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1.
Freeze-dried ectomycorrhizal fungus cultures were tested for qualitative enzymatic activity and compared with that of the non-lyophilized culture. Enzymes involved in the utilization of starch, cellulose, lipid, lignin and urea were tested for their qualitative presence/activity. Expression of amylase and urease was stronger than that of lipase and lignin-degrading activity for the isolates tested. Variation among the species of Laccaria was low and prominently seen only for cellulase and urease. Amanita muscaria showed significant variation relative to other members of the Agaricales reported in the present study, except for β-glucosidase activity. All the enzymatic tests showed an unequivocal uniformity between the lyophilized vegetative mycelium (L) and the respective non-lyophilized mycelial cultures (NL), indicating that the lyophilization procedure maintained stable enzyme activity. This revised version was published online in November 2006 with corrections to the Cover Date.  相似文献   

2.
Abstract β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β- d -glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity ( V max) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity( K m)was higher in the suspended sediments. V maxand K m both increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. V max was uninfluenced by whether ground or stream water was perfused through the sediments, but K m was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.  相似文献   

3.
Summary The desymmetrisation ofendo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrication adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating anendo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of β-turns and parallel β-sheets.  相似文献   

4.
A combination of the phytohormones naphthalene acetic acid and benzylaminopurine (5 μM each) allows lignification in various plant cell cultures. This system has been used to investigate the relationship between the coniferin-hydrolyzingβ-glucosidase activity and lignification. InPetroselinum hortense andTriticum aestipum cell cultures the appearance of this enzymatic activity coincided with lignification. In parsley cell cultures it was moreover shown that this activity appears concomitantly with other lignin biosynthetic enzymes. The unique enzymes of the flavonoid pathway did not appear by this phytohormone treatment. In other cell cultures investigated the correlation between the coniferin-hydrolyzing activity and lignification was not as evident as in the above two cases. This was probably due to the high activity of coniferin glucosidase already present in the normally grown cultures. Coniferinβ-glucosidase was found in all lignified cell cultures.  相似文献   

5.
Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.  相似文献   

6.
AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.  相似文献   

7.
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.  相似文献   

8.
A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.  相似文献   

9.
A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.  相似文献   

10.
A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.  相似文献   

11.
Decrease in seed viability and germination rate may be caused by biochemical changes associated with seed ageing. Different biochemical assays were conducted to investigate the changes occurring at the ageing of Bambusa bambos seeds. A reduction in the total content of food reserves such as sugars, proteins and lipids were recorded. Decreased activity of peroxidase, acid phosphatase, alkaline phosphatase were also noticed during accelerated ageing. A substantial increase in total free amino acids and the activity of amylases confirms the degradation of stored biomolecules in seeds during ageing. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

12.
Summary. For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO2-Phe-OMe (8a), For-Met-NH-mC6H4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. 1H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.  相似文献   

13.
Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s Km was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).  相似文献   

14.
A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed β-glucosidase resisted pepsin digestion compared with secreted, free β-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted β-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed β-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted β-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed β-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.  相似文献   

15.
Cellobiose metabolism was studied in Alkaliflexus imshenetskii, a haloalkaliphilic hydrolytic bacterium capable of utilizing certain polymers of plant origin, as well as mono- and disaccharides. The major products of cellobiose fermentation by the bacterium were succinate and acetate, and formate was a minor product. Cellobiose could be split into glucose molecules by both β-glucosidase (hydrolytic pathway) and phosphorylase (phosphorolytic pathway); the activity of the former enzyme was two orders of magnitude higher (3600 nmol/(min mg) versus 36 nmol/(min mg)). In cell extracts of the bacterium, high activities of the Embden-Meyerhof-Parnas pathway enzymes—hexokinase, glucose-phosphate isomerase, and phosphofructokinase—were revealed, as well as the activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and key enzymes of the Entner-Doudoroff pathway—6-phospho-gluconate dehydratase and 2-keto-3-deoxy-6-phospho-gluconate aldolase. Neither the activity of the key enzyme of the hexose-mono-phosphate pathway, 6-phospho-gluconate dehydrogenase, nor the activities of the key enzymes of the modified Entner-Doudoroff pathway, glucose dehydrogenase and 2-keto-3-deoxy-gluconate kinase, were revealed.  相似文献   

16.
Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λmax ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λmax ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.  相似文献   

17.
Fat facets is a Drosophila deubiquitinating enzyme required for eye development and early embryogenesis. Genetic evidence suggests that Fat facets deubiquitinates and thereby prevents the proteasomal degradation of specific substrates. The Drosophila Liquid facets protein is implicated as the critical substrate of Fat facets in the eye. A mouse homolog of Fat facets, called Fam, has been identified. The results of biochemical experiments implicate two different proteins, Af-6 and β-catenin, as substrates for Fam. Here, the functional relationship between Fat facets and Fam is explored. We show that Fam can substitute for Fat facets in all of its essential functions in Drosophila. In addition, we tested the hypothesis that Canoe and Armadillo, the Drosophila homologs of Af-6 and β-catenin, respectively, are important substrates for Fat facets in the Drosophila eye. We found no genetic evidence to support a role for either Canoe or Armadillo in the essential Fat facets pathways in Drosophila eye development. The significance of these results is discussed in light of the biochemical experiments that suggest that Af-6 and β-catenin are substrates of Fam. Received: 23 May 2000 / Accepted: 21 August 2000  相似文献   

18.
The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (α,α-diphenyl-β-picrylhydrazyl) assay, and higher than those of the positive controls in β-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the β-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1–F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg·g−1 seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1–F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.  相似文献   

19.
Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. This system enabled coagulation to be monitored in a kinetic fashion, and the use of fluorescence enabled a wide range of samples to be analyzed including lyophilized plasma containing fibrin, fresh platelet-poor plasma, platelet-rich plasma, and even whole blood. Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood.  相似文献   

20.
Understanding the relationship between prokaryotic traits and phylogeny is important for predicting and modeling ecological processes. Microbial extracellular enzymes have a pivotal role in nutrient cycling and the decomposition of organic matter, yet little is known about the phylogenetic distribution of genes encoding these enzymes. In this study, we analyzed 3058 annotated prokaryotic genomes to determine which taxa have the genetic potential to produce alkaline phosphatase, chitinase and β-N-acetyl-glucosaminidase enzymes. We then evaluated the relationship between the genetic potential for enzyme production and 16S rRNA phylogeny using the consenTRAIT algorithm, which calculated the phylogenetic depth and corresponding 16S rRNA sequence identity of clades of potential enzyme producers. Nearly half (49.2%) of the genomes analyzed were found to be capable of extracellular enzyme production, and these were non-randomly distributed across most prokaryotic phyla. On average, clades of potential enzyme-producing organisms had a maximum phylogenetic depth of 0.008004–0.009780, though individual clades varied broadly in both size and depth. These values correspond to a minimum 16S rRNA sequence identity of 98.04–98.40%. The distribution pattern we found is an indication of microdiversity, the occurrence of ecologically or physiologically distinct populations within phylogenetically related groups. Additionally, we found positive correlations among the genes encoding different extracellular enzymes. Our results suggest that the capacity to produce extracellular enzymes varies at relatively fine-scale phylogenetic resolution. This variation is consistent with other traits that require a small number of genes and provides insight into the relationship between taxonomy and traits that may be useful for predicting ecological function.  相似文献   

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