook in courses in biodegradation, this important new volume details both the fundamental concepts of the microbial transformation of organic compounds as well as its application for biotechnology and biodegradation. It offers comprehensive coverage of microbial catabolism from the group that developed the Biocatalysis/Biodegradation database on the Web and discusses the logic of catabolism use in predicting biodegradation.
Hardcover, 379 pages, illustrations, index.
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Chapter 1 : General Concepts in Biodegradation and Biocatalysis
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Abstract:
Bacteria take relentlessly from their environments, but they do so selectively, because the chemical composition of a bacterium differs markedly from that of its environment. Carbon is what we often think of as the building block of life. The genetic tape, DNA or RNA, is carbon based, as are the cell membrane; the catalysts, or enzymes and the energy storage molecules, such as ATP. The carbon cycle on Earth is largely dependent on microbiological processes, and biodegradation constitutes one-half of the carbon cycle. At the fundamental level, biodegradation and biocatalysis are inextricably linked by the biotransformation reactions and the enzymes catalyzing the reactions. Naphthalene dioxygenase is prototypical of an important class of aromatic hydrocarbon dioxygenases which initiate the biotransformation of thousands of naturally occurring and industrial hydrocarbons. The reaction with naphthalene and dioxygen yields cis-1,2-dihydroxydihydronaphthalene. This is thought of as a biodegradation reaction, as it typically initiates the metabolism of naphthalene and related compounds to produce carbon dioxide, cell carbon, and chemical energy captured as ATP. Nitrile hydratase enzyme has been studied for its role in biodegradation of the nitrile-containing herbicide bromoxynil.
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Chapter 2 : A History of Concepts in Biodegradation and Microbial Catalysis
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This chapter presents a historical view of microbial biodegradation and biocatalysis. One indicator of the importance of oxygenases in microbial catabolism, and in the reactions principally studied for biodegradation and biocatalysis, is their high level of occurrence in the Biocatalysis/Biodegradation Database. As the 20th century proceeded, biochemical investigations of microbes revealed two important and seemingly contradictory views of the biochemistry of life. First, the idea of the unity of biochemistry was established. At the same time, the diversity of microbial metabolism was continuously recorded as new microbes were identified and studied biochemically. The role of oxygen in microbially mediated transformation of chemicals was perhaps first suggested by the work of Jean Jacques Schloesing and A. Muntz. A classic treatment of the thermodynamics of anaerobic metabolism was published by Rolf Thauer and colleagues in 1977. This review also elegantly described the thermodynamic logic behind interspecies hydrogen transfer. While anaerobic metabolism was important in commercial fermentation processes, the details of anaerobic reactions were not well known because of difficulties in culturing anaerobic bacteria in pure culture. This derived partly from the predilection of microbiologists for agar plate culturing and the difficulty of maintaining plates under sufficiently anoxic conditions. Experiments examining the expression of metabolic activities in microbial cells led to the idea of catabolite control, or induction, of enzyme activity.
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Chapter 3 : Identifying Novel Microbial Catalysis by Enrichment Culture and Screening
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Enrichment culture is an experimental method which has been used extensively by microbiologists to obtain bacteria in monotypic, or pure, culture. Enrichment culture has yielded key information on the metabolic activities and genes of thousands of microorganisms. A pure culture offers a range of techniques for revealing the molecular details of biodegradation. Enrichment culture techniques allow selective cultivation of one or more bacterial strains obtained from a complex mixture such as that found in most soils. The method typically relies on using a particular organic compound as the sole carbon source or, less frequently, as the nitrogen, sulfur, or phosphorus source. In enrichment cultures conducted for obtaining a desired biocatalyst, a given pathway or reaction might be assumed; the goal is to obtain an enzyme of a known type which is highly active with a given substrate or under a specific set of conditions. In this case, the screening method is important, as it might be necessary to look at hundreds or thousands of enzymes yielded in the first round of screening. Another approach is to test existing strains to determine if they have the enzyme of interest and will show high activity with the substrate of interest. In this context, a limited taxonomic range of organisms is typically screened. The fungi screened include Aspergillus, Fusariiim, Trichoderma, Mucor, and Rhizomucor. These genera are known to produce a host of hydrolytic biodegradative enzymes of importance in industry. The enzymes include cellulase, lipase, and rennilase.
