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1.
Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH14CO3 and [methyl-3H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter−1 h−1. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter−1 h−1. During holomixis, production throughout the water column was in excess of 100 μg of C liter−1 h−1 and above 150 μg of C liter−1 h−1 near the surface. Annual areal primary production was 588 g of C m−2. Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h−1, and production rates were typically 5 μg of C liter−1 h−1. Growth rates were higher during warmer parts of the year and reached 0.03 h−1 by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter−1 h−1. Annual areal bacterial production was 125 g of C m−2. Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002).  相似文献   

2.
Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter−1 at night and 1 atm pO2 during the day. During experimental H2S to O2 transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H2S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter−1, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter−1 (25 to 75 μmol of H2S liter−1). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter−1 (500 μmol of H2S liter−1) in the overlying water. The implications of such a localized O2 production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.  相似文献   

3.
A β-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical β-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-β-N-acetylglucosamine were 425.7 μM and 24.8 μmol min−1 mg of protein−1, respectively.Streptomycetes are gram-positive, mycelial soil bacteria with a high G+C content. In addition to having the ability to synthesize a wide variety of antibiotics and chemotherapeutic agents, they produce extracellular hydrolytic enzymes to obtain nutrients and energy by solubilizing polymeric compounds in soil. These enzymes include proteases, nucleases, lipases, and a variety of enzymes that hydrolyze different types of polysaccharides such as cellulose, chitin, and xylan (13). This last class of enzymes has received considerable attention not only from the standpoint of the utilization of renewable resources but also from that of basic research. Among actinomycetes, Streptomyces spp. make up one group regarded as particularly efficient in the breakdown of chitin (10). Following cellulose, chitin is the second most abundant polymer (β-1,4-linked polymer of N-acetylglucosamine) in nature. Efficient degradation of chitin by microorganisms is achieved by the concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) (1, 19, 20).We have been studying the chitinolytic system of Streptomyces thermoviolaceus OPC-520 to clarify the roles of individual enzymes involved in chitin degradation, the relationship between structure and function, and the regulation of gene expression. When S. thermoviolaceus OPC-520 is cultivated in the presence of chitin, this strain secretes three different chitinases and only one β-N-acetylglucosaminidase and the production is repressed by glucose (unpublished data). Previously, we purified and characterized a major chitinase (Chi40) produced by the strain, which shows a high optimum temperature (70 to 80°C), high optimum pH (pH 8.0 to 10.0), and heat stability (22), and recently reported the cloning and expression of the Chi40 gene (23).While a number of chitinase genes have been isolated from a wide variety of organisms, including bacteria, fungi, insects, plants, and animals, examples of cloning of the β-N-acetylglucosaminidase gene involved in a chitinolytic system are few. To understand the role of β-N-acetylglucosaminidase in chitin degradation by strain OPC-520, its relationship to similar proteins isolated from other sources, and the regulatory system involved in the induction of the enzyme, we have isolated and expressed the gene encoding β-N-acetylglucosaminidase. Here we report the molecular cloning and biochemical characterization of a β-N-acetylglucosaminidase, designated NagA, from S. thermoviolaceus OPC-520. This novel enzyme, which is clearly different from the N-acetylglucosaminidases so far reported, is assigned to family 3 of the glycosyl hydrolases on the basis of sequence comparison. This is the first report of a β-N-acetylglucosaminidase gene isolated from the genus Streptomyces.  相似文献   

4.
A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the Km values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with Ki and Ki′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn2+ or Co2+ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH2-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.  相似文献   

5.
An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcarAt) has been characterized. βcarAt is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn2+, Ni2+, and Co2+. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcarAt also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcarAt is able to produce monosubstituted β2- and β3-amino acids, showing better catalytic efficiency (kcat/Km) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcarAt an outstanding candidate for application in the biotechnology industry.  相似文献   

6.
The construction of a microsensor which can be used to measure O2 and N2O simultaneously is described. The microsensor exhibited a linear response to both O2 and N2O, and the response to N2O was independent of the O2 concentration and vice versa. The N2O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter−1. The signals for O2 and N2O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O2 and N2O were measured in a biofilm which was exposed to acetylene to block the N2O reductase activity of denitrifying bacteria. O2 penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N2O concentration at 1.4 mm depth increased from 32 to 411 μmol liter−1 after the addition of the inhibitor. The shape of the N2O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm.  相似文献   

7.
The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of Aeromonas hydrophila challenged with pH, NaNO2, and salt combinations at 30°C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO2 and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter−1), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter−1). This change is not due to a “synergy” between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO2, and salt act independently on the growth of A. hydrophila. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot. 54:429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.  相似文献   

