Assays Of Ectoenzyme Activity A Methods

There are significant difficulties in measuring ectoenzyme activities in heterogeneous environments such as natural waters and soil, which include questions about methodology and data interpretation. For example, should assays be performed according to the well-established principles of enzymology (e.g., excess substrate, optimal pH and temperature, shaking of reaction mixtures) or in situ conditions encountered in an aquatic environment (e.g., limiting and unevenly distributed substrate,...

References

Bacterial colonization and ectoenzymatic activity in phytoplank-ton-derived model particles. Part II. Cleavage and uptake of carbohydrates. Microb Ecol 36 66-74, 1998. 2. S Alvarez, MC Guerrero. Enzymatic activities associated with decomposition of particulate organic matter in two shallow ponds. Soil Biol and Biochem 32 1941-1951. 3. RA Blanchette. Delignification by wood-decay fungi. Annu Rev Phytopathol 29 381-398, 1991. 4. P Biely. Microbial xylanolytic...

Role Of Chemistry In Enzyme Activity Measurement

One of the fundamental requirements of enzyme measurements is a thorough understanding of the reactions involved, quantitative extraction of the product(s) released, and a suitable analytical method for measuring quantitatively the extracted compound. Therefore, knowledge of analytical chemistry and chemical kinetics are essential in soil enzyme research. In addition, because soils contain both organic constituents and mineral components, a thorough understanding of the potential reactions...

Oh

Poljchlorinated Bipheny Lindane Figure 1 Persistent halogenated compounds in the environment. DDT, dichlorodiphenyltrichloro-ethane, Lindane, y-hexachlorocyclohexane. Poljchlorinated Bipheny Lindane Figure 1 Persistent halogenated compounds in the environment. DDT, dichlorodiphenyltrichloro-ethane, Lindane, y-hexachlorocyclohexane. bromine. Generally, as the number of halogens per molecule increases, so does the recalcitrance of the compound. Chlorinated aromatics, including polychlorinated...

Function Of Biofilm Structure And Architecture

It would be naive to assume that a biofilm community is simply defined as microorganisms residing and growing at an interface. Microbes are, in fact, components of complex communities continuously responding to both their immediate microenvironment and their surrounding habitat. This is reflected in the range of biofilm structures from thin layers of attached cells, as seen with monocultures of some Pseudomonads or smooth colony variants of Vibrio cholerae (31), to more complex forms of...

Info

B Dassi, E Dumas-Gaudot, A Asselin, C Richard, S Gianinazzi. Chitinase and -1,3-gluca-nase isoforms expressed in pea roots inoculated with arbuscular mycorrhizal or pathogenic fungi. Eur J Plant Pathol 102 105-108, 1996. 125. MJ Pozo, E Dumas-Gaudot, S Slezack, C Cordier, A Asselin, S Gianinazzi, V Gianinazzi-Pearson, C Azcon-Aguilar, JM Barea. Induction of new chitinase isoforms in tomato roots during interactions with Glomus mosseae and or Phytophthora nicotianae var, parasitica....

To Arbuscular Mycorrhizal Fungal Colonization

Arbuscular mycorrhizal fungal penetration and establishment in the host roots involve a complex sequence of events and intracellular modifications that influence root colonization (25). Genotype and environmental factors influence the infection intensity or even the host compatibility and or resistance (33,105). The key to understanding the phenomenon of compatibility is to study recognition mechanisms and molecules involved in early stages of the AM interaction. In this sense, the formation of...

The Role Of Fungal Enzymes In The Biological Control Of Plant Diseases

Gliocladium and Trichoderma Species Systems The fungus Gliocladium virens Miller, Giddens and Foster ( Trichoderma virens, Miller, Giddens, Foster, and von Ark) is a common soil saprophyte and one of the most promising and studied fungal biocontrol agents. It originally was isolated from a sclerotium of the plant pathogenic fungus Sclerotinia minor and then was found to be active against several fungal plant pathogens. Trichoderma, a genus of hyphomycetes that is an anamorphic Hypocreaceae...

