Measurement of bacterial production in stream-bed sediments via leucine incorporation

Abstract: The leucine incorporation technique was evaluated and optimised for measuring bacterial production in stream-bed sediments. The original procedure was modified in order to obtain reliable production estimates in this habitat. This included the use of higher leucine concentrations (50 μmol l⁻¹) for obtaining substrate saturation, and an enhanced protein extraction procedure after sample fixation. The leucine method was combined with a perfused core technique. Water containing ¹⁴C-labelled leucine was perfused up through sediment cores, enabling the measurement of bacterial production in an experimental situation resembling natural conditions in the stream bed. Bacterial production in the Breitenbach, a small upland stream in Central Germany, showed a high degree of spatial variability in the sandy stream bed. It was related closely to sediment organic matter content, whereas varying perfusion rates had less influence. Annual bacterial production was estimated at about 200 g C m⁻², demonstrating the potential for bacteria to act as a food resource for benthic fauna in this stream.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

Selection and study of mutants of Dekkera intermedia and Candida wickerhamii derepressed for β-glucosidase production

Abstract Mutants of Candida wickerhamii and Dekkera intermedia , derepressed for β-glucosidase biosynthesis, were isolated. These mutants were also shown to hyperproduce this enzyme. In anaerobic culture, the C. wickerhamii mutant still hyperproduced β-glucosidase and was derepressed. Glucose-cellobiose anaerobic fermentation by this strain was thus improved. On the other hand, the D. intermedia mutant did not show any difference from the wild-type strain in anaerobic culture.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

The use of fluorogenic substrates for measuring enzyme activity in peatlands

The fluorogenic model substrates, methylumbelliferyl [MUF]-β-D-glucoside, MUF-phosphate and MUF-sulphate, were used to investigate the activities of β-glucosidase, phosphatase and sulphatase, respectively, in Welsh peatland soils. The method was used to investigate depth dependent variations in enzyme activity in a riparian wetland, and flush channel wetland. The highest activities were found at depths of less than 10 cm, thus confirming other studies which suggest this upper depth to be the site of greatest microbial activity. The most serious limitation to the technique was found to be the (fluorescence) quenching effects of the phenolic materials that dominate peatland dissolved organic carbon. The problem necessitates the adoption of a time consuming quench correction procedure with every sample. Fluorogenic substrates have led to a greater understanding of the role of enzymes in other aquatic systems. It seems likely that they will prove of equal value in elucidating their role in nutrient cycling and the biogeochemistry of peatlands.     

Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

Decomposition of plant material is a complex process that requiresinteraction among a diversity of microorganisms whose presence and activity issubject to regulation by a wide range of environmental factors. Analysis ofextracellular enzyme activity (EEA) provides a way to relate the functionalorganization of microdecomposer communities to environmental variables. In thisstudy, we examined EEA in relation to litter composition and nitrogendeposition. Mesh bags containing senescent leaves of Quercusborealis (red oak), Acer rubrum (red maple) andCornus florida (flowering dogwood) were placed on forestfloor plots in southeastern New York. One-third of the plots were sprayedmonthly with distilled water. The other plots were sprayed monthly withNH₄NO₃ solution at dose rates equivalent to 2 or 8 g N m^–2 y^–1. Mass loss, litter composition, fungal mass, and the activities ofeight enzymes were measured on 13 dates for each litter type. Dogwood wasfollowed for one year, maple for two, oak for three. For each litter type andtreatment, enzymatic turnover activities were calculated from regressions of LN(%mass remaining) vs. cumulative activity. The decomposition of dogwood litterwas more efficient than that of maple and oak. Maple litter had the lowestfungal mass and required the most enzymatic work to decompose, even though itsmass loss rate was twice that of oak. Across litter types, N amendment reducedapparent enzymatic efficiencies and shifted EEA away from N acquisition andtoward P acquisition, and away from polyphenol oxidation and towardpolysaccharide hydrolysis. The effect of these shifts on decomposition ratevaried with litter composition: dogwood was stimulated, oak was inhibited andmaple showed mixed effects. The results show that relatively small shifts intheactivity of one or two critical enzymes can significantly alter decompositionrates.     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Short-term response of soil enzyme activities in a chlorpyrifos-treated mesocosm: Use of enzyme-based indexes

