灵芝悬浮培养中添加微颗粒Talc对灵芝酸产生的影响

灵芝酸是灵芝中重要的药理活性物质,其低产量限制了它的广泛应用和深入研究,高效提高液体发酵中灵芝酸的含量十分必要。以CGMCC 5.65为材料,在悬浮培养条件下,研究添加微颗粒Talc对灵芝酸产生的影响。结果表明,微颗粒Talc添加显著减小了灵芝细胞粒径,对照组为(3.33±0.16)mm,15g/L微颗粒Talc添加组为(2.04±0.12)mm。灵芝细胞中单体灵芝酸和总灵芝酸的含量在微颗粒Talc添加条件下也显著提高。15g/L微颗粒Talc添加组的总灵芝酸含量达到(1.51±0.02)mg/100mg细胞干重,GA-Mk、GA-T和GA-Me的含量最高为(6.02±0.29)、(5.08±0.14)和(1.71±0.09)μg/100mg细胞干重,分别是对照的1.6、4、1.9和1.4倍。另外,15g/L微颗粒Talc添加条件下鲨烯和羊毛甾醇的最大积累量分别为(3.69±0.23)和(34.86±6.41)μg/100mg细胞干重,是对照的2.6和4.2倍;灵芝酸生物合成途径关键基因fps和cyp-5150l8的表达量最高为对照的2.35和1.53倍。     

Stimulatory effects of Coix lacryma-jobi oil on the mycelial growth and metabolites biosynthesis by the submerged culture of Ganoderma lucidum

Effects of Coix lacryma-jobi oil (CLO) addition on the mycelia growth and production of bioactive metabolites, such as triterpenoids, exopolysaccharide (EPS), and intracellular polysaccharide (IPS) in the submerged culture of Ganoderma lucidum were studied. The results showed that when a level of 2% CLO was added at the beginning of culture, the biomass, triterpenoids, EPS, and IPS productions reached a maximum of 10.71 g/L, 92.94 mg/L, 0.33 g/L, and 0.389 g/L, respectively, that were 3.34-fold, 2.76-fold, 2.2-fold, and 2.23-fold compared to that of control. Analysis of fermentation kinetics of G. lucidum suggested that glucose concentration in the culture of CLO-added group decreased more quickly as compared to the control group from day 2 to day 7 of fermentation process, while the triterpenoids and polysaccharides biosynthesis were promoted at the same culture period. However, the culture pH profile was not affected by the addition of CLO. There were no new components in the two types of polysaccharides obtained by the addition of CLO. Enzyme activities analysis indicated CLO or its fatty acids affected the synthesis level of phosphoglucose isomerase and α-phosphoglucomutase at different stage.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum

Control of inoculation density was significant for cell growth, morphology, and production of polysaccharide and ganoderic acid in submerged culture of the higher fungus Ganoderma lucidum. A maximal cell concentration of 15.7 g dry cell weight (DW)/l was obtained at an inoculation density of 330 mg DW/l. For inoculation density within the range of 70–670 mg DW/l, a large inoculation density led to a small pellet size and high production of extracellular and intracellular polysaccharides, while a relatively big pellet size and high accumulation of ganoderic acid were observed at a low inoculation density. It was also shown that small pellet size resulted in high polysaccharide production, while large pellet size led to high production of ganoderic acid.     

Improving of red colorants production by a new Penicillium purpurogenum strain in submerged culture and the effect of different parameters in their stability

There is a worldwide interest in the development of processes for colorants production from natural sources such as microorganism. The aim of this study was to optimize red colorants production by Penicillium purpurogenum DPUA 1275 and to evaluate the effect of pH, temperature, salts and polymers on the stability of these colorants. Under optimized conditions, a 78% increase in red colorants production was achieved. The best pH and temperature conditions were obtained at pH 8.0 and 70°C, respectively. In the presence of salts NaCl and Na₂SO₄, both at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 8.0), the red colorants showed good stability. In the presence of both polymers polyethylene glycol and sodium polyacrylate, the red colorants kept their color intensity. Thus, this study presents characteristics of red colorants produced by P. purpurogenum that can be applied in different industries after toxicological examination. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:778–785, 2013     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

Summary Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.     

Possible role of a regulatory gene product upon the myo-inositol-1-phosphate synthase production in Neurospora crassa

The regulatory effect of inositol on inositol-1-phosphate synthase in Neurospora crassa strains was studied. Inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22°C. Enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. Inositol-requiring strains used in our experiments can be divided into two groups. The first group produces a protein related immunologically to inositol phosphate synthase, but which is enzymatically inactive. The synthesis of this defective enzyme was also repressed by inositol. In the second group, this protein was found to be completely lacking, in both the thermosensitive mutant grown at 37°C, and in a strain requiring inositol due to a reciprocal translocation. The thermostability and the cross immunoelectrophoresis of the enzyme suggest that in the case of the thermosensitive inositol-requiring mutant, the mutation did not occur in the structural gene of the enzyme, but its regulation was probably affected.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

Chloroplast culture VIII a new effect of kinetin in enhancing the synthesis and accumulation of protochlorophyllide invitro

By pretreating etiolated cucumber cotyledons with kinetin in the dark, it was observed that the plastids isolated from such tissues were 400% more active in the conversion of δ-aminolevulinic acid into protochlorophyllide, than plastids prepared from water-treated controls. The experimental evidence is consistent with the hypothesis that (a) the kinetin dark-pretreatment of the etiolated tissue, uncouples the joint biosynthesis of prothylakoids and protochlorophyll and results in the accumulation of excess prothylakoid membranes poorly supplied with protochlorophyllide (b) upon isolation of the plastids and incubation with δ-aminolevulinic acid, the latter is very rapidly converted into membrane-bound protochlorophyllide.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and omega-agatoxin IVA (omega-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCl-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (+/-)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (+/-)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca2+ channels involving KCl-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCl-stimulated cyclic GMP formation measured in the presence of omega-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCl-induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L-type and P/Q-type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.