Selenium

Selenium belongs to group VIA of the periodic table and has been classified as a metalloid. In the environment Se exits in four oxidation states, +2, 0, +4, and +6, forming a variety of compounds. Selenate (SeO42 , Se6+) and selenite (SeO32 , Se4+) are the most common ions found in soil solution and natural waters. Organic Se-containing compounds include Se-substituted amino acids, such as selenomethionine, selenocysteine, and selenocystine, and volatile methyl species such as dimethylselenide...

Evolution Of Dehalogenating Enzymes

How did the genes that code for dehalogenases evolve and become distributed in the environment It is possible that the dehalogenases are at an early stage of evolution driven by the use of large quantities of both natural and synthetic halogenated organic compounds in the environment over the last 50 years. Are there different evolutionary explanations for various dehalogenases Given the information that we have determined to date, there are no absolute answers to these questions. Four possible...

Conclusions

Evidence has been provided that exoenzymes produced by many fungi and bacteria are strictly involved in the microbes' antagonism toward plant pathogens and pests. These exoenzymes, possessing chitinolytic, glucanolytic, cellulolytic, or proteolytic activities, can be used individually or in combination to provide an enzymatic basis for a number of processes, which ultimately lead to a biocontrol effect. Among these processes, one of the most intensively studied is the mycoparasitism seen in...

Soil Enzymes As Functional Indicators

Soil enzyme measurements are extremely important in assessing the status or the condition of the soil environment. This is because enzymes are essential to the cycling of nutrients in soil and are thus critical to the availability of nutrients to both microbiota and plants. Frequently enzymes are regulated and externalized as a response to exogenous soil conditions, such as phosphatase secretion due to phosphate deficiency (37,38) or plant chitinase as a result of fungal or insect attack (11),...

Of Nematodes

Plant-parasitic nematodes are major agronomic pests. The economic loss caused by nematodes is estimated at U.S. 100 billion worldwide (265) the nematocide market share is currently estimated at 500 million, which is only 1 40 of the world pesticide market (266). Bacteria are omnipresent and destroy nematodes in virtually all soils because of their constant association in the rhizosphere. Parasitoid endospore-forming bacteria, such as Pasteuria penetrans destroy nematodes by parasitic action,...

H2o

Trichloroacetic acid Ace lie acid formed by several anaerobic bacteria. It is coupled to energy conservation and therefore usually is termed dehalorespiration (50,51). A reduced organic substrate or H2 is required as the source of the two electrons and the protons. In vitro studies of reductive dehalogenation usually have used methyl viologen as the artificial electron donor. Methyl viologen is transformed from a reduced blue to an oxidized colorless form, thus allowing the progress of the...

Comparative Analyses

In the context of the successional loop model (Fig. 1), there are three dimensions for comparing studies of enzymatic decomposition enzyme activity and litter composition, enzyme activity and mass loss rate, enzyme activity and community composition. A fourth dimension is large-scale patterns in relation to ecosystem type or disturbance type. These comparisons are external to the model but integrate enzymatic decomposition into large-scale perspectives. At present, there are too few studies in...

Soil Hydrolase Enzymes To Assess Soil Contamination

Contamination by Crude Oil and Oil By-Products Because of the huge volumes of oil and its by-products that are produced, transported, and stored, there is a very serious threat of soil contamination in the vicinity of oil fields, refineries, and storage and distribution facilities. The effects of oil pollution on the activities of soil enzymes have been extensively reviewed by Kiss et al. (14). We therefore give only a synopsis of the data presented in that review and limit our discussion to...

Bioremediation of Mercury

A number of naturally occurring Hg biotransformations may have applications in the bio-remediation of Hg-contaminated soil and water. Mercury-resistant bacteria have been isolated by Glombitza at the Akademie der Wissenschaften (137,138). These Hg-resistant bacteria can accumulate up to 2 -4 by weight of nonvolatile Hg from aerated solutions containing 50-100 mg LHg2+ (139). Biomass can be suspended in a feed solution within a bioreactor and provided a C source (e.g., methanol or acetate) plus...

Microbial Foraging Using Extracellular Enzymes

The quantitative modeling work of Vetter and associates (10) addresses the specific use of extracellular enzymes as a bacterial foraging strategy in moderate-temperature microenvironments rich in particulate organic material (POM), of a size too large to pass the cell membrane without extracellular hydrolysis. The typical marine environment where this strategy is demonstrably advantageous involves an aggregation of POM-rich particles, either mineral grains with sorbed POM (as in marine...

