Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.     

Accelerated Germination of Maize Seeds Under Chilling Stress by Osmotic Priming and Associated Changes in Embryo Phospholipids

Osmotic priming of maize seeds (Zea mays L. cv. Partap) usingpolyethylene glycol or potassium salts (K₂HPO₄, KH₂PO₄, KNO₃and K₂HPO₄+KNO₃) resulted in accelerated germination at a chillingtemperature (10 °C). The response of seeds primed in solutionsof either 2.5% K₂HPO₄ or 2.5% K₂HPO₄+ KNO₃ was particularlymarked compared with the untreated seeds, and the effect ofpriming was largely retained after seeds had been dried back.All embryo phospholipid fractions and sterols increased duringsalt-priming and the proportion of phospholipid which was diphosphotidylglycerol(DPG) also increased. It is suggested that the marked increasein the DPG content of primed embryos may be due to enhancedinternal organization of their mitochondrial membranes, andthat the benefit of osmotic priming may be at least partly dueto an increased potential for ATP accumulation. Germination, Zea mays L., osmotic priming, phospholipid changes     

Downstream processing of luciferase from fireflies (Photinus pyralis) using aqueous two-phase extraction

The firefly luciferase has been extensively used for sensitive detection of bacteria, gene expression and environmental toxins (biosensors). The aim of the present study was to design a simple and more efficient method for the purification and concentration of luciferase using aqueous two-phase extraction (ATPE). Downstream processing of luciferase from North American Firefly Photinus pyralis was carried out, for the first time, using polymer/salt aqueous two phase system (ATPS) at 4 °C. The enzyme was observed to preferentially partition to the polyethylene glycol (PEG) rich top phase. The best results of purification (13.69 fold) and enzyme activity recovery (118.34%) were observed in the system containing 4.0% (w/w) PEG (1500) and 20.5% (w/w) (NH₄)₂SO₄ with a phase volume ratio of 0.21.     

Influence of milk whey, nitrogen and phosphorus concentration on oxalic acid production by Aspergillus niger

A factorial design at two levels was used to determine the effect of milk whey concentration and the addition of nitrogen (as NH₄NO₃) and phosphorus (as KH₂PO₄) on the oxalic acid production by Aspergillus niger. The results of the experiments indicated that milk whey contains enough nutrients for fungus growth, therefore medium supplementing with N and P is not necessary. The optimum milk whey concentration was 100 kg/m³ reaching a final oxalic acid concentration of 37 kg/m³ and a maximum production rate of 3.4 kg/m³ · d. The yield of oxalic acid was 0.4, a very high value compared to previous works.     

Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.     

Partitioning of xylanolitic complex from Penicillium janthinellum by an aqueous two-phase system

This work evaluates the influences of five parameters (pH, PEG molecular mass, PEG concentration, concentration of buffer K₂HPO₄–KH₂PO₄ and NaCl concentration) on xylanolitic complex partitioning produced by P. janthinellum in aqueous two-phase systems, using a 2⁵ factorial experimental design. A mathematical model to quantify the influence of these parameters was attained and statistically tested. The optimum point for total protein extraction was obtained under the following conditions: pH 7.0, PEG 10 000, 3.67% PEG, 10% potassium phosphate and 12.4% NaCl. The partition coefficient (K) value experimentally obtained was 5.25 and that predicted by the model was 5.89.     

产脂肪酶菌株筛选及柠檬酸杆菌产酶条件优化

脂肪酶可以催化甘油三酯水解成脂肪酸和甘油,已广泛应用在工业领域,而获得产酶微生物是研究的基础。采用油脂平板法筛选出1株脂肪酶产生菌。经16S rRNA序列分析可知,该菌株属于柠檬酸杆菌(Citrobacter werkman and Gillen)。单因素试验对其进行产酶条件优化,优化后产酶条件（g/L）：淀粉2.0,KH₂PO₄ 1.0,K₂HPO₄·3H₂O 2.2,(NH₄)₂SO₄ 1.0,MgSO₄·7H₂O 0.1,牛肉膏2.0,橄榄油10.0 mL,pH 7.5,接种量1.5%(v/v),37 ℃培养43 h。获得最大酶活为384 U/mL,是优化前的13倍。可以利用该菌制备脂肪酶。     

Effects of Na2SO4, K2SO4 and KCl on Growth and Ion Uptake of Callus Cultures of Vaccinium corymbosum L. cv. Blue Crop

