吐温-80对野葛毛状根生长及异黄酮含量的影响

将不同浓度的吐温-80添加到野葛毛状根悬浮培养液中,研究在一定的作用时间内其对毛状根生长及次生代谢物合成与分泌的影响。结果表明,采用2％浓度处理较为适宜,不仅可以提高毛状根内葛根素的含量,而且有利于培养液中葛根素、大豆甙元及总异黄酮的积累,与对照相比,其含量可分别提高24．2％、50％和46．7％。在该浓度下连续处理毛状根72h后,发现毛状根仍生长旺盛,其生长量已是对照的1．5倍。但不同时间的连续处理对毛状根及培养液中几种异黄酮物质的积累与释放作用不同,其中以处理48h最有利于培养液中总异黄酮的累积,其含量是毛状根中的38倍。     

Effect of different non-ionic surfactants on the biodegradation of PAHs by diverse aerobic bacteria

The aim of this work was to evaluate the effect of several non-ionic surfactants (Tween-80, Triton X-100 and Tergitol NP-10) on the ability of different bacteria (Enterobacter sp., Pseudomonas sp. and Stenotrophomonas sp.) to degrade polycyclic aromatic hydrocarbons (PAHs). Bacterial cultures were performed at 25 °C in an orbital shaker under dark conditions in BHB medium containing 1% of surfactant and 500 mg l⁻¹ of each PAH. Experiments performed with Tween-80 showed the highest cell density values and maximum specific growth rate because this surfactant was used as a carbon source by all bacteria. High degree of PAHs degradation (>90%) was reached in 15 days in all experiments. Toxicity increased at early times using Tween-80 but decreased to low levels in a short time after the firsts 24 h. On the other hand, Triton X-100 and Tergitol NP-10 were not biodegraded and toxicity kept constant along time. However, PAHs-degradation rate was higher, especially by the action of Enterobacter sp. with Tween-80 or Triton X-100. Control experiments performed without surfactant showed a significant decrease in biomass growth rate with a subsequent loss of biodegradation activity likely due to a reduced solubility and bioavailability of PAHs in absence of surfactant.     

Polyamine and methyl jasmonate-influenced enhancement of betalaine production in hairy root cultures of Beta vulgaris grown in a bubble column reactor and studies on efflux of pigments

Hairy root cultures of red beet (Beta vulgaris) were grown in 3 l bubble column reactor for studying growth and pigment production under the influence of polyamines (PA) and elicitor treatment. Earlier studies with shake flask cultures had shown that combined feeding of spermidine (spd) and putrescine (put) (each 0.75 mM) significantly enhanced betalaine productivity in hairy root cultures of red beet. The present study has been focused on betalaine production in 3 l bubble column bioreactor where the growth pattern and betalaine synthesis under the influence of similar levels of polyamines were followed. A combination of spermidine and putrescine fed to the roots each at levels of 0.75 mM efficiently increased growth and pigment production resulting in 1.23-fold higher biomass (39.2 g FW l⁻¹) and 1.27-fold higher betalaine content (32.9 mg g⁻¹ DW) than control. Treatments with various levels of elicitor-methyl jasmonate (MJ), though progressively retarded biomass, at 40 μM level resulted in a significant increase in betalaine content resulting in 36.13 mg g⁻¹ DW which was 1.4-fold higher than the control. Further higher concentrations of methyl jasmonate treatments supported high as well as rapid accumulation of betalaines, the overall betalaine productivity was hampered mainly because of the inhibitory action on biomass. Pigment release studies with cetyl trimethyl ammonium bromide (CTAB) resulted in optimization of concentration for better efflux of betalaines without showing any inhibitory effect on hairy root viability. These studies on product enhancement and on-line extraction of pigment are useful for developing a bioreactor system for betalaine production using B. vulgaris hairy root cultures. In particular the use of elicitors and efflux studies provide an insight for integrating unit operations and developing a process for continuous operation and higher production of phytochemicals.     

Production and release of anthraquinone pigments by hairy roots of madder (Rubia tinctorum L.) under improved culture conditions

Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O₂ starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 mg/dm³, representing an average production rate of 0.72 mg/(dm³·d).     