Status Of The Search For Hyperthermophilic Microorganisms And Enzymes

Euryarchaeota Bacteria Under Microscope

Focus on Culturable Hyperthermophiles Although the discovery of hyperthermophilic microorganisms at marine hydrothermal vents was reported in 1982 (53,54), their potentially exciting activities in situ have been studied by few and remain poorly constrained (1, 28). The in situ activities of enzymes that hyperthermophiles may release into their surroundings are completely unknown. This general lack of ecological information on the functioning of either hyperthermophilic organisms or enzymes...

In Situ Enzyme Activity Direct Approach

True ecological information requires the detection of environmental processes under in situ conditions, which cannot be fully controlled and, therefore, cannot be simulated in the laboratory. The composition of naturally occurring substrates in water samples usually is unknown, and concentrations may vary widely over short sampling times. This condition complicates the choice of the substrate concentration being monitored in ectoenzyme assays because of the potential interference or competition...

Lake Microplankton

Currently, there is a large amount of scientific information indicating that besides biotic relations, the diversity of microbial communities, their biomass, and their metabolic activity depend strongly on the composition and biological availability of inorganic nutrients and or organic substrates in aquatic ecosystems (131-134). It is widely accepted that microorganisms, especially heterotrophic bacteria, play a crucial role in nutrient cycling and organic matter decomposition. The majority of...

Exploitation Of Microbial Enzymes From Plant Surfaces

The repertoire of enzymes possessed by microorganisms that colonize and proliferate on the aerial parts of plants is extensive and in many respects distinguishable from those that occur in other less extreme habitats. The enzymes must be able to function in environmental extremes such as temperature and moisture that are also prone to sudden fluctuations. The plant tissues (waxes, cutin, and cellulose) on which the microbial colonizers have to survive and from which they derive nutrients are...

Soil Minerals as Catalysts Pseudoenzymes in Biochemical Reactions

Soil minerals can affect the fate of biochemical compounds in soil in at least three main ways (1) incorporation of N-, P-, and S-bearing organics into the structural network of mineral colloids and adsorption of these organics to their surface consequently the dynam ics and bioavailability of these nutrients may be modified (124) (2) adsorption of enzymes on clay and or clay-organic complexes these immobilized enzymes are active and stable, but they exhibit activities quite different from...

Enzyme Assays And Their Effect On Data Interpretation

The two primary questions that must be addressed in assessing carbon and nitrogen metabolic processes in soil are, How can the activity be quantified and what is an appropriate assay method and How does the activity vary both in a relatively defined system in the test tube as well as in the more complex, heterogeneous environment of the soil A primary property that is intimately linked to the latter question is the kinetics of the reaction. Although a sound assay method based on a clear...

Mechanisms Of Dehalogenation

Dehalogenation Post Cleavage

It is recognized that compounds may be dehalogenated through a variety of pathways. For example, Ramanand et al. (18) followed the degradation in slurry microcosms of hexachlorobenzene, pentachlorobenzene, and 1,2,4-trichlorobenzene to chlorobenzene. Through an examination of intermediates that were degraded and those that remained recalcitrant, the authors argued that multiple pathways for degradation existed and that these pathways reflected the microorganisms present and the local physical...

Ectoenzyme Activity And Lake Water Eutrophication

The importance of organic matter as a variable for evaluating the trophic status of lakes has been recognized since the beginning of the 20th century (145,146). Increasing concentrations of organic constituents in water are the distinct indicators of accelerated eutrophi-cation processes in many lakes (147-149). Our studies clearly demonstrated that enzyme activities were significantly positively proportional to DOC content of lakes (Fig. 13C). As described earlier in this chapter, several...

Enzyme Indexes and Multivariate Analysis

As mentioned, microbial functional diversity depends on many metabolic reactions and interactions of microbiota. Therefore, it is unrealistic to assume that a simple relationship exists between measurements of a single enzyme activity and microbial functional diversity of soil. The simultaneous measurement of the activities of a range of enzymes is needed. The next step is proper analysis to allow interpretation of enzyme activities for assessing microbial functional diversity across different...

