Production of polysaccharides by Silene vulgaris callus culture depending on carbohydrates of the medium

Sources of carbohydrate nutrition such as sucrose, glucose, and galactose, with the exception of arabinose, were shown to influence positively callus growth and polysaccharide (pectin silenan and acidic arabinogalactan) biosynthesis. Galactose was found to cause a stimulatory effect on yield and productivity of arabinogalactan. Low concentrations of sucrose failed to support the cell growth and polysaccharide biosynthesis. Increasing sucrose concentrations led to biomass accumulation but failed to enhance efficiency of the substrate utilization. The optimal medium for the campion cell culture growth was found to be one containing 30 g/liter of sucrose or a mixture of sucrose with glucose (in 15 g/liter). Increasing sucrose concentrations in the medium from 30 to 100 g/liter failed to significantly influence the polysaccharide yields while the polysaccharide productivity per liter of the medium grew due to promotion of culture productivity in biomass. Variations of the carbon sources in the nutrient media were shown to influence insignificantly the biochemical characteristics of arabinogalactan and silenan while an increase in the sucrose concentration to 50-100 g/liter led to a diminution of the galacturonic acid content in silenan and to changes in contents of the neutral monosaccharide residues in silenan and arabinogalactan.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.     

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.     

Influence of the fungus Trichoderma harzianum on the enzyme and polysaccharide composition of Silene vulgaris callus

Activities of polygalacturonase and 1,3-β-glucanase increased in campion (Silene vulgaris) callus cells during co-cultivation with the fungus Trichoderma harzianum. This was associated with a decrease in galacturonic acid residues in the pectic polysaccharide of campion silenan and also in the production of pectin by the callus. Co-cultivation of the callus and the fungus resulted in an increase in contents of arabinose residues in the intracellular arabinogalactan and in contents of galactose residues in the extracellular arabinogalactan.     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280-315 nm; power, 0.2-13.0 W/m2; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m2) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1 : (3.4-8.3).     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280–315 nm; power, 0.2–13.0 W/m²; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m²) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1: (3.4–8.3).     

The effects of ultraviolet irradiation upon structure and antioxidant activity of silenan from bladder campion callus

Effects of UV-B (280–315 nm) and UV-C (254 nm) at various doses upon callus of bladder campion (Silene vulgaris (M.) G. were studied. It was revealed that UV irradiation results in the decrease in arabinose and galactose residues in the silenan—the pectin fraction isolated from callus. The silenan possesses antioxidant activity (AOA) as assessed by the reaction with a stable radical. At the irradiation of callus by UV, the AOA of the silenan and the relative content of phenolic compounds in it increased; the highest increase was observed after the irradiation of callus by UV-B. Positive correlation between the AOA of the pectin fraction and an increase in phenolic compounds was revealed. This evidences that the AOA of the silenan relates to and is partially determined by phenolic compounds in its composition. The UV irradiation may be used as a tool to modify the structural features of the cell walls’ polysaccharides in order to produce physiologically-active polysaccharides with desired properties.     

Polysaccharides of cell cultures of Silene vulgaris

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Polysaccharides of cell cultures of <Emphasis Type="Italic">Silene vulgaris</Emphasis>

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3–6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5–1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Summary Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non-embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gaschromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.Abbreviations EC embryogenic callus - NC non-embryogenic callus - SE somatic embryo - MS Murashige and Skoog - PAS periodic acid-Schiff s reagent     

Isolation of polysaccharides from callus culture of Lemna minor L

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minor L.) with water and ammonium oxalate. Residues of galactose and arabinose in the 2.0-2.5:1 ratio were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glucuronic acids, galactose, and arabinose. The percentage of arabinogalactan and pectin was similar. The yield of polysaccharide fractions did not depend on the method for their isolation. Extraction with water, treatment of the biomass with an aqueous solution of formalin and diluted hydrochloric acid, and extraction with an aqueous solution of ammonium oxalate allowed us to obtain the highest-purity pectin polysaccharide.     

