Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture

Summary The simultaneous production of endoglucanase and -glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and -glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and -glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C     

Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales,Rhodophyta)

Summary The effect of age, pH of the culture medium, pre-treatment of tissues, enzymes sources and enzymatic adaptability of phycophages fed with a monospecific diet were analyzed on the protoplast yields of the red seaweed Solieria filiformis (Kützing) Gabrielson. New apices from fast growing plants showed the highest protoplast yields. The protoplast yield decreased when the pH of the culture medium increased from 6.0 to 9.0. Crude extracts from the abalone Haliotis coccinea canariensis Nordsieck, fed with Solieria filiformis thalli for three months in combination with cellulysin, released the highest number of viable cells and protoplasts. Yields ranged from 1.0 to 8.5 x 10⁶ protoplasts per gram of fresh weight.Abbreviations AAP abalone acetone powder - Bis-Tris Bis(2hydroxyethyl)imino-tris(hydroxymethyl)methane - CMC carboxymethyl cellulose - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(ß-aminoethyl ether) NNNN-tetraacetic acid - FDA fluorescein diacetate - FW fresh weight - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane     

Chemolithotrophic metabolism of the newly-isolated moderately thermophilic,obligately autotrophic Thiobacillus tepidarius

Thiobacillus tepidarius, isolated from the hot springs at Bath, Avon, UK, grew optimally at 43–45°C and pH 6.0–7.5 on thiosulphate or tetrathionate. In batch culture, thiosulphate was oxidized stoichiometrically to tetrathionate, with a rise in pH. The tetrathionate was then oxidized to sulphate, supporting growth and producing a fall in pH to a minimum of ph 4.8. The organism contained high levels of thiosulphate-oxidizing enzyme, rhodanese and ribulose bisphosphate carboxylase. It was obligately chemolithotrophic and autotrophic. In chemostat culture, T. tepidarius grew autotrophically with the following sole energy-substrates: sulphide, thiosulphate, trithionate, tetrathionate, hexathionate or heptathionate. Thiocyanate, dithionate and sulphite were not used as sole substrates, although sulphite enhanced growth yields in the presence of thiosulphate. Maximum specific growth rate on tetrathionate was 0.44 h^-1. True growth yields (Y _max) and maintenance coefficients (m) were calculated for sulphide, thiosulphate, trithionate and tetrathionate and observed yields at a single fixed dilution rate compared with those on hexathionate and heptathionate. Mean values for Y _max, determined from measurements of absorbance, dry wt, total organic carbon and cell protein, were similar for sulphide, thiosulphate and trithionate (10.9 g dry wt/mol substrate) as expected from their equivalent oxygen consumption for oxidation. Y _max for tetrathionate (20.5) and the relative Y _o values (as g dry wt/g atom oxygen consumed) for thiosulphate and all four polythionates indicated that substrate level phosphorylation did not contribute significantly to energy conservation. These Y _max values were 40–70% higher than any of those previously reported for obligately aerobic thiobacilli. Mean values for m were 6.7 mmol substrate oxidized/g dry wt·h for sulphide, thiosulphate and trithionate, and 2.6 for tetrathionate.Abbreviation PIPES Piperazine-N,N-bis(ethane sulphonic acid)     

Increased β-carotene synthesis in Blakeslea trispora under strong alkaline culture condition

Summary A high yield of -carotene, 48 mg/l, was obtained by cultivation of a strain of Blakeslea trispora under a strong alkaline culture condition (pH 10), which was significantly higher than those obtained under milder pH conditions (3.26–7.80 mg/l at pH 4 to 8). The formation of small pellets at pH 10 was observed, whereas a single large cluster was formed for the pHs ranged from 4 to 8. Span 20 at 0.1 to 1% in the culture medium changed cellular morphology to micro-pellets regardless of the culture pH. The same trend of -carotene production, depending on the alkalinity of the culture broth, was also observed in the presence of 1% of Span 20 yielding much higher productivity. The amounts of -carotene yielded at pH 10 and 11 were 92.0 and 100.9 mg/l, respectively, which were about two-times higher than those obtained at pH 4 to 9.     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

β-hydroxypropionic acid production by Byssochlamys sp. grown on acrylic acid

A strain of Byssochlamys sp. produced -hydroxypropionic acid when grown on media containing high concentrations of acrylic acid. The maximal production of -hydroxypropionic acid was 4.8% when the initial culture medium contained 7% acrylic acid and 2% glucose, and the initial culture pH was adjusted to 7.0. -Hydroxypropionic acid production from acrylic acid depended greatly on the pH of the culture medium. Calcium hydroxide was the best neutralizer. Correspondence to: K. Takamizawa     