Enzyme Protein Concentration In Soils

Numerous attempts have been made to extract pure enzymes from soils, but in reality the best that has been achieved is the extraction of enzyme-containing substances and complexes (68). The reagents used in the extraction procedures range from water to salt solutions or buffers to strong organic matter-solubilization reagents, such as NaOH or sodium pyrophosphate. The extracted activities are usually associated with carbohydrate-enzyme protein complexes and are often difficult to purify. Modern...

Enzymes In Soils

Buffering Capacit Soils

The first known report on enzymes in soils was presented by Woods at the 1899 Annual Meeting of the American Association for the Advancement of Science in Columbus, Ohio (1). However, little significant progress occurred in the area of soil enzymology until the 1950s. This was mainly due to a lack of appropriate methodologies and understanding of the true nature of enzymes. Although Sumner first isolated urease in crystalline form from jack bean (Canavalia ensiformis) meal in 1926, for which he...

Origin And Association Of Enzymes With Aquatic Microorganisms

Three common terms are used for the enzymes involved in the transformation and degradation of polymeric substrates outside the cell membrane ectoenzymes (19), extracellular enzymes (20), and exoenzymes (21). In this chapter, the term ectoenzyme is used to refer to any enzyme that is secreted and actively crosses the cytoplasmic membrane and remains associated with its producer. Ectoenzymes are cell-surface-bound or periplasmic enzymes that react outside the cytoplasmic membrane with polymeric...

1

Figure 1 Diagram of steps employed in genetic fingerprinting of soil bacterial communities. Genetic fingerprinting techniques (Fig. 1) involve extraction and purification of nucleic acids directly from the soil, although some investigators prefer to separate the cells from the environment prior to cell lysis (12-14). Extraction is followed by amplification of specific target sequences by using the polymerase chain reaction (PCR). When RNA is the molecule of interest, reverse transcriptase is...

Enzymes Of Entomopathogenic Fungi And Bacteria For The Biocontrol Of Insect Pests

Bioinsecticides are used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. The agrochemical industry recently introduced highly insect-pest-specific insecticides of microbial origin, with modes of action that are targeted to a variety of insect species. Most of them are based on the use of Bacillus thuringiensis and Bacillus sphaericus, both of which produce insecticidal crystal protein toxin (Cry) of Streptomyces spp. producing...

Chit 67 Chit 58 Chit

Figure 3 Detection of chitinolytic activity (A) and Coomassie blue staining (B) of extracellular proteins produced by an E. agglomerans strain grown on minimal media with chitin, after separation by SDS-PAGE. Chitinolytic activity was detected with the (4-MU-(GlcNAc)2). The bands on lane B corresponding to chitinolytic enzymes visible on lane A are indicated by arrows. the ChiA endochitinase of S. marcescens (174). A soil-borne chitinolytic E. agglomerans strain IC1270 was found to be a strong...

Arsenic

Arsenic (As) is a metalloid of group VA of the periodic table and exists in four oxidation states, +5, +3, 0, and -3. It occurs naturally in the environment as well through anthropogenic discharge in a variety of chemical states. Arsenic forms alloys with various metals and covalently bonds with carbon, hydrogen, oxygen, and sulfur (59). Arsenate (AsO43-), a biochemical analog of phosphate, is transported by highly specific energy-dependent membrane pumps into the cell during assimilation of...

Determination of the Extracellular Enzyme Activity in Soil

Bacteriostatic agents have been used in enzyme assays to prevent microbial growth and thus synthesis of intracellular enzymes during incubation. This approach has been unsatisfactory because of changes in cell wall permeability and induction of plasmolysis with release of intracellular enzymes. In addition, the effect of bacteriostatic agents (usually toluene), and irradiation with gamma rays or electron beams have shown variable effects, depending on soil and enzyme assay conditions...

In Seawater

Enzymes in Coastal Regions, Lagoons, and Estuaries Coastal regions represent the transition zone between land and the open sea. Thus they are frequently characterized by local morphological and hydrographical patterns and by gradients of salinity, eutrophication, pollution, and sediment resuspension. These conditions are clearly reflected by specific patterns of extracellular organic matter degradation. In general, the enzymatic potential within the coastal regions affects the export of...

