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1.
One strain each of Xanthomonas campestris and 'Xanthomonas phaseoli', three strains of 'Xanthomonas oryzae', and five strains each of Escherichia coli and Klebsiella pneumoniae were found to produce a mixture of (1----2)-beta-D-gluco-oligosaccharides and/or low-Mr (1----2)-beta-D-glucans in their culture media. The saccharides from the strains of Xanthomonas were all composed of unbranched, linear (1----2)-beta-D-glucosaccharides with degrees of polymerization (DPs) of 8 to about 20, and a cyclic (1----2)-beta-D-glucan (DP16) containing one (1----6)-linkage and one alpha-linkage. The saccharides produced by the five strains of E. coli and four strains of K. pneumoniae were glucans with branches at O-6, and DPs of 10 to 15, whereas one strain of K. pneumoniae produced unbranched linear (1----2)-beta-D-glucosaccharides with DPs of 6 to about 20. 相似文献
2.
Figueiredo Douglas B. Carvalho Eneas Santos Mauricio P. Kraschowetz Stefanie Zanardo Rafaela T. Campani Gilson Silva Gabriel G. Sargo Cíntia R. Horta Antonio Carlos L. de C. Giordano Roberto Miyaji Eliane N. Zangirolami Teresa C. Cabrera-Crespo Joaquin Gonçalves Viviane Maimoni 《Applied microbiology and biotechnology》2017,101(6):2305-2317
Applied Microbiology and Biotechnology - Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The... 相似文献
3.
Carvalho RJ Cabrera-Crespo J Tanizaki MM Gonçalves VM 《Applied microbiology and biotechnology》2012,94(3):683-694
Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection
and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of
His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon
source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of
both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A
study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography
step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed
by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography
steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation
exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the
protein was shown to have the expected alpha-helix secondary structure. 相似文献
4.
Lasaro MA Rodrigues JF Mathias-Santos C Guth BE Régua-Mangia A Piantino Ferreira AJ Takagi M Cabrera-Crespo J Sbrogio-Almeida ME de Souza Ferreira LC 《FEMS immunology and medical microbiology》2006,48(1):123-131
Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals. 相似文献
5.
Ferrer-Miralles N Feliu JX Vandevuer S Müller A Cabrera-Crespo J Ortmans I Hoffmann F Cazorla D Rinas U Prévost M Villaverde A 《The Journal of biological chemistry》2001,276(43):40087-40095
The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties. 相似文献
6.
Takagi M Lima RB Albani SM Zangirolami TC Tanizaki MM Cabrera-Crespo J 《Journal of industrial microbiology & biotechnology》2008,35(11):1217-1222
Haemophilus influenzae type b, an encapsulated bacterium, causes meningitis in infants worldwide. The capsular polysaccharide conjugated to a carrier
protein is effective in the prevention of such infections. The traditional purification process of polysaccharide from bacterial
cultures for vaccine production is based on several selective precipitations with solvents such as: ethanol, phenol, and cationic
detergents. The separations of solid and liquid phases are based on continuous centrifugation in explosion proof installations.
The lipopolysaccharides are separated by ultracentrifugation. A simple and efficient method that can easily be scaled-up was
developed for purification of polysaccharides. The ethanol precipitation was reduced to only two steps. The phenol treatment
was substituted by ultrafiltration and enzymatic digestion. Lipopolysaccharide was removed by ultrafiltration together with
addition of detergent and chelating agent. 相似文献
7.
Rodrigues JF Mathias-Santos C Sbrogio-Almeida ME Amorim JH Cabrera-Crespo J Balan A Ferreira LC 《The Journal of biological chemistry》2011,286(7):5222-5233
Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an ~10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants. 相似文献
8.
Silva M Cabrera-Crespo J Sbrogio-Almeida ME Miyaji EN Ho PL Leite LC Lopes AP 《Molecular biotechnology》2007,37(2):146-154
Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family. 相似文献
9.
Akinori Amemura Joaquin Cabrera-Crespo Masatomo Maeda Masamitsu Futai 《Bioscience, biotechnology, and biochemistry》2013,77(10):2649-2656
In an attempt to finding polysaccharides with immunostimulating activity, we isolated 14 bacteria that produced polysaccharides from soil. The polysaccharides produced by these bacteria and by strains kept in this laboratory and commercial polysaccharides were tested for ability to activate J774.1 macrophage-like mouse cells. Acidic polysaccharides, especially those from the newly isolated bacteria, induced spreading of the cells and release of prostaglandin E2 from the cells, which are manifestations of a state of activation. The prostaglandin E2-releasing activity of the polysaccharides was 1/10 to 1/20 on a weight basis of that of Escherichia coli lipopolysaccharide, a potent immunostimulant. 相似文献
10.
Anita Mitico Tanaka Aparecida Sadae Tanaka Alexandre Paulo Yague Lopes Mickie Takagi Joaquin Cabrera-Crespo Isaias Raw 《Biotechnology letters》2000,22(4):257-260
Factor VIII was purified from porcine plasma using adsorption on aluminium hydroxide with CM-cellulose as a filtration aid, cold ethanol precipitation, and two anion-exchange (Q-Sepharose fast flow) chromatographies. The final product was purified 264-fold and had a specific activity of 10 U mg–1. The method is suitable to produce purified porcine FVIII by an easy process where all steps can be scaled up. The final product is free of von Willebrand factor that is responsible for the main side effects in patients. Finally, this method can be used to obtain purified porcine plasma FVIII for use in haemophilic patients with inhibitors. 相似文献