Decarboxylase activity of bacteria of the genus Enterobacter depending on the growing conditions

The activity of decarboxylase in the cells of two bacterial species belonging to the genus Enterobacter was found to depend on the carbon source of the growth medium and on the substrate used to determine the enzyme activity. Cells grown on a medium containing glucose and incubated in solutions of glucose and sodium pyruvate produced 1.4 to 2.1 times more CO2 than cells utilizing glycerol. The highest amount of CO2 was formed on pyruvate, the substrate of the first reaction of decarboxylation.     

Influence of sugars and light on rhizosecretion of α-glucosidase and acid phosphatase during micropropagation of potato

The effects of carbohydrate supply and light on rhizosecretion during micropropagation of potato plantlets (Solanum tuberosum L., cv. ‘Iwa’) in liquid medium were investigated. Soluble protein content was higher in the spent medium for plantlets grown under light conditions than in the dark. For those plantlets grown under light conditions and on different sugar-supplemented media, they rhizosecreted the highest amount of soluble protein when grown in the presence of maltose, while they rhizosecreted the lowest amount of soluble protein when grown on medium containing glucose. Moreover, plantlets grown under light and on a medium containing sucrose were the most vigorous, and exhibited the highest levels of rhizosecreted acid phosphatase activity. However, there was no direct relationship between plantlet growth and rhizosecretion. When plantlets were grown in the dark and on medium containing maltose, a higher α-glucosidase activity was detected than those grown on medium containing sucrose. These results suggested that rhizosecretion of certain proteins from plantlets grown in vitro might not require exposure to light conditions.     

Interaction between proteinases secreted by the fungal plant pathogen Rhizoctonia solani and natural proteinase inhibitors produced by plants

The fungal plant pathogen Rhizoctonia solani Kuhn. grown in a medium containing thermostable potato tuber proteins produced proteinases active at moderately alkaline pH values. Electrophoretic analysis in polyacrylamide gel with SDS and copolymerized gelatin showed that the extracellular proteinase complex contained four components that differed in molecular weight. Studies on the action of the exoenzymes on various synthetic substrates indicated that the culture liquid of R. solani contained mainly trypsin-like proteinases. The exoproteinase activity was virtually completely suppressed by trypsin inhibitor proteins isolated from potato tubers and seeds of various legume species. The results suggest that the extracellular proteinases produced by R. solani play a significant role in attacking plant tissue, and natural inhibitors contribute to the protection of Solanaceae and Leguminosae from this fungal pathogen.     

白腐菌液体和固体培养产生木质纤维素降解酶的比较研究

侧耳sp2(Pleurotus sp．2)和粗毛栓菌(Trametes gallica)是产木质纤维素降解酶能力强,且产酶较快的菌株。对其在液体培养基、固体培养基中产生木质纤维素降解酶能力和行为进行了比较分析和研究。结果表明,Pleurotus sp．2在低氮高碳高无机盐培养基中的锰过氧化物酶(Manganese peroxidases, MnPs)、木质素过氧化物酶(Lignin peroxidases．LiPs)、漆酶(laccases,Lacs)和半纤维素酶(Hemicellulases, Hcels)的活性最高。当该菌株培养在含有低氮无碳高无机盐液体培养基的麦草粉中时,MnPs和Lacs的活性峰值均出现在10d,而Hcels的活性在40d时达到峰值。Trametes gallica在高氮低碳高无机盐培养基中的Lacs和LiPs的活性最高,在低氮高碳高无机盐培养基中的MnPs和Hcels的活性最高。当该菌株培养在含有高氮无碳高无机盐和低氮无碳高无机盐液体培养基的麦草粉中时,MnPs存10d、Lacs和Hcels在40d、LiPs存50d,分别达到峰值。Pleurotus sp．2和Trametes gallica在液体培养基中具有很强的木质纤维素降解酶产生能力且产酶速度较快,在固体培养基中具有很强的降解麦秸生物质能力,但这两株菌在液体和固体培养基中,产木质纤维素降解酶的能力和行为都有较大的差异,相关性小。     

How important is secretion of exoenzymes through apical cell walls of fungi?

A hypothesis is proposed that the passage of exoenzymes through cell walls occurs more easily through the more plastic and porous nascent cell wall, e. g., the apical region of fungal hyphae. It also accounts for the occurrence of some exoenzymes in cell walls. As the porous and nascent apical wall of fungi is transformed to the less porous lateral wall during growth, some exoenzymes are trapped in transit, thus becoming bound into the wall. Enzymes with binding sites in the wall are not considered in the hypothesis. Several experimental tests performed on Neurospora crassa yield results consistent with its predictions: 1. under selected growth conditions, a group of three exoenzymes of high molecular weight has a significantly higher percent of the total cellular enzyme activity in the wall fraction than another group of three exoenzymes of low molecular weight; this complies with the prediction that larger molecules are more easily trapped in transit, 2. during germ tube outgrowth and early log phase, when the relative amount of surface area occupied by hyphal tips is larger than in older cultures, there is decreased molecular sieving of secreted exoenzymes as judged by a) a smaller proportion of the secreted invertase, comprising light invertase (mol wt=51,500) and heavy invertase (mol wt=210,000), being in the light form, and b) a larger amount of proteins with molecular weights over 40,000 than those of 20,000–40,000 in the culture filtrate. Some of the possible applications of the hypothesis to other microorganisms are discussed.     