Enzymes In Biofilms

A number of different processes that occur within a microbial biofilm contribute to the creation of a heterogenous, dynamic environment. These processes, among many others, include cycling and exchange of nutrients, plasmid transfer, communication via chemical signals, and frequent deterioration of the surface (corrosion or degradation). The biofilm microorganisms respond by having different physiological characteristics, metabolic activity, and growth rates from those of unattached organisms...

Enzymatic Mechanisms Of Penetration And Formation Of The Symbiosis

The mechanisms by which endomycorrhizal fungi enter and spread through host tissues are unknown. Different steps in the infection process (e.g., formation of entry points, inter-and intracellular colonization) necessitate the growth of hyphae along the middle lamella or through cell walls of the host root. Only localized changes in wall texture have been observed as endomycorrhizal fungi penetrate epidermal cells or develop through the middle lamella of parenchymal tissue, suggesting that...

Chromium

Chromium (Cr) has many industrial uses, and, as a result, large volumes of Cr waste in various chemical forms are discharged into the environment. Chromium can exist in oxidation states ranging from 2 to +6. However, only +3 and +6 are normally found within the range of pH and redox potentials common in environmental systems. Hexavalent Cr (Cr6+) forms chromate (CrO42) and dichromate (Cr2O72), which are toxic and muta-genic, soluble over a wide pH range, and mobile in the environment....

Diversity Among Dehalogenase Enzymes

The diverse nature of the dehalogenases is apparent from the information already presented. Dehalogenases clearly illustrate that enzymatic similarity and diversity exists at several levels. Certain dehalogenases have a narrow substrate specificity, including the 2HAA dehalogenases (Table 1). The presence of genetically, immunologically, and mechanistically different dehalogenases that dehalogenate the same compound illustrates the diversity of structure and function that has been observed for...

Soil Hydrolase Enzymes To Assess Soil Recovery And Development After Mining

Many studies have demonstrated the decline of organic C, microbial biomass, and enzyme activities with increasing soil depth. Ross et al. (39) showed the removal of 10 cm, and especially 20 cm, of topsoil from temperate pasture plots markedly lowered activities of a number of enzymes. This finding is not at all surprising, since the top centimetres of a soil are the major loci of biological activity and organic matter. What is especially interesting, however, is that removal of 10 cm of topsoil...

Microbial Inoculation And Soil Enzyme Activities

A proposed target for genetic manipulation is the insertion or enhancement of genes encoding specific enzymes a prime candidate for this is chitinase because of its postulated role in the biocontrol of fungal crop pathogens. Ridout and colleagues (22) investigated the protein production induced in a Trichoderma species when cell wall fragments of the crop plant pathogen Rhizoctonia solani (to which the Trichoderma species was an antagonist) were used as the sole carbon source. Both P-glucanase...

Models of Interaction

Four different mechanisms currently are invoked to explain the modification of properties with pH between the free and adsorbed enzymes they are based either on the activity of the protons or the substrates in the microenvironment of the enzyme (interfacial pH effects, diffusional effects) or on the state of the adsorbed enzyme itself (orientation effects, conformational effects). Although increasingly contested, this hypothesis is the most frequently invoked to explain the pH shift in the...

Microbes and Enzymes in Biofilms

University of Illinois-Chicago, Chicago, Illinois Microbial enzymes and their activities have been studied primarily in pure liquid cultures under laboratory conditions. However, in natural environments microorganisms grow at interfaces as attached (sessile) mixed communities rather than as suspended planktonic populations (1). Studies of microbial enzymes in soil go some way to recognizing this, but data interpretation has often been difficult because the methodologies do not easily...

Bacterial Enzymes In The Biocontrol Of Plant Pathogens And Pests

Lytic Enzymes of Soil-Borne and Rhizospheric Bacteria in Plant-Pathogen Biocontrol Chitinase activity has been found in a wide variety of bacteria (59). Bacteria produce chitinase to digest chitin, primarily to utilize it as a C and energy source. The ability to produce lytic enzymes is a widely distributed property of soil, marine, and rhizosphere bacteria. Many of these are potential biocontrol agents of chitin-containing plant pathogens. The list of such bacterial antagonists includes...