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Chapter 4 : Microbial Diversity: Catabolism of Organic Compounds Is Broadly Distributed
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This chapter focuses on microbial diversity and the catabolism of nonintermediary chemical compounds by disparate genera of prokaryotes. It describes the role that prokaryotes and fungi play collectively in biodegradation and how their extensive biocatalytic potential derives from a long evolutionary history. But microbial diversity also implies that individual bacteria and fungi are metabolically unique. In this context, it is useful to think of microbes as metabolic machines, dependent on gathering chemicals from their environment to obtain carbon, other elements, and energy to compete favorably against other microbes. Fungi are prominent in many environmental biodegradation processes and also in industrial biocatalysis. Four of the five major phyla of fungi are commonly used in industry. Of the ascomycota, Saccharomyces and Schizosaccharomyces are the major fungal genera used in alcoholic-beverage fermentations. Many of the best-studied prokaryotes are aerobic proteobacteria and the high-G + C gram-positive bacteria. These, along with the fungus Cunninghamella elegans, are the examples discussed in the chapter. It is important to point out that some of the extensive catabolic activities of this class are based on the broad substrate specificities of a few oxygenases and related enzymes which handle the oxygenated intermediates.
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Chapter 5 : Organic Functional Group Diversity: the Unity of Biochemistry Is Dwarfed by Its Diversity
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The compounds to be considered in the context of biodegradation and biocatalysis are the known organic molecules, an ever-expanding set of over 10 million compounds. This chapter presents evidence from the natural-product literature to support the idea that many functional groups typically referred to as xenobiotic are in fact found in the natural biological world, exclusive of organic synthesis. A common practice in correlating the types of organic molecule with their ease of biodegradation is to define them as (i) natural products or (ii) industrial chemicals. Many organic functional groups are acted upon by the individual enzymes of biodegradation. A given enzyme typically transforms one organic functional group in isolation, for example, oxidizing an alcohol to an aldehyde or hydrolyzing an amide to a carboxylic acid and an amine. The chapter highlights the impressive diversity of biologically relevant chemical structures and provides a framework for categorizing their microbial metabolism. The diversity of organic compounds is effectively infinite, and over 10 million compounds are currently described in the chemical literature. Understanding the microbial metabolism of such a broad range of compounds necessitates an efficient categorization of microbial reactions.
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Chapter 6 : Physiological Processes: Enzymes, Emulsification, Uptake, and Chemotaxis
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This chapter focuses on single-enzyme-catalyzed biodegradation reactions and other physiological processes that microbes use to compete successfully for scarce nutritional resources in soil and water. It is likely that a lot of reactions that fall under the general heading of biodegradation are fortuitous. There are numerous examples of this with insects or fungi that biosynthesize broad-specificity enzymes, such as cytochrome P450 monooxygenases, for detoxifying biological toxins. For example, plant-pathogenic fungi are sometimes warded off with toxic chemicals manufactured in the leaves of the plant being attacked. Fungal cytochrome P450 monooxygenases oxidize an enormous array of compounds, some of which are unlikely to prove toxic, and thus these reactions may well fall into the fortuitous category. Catabolic enzymes are so useful in large part because many have been found. In fact, catabolic enzymes may be the major group of enzymes catalyzing unique reactions found on the Earth. Additionally, there are the known catabolic enzymes which catabolize industrial chemicals. The role of biosurfactants in microbial metabolism has been investigated primarily with petroleum or with purified alkanes. Surfactants are compounds which are amphipathic; that is, they contain hydrophilic and hydrophobic chemical groups linked together in the same molecule. People use surfactants as soaps and detergents and as emulsifying agents in food. The sensing of chemical compounds starts with binding at the cell membrane to a methyl-accepting chemotaxis protein (MCP). The extracellular sensing is transmitted through the MCP, which spans the membrane, to its cytoplasmic domain.