Non-selected and Na₂SO-, K₂SO₄- or KCl-selected callus culturesof Vaccinium corymbosum L. cv. Blue Crop were grown on mediasupplemented with 0, 25 and 50 mM Na₂SO₄ (non-selected and Na₂SO(-selectedonly), 0, 25 and 50mMK₂SO₄ (non-selected and K₂SO₄-selectedonly) or 0, 50 and 100 mM KCl (non-selected and KCl-selectedonly). On all media, growth of selected callus (on a fresh-weightor dry-weight basis) was greater than that of non-selected callus,and selected callus grew optimally on the level and type ofsalt on which it was selected. Selected callus was friable andmaintained a higher f. wt:d. wt ratio. Tissue water potentialin selected callus was more negative than in non-selected callus. Flame photometry and chloridometry showed Na⁺, K⁺ and Cl^–accumulated in callus to concentrations equal to or greaterthan the initial concentration in the medium. Turbidometry showedthat tissue SO₄^2- concentration was lower than the concentrationin the medium. In most cases selected callus accumulated moreNa⁺, K^sup, SO₄^2– or Cl^– than non-selected callus.Vacuolar ion concentration was measured by electronprobe X-raymicroanalysis, and on most media selected callus had highervacuolar ion concentrations than non-selected callus. SO₄^2–and Cl^– were accumulated in the vacuoles at concentrationshigher than the external medium, but vacuolar Na⁺ concentrationdid not reach external concentration on Na₂SO₄ and on potassiumsalts was maintained between 12 and 17 mM. Vacuolar K⁺ concentration(approx. 142–191 mM on no salt) decreased on Na₂SO₄ andincreased on K₂SO₄ and KCl. There was no precise correlation between total or specific ionaccumulation (Na⁺, K⁺, SO₄^2– and Cl^– and fresh-weightyield. Results suggest that selection results in adaptationin response to decreased water potential of the medium. Vaccinium corymbosum, blueberry, electronprobe X-ray microanalysis, callus, in vitro selection, salt tolerance, KCl, K₂SO₄, Na₂SO₄     

Recovery of antibiotics by aqueous two-phase partition —Partitioning behavior of pure acetylspiramycin solution in polyethylene glycol/potassium phosphate aqueous two-phase systems

Summary The effects of average molecular weight of PEG, concentrations of PEG and KH₂PO₄ and pH on the partition equilibrium of acetylspiramycin in PEG/KH₂PO₄ aqueous two-phase systems were studied in detail. The partition coefficients of acetylspiramycin in PEG/ KH₂PO₄ systems were measured at room temperature 25 °C. It was found that acetylspiramycin partitioned unevenly in the aqueous two-phase systems composed of PEG and KH₂PO₄ and could be purified by this technique. A suitable phase-forming system (pH=6.7, 12w/w% PEG2000, 11w/w% KH₂PO₄) was found out after partition coefficient (Kp=42) , extraction ratio (=96%) and recovery ratio(R=98.8%) were investigated comprehensively in this paper.Hua qiang is one of the cooperators of the experimetal.     

The Growth and Intracellular Ionic Composition of Dunaliella tertiolecta in Magnesium-Rich Media

Dunaliella tertiolecta grew in a medium that contained MgSO₄(MgSO₄ medium), while this alga did not grow at all in mediathat contained MgCl₂ or Mg(NO₃)₂. The growth in the MgSO₄mediumwas inhibited in comparison with that in media that containedsodium salts, such as NaCl, NaNO₃, and Na₂SO₄. The energy chargeobtained from measurements of levels of adenine nucleotidesby HPLC were almost constant in Na- and Mg-containing media(about 0.87), indicating that the failure of growth in MgCl₂medium and Mg(NO₃)₂ medium was not directly related to.changesin the energy metabolism. K⁺ and Mg²⁺ were the dominant intracellularcations not only in Na-containing media (Na-media) but alsoin Mg-cohtaining media (Mg-media). The intracellular concentrationof Ca²⁺ was lower in Mg-media (1.6 mM) than that in Na-media(6mM). The concentrations of HPO₄^2– in cells incubatedin Mg-media were lower (less than 60 mM) than those in Na-media(greater than 110 mM). By contrast, the intracellular concentrationof SO₄^2– was higher in a MgSO₄ medium (26 mM) than thatin a Na₂SO₄ medium (4 mM) which, at least, compensated by 40%for the decrease in HPO₄^2–. The ability to grow in a MgSO₄medium may be related to the high intracellular concentrationof SO₄^2–. (Received September 20, 1990; Accepted March 22, 1991)     

Sequential liquid-liquid extraction of d-amino acid oxidase from Trigonopsis variabilis

A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l^–1), potassium phosphate (110 g l^–1, K₂HPO₄ + KH₂PO₄ = 10.1:1, mol mol^–1) and dl-methionine (11 g l^–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein^–1.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Solubility of phosphorus added to four Manitoba soils with different calcium and magnesium contents

