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61.
Béki E Nagy I Vanderleyden J Jäger S Kiss L Fülöp L Hornok L Kukolya J 《Applied and environmental microbiology》2003,69(4):1944-1952
Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides. 相似文献
62.
Tóth I Schmidt H Dow M Malik A Oswald E Nagy B 《Applied and environmental microbiology》2003,69(12):7242-7247
In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Phi3538 (Deltastx(2)::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Phi3538 (Deltastx(2)::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions. 相似文献
63.
Biological denitrification in a continuous-flow pilot bioreactor containing immobilized Pseudomonas butanovora cells 总被引:6,自引:0,他引:6
Pseudomonas butanovora, a novel denitrifying bacterium, was immobilized in composite beads and filled into a reactor system. The pilot bioreactor average denitrification activity was at ethanol-C:nitrate-N ratios of 3:1 and 1.5:1 0.88 and 0.54 kg NO3(-)-Nm(-3) d(-1), respectively. The denitrification was stable in spite of the relatively low hydraulic retention times of 2.47 and 3 h. The nitrate content of the influent was almost completely reduced at the first level of the bioreactor and the nitrite formed underwent reduction in the upper part of the reactor. The experimentally determined optimum ethanol-C:nitrate-N ratio was 1.41 +/- 0.41. In consequence of the aerobic conditions, the acetic acid produced by the oxygenation of ethanol was also detectable in the reactor effluent. The pH of the effluent (7.58) never exceeded the acceptable maximum (8.5). The nitrate removal efficiency of the cells was nearly 1000% at both C:N ratios, and the nitrite content of the effluent was around the prescribed limit throughout the continuous operation. This continuous-flow pilot bioreactor containing immobilized P. butanovora cells proved an efficient denitrification system with a relatively low retention time. 相似文献
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66.
János Bajdik Zsolt Makai Ottó Berkesi Károly Süvegh Tamás Marek István Erős Klára Pintye-Hódi 《Carbohydrate polymers》2009,77(3):530-535
The aim of this study was to evaluate the interaction between the film-forming sodium alginate and lactose monohydrate. This combination is used in the co-spray-drying technique for microencapsulation, but no respect on the structure of the film formed has not been published previously. From mechanical tests, positronium lifetime measurements and FT-IR studies on free films containing different ratios of film-former and lactose, we concluded that the mechanical strength of the sodium alginate film decreased with the increasing proportion of lactose. The free volume in the polymer matrix decreased to a minimum as the lactose content was progressively increased to 40%, but subsequently increased at higher lactose contents. The explanation of this phenomenon is the filling of the holes with the sugar. As lactose became predominant component, the structure of the polymer network weakened. These conclusions were supported by the FT-IR findings. The present results permit a clear explanation of the previously reported favourable effects of this film-forming combination on the dissolution of the active agent from the microcapsules. 相似文献
67.
Gerg? Gulyás József T. Tóth Dániel J. Tóth István Kurucz László Hunyady Tamas Balla Péter Várnai 《PloS one》2015,10(5)
Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. 相似文献
68.
Ágnes Jakab Károly Antal Ágnes Kiss Tamás Emri István Pócsi 《Folia microbiologica》2014,59(4):333-340
A selection of tert-butylhydroperoxide (tBOOH)-tolerant Candida albicans mutants showed increased tolerances to 19 different stress conditions. These mutants are characterized by a constitutively upregulated antioxidative defense system and, therefore, adaptation to oxidative stress may play an important role in gaining general stress tolerance in C. albicans. Although C. albicans cells may undergo morphological transitions under various stress treatments, this ability shows considerable stress-specific and strain-specific variability and, hence, it is independent of mounting stress cross protections. 相似文献
69.
János Galambos Gábor Wágner Katalin Nógrádi Attila Bielik László Molnár Amrita Bobok Attila Horváth Béla Kiss Sándor Kolok József Nagy Dalma Kurkó Mónika L. Bakk Mónika Vastag Katalin Sághy István Gyertyán Krisztina Gál István Greiner Zsolt Szombathelyi György M. Keserű György Domány 《Bioorganic & medicinal chemistry letters》2010,20(15):4371-4375
Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully eliminated. Systematic exploration of different parts of the advanced hit led us to some promising lead compounds with mGluR5 affinities comparable to that of MPEP. 相似文献
70.
Gábor Wágner Csaba Wéber Olga Nyéki Katalin Nógrádi Attila Bielik László Molnár Amrita Bobok Attila Horváth Béla Kiss Sándor Kolok József Nagy Dalma Kurkó Krisztina Gál István Greiner Zsolt Szombathelyi György M. Keserű György Domány 《Bioorganic & medicinal chemistry letters》2010,20(12):3737-3741
Here we report the discovery and early SAR of a series of mGluR5 negative allosteric modulators (NAMs). Starting from a moderately active HTS hit we synthesized 3,5-disubstituted-oxadiazoles and tetrazoles as mGluR5 NAMs. Based on the analysis of ligand efficiency and lipophilic efficiency metrics we identified a promising lead candidate as a starting point for further optimization. 相似文献