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Chapter 7 : Evolution of Catabolic Enzymes and Pathways
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This chapter focuses on prokaryotic evolution in the context of biodegradation and microbial biocatalysis. It discusses the major evolutionary families of microbial catabolic enzymes, current ideas as to how genes are recruited and acquire new functions, and how new metabolic pathways arise and are disseminated among different prokaryotes. The discussion starts with a brief history of molecular-evolution studies. Then, it will be developed using primarily one example: how soil bacteria have evolved to use the herbicide atrazine as their sole source of nitrogen. Some microbial protein superfamilies important in biodegradation and biocatalysis are provided in a tabular form in which the major headings generally follow the major Enzyme Commission (EC) headings: (i) oxidoreductases, (ii) transferases, (iii) hydrolases, (iv) lyases, (v) isomerases, and (vi) ligases; except that lyases and isomerases are clustered together. The scientific literature suggests that in some cases, industrial chemicals initially evade microbial catabolism, as evidenced by their persistence in the environment, but are later found to be readily biodegraded. Evolution is especially instructive to study microbial catabolic enzymes to learn how new enzymes evolve and are transferred globally. In this context, the study of evolution is expanding from its historical role of providing fundamental explanations to become a biological tool that offers great promise for using microbes in our efforts to develop a sustainable human society.
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Chapter 8 : Metabolic Logic and Pathway Maps
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This chapter makes extensive use of visual images of chemical compounds, in a familiar format, to highlight the underlying patterns of catabolism. Intermediary metabolism is defined as enzyme-catalyzed reactions common to most living things: bacteria, eukaryotic single-celled life, plants, and animals. More specifically, intermediary metabolism deals with (i) common energy metabolism—for example, the catabolism of glucose by the glycolytic, or Embden-Meyerhof, pathway—and (ii) common biosynthetic pathways, such as those that generate necessary amino acids, lipids, and carbohydrates. This is illustrated, in the form of an electronic circuit diagram of the intermediary metabolism of Escherichia coli. Catabolic pathways funnel into a limited set of key intermediates, getting the most metabolic bang from the limited genetic buck. Thus, a new pathway is not required for each compound the organism might metabolize if there is redundancy by generating a common intermediate. This is well illustrated in aromatic-ring metabolism, which often proceeds through catecholic intermediates aerobically and benzoylcoenzyme A (CoA) anaerobically. These points are illustrated in the chapter by using a series of metamaps. The metamaps discussed include C1 metamap, C2 metamap, cycloalkane metamap, BTEX metamap, PAH metamap, heterocyclic-ring metamap, triazine-ring metamap, organohalogen metamap, and organometallic metamap.
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Chapter 9 : Predicting Microbial Biocatalysis and Biodegradation
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The ability to accurately predict biodegradation has enormous implications. Industries will continue to synthesize new materials faster than they, or regulatory agencies and academic researchers, can study their environmental fates. This chapter focuses largely on the prediction of biodegradation pathways. It is recognized that the use of the term "metabolic pathway" is coming under criticism, as pathways do not exist in isolation and occur as many variations on a theme. One approach to prediction is the quantitative structure activity relationships (QSAR) method, in which certain structural features of a molecule are correlated with some outcome, in this case biodegradability. Another set of systems have had the goal of predicting metabolic pathways for biodegradation. The basic principles needed for prediction are defining (i) the metabolic trunk pathways that initial biodegradation reactions funnel into and (ii) the enzymatic mechanisms by which different organic functional groups are metabolized. It is readily apparent that many compounds in anaerobic benzenoid ring metabolism feed into the common intermediate benzoyl-coenzyme A (CoA). Benzoyl-CoA is further metabolized reductively, and the ring is cleaved to produce 3-hydroxypimelyl-CoA.
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Chapter 10 : Microbial Biotechnology: Chemical Production and Bioremediation
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Certain large-scale fermentation processes persisted for some time, but after World War II, there was a tremendous rise in the size and influence of the commodity chemical industry. The industry has increasingly looked into alternatives, and slowly but surely, biotechnology is making inroads into commodity chemical production. For specialty chemical production, many different microorganisms are used, for example, Streptomyces sp. for antibiotics and Corynebateria sp. for amino acids. For many years the herbicide was marketed as the racemate, (R,S)- dichloroprop. The (S)-isomer is inactive as a herbicide. As a result, application of the racemate imposed an additional and unnecessary burden on the environment for the biodegradation of the herbicide. In the South Dakota field test, it was decided to implement several treatments. The statistical software used to deal with the large variations was MacAnova, a package developed at the University of Minnesota. The goal of this statistical treatment was to attain a final atrazine level in the treated soils of 2,000 ppm or lower. This study demonstrated that a combination of laboratory pretesting, innovative technology, and regulatory agency-industry cooperation can result in the management of an accidental spill. In the past, applications of biodegradation and biocatalysis for chemical manufacture, even while using similar enzymes, have been carried out by different practitioners. This is changing as industry seeks cleaner practices and strives for greater competitiveness.