DMSO、Tween-20和Triton X-100对野葛悬浮细胞异黄酮生产的影响

二甲基亚砜(DMSO)、Tween-20和Triton X-100可以增大悬浮培养细胞的细胞膜和液泡膜的透性,促进细胞内次生代谢物的释放,从而影响这些次生代谢产物的产量。为了提高野葛悬浮细胞中异黄酮类化合物的产量,以1％、3％和5％的DMSO、Tween-20和Triton X-100分别处理野葛叶悬浮细胞,结果显示,Tween-20和Triton X-100皆明显促进细胞生物量和葛根素和异黄酮化合物的释放。5％的Triton X-100处理3d,促进细胞产生总异黄酮化合物,增产率达40．6％。     

A study of nicotine release from tobacco hairy roots by transient technique

A material balancing method for determining the productivity of nicotine-releasing Nicotiana tabacum hairy roots is presented. Diffusive nicotine release from the intracellular environment of N. tabacum to its medium is based upon an equilibrium partitioning process. By removing nicotine from a culture's medium, subsequent transient release from the tissue is induced, as is the heightened production of nicotine. Under normal conditions, sixteen hours is required for releasing tissue to re-establish equilibrium with the medium. 100 ppm of the surfactant Triton X-100 was used to enhance the rate of nicotine release and to reduce the return to equilibrium to four hours without toxic side-effects.     

表面活性剂对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

研究了不同浓度表面活性剂Tween-80,Triton X-100,SDS对大肠杆菌生产α-环糊精葡萄糖基转移酶（α-CGT酶）的影响。结果表明：发酵初始添加Tween-80和Triton X-100的最适浓度分别为2％,0.5％,最终胞外酶活分别达2.03U/ml和4.92U/ml,相对于未添加表面活性剂时提高4.6倍和12.67倍,且改变添加时间不能提高酶的产量;发酵36 h添加0.02％SDS对α-CGT酶产量促进最大,最终胞外酶活达5.31U/ml,较对照组提高12.75倍。表面活性剂对α-CGT酶生产的促进作用可能是由大肠杆菌细胞内外膜渗透性增加所致,使细胞周质空间中α-CGT酶能更加快速地渗透到胞外。     

α-淀粉酶AmyP对有机溶剂和表面活性剂的耐受性

AmyP是一个来自海洋宏基因组文库的α-淀粉酶。AmyP不仅对log Pow值从4．5到-0．24的各种有机溶剂均具有良好的耐受性,而且能被正辛醇、正辛烷和甲苯提高活性为139％、118％和119％。正辛醇影响AmyP的淀粉水解产物、葡萄糖的含量增加、麦芽三糖的含量降低。非离子型的表面活性剂Tween-20、Tween-80和Triton X-100存在条件下,AmyP的活性反而有不同程度的提高。但是,AmyP对阴离子型的SDS和阳离子型CTAB的耐受性稍差。结果表明AmyP是一个同时具有有机溶剂和表面活性剂耐受性的新型α-淀粉酶。     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.     

Enhanced production of thermostable β-amylase and pullulanase in the presence of surfactants by Clostridium thermosulfurogenes SV2

The addition of the surfactants Triton X-100, CHAPS, Tween-80 and sodium taurocholate to Clostridium thermosulfurogenes SV2 culture individually resulted in a marked increase in the yields of thermostable β-amylase and pullulanase. The stimulation of enzymes production was greater when the surfactants were added after 18 h of incubation of the culture. Upon treatment with 1.0 mM Triton X-100, 0.1 mM CHAPS, 0.1 mM Tween-80 and 0.1 mM sodium taurocholate, C. thermosulfurogenes SV2 produced 140, 34, 88 and 28% more β-amylase and 114, 146, 47 and 28% more pullulanase than the control (lacking surfactants), respectively. Besides stimulation, the surfactants caused an increased secretion of the enzymes into the extracellular fluid. These surfactants also further enhanced the stability of the enzymes. All the surfactants tested were found to have a little inhibitory effect on the growth of the bacterium.     

Umbelliferone released from hairy root cultures of Pharbitis nil treated with copper sulfate and its subsequent glucosylation

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a beta-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal byAspergillus carbonarius during a solid state fermentation process

Summary During the growth of A. carbonarius, the rates of biomass growth, phytase production and phytic acid content reduction in canola meal media during solid state fermentation were higher in the presence of Na-oleate or Tween-80 than in the control medium which was not supplemented with these surfactants. Addition of Triton X-100 had a negative effect on the studied processes.The optimum concentration of Na-oleate in solid state culture media was 1%.     