Integrated immobilized cell reactor-adsorption system for β-cyclodextrin production: a model study using PVA-cryogel entrapped Bacillus agaradhaerens cells

Production of cyclodextrins (CDs) by immobilized cells of the alkaliphilic Bacillus agaradhaerens LS-3C with integrated product recovery was studied. The microorganism was entrapped in polyvinyl alcohol-cryogel beads and used as a convenient source of immobilized cyclodextrin glycosyltransferase (CGTase). On activation by incubation in the cultivation medium containing 1% (w/v) starch, the entrapped cells multiplied and secreted CGTase with an activity of 2–3 mg -cyclodextrin h^–1 g^–1 beads. The immobilized biocatalyst exhibited maximum activity at pH 9 and 50 °C, and formed cyclodextrins comprising 92–94% -CD and remaining -CD. The cyclodextrin product from the immobilized cell bioreactor was continuously recovered by adsorption to Amberlite XAD-4 in a recycle batch mode. The product adsorption was facilitated at low temperature while hot water was used for elution.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 M Al at pH 5.5. Three strains were tolerant of 20 M Al at pH 5.5 and 10 M Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 M Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 M Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Evaluating the contribution of magnesium deficiency in the aluminium toxicity syndrome in twelve sorghum genotypes

The contribution of Mg deficiency to Al stress in twelve different sorghum (Sorghum bicolor (L.) Moench) genotypes was investigated in nutrient solution culture under conditions of low Mg supply (between 50 and 1000 M) at two pH values. At pH 4.2, 30 M Al strongly inhibited Mg uptake. When dry matter yield was plotted as a function of the plant Mg concentration, similar response curves were obtained in the absence and the presence of Al with three genotypes. With many other genotypes dry matter yields of the control (without Al treatment) and Al-stressed plants were remarkably different at similar internal Mg concentrations, suggesting that growth had been suppressed not by Mg deficiency but by another factor, i.e. Al-induced root damage. At pH 4.8, 30 M Al hardly induced root damage but reduced Mg uptake and Al-induced Mg deficiency could almost completely account for the growth reaction of all genotypes. Therefore, at this pH the efficiency of uptake or use of Mg in different genotypes was the basis of their respective susceptibility to Al toxicity. When specific root length surpassed a certain critical range below 80–100 m per g dry root, growth control by Al-induced Mg deficiency was nearly abolished. The pH and Al concentration where this range was reached depended on the Al sensitivity of the genotypes.     

Differential responses of soybean genotypes to excess manganese in an acid soil

To determine the tolerance of soybean genotypes to Mn toxicity, a green house study was conducted. Hayesville sandy loam (clayey, oxidic, mesic, Typic Hapludult), high in manganese, was used for the experiment. The experimental design was split-plot with three replications. Forty-one different soybean genotypes were planted in pots at two different pH levels: 5.2 (original soil pH) and 6.4 (amended with lime). Soybean genotypes were allowed to grow to the dry pod stage.Soil pH levels affected the soybean genotypes yields significantly (p < 0.01). Tolerant genotypes showed a higher or similar seed yield at pH 5.2 compared to pH 6.4. Sensitive genotype yields were lower at pH 5.2 than at pH 6.4. In general, Mn in leaves was higher at pH 5.2 than at pH 6.4. Some of the sensitive genotypes at pH 5.2. showed severe chlorosis and crinkle leaf symptoms as a result of Mn toxicity. Excess available Mn at pH 5.2. induced Ca deficiency. Soybean genotypes PI423758, PI417440, Aoda, Kingston, Rokusum and some others were tolerant to Mn toxicity, whereas PI417288, Verde, Wilson 5, Sango, Funk Delicious and some others were sensitive to Mn toxicity. The genotypes found to be tolerant can be recommended to plant breeders for development of Mn-tolerant cultivars.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature

Summary High pressure/high temperature investigations on thermophilic methanogens require specific precautions to provide well-defined pH conditions in their culture media. Applying CO₂ as carbon source, sufficient buffering capacity of the culture medium is of crucial importance in investigations involving elevated pressures. In order to separate pressure effects on the growth and reproduction of thermophilic methanogens from pressure-induced protonation/deprotonation and increased solubility of gaseous components, direct pH measurements in common culture media in the absence and in the presence of CO₂ were performed at elevated temperature (65° C), and at pressures up to 100 MPa. Neutral phosphate buffer at high pressure shows a significant downward shift of its pH which is strongly enhanced in the presence of CO₂. In minimal media containing acetate, carbonate, formate and phosphate in 100 mM concentrations, 120 mM HEPES is found to provide optimum pH stability: near neutrality the pH change upon CO₂ saturation in the absence and in the presence of HEPES amounts to pH=2.10 and 0.41, respectively; the corresponding pressure dependences are pH/100 MPa=-0.26 and -0.07. As taken from these results, the apparent pressure dependence of the optimum growth ofMethanococcus thermolithotrophicus at 65° C in minimal medium reflects the pH shift below the cutoff point of growth (pH 5.5), rather than pressure-induced growth inhibition. At constant pH, elevated pressure up to 400 MPa is found to increase the rate and yield of growth; at the same time, alterations in the phenotype of the bacterium are observed.     

Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the -amylases. The optimum pH, specific activity and K _m value for -cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mm, respectively. These values were almost identical to those from B. sphaericus E-244. Correspondence to: T. Oguma     

Effects of pH on expression and stabilization of β-galactosidase by recombinantE. coli with a thermally-inducible expression system

Summary Effects of pH on -galactosidase expression and stabilization were investigated using recombinantE. coli harboring an expression vector with a thermally-inducible P_L promoter. Expression of -galactosidase was strongly promoted by lowering culture pH when culture temperature was raised to the induction temperature. Optimal pH for induction ranged from 5.4–5.8. The degradation of expressed -galactosidase could be reduced by lowering the culture temperature while at the same time slightly increasing the culture pH in the -gal degradation stage.     

Molecular mass control using polyanionic cyclodextrin derivatives for the epsilon-poly-l-lysine biosynthesis by Streptomyces

To control the molecular mass of a natural polycationic, antimicrobial, Streptomyces-biosynthesized polymer, epsilon-poly-l-lysine, addition of polyanionic cyclodextrin derivatives to the culture medium was evaluated. Chemically modified cyclodextrins such as a sulfated cyclodextrin caused a notable shortening of the polymer length of epsilon-poly-l-lysine from 3.5 to 4.5 kDa to less than 2.5 kDa by the enforcing action of glycerol, which has a weak potential to inhibit polymer elongation by terminal esterification. Meanwhile, polyanionic cyclodextrin inhibited the shortening action with n-octanol, which has a strong ability to inhibit polymer elongation through an undetermined function. The design of chemical structures of polyanionic cyclodextrin, optimization of their addition concentrations, and selection of hydroxyl compounds coexisting with them are critical for this simple and easy method for polymer length control and terminal modification of epsilon-poly-l-lysine.     

Stimulation of the gibberellic acid synthesis by Aspergillus niger in submerged culture using a precursor

Stimulation of Aspergillus niger in submerged culture using a commonly known precursor, mevalonic acid (MVA), was investigated in terms of growth and gibberellic acid production. Increasing concentrations of MVA up to 60 M enhanced product and growth yields. Above this amount, gibberellic acid yields and growth were gradually decreased.     

The production of α-amylase in batch and chemostat culture by Bacillus stearothermophilus

The production of -amylase (-1,4-glucan 4-glucanohydrolase; EC. 3.2.1.1) by a strain of Bacillus stearothermophilus isolated from leaf litter was investigated in a tryptone-maltose medium at 55°C in batch and chemostat culture. Amylase production was growth-limited and restricted to the exponential phase in batch culture. The enzyme yield was reduced by 40% when the culture pH was maintained at pH 7.2. Amylase production in chemostat culture was influenced by the growth rate throughout the dilution rate range used.     

Studies on the extracellular xylanase activity of some thermophilic actinomycetes

Summary An agar plate-clearing assay was used to screen 37 thermophilic actinomycete strains for extracellular xylanase production. The xylanase activity in culture supernatants of strains representing Saccharomonospora viridis and three Thermomonospora spp. was characterised by measurement of reducing sugar released from oat spelt xylan and analysis of degradation products by thin-layer chromatography. In all four species, xylanase activity was optimal within the temperature range 60–75°C and between pH 5 and pH 8. While culture supernatants of Thermomonospora strains incubated at 70°C for 60 min retained >80% of their activity, that of S. viridis was almost, totally inactivated.All of the culture supernatants initially hydrolysed xylan to a mixture of oligomeric products, indicating that the main activity was of the endoxylanase type. Prolonged incubation for 24h resulted in the hydrolysis of xylan to d-xylose by T curvata and T. fusca preparations, indicating the additional presence of exoxylanase or -xylosidase activity. Xylanase production was induced by growth on xylan although low levels of activity were also detected in glucose-grown cultures. Thermomonospora curvata MT815 culture supernatant was the most active and produced d-xylose from milled wheat straw in yields approximately 10% of those from oat spelt xylan.