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Chapter 11 : The Impact of Genomics on Microbial Catalysis
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This chapter gives a brief introduction and then focuses on the projected major influence of genomics on the field of biocatalysis and biodegradation. The clustering of prokaryotic genomes within an order of magnitude contrasts with those of different multicellular eukaryotes, which vary by 4 orders of magnitude. While the lifestyles of prokaryotes differ widely, it is likely that they have an upper limit of genome size based on the need to replicate efficiently and thus have fairly compact genomes. While DNA sequence analysis is yielding important patterns, the greatest genomic richness results from assigning metabolic functions to individual genes and deriving a biological usefulness of the gene in the context of the organism and its environment. The process requires a comparison of new DNA sequences with sequences in databases, and as such is a very computationally intense exercise. The ultimate goal is to map a sequence back to one whose biological function has been well established by a variety of methods. Most assignments of new gene sequences match known sequence types with high confidence about 60% of the time. Genes can be assigned to a previously elucidated function based on sequence homology arguments. However, if a gene is discovered which encodes an enzyme catalyzing a fundamentally new reaction, that biological function will never be deduced from the sequence alone.
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Chapter 12 : The Extent of Microbial Catalysis and Biodegradation: Are Microbes Infallible?
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It is often speculated that every non-polymeric compound conceived of by humans and occurring in nature will be metabolized by some microorganism somewhere in the soil or water of the Earth. Is this true? Can it be studied systematically? This chapter provides answers to these questions. The beginnings of an answer are first addressed with existing knowledge. Then, ideas are presented as to how the far reaches of microbial metabolism can be further identified. This effort is also linked to the widespread enterprise of microbial functional genomics. The total extent of genomic diversity is unknown, but comparative genomic analysis to date suggests that genetic diversity in prokaryotes is greater than previously anticipated. This, along with other evidence presented in the chapter, suggests that there is much catalytic diversity yet to be discovered. In the wealth of catalytic diversity possessed by microbial enzymes, two issues are relevant. First, it is unlikely that we have uncovered all of the catalytic potential of these known cofactors. Moreover, it is unclear how many new cofactors, metals, and modified amino acids remain to be discovered in microbial systems. To determine the potential for microbes to transform the exotic compounds for which metabolism is unknown, several compounds containing such functional groups were put into standard enrichment cultures in which the compounds were used as sole carbon or nitrogen sources to support growth. Most of these yielded actively growing cultures of microorganisms, and the target compound was transformed in the cultures, as demonstrated by high-pressure liquid chromatography.
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Chapter 13 : Big Questions and Future Prospects
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This chapter discusses questions related to microbial biocatalysis and biodegradation. These questions were selected for their important impact on applications in biocatalysis and biodegradation and on one's basic understanding of microbial physiology, genomics, and ecology. The preponderance of Pseudomonas and related species in the biodegradation literature is due, at least in part, to the conditions used for their enrichment and cultivation. Comparative genomics is beginning to show, not unexpectedly, that the amount of catabolic metabolism encoded in the genome varies considerably, depending on whether the bacterium is an obligate parasitic pathogen or a soil organism known to have extensive biodegradative capabilities, such as Pseudomonas spp. and Sphingomonas spp. It is unclear how rapidly new metabolism can evolve, practically and theoretically. One approach that promises to shed light on this is the use of directed evolution in the laboratory. This can reveal the plasticity of microbial enzymes and pathways and point the way toward a better understanding of what happens in the soils of the world. It seems very likely that even more challenges will be posed for microbial metabolism, and one will need to continually discover new microbial metabolism to match the discovery of new organic compounds.
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