Bacterial decolorization of acid orange 7 in the presence of ionic and non-ionic surfactants

The effects of the non-ionic surfactant Triton X-100, the cationic surfactant cetyltri-methylammonium bromide (CTAB) and the anionic surfactant sodium N-lauroyl sarcosinate (SLS) on the decolorization of the reaction medium containing the monoazo dye Acid Orange 7 (AO7) by Alcaligenes faecalis and Rhodococcus erythropolis were studied. It was found that the surfactants influenced in different ways the rate of decolorization. At all concentrations tested the non-ionic surfactant Triton X-100 decreased the decolorization rate of R. erythropolis. At concentrations above the critical micelle concentration (CMC) Triton X-100 upset the usually observed exponential decay of the dye with A. faecalis due probably to the existence of an outer membrane in this organism. In concentrations above the CMC the anionic surfactant SLS inhibited the decolorization and, at prolonged incubation, caused partial release of the bound dye. The cationic surfactant CTAB in concentrations above and below the CMC accelerated drastically the binding of AO7 to the cells causing a rapid staining of the biomass and complete decolorization of the reaction medium. An attempt was made for explanation of the observed differences by the negative electrostatic charge of the living bacterial cell.     

Rapid protein release from Escherichia coli by chemical permeabilization under fermentation conditions

Overall protein release greater than 75% in less than 1 h can be attained by exposing exponentially growing Escherichia coli cells to 0.4 M guanidine plus 0.5% Triton X-100 at 37 degrees C in medium. Cell growth stops immediately upon addition of the chemicals, but the cells are not lysed. Guanidine concentrations lower than 0.2 M, in conjunction with 0.5% Triton X-100, do not release significant intracellular protein, nor do they inhibit cell growth. Under these conditions, the cells undergo an adaptation that confers resistance to protein release by further treatment with guanidine and Triton X-100. Cells treated with 0.2 M guanidine plus 0.5% Triton X-100 display intermediate behavior. Protein release is approximately 35%, and growth is temporarily interrupted by an extended lag phase. Subsequent resumption of cell growth results in resistant cells and no additional protein release. This resistance is shown to be reversible and is most likely due to physiological adaptation rather than genetic mutation.     

Characterization of lucidin formation in Rubia tinctorum L.

In order to approach lucidin formation (a strong mutagen or a carcinogen) from a physiological standpoint, hairy roots of Rubia tinctorum L. were established by a transformation of Agrobacterium rhizogenes strain 15834 and cultured in a liquid woody plant medium without plant hormones. The anthraquinone pigment composition of the intact hairy roots was essentially the same as that of the intact non-transformed (normal) roots, in which lucidin O-beta-D-primeveroside (LuP) was one of the major pigments. Lucidin was scarcely detected in the intact hairy roots, but was a main pigment after the squash treatment. The crude protein extract of intact hairy roots exhibited LuP-glycosidase activity (an activity converting LuP to lucidin). This activity was also detected in the roots of the normal plants at a high level, but slightly in the stems and not in the leaves. Methyl jasmonate enhanced the LuP production and LuP-glycosidase activity in the hairy roots. On the other hand, ethephon or salicylic acid had either no effect or rather an inhibitory effect on them. After partial purification of LuP glycosidase, the resultant active fraction producing a major band with an apparent Mr of 68 kDa exhibited the substrate specificity for both aglycon and sugar-moiety. The sugar released from LuP by this fraction was neither D-glucose nor D-xylose and was hydrolyzed into them. These results suggest that LuP specific beta-primeverosidase (EC 3.2.1.149) exists in the roots of R. tinctorum and is involved in the systematic defense system.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Influence of medium constituents on enhancement of pigment production by batch culture of red beet hairy roots

For the enhancement of pigment production by red beet hairy roots, the effects of medium constituents (Murashige-Skoog (MS) medium) on hairy root cultures were investigated in flasks. In a series of cultures using media with diluted medium components, it was found that phosphate was a key nutrient involved in pigment accumulation in the hairy roots, and that higher pigment contents in the roots were obtained at lower phosphate concentrations (range of 0–2.5 mol/m³). In an 18 d batch culture using phosphate-free medium, the total amount of pigment production was 4.8 times that obtained in a control culture using normal MS medium with 1.25 mol/m³ phosphate.     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.