Identification of mono-, oligo-, and polysaccharides secreted from soybean roots

The mist culture system was conducted to study secreted polysaccharides from soybean ( Glycine max) roots grown for 15 days. Roots were rinsed with distilled water (DW) for 15 min, then with 30 mM oxalic acid (OXA) for 15 min to remove ionically bound sugar. Released sugars were further fractionated into low (L) and high (H) molecular weight fractions with Sephadex G-10. DW rinsing released 190 microg neutral sugar (NS) and 62 microg uronic acid (UA) per plant, while 374 microg NS and 70 microg UA per plant were released by OXA rinsing. Acetylation analysis revealed that the L fraction by DW and OXA mainly consisted of glucose (Glc), pinitol, and UA, whereas the H fraction mainly consisted of arabinose (Ara), galactose (Gal), Glc, and UA. The presence of rhamnose (2%-6%) in both fractions suggests secretion of rhamnogalacturonans. Methylation analysis revealed that the H fraction by DW and OXA contained T-Ara, 3-, 6-, and 3,6-Gal, suggesting the presence of type II arabinogalactan and arabinogalactan proteins. HPLC analysis detected mono-, di-, and tri-GalA in the L fraction by DW and OXA. Substances corresponding to sucrose, kojibiose, cello- and laminari-oligosaccharides were also found in root exudates.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Isolation and structural study of polysaccharides from campion Silene vulgaris

Silenan SV, a pectic polysaccharide, was isolated from the aerial part of Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn.), widespread through the European North of Russia. The polysaccharide was found to contain residues of galacturonic acid (63%), arabinose, galactose, and rhamnose as the main constituents. The results of a partial acidic hydrolysis, pectinase digestion, and NMR studies of silenan SV indicated that its molecule contains a linear alpha-1,4-D-galacturonan backbone and ramified regions. The core of the ramified regions is composed of residues of alpha-1,4-D-galacturonic acid along with 2-substituted alpha-rhamnopyranose residues. The NMR data showed that the silenan SV side chains are composed of the blocks built from the terminal alpha-1,5-linked arabinofuranose and beta-1,4-linked galactopyranose residues; these most likely are the side chains of rhamnogalacturonan, characteristic of other pectic polysaccharides. The nonreducing ends of these side chains contain alpha-arabinofuranose residues.     

Modified arabinogalactans from callus culture <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G.

The cultivation of Silene vulgaris (M.) G. callus culture on the nutrient mediums contained carbohydrates, phytohormones, nitrogen, and phosphate has led to the modification of the arabinogalactan structure from the cell walls. It was noticed that a sucrose concentration increase in the cultivation medium led to an increase of the arabinogalactan fragment yield with a molecular weight more than 300 kDa and a decrease of the yield of fragments with molecular weight less than 300 kDa. The sucrose concentration increase in the nutrient medium entailed the increase of arabinose and galactose content in the fragment with the molecular weight more than 300 kDa and a decrease in the fragment with a molecular weight of 100–300 kDa. On the nutrient medium containing a mix of sucrose and arabinose, the yield of the fraction with a molecular weight more than 300 kDa and the amount of arabinose residues increased, and the yield of minor fragments and the content of arabinose and galactose residues, included in these, decreased. On the medium containing an increased concentration of 2,4-dichlorphenoxyacetic acid, the yield of high-molecular fragment and the content of arabinose residues are two times increased. The decreasing of the amount of arabinose and galactose residues in the fragment with a molecular weight more than 300 kDa was observed at a lack of nitrogen or phosphate in the nutrient medium.     

Isolation of Polysaccharides from the Callus Culture of Lemna minor L.

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minorL.) with water and ammonium oxalate. Residues of galactose and arabinose (ratio, (2.0–2.5) : 1) were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glycuronic acids, galactose, and arabinose. The percentages of arabinogalactan and pectin were similar. The yield of polysaccharide fractions did not depend on the method used for their isolation. Extraction with water, treatment of the biomass with aqueous formalin and dilute hydrochloric acid, and extraction with aqueous ammonium oxalate allowed us to obtain the pectin polysaccharide with the highest purity.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Utilization of Waste Products of Dehydrated Onion Industry for Production of Fodder Yeast

An organism producing extracellular polysaccharide was isolated from soil and identified as Aeromonas hydrophila (Chester) Stanier. The effects of medium components and cultural conditions on production of the polysaccharide were studied. The optimal concentrations of carbon and nitrogen sources were 5% and 0.3%, respectively, for production of the polysaccharide. The optimal initial pH was 7~9. The maximum polysaccharide yield was obtained at 4~8 days of fermentation. From sucrose and raffinose as carbon source, the organism produced levan and acidic polysac-charide in the ratio of 7:3 and 4:6, respectively. From glucose, galactose, fructose, mannose, maltose and lactose, mainly acidic polysaccharide was produced. The acidic polysaccharide was found to contain galactose, mannose and glucuronic acid in a ratio of 5:4:2. The acidic polysaccharides obtained from sucrose and lactose seemed to be the same polysaccharide.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.