Examples Of Using Enzymes For Bioremediation

As evident from the previous subsections, many enzymes that are able to transform hazardous substances are now available in a purified form. Catalytic, molecular, and biochemical features are known for several of these enzymes. Studies have been dedicated to the use of these enzymes for practical applications. Both free enzymes and enzymes immobilized on diverse matrices by different means have been applied for the transformation of polluting and nonpolluting compounds. Batch as well as reactor...

Isolated Enzymes for the Transformation and Detoxification of Organic Pollutants

University of Naples Federico II, Portici, Naples, Italy Center for Bioremediation and Detoxification, The Pennsylvania State University, University Park, Pennsylvania I. INTRODUCTION A. Pollution of the Environment Pollution of the environment has been one of the largest concerns for science and the general public in the last 50 years. The rapid industrialization of agriculture, expansions in the chemical industry, and the need to generate cheap forms of energy have all caused the continuous...

Biofilm Life Cycle

Biofilm Catheter

It is often easier to understand what a biofilm is in terms of the events that lead to its formation. Most studies of biofilms in natural environments have concentrated on events at solid-liquid interfaces, the colonization of a submerged abiotic surface is depicted in Fig. 1. Upon immersion of a nonbiological material, such as glass or silica, the surface becomes coated rapidly with a layer of proteinacous material called a conditioning layer (15-17). Ions and other nutrient sources accumulate...

Biosensors Of Soil Health

A biosensor is ''any biological material which, when exposed to an analyte (e.g. air, soil, water), provides an information linked response via a suitable transducer'' (38). The biological material used in a biosensor can comprise plants (whole plants, organs, or cells), vertebrates, invertebrates, microorganisms), microbial tissue, enzymes, nucleic acid probes, antibodies, as well as other kinds of biological receptor. In using biosensors to test for soil health, the analyte is the soil or...

Ljubljana Cezares

Origin, composition and microbial utilization of dissolved organic matter. In J Overbeck, RJ Chrost, eds. Aquatic Microbial Ecology Biochemical and Molecular Approaches. New York Springer-Verlag, 1990, pp 8-46. 2. RJ Chrost, H Rai. Bacterial secondary production. In J Overbeck, RJ Chrost, eds. Microbial Ecology of Lake Plu see. New York Springer-Verlag, 1994, pp 92-117. 3. RJ Chrost. Microbial enzymatic degradation and utilization of organic matter. In J Overbeck, RJ...

Bioremediation of Seleniferous Water and Sediment

Since the 1990s attention has been given to the development of an effective remediation technology for the permanent removal of Se oxyanions from seleniferous soil and water. A majority of the focus has been applied to contaminated agricultural drainage water, which has been responsible for a number of well-documented ecotoxicological problems. Since Se undergoes microbial transformations, their application may be potentially useful as bioremediation strategies. Several different bioremedial...

Limitations of a Single Enzyme Activity as an Index of Microbial Activity

Enzyme activities can be measured and used as an index of microbiological functional diversity if they reflect changes in microbial activities. Since microbial functional diversity includes many different metabolic processes, theoretically a large number of different enzymes should be measured. Data should be integrated in an attempt to calculate an index of microbial functional diversity and to compare the microbial functional diversity of different soil samples. Because this task is not...

Carbon Substrate Utilization Patterns And Microbial Functional Diversity

The BIOLOG system, originally developed to assist with the taxonomic description of bacteria in axenic culture, is based on the ability of bacterial cells to oxidize up to 95 different carbon substrates in microtiter plates 16 . The plates are incubated for a suitable period of time generally 72 h , and the oxidation of the substrate is monitored by measuring the reduction of a tetrazolium dye. The rate and density of the color change depend upon the number and activity of microbial cells in...

Enzyme Activities Methodology and Interpretation

According to the review by Skujins 40 , Woods in 1899 suggested that extracellular enzymes could be present and active in soil. The first measurements of enzyme activities in soil were done on catalase and peroxidase activities from 1905 to 1910 40 . Since then, the activity of dozens of enzymes has been detected in soil. Obviously the number of enzymes is considerably greater than those measured because of the multitude of microbial, faunal, and plant species inhabiting soil 46 . In addition,...