8.
Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.  相似文献   

9.
A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphEal) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter−1 citrate, pH 6.5, and 37°C are ideal parameters for CphEal production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter−1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter−1 induced CphEal production with only about 30% efficiency in comparison to that with CGP. CphEal was purified utilizing its affinity for the substrate and its specific binding to CGP. CphEal turned out to be a serine protease with maximum activity at 50°C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphEal preparation. Optimum degradation parameters were 100 g liter−1 CGP, 10 g liter−1 crude CphEal powder, and 4 h of incubation at 50°C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphEal powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.  相似文献   

10.
The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The 14C and O2 methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO3 N assimilation ratio was 1.57. The total integrated incorporation of [14C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 109 to 1.52 × 109 cells liter−1 (~0.5 μg of C liter−1 day−1). Estimates of bacterioplankton production from rates of [3H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter−1 day−1. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter−1 day−1, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [3H]thymidine incorporation method.  相似文献   

11.
Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain β-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-Å resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His234 of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp232 for proper function. Replacement of both His234 and Asp232 with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4′-methylumbelliferyl N-acetyl-β-d-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and β-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within β-N-acetylglucosaminidases but otherwise absent in β-glycosidases of family 3, which instead carry a “classical” glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His234 of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 β-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of β-lactamase.  相似文献   

12.
Kinetic Parameters of Denitrification in a River Continuum   总被引:4,自引:0,他引:4       下载免费PDF全文
Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (Kmap) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (Vmaxap) ranged between 35.8 and 324 μmol of N m−2 h−1 in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m−2 h−1). A study of nitrous oxide (N2O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N2O-N m−2 h−1) at the headwater site to 27 μmol of N2O-N m−2 h−1 at the downstream site. In the Wiske the rate was up to 570 μmol of N2O-N m−2 h−1, accounting for up to 80% of total N gas production.  相似文献   

13.
Flocs consisting of Anabaena and Zoogloea spp. were used as a model system for the study of planktonic phototroph-heterotroph interactions. In CO2-limited continuous culture (3.2 μmol of NaHCO3 liter−1 h−1, 1.5 μmol of glucose liter−1 h−1, pH 8.5, D = 0.026 h−1), the biomass of the phototroph increased 8.6-fold due to association. However, direct CO2 exchange accounted for only a 3.8-fold increase. When the glucose supply rate was increased to 7.5 μmol liter−1 h−1, there was a 26-fold increase in biomass. When CO2 was supplied in excess, there was no difference due to association. In batch culture, using the same medium, the specific growth rate was 0.029 h−1 for the phototroph alone and 0.047 h−1 for the phototroph in association with the heterotroph. The stimulatory effect of the heterotroph was found only under CO2-limiting conditions and was directly related to the concentration of organic matter supplied in the medium. Both the biomass and the growth rate of the Anabaena sp. were increased by association with the Zoogloea sp. Thus, dissolved organic matter may substitute for CO2 to maximize both growth rate and biomass production by phototrophs when heterotrophic bacteria are present.  相似文献   

14.
Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s−1, 3.7 ± 0.3 s−1, 4.5 ± 0.5 s−1, and 7.1 ± 0.2 s−1, respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.  相似文献   

15.
Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced β-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher β-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had β-N-acetylgalactosaminidase activity equivalent to about 40% of its β-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for β-N-acetylglucosaminidase activity and 5.5 to 6.5 for β-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.  相似文献   

16.
Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.  相似文献   

17.
Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein “multisubstrate glycosidase A” (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.  相似文献   

18.
The carbon monoxide consumption rates of the carboxydobacteria Pseudomonas (Seliberia) carboxydohydrogena, P. carboxydovorans, and P. carboxydoflava were measured at high (50%) and low (0.5 μl liter−1) mixing ratios of CO in air. CO was only consumed when the bacteria had been grown under CO-autotrophic conditions. As an exception, P. carboxydoflava consumed CO also after heterotrophic growth on pyruvate. At low cell densities the CO consumption rates measured at low CO mixing ratios were similar in cell suspensions and in mixtures of bacteria in soil. CO consumption observed in natural soil (loess, eolian sand, chernozem) as well as in suspensions or soil mixtures of carboxydobacteria showed Michaelis-Menten kinetics. The Km values for CO of the carboxydobacteria (Km = 465 to 1,110 μl of CO liter−1) were much higher than those of the natural soils (Km = 5 to 8 μl of CO liter−1). Considering the difference of the Km values and the observed Vmax values, carboxydobacteria cannot contribute significantly to the consumption of atmospheric CO.  相似文献   

19.
Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO2 dark fixation rates, ranging from 0.7 to 1.4 μmol liter−1 day−1, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter−1 CO2 day−1. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter−1 NO3 day−1. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.  相似文献   

20.
1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-14C]adenine, [8-14C]guanine and [8-14C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 4·7 μm) and hypoxanthine phosphoribosyltransferase (Ki 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of Ki for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.  相似文献   

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