Soil enzyme activities have been long used as indicators of soil contamination, and their integration into numerical indexes of microbial functional diversity is a practical approach in the environmental risk assessment of soil pollutants. However, suitable numerical indexes need to be developed and standardized for monitoring deterioration of soil quality by agrochemicals. Herein, a mesocosm study was performed to examine short-term responses of selected soil enzyme activities to chlorpyrifos (Lorsban^® 4E). Hydrolases (carboxylesterase, acid phosphatase, β-glucosidase, urease and protease) and oxidoreductases (dehydrogenase and catalase) were measured in Andisols 14 d after an application with two doses (4.8 and 24 kg a.i. ha⁻¹) of chlorpyrifos. Both application rates caused a strong inhibition of carboxylesterase (62–78% of controls), acid phosphatase (56–60%) and β-glucosidase (43–58%) activities. Soil microbial activity was also reduced in pesticide-sprayed soils as indicated by the decreased dehydrogenase (47%) and catalase (38%) activities compared with control soils. However, only carboxylesterase activity showed a dose-dependent response with the chlorpyrifos application rate. An in vitro trial was further performed to provide evidence of a direct interaction between the enzyme (carboxylesterase, acid phosphatase and β-glucosidase) and the pesticide (chlorpyrifos and its main metabolites chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol). Results of these in vitro assays showed that the activity of carboxylesterase was directly affected by chlorpyrifos-oxon and, at less extend, by chlorpyrifos, whereas variations of both acid phosphatase and β-glucosidase activities were likely dependent on changes in microbial activity. Urease and protease activities did not change in pesticide-treated soils compared with pesticide-free soils. Despite the absence of response in these two N-cycling enzyme activities, four enzymatic indexes (geometric mean, weighted mean, “treated-soil quality index” [T-SQI] and “integrated biological response” [IBRv2] index) were significantly lower in the chlorpyrifos-sprayed soils compared with controls. Moreover, there was a significant (r² = 0.87, P < 0.0001) correlation between T-SQI and IBRv2 scores, which suggested that the IBRv2 index (an index used for assessing animal’s health inhabiting contaminated sites) may be a complementary index in soil quality assessment.     

Chitinolytic activity in the autolysis of Aspergillus nidulans

Abstract Chitinolytic activity in filtrates of Aspergillus nidulans cultures was studied at the start of the autolysis (maximum dry weight of mycelium) and during autolysis in 24 different media. During the growth the chitinolytic activity was induced only by the presence of ascorbic acid or colloidal chitin in the medium. During autolysis an increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β - N -acetylgucosaminidase and endochitinase was detected. The possible induction of these enzymes during A. nidulans autolysis is established.     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.     

Use of 13C nuclear magnetic resonance spectroscopy for the evaluation of hepatic drug-metabolizing enzyme activity in perfused mouse livers

Experiments were performed to determine whether the activity of hepatic drug-metabolizing enzymes could be estimated in intact livers by 13C NMR spectroscopy. 13C-labelled aminopyrine was administered to isolated perfused mouse livers and the rate of decline in the NMR signal arising from the drug was used to estimate the half-life of aminopyrine. The technique was sufficiently sensitive to discriminate between the enzyme activities in normal livers and in livers whose enzyme activity was enhanced by treatment with sodium phenobarbitone. It was also demonstrated that demethylation of aminopyrine was not impaired in perfused livers from mice infected with Plasmodium berghei.     

Distribution and morphology of extrafloral nectaries in some Cucurbitaceae

A study of extrafloral nectaries has been made in the Cucurbitaceae to ascertain their structure and assess their taxonomic potential. Nineteen species representing nine Old World genera and one New World genus were examined. These included Telfairia occidentalis, Telfairia pedata, Momordica charantia, Lagenaria siceraria, Citrullus lanatus, Luffa aegyptiaca, Cucurbita moschata and Trichosanthes cucumerina , which are of economic importance and cultivated in Nigeria for their leaves and/or fruits.
Observation of the regularity of ant and insect-visitors, along with tests for glucose and β-glucosidase enzymes, revealed the presence of extrafloral nectaries in nine species. Considerable variation exists in the distribution and morphology of nectaries between genera, especially in the tribe Benincaseae. The nutritional and ecological significance of the occurrence of extrafloral nectaries in Telfairia occidentalis is discussed.     

Fluxes of mercury to lake sediments in central and northern Canada inferred from dated sediment cores

Sediment cores were collected from lakes in central and northern Canada and from Hudson Bay to compare current and historic net depositions of mercury. Cores from most locations were enriched in mercury in the upper layers deposited recently relative to deeper, historic layers. The lakes with the greatest enrichments in mercury were located in central/southern Canada. This enrichment was interpreted as being of anthropogenic origin. Mercury inputs at the Experimental Lakes Area (ELA) in northwestern Ontario inferred from a core profile agreed well with inputs calculated independently from precipitation and runoff data. Anthropogenic inputs of mercury to northwestern Ontario were calculated to be about 9 µg m^-2 y^-1. Considering all the locations over the geographic range, the core profiles infer that fluxes of mercury have increased on average by about 2 fold over the past half century. This is consistent with results from other sites in North America and Europe.     

Insertion of a 27 amino acid viral peptide in different zones of Escherichia coliβ-galactosidase: Effects on the enzyme activity

Abstract Seven internal, putatively exposed regions of Escherichia coli β-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique Bam HI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate incorporate and study biological properties of heterologous peptides in correctly folded β-galactosidase chimeric proteins.