对黄豆苷原具转化作用的耐氧突变株Aeroto-AUH-JLC140未知代谢产物分离、鉴定及抗氧化活性测定

【目的】与原出发菌株AUH-JLC140相比,耐氧突变株Aeroto-AUH-JLC140在其生长过程中产生一种未知物质,且该未知物质的产生与添加底物黄豆苷原无关。对该未知物质进行分离纯化和结构鉴定,并测定其产生动态及抗氧化活性。【方法】利用高效液相色谱对未知物质进行分离,经紫外吸收图谱、质谱、核磁共振氢谱和碳谱等分析,对未知物质进行结构鉴定;通过1,1-二苯基-2-苦味酰基自由基(DPPH)清除试验测定其抗氧化活性。【结果】Aeroto-AUH-JLC140产生的未知物质被鉴定为吲哚,接种后15 h菌株产吲哚最高,所产吲哚量为19.89 mg/L。浓度为0.2 mmol/L(即23.40 mg/L)的吲哚除对DPPH自由基具有明显清除作用外,还能有效降低脑心浸液(BHI)液体培养基的氧化-还原电位。【结论】耐氧突变株Aeroto-AUH-JLC140产生的未知代谢产物为吲哚,菌株通过产生吲哚降低培养基氧化-还原电位,进而为该菌株的生长提供适宜的低氧微环境。     

Interaction of proteinases secreted by the fungal plant pathogen Rhizoctonia solani with natural proteinase inhibitors produced by plants

The fungal plant pathogen Rhizoctonia solani Kuhn. grown in a medium containing thermostable potato tuber proteins produced proteinases active at moderately alkaline pH values. Electrophoretic analysis in polyacrylamide gel with SDS and copolymerized gelatin showed that the extracellular proteinase complex contained four components that differed in molecular weight. Studies on the action of the exoenzymes on various synthetic substrates indicated that the culture liquid of R. solani contained mainly trypsin-like proteinases. The exoproteinase activity was virtually completely suppressed by trypsin inhibitor proteins isolated from potato tubers and seeds of various legume species. The results suggest that the extracellular proteinases produced by R. solani play a significant role in attacking plant tissue, and natural inhibitors contribute to the protection of Solanaceae and Leguminosae from this fungal pathogen.     

Effect of Penicillium expansum culture conditions on patulin production.

Penicillium expansum strains grown on Capek-Dox liquid medium excreted patulin to the medium. Its amount increased until day 12, then smaller amounts of the toxin were observed. Maximum patulin excretion took place at 25 degrees C, pH 6, although at 5 degrees C, pH 3 the presence of this mycotoxin was also observed. The highest amount of patulin produced was observed in medium containing fructose.     

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.     

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

The major objectives of this study were to investigate an efficient rapid protocol for mass propagation of adventitious shoots of brahmi using semisolid and liquid cultures; and to assess the amount of bacoside A accumulated in the regenerated shoots. Leaf explants were grown in vitro on Murashige and Skoog semisolid medium supplemented with 0.5, 1.0, 1.5, 2.0 and 2.5 mg l⁻¹ 6-benzyladenine or kinetin (KN) or thidiazuron (TDZ) for 4 weeks. Adventitious shoots developed from leaf explants on all cytokinin supplemented media. After 4 weeks of incubation, leaf explants were split into two batches and one set was subcultured on semisolid medium and another set in liquid medium containing same concentration of cytokinins where they have come from. Highest rate of shoot regeneration was observed for explants cultured on medium with 2 mg l⁻¹ KN. The fresh and dry weight of shoots was also highest with this treatment. Liquid cultures were found suitable for proliferation of shoots (155.6 shoots per explant) and they also favored highest biomass accumulation (8.60 g fresh and 0.35 g dry biomass). The bacoside A contents were determined in shoots using HPLC. Analysis revealed that, the contents were highest with shoots regenerated on medium supplemented with 2 mg l⁻¹ KN. The amount of bacoside A was highest in the shoots regenerated in liquid medium (11.92 mg g⁻¹ DW) and it was 2.2-fold higher compared to shoots grown on semisolid cultures.     

Apparent phosphate retrieval system in Bacillus cereus.

