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At least 667 records · Page 37Linked to original sources
PubMed2001

An improved procedure for production of human epidermal growth factor from recombinant E. coli.

An improved procedure for the fermentation and purification of human epidermal growth factor (hEGF) was developed. Recombinant Escherichia coli HB-101 [lacUV5omp08hEGF] harboring plasmid lacUV5omp08hEGF encoding hEGF was used in fermentation to increase levels of hEGF. Medium composition, and the levels of inoculum, inducer (isopropyl-beta-D-thiogalactoside) and ampicillin were optimized with respect to volumetric fermentation of hEGF. As a result, the hEGF concentration reached a high value of 242 mg l(-1) and the amount of heterogeneous protein decreased by 62% compared with that before optimization in batch fermentation. High-quality hEGF was purified from the fermentation culture by centrifugation, salting-out, resuspension, recentrifugation and finally gel chromatography on a Grad-iFrac System using Sephadex G-50 superfine. The purity of hEGF and the total yield were more than 94% and higher than 36%, respectively, and SDS-PAGE of the purified hEGF demonstrated a single band corresponding to an hEGF standard. In particular, a very important phenomenon was found, i.e. that the amount of heterogenous protein in fermentation broths cultured in media with high concentrations of lactose is far less than that cultured in media with high concentrations of glucose.

Biotechnology↗
PubMed1995

Taxonomic diversity of anaerobic glycerol dissimilation in the Enterobacteriaceae.

A total of 1,123 strains representing 128 taxa in the Enterobacteriaceae (named species or subspecies and genomic species) were screened for the presence of glycerol dehydrogenases and 1,3-propanediol dehydrogenase. Only eight taxa, Citrobacter freundii sensu stricto, C. youngae, C. braakii, C. werkmanii, Citrobacter genomospecies 10 and 11, Enterobacter gergoviae and Klebsiella pneumoniae subsp. pneumoniae could grow fermentatively on glycerol and possessed both glycerol dehydrogenase type I (induced by glycerol and dihydroxyacetone) and 1,3-propanediol dehydrogenase which are typical enzymes of the anaerobic glycerol dissimilation pathway. Six other species, C. koseri, E. aerogenes, E. intermedium, K. oxytoca, K. planticola and K. terrigena could not grow fermentatively on glycerol and possessed a glycerol dehydrogenase type I but no 1,3-propanediol dehydrogenase. Other glycerol dehydrogenases types were found: type II (induced by glycerol and hydroxyacetone), type III (induced by glycerol only) and type IV (induced by hydroxyacetone only). They were widely distributed among the Enterobacteriaceae. Classification and identification may take advantage of tests exploring the dissimilation of glycerol.

Enterobacteriaceae↗
PubMed1999

Physico-chemical characterization of recombinant hepatitis B surface antigen by a multidimensional approach.

Initially, our work was directed to respond to the question: why hepatitis B surface antigen (HBsAg) produces a very broad peak in preparative size-exclusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the individual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay and transmission electron microscopy. As a result, HBsAg particles were shown to be heterogeneous in terms of particle assembly. In order to elucidate the origin of HBsAg heterogeneity, we included here the denaturing SEC into a multidimensional approach. The data from denaturing SEC evidenced the fragmentation of protein monomers within the HBsAg particle that, probably, occurs during fermentation broth, rather than during in vitro HBsAg processing. The fractions isolated from widely separated regions of HBsAg peak differed in the extent of protein fragmentation, suggesting that the variable extent of protein degradation within HBsAg particles may be one of the factors responsible for broadening of the HBsAg peak in SEC.

Chemical Phenomena↗
PubMed1996

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases.

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.

Amino Acid Sequence↗
PubMed1981

Effect of monensin on the fermentation of basal rations in the Rumen Simulation Technique (Rusitec).

1. A long-term experiment was made with the Rumen Simulation Technique (Rusitec), in which the fermentation of a mixed ration of hay (10 g/d) and bruised barley (5 g/d) was compared with the fermentation of the same diet in the presence of 2, 10 and 50 mg monensin/d. 2. Monensin depressed the production of acetic and butyric acids, markedly increased the production of propionic acid and virtually, eliminated the production of isovaleric acid. The production of methane was decreased in the presence of monensin, but this decrease could be accounted for entirely by the changes in the production of volatile fatty acids and redistribution of metabolic hydrogen. 3. The digestibility of dry matter (DM) in the rations declined in the presence of monensin. Determinations of the rates of digestion showed that the digestion of the readily-fermented food in the initial stages was not affected by monensin, but that at 24 h digestion had been inhibited by monensin. The inhibition was due entirely to its effect on the digestion of the fibrous components. Digestion of non-fibrous material was not affected. 4. The efficiency of microbial growth, expressed as g dry weight/mol ATP formed (YATP) and in terms of DM digested, tended to be increased by monensin. This however occurred only at high, non-practical doses. 5. Urease (EC 3. 5. 1. 5) was induced by the addition of urea of the fermentation, but monensin had no effect on urease activity. Although monensin increased the activity of protease in washed suspensions, more food protein apparently escaped degradation. This may have been due to decreased deaminative activity. 6. Monensin altered the microscopic appearance of the fermentation fluid, and changed the activity of some enzymes in sonicated extracts, including alkaline phosphatase (EC 3. 1. 3. 1), acetate kinase (EC 2. 7. 2. 1) and succinate dehydrogenase (EC 1. 3. 99. 1). These results are discussed in terms of known sensitivities of rumen microbes to monensin and their contribution to the fermentation as a whole.

