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Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM
flow cytometry
- FITC
fluorescein-iso-thiocyanate
- LB
Luria broth
- MM
minimal salt medium
- PBS
phosphate buffered saline
- PMSF
phenylmethylsulfonyl fluoride 相似文献
2.
Dams-Kozlowska H Mercaldi MP Panilaitis BJ Kaplan DL 《Applied microbiology and biotechnology》2008,81(2):201-210
Since its discovery in the late 1970s, emulsan has been the subject of significant interest for fundamental biosynthesis and
structure–function relationships as well as for its potential industrial applications. These studies initially examined the
emulsification properties of the compound, while more recent efforts have focused on potential biomedical applications. As
a result of this change of focus, it became necessary to more completely characterize the structure of the emulsan molecule
and to develop a more reproducible purification process. We review previous studies with emulsan and explain how prior notions
were recently shown to be incorrect through the development of a new purification process. More recent genetic modification
of the relevant operon is also reviewed. Finally, the potential applications for the new purified polymer will be discussed. 相似文献
3.
The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 106) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain. 相似文献
4.
Saleha Husain 《World journal of microbiology & biotechnology》2008,24(11):2411-2419
The effect of surfactants on pyrene degradation in Pseudomonas fluorescens 29L was investigated. This strain produced 30.1 μM of rhamnolipid equivalents (RE) of biosurfactants on 50 mg of pyrene per
liter of medium. The production of biosurfactants was significantly correlated with the water solubility (S
w) of the substrate and the growth rate on it. When chrysene, with a S
w of 2.8 × 10−3 mg per liter of water, was the carbon source, 13.1 μM of RE of biosurfactants were produced compared to 10.3 μM of RE of
biosurfactants on acenaphthene with a S
w of 1.9 mg per liter of water. No biosurfactants were produced on salicylic acid, catechol, and citrate. All of the strain
29L mutants which grew on pyrene produced biosurfactants while among the mutants which grew on naphthalene, only 88.4% produced
biosurfactants. The rhamnolipid mixture, JBR425, inhibited the growth of Strain 29L wild type (WT) and all of its mutants
on pyrene. However, these mutants were able to grow in the presence of pyrene when the growth medium was supplemented with
10−6 mg of emulsan per milliliter of medium. This study implies biosurfactants are produced by Strain 29L as a physiological response
to the hydrophobicity of pyrene. The combined use of indigenous biosurfactants and the added biosurfactant, emulsan, is a
biotechnology to enhance pyrene degradation by Pseudomonas fluorescens 29L. 相似文献
5.
Gawon Yi Jihwan Son Jihye Yoo Changhee Park Heebeom Koo 《Biochemical and biophysical research communications》2019,508(1):326-331
Nanoparticles have been widely used as drug carriers, and finding new materials for them is important for efficient drug delivery. Herein, we developed a new nanoparticle using emulsan and flax seed oil. Emulsan is one of the representative biosurfactants obtained from Acinetobacter calcoaceticus RAG-1. The resulting nanoparticles have an emulsan shell and a hydrophobic oil core, into which pheophorbide a (Pba) was loaded as a model drug. The nanoparticles were about 165.7?nm and were stably dispersed in an aqueous condition for more than one week. They demonstrated fast uptake in SCC7 mouse squamous cell carcinoma cells and killed the tumor cells after laser irradiation due to the photodynamic effect of Pba. After injection into SCC7 tumor-bearing mice via the tail vein, the particles showed longer blood circulation and 3.04-fold higher tumor accumulation in tissue than free Pba. These results demonstrate that emulsan-based nanoparticles have promising potential in drug delivery. 相似文献
6.
Alternate hydrophobic sites on the cell surface of Acinetobacter calcoaceticus RAG-1 总被引:2,自引:0,他引:2
Abstract Mutants of A. calcoaceticus RAG-1 lacking thin fimbriae (35 Å) do not adhere to hydrophobic surfaces [5], or grow on hydrocarbons under conditions of weak agitation and small inocula. Emulsan-deficient derivatives of such mutants, isolated in the present study, (i) lacked cell-surface emulsan, (ii) adhered avidly to hydrocarbons, (iii) lacked thin fimbriae, and (iv) regained the capacity to grow on hydrocarbons. The results show that emulsan masks an alternate hydrophobic site(s) on the cell surface of RAG-1. 相似文献
7.
Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the
optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent
and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L
ethanol, 8.2 g/L KH2PO4, 23.32 g/L K2HPO4, 5.77 g/L (NH4)2SO4 and 0.354 g/L MgSO4•7H2O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L
for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents
for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production. 相似文献
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