Bacillus cereus secretes three different phospholipases C. We studied the effect of Pi levels in the growth medium on the production of these exoenzymes. Production of both phosphatidylcholine-preferring phospholipase C and sphingomyelinase C was repressed by Pi in the growth medium, whereas production of phosphatidylinositol phospholipase C was unaffected. We also found that B. cereus secretes a phosphate-repressed alkaline phosphatase activity. Together with a previously reported highly efficient, active uptake system for Pi, these three phosphate-repressed exoenzyme activities seem to be part of a phosphate retrieval mechanism that operates under growth-limiting concentrations of Pi. In natural soil systems, which are the natural habitats of B. cereus, the scarcity of Pi is the major growth-limiting factor. A phosphate-repressed metalloprotease activity was also detected in culture supernatants of B. cereus. It is unclear whether this exoenzyme activity also participates in the proposed phosphate-scavenging system.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Violacein biotransformation by basidiomycetes and bacteria

AIMS: The biotransformation study of the pigment (violacein) produced by Chromobacterium violaceum, was verified by its decolorization utilizing six different organisms in liquid and solid media. METHODS AND RESULTS: Lentinus edodes showed the fastest violacein decolorization (8th d) in solid medium whereas in a liquid medium, violacein was rapidly decolorized in 2 h by Azotobacter vinelandii. Adsorption experiments indicated no significant contribution for the decolorization process for all organisms in both media. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed poor lignin peroxidase activity in both media but manganese peroxidase and laccase activity varied according to the organism used. Siderophores presence was also detected in all organisms mainly in the liquid medium experiment.     

Effect of lincomycin on its own producer, Actinomyces roseolus, in periodic cultivation in a liquid medium

Lincomycin added to the cultivation medium induced a number of changes in the organism producing it during its ontogenesis when grown recurrently on liquid media. It was found that lincomycin inhibited the culture growth and decreased the absolute amount of the antibiotic synthesized while the specific activity of the culture increased. A number of cytomorphological rearrangements relevant to the adaptive protective reactions was found. It is suggested that an increase in the resistance of the culture to the antibiotic produced by it at the late developmental stages is the result of the above protective reactions.     

The influence of colouring and pungent agents of red Chilli (Capsicum annum) on growth and aflatoxin production by Aspergillus flavus

Capsanthin and capsaicin, the colouring and pungent principles of red chilli Capsicum annum , respectively, were tested against the growth and aflatoxin producing potentials of Aspergillus flavus in SMKY liquid medium. Capsanthin completely checked both the growth and toxin production at all the concentrations viz. 0.2, 0.6 and 1.0 mg ml^-1, till the fourth day of incubation. On the 10th day growth of the fungus and toxin biosynthesis were 39 and 22% of the control, respectively, at 1.0 mg ml^-1. Capsaicin showed some inhibitory efficacy only up to the fourth day of incubation. The fungus grew thereafter with a marginal inhibition in growth at the highest concentration. The amount of the toxin in the medium was also higher.     

Growth media effects on morphology and 17β-HSD activity in the fungusCurvularia lunata

The filamentous fungusCurvularia lunata has enzymatic activities of biotechnological significance. Its 11β-hydroxylase actvity produces hydrocortisone, and its 17-ketosteroid reductase (17β-HSD) activity is important in the production of 17β-hydroxysteroids. We have examined the effects of different media on the 17β-HSD activity, and on the morphology and ultrastructure ofC. lunata in the search for a correlation between high enzymatic activity, morphology and ultrastructure. The highest 17β-HSD activity per dry weight ofC. lunata was seen in Czapek medium, while more product per volume was formed in malt extract broth. In all growth media tested, the highest 17β-HSD activity coincided with the exponential phase of growth. In Czapek broth medium, fungal hyphae formed pellets, while in malt extract and yeast nitrogen base broth media, the fungus formed branched hyphae dispersed throughout the media. Scanning electron microscopy revealed differences in the thickness of hyphal cells; those of the fungus grown in Czapek and yeast nitrogen base media were much thicker than those in malt extract medium. Transmission electron microscopy showed melanized cell walls in the fungus grown in malt extract and Czapek media, but not in yeast nitrogen base medium. Our results show that theC. lunata with the highest 17β-HSD activity grow as dispersed, melanized mycelia with thin hyphae.     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Enzymatic activity in the activated-sludge floc matrix

The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.     

Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type backgroud, both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.     

Gelling agents for tissue culture of the seaweed Hizikia fusiformis

Callus and blade formation of the seaweed Hizikia fusiformis depended on the gelling agents used under axenic culture conditions. Excised cylindrical pieces (5 mm) of the hold fast were cultured on seven different gelling agents in seawater with added Provasoli's enrichment (PESI), at 40 μmol m⁻² s⁻¹ light intensity, 18 −C for 1 month. The highest percent of callus formation (47%), from holdfast pieces, was produced on solid medium composed of 2.0% high gel strength agar. No callus was formed in liquid medium. Blades, from holdfast pieces, were formed in PESI liquid medium at the rate of 45%, while the high level of axenic blade formation (30%) on solid support was observed on 0.5% high gel strength agar. Callus and blade were identified with the original strain, at the DNA level, using random amplified polymorphic DNAs. This revised version was published online in August 2006 with corrections to the Cover Date.