Animal Feed↗
PubMed1988

Intestinal microflora and gastrointestinal adaptation in the rat in response to non-digestible dietary polysaccharides.

1. A comparison was made of the effect of a fibre-free diet and diets containing non-digestible polysaccharides on rat caecal and colonic physiology and microflora. 2. All polysaccharide-containing diets led to enlargement of the caecum and colon, associated with increased weight of contents, and of tissue. Carboxymethylcellulose (CMC) had the most marked effect and animals given this also had watery faeces. 3. The density of bacteria in the caecum and colon varied significantly with diet and the proportion of aerobic bacteria in the flora was increased by the CMC diet. 4. In vitro, CMC and hydroxypropylmethylcellulose were poorly fermented. 5. There was a high correlation (caecum r 0.93; colon r 0.94) between tissue weight and wet weight of organ contents but no correlation with bacterial density, number of bacteria per organ, moisture content or short-chain fatty acid content. 6. It is concluded that caecal and colonic enlargement is due to tissue hypertrophy in response to increased bulk of contents, irrespective of the nature of that bulk which varies with diet; it is unlikely that short-chain fatty acids or other microbial metabolites are the stimulus for the trophic response seen when non-digestible dietary polysaccharides are fed to rats.

Adaptation, Physiological↗
PubMed2002

Effect of administration of Lactobacillus crispatus, Clostridium lactatifermentans and dietary lactose on the development of the normal microflora and volatile fatty acids in the caeca of broiler chicks.

1. Lactobacillus crispatus and Clostridium lactatifermentans, both isolated from the caeca of chickens, grown together in an in vitro model system are able to ferment lactose to acetate and propionate. In this study, the capabilities of these organisms were studied in vivo. 2. The effect on concentrations of volatile fatty acids and lactate, together with the development of some bacterial groups in the caeca of chicks, was studied after oral inoculation with L. crispatus and C. lactatifermentans together with dietary lactose. For this purpose, chicks were divided into 4 groups: (i) control group, (ii) dietary lactose, (iii) L. crispatus and C. lactatifermentans, and (iv) dietary lactose together with L. crispitaus and C. lactatifermentans. 3. In general, concentrations of (undissociated) volatile fatty acids in the caeca were not significantly different in broilers receiving both bacteria and dietary lactose compared with control broilers. Concentrations of lactate in the caeca of 14-d-old broilers treated with any of the three treatments were significantly higher than in the caeca of control broilers. 4. This indicates that L. crispatus or other lactate-producing organisms were responsible for the elevated concentrations of lactic acid. Clostridium lactatifermentans has probably not colonised the caeca sufficiently to ferment this lactate further to acetate and propionate. 5. Numbers of Enterobacteriaceae and enterococci in the caeca of broilers receiving both bacteria and dietary lactose were not different from control broilers. 6. We conclude from these results that under the conditions applied in this study a mixture of L. crispatus and C. lactatifermentans with dietary lactose was able to increase lactate concentrations but was unable to increase concentrates of acetate and propionate in the caeca of broiler chicks.

Animals↗
PubMed2001

Research note: effect of increasing dietary concentrate levels on microbial biotin metabolism in the artificial rumen simulation system (RUSITEC).

The effect of varying hay/barley-proportions in the feed ration on biotin metabolism of rumen microbes was studied by means of the rumen simulation technique RUSITEC. The stepwise replacement of hay by barley decreased dietary biotin and the net output of biotin by the microbial metabolism. It is concluded that rumen microbes utilise more and/or synthesize less biotin with increasing proportions of dietary barley. These results indicate that a critical reconsideration of current views with regard to the supply and requirement of the high yielding dairy cow for biotin is necessary.

Animal Feed↗
PubMedDate not supplied

In vitro study of the response of rumen microorganisms to different doses of abierixin, a new antibiotic ionophore, according to the nature of nitrogen supplies.

An in vitro study on a "batch" type fermentation system was carried out in order to evaluate the effect of different doses of a new ionophore antibiotic, abierixin, on the rumen fermentation parameters. The reaction of microorganisms to the antibiotic were determined according to the nature of the nitrogen introduced into the fermenter: ammonium sulfate alone or complemented with protein nitrogen (peanut meal or a mixed pelleted feed). This molecule had a protective effect with respect to dietary protein degradation in the rumen, at the lowest dose tested (13.5 ppm). The dose (8 ppm) used with the pelleted feed led to a decrease in the protein degradability but the significant threshold was not reached. Abierixin failed to modify bacterial ammonia uptake with the nitrogen sources used in our experiment. In addition, it had no effect on rumen fermentations. It neither led to any alteration in the total VFA and gas productions during 6 hours of fermentation, nor on their molar composition, whatever the dose of antibiotic used and the nitrogen source considered.

Animal Feed↗