Summary The solubility of phosphorus was found to approximate that of dicalcium phosphate dihydrate and/or dimagnesium phosphate trihydrate when KH₂-PO₄, H₃PO₄ and K₂HPO₄ were added to four Manitoba soils. Eighty to one hundred, seventy to ninety and sixty to eighty per cent of the phosphorus added remained in solution when H₃PO₄, KH₂PO₄ and K₂HPO₄ were added, respectively. The solubility of the added phosphorus was high in all samples and relatively soluble compounds, dicalcium phosphate dihydrate and dimagnesium phosphate trihydrate, were most likely formed in the samples indicating that phosphorus added to these soils would be readily available to plants. Associate Professor and Professor respectively.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe²⁺, BO₄^3-, Ca²⁺, Cu²⁺ and Zn²⁺, very low in PO₄^3- and lacking MoO₄^2-, NH₄⁺ and Co²⁺), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.     

Statistical optimization of medium components for improved synthesis of riboflavin by Eremothecium ashbyii

Statistical designs were used to determine optimum levels of medium nutrients for riboflavin production in shake flask fermentation. The medium components considered include molasses, sesame seed cake (SSC), yeast extract, KH₂PO₄, MgSO₄·7H₂O, NaCl and Tween-80. Information about the effects of different medium components on riboflavin production were investigated by using the Plackett-Burman experimental design. MgSO₄·7H₂O and NaCl were found to significantly influence the riboflavin production (confidence levels above 70%). For obtaining the mutual interaction between the variables required, for achieving the optimal concentrations, a 2²-factorial central composite design was employed. The optimal concentrations for the enhanced production of riboflavin were (g/l): molasses =50.0 (glucose equivalents); SSC =50.0; yeast extract =2.0; KH₂PO₄=2.0; MgSO₄·7H₂O=0.117 and NaCl =1.13.     

Optimization of chitinase production by a new <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> C9 isolate using response surface methodology

The purpose of this article is to use statistical Plackett–Burman and Box–Wilson response surface methodology to optimize the medium components and, thus, improve chitinase production by Streptomyces griseorubens C9. This strain was previously isolated and identified from a semi-arid soil of Laghouat region (Algeria). First, syrup of date, colloidal chitin, yeast extract and K₂HPO₄, KH₂PO₄ were proved to have significant effects on chitinase activity using the Plackett–Burman design. Then, an optimal medium was obtained by a Box–Wilson factorial design of response surface methodology in liquid culture. Maximum chitinase production was predicted in medium containing 2% colloidal chitin, 0.47% syrup of date, 0.25 g/l yeast extract and 1.81 g/l K₂HPO₄, KH₂PO₄ using response surface plots of the STATISTICA software v.12.0.     

Influence of micronutrients on yeast growth and β-<Emphasis Type="SmallCaps">d</Emphasis>-fructofuranosidase production

β-d-fructofuranosidase is one of the most important enzymes of the food industry especially due to its application in preparation of soft centered candies, confectioneries and to produce fructose syrups. Although several bacteria and filamentous fungi are reported for its production, yeasts are the most preferred source for this enzyme. In the present study mineral nutrients were screened for their effect on yeast growth and the enzyme production using Plackett-Burman design. Of the 11 nutrients screened, three variables (KH₂PO₄, FeCl₃ and CoCl₂) were significantly effecting yeast growth while six variables, i.e. KH₂PO₄, CaCl₂, MnSO₄, Na₂MoO₄, ZnSO₄ and CoCl₂ had significant effect on enzyme production.     

Modeling some mineral nutrient requirements for micropropagated wild apricot shoot cultures

A response surface methodology (RSM) experimental design was applied for improving micropropagation of a wild apricot, Prunus armeniaca Lam., from the mountains of Kazakhstan. In an initial study, woody plant medium (WPM) mineral nutrients [calcium nitrate, ammonium nitrate, mesos (calcium chloride, potassium phosphate and magnesium sulfate) potassium sulfate and minor nutrients] were tested in a response surface methodology (RSM) experiment. Shoot quality was the best when nitrogen and mesos (CaCl₂, MgSO₄, K₂SO₄, KH₂PO₄) compounds were altered. In this study an expanded mesos optimization experiment was run. Data taken included a subjective quality rating, shoot length, shoot number, leaf color and size, callus and physiological disorders. Data were analyzed by Classification and Regression Tree Analysis (CART), a data mining technique that provides specific cutoff values for data and easy to interpret data trees. The CART analysis indicated that the best quality would be with ≤2.4× WPM levels of KH₂PO₄ and ≤0.75× MgSO₄. Shoot length was affected by K₂SO₄, but most shoots were of good size at any concentration. Shoot multiplication was affected by KH₂PO₄, but there were >5 shoots at any concentration. Leaf color was best with ≤2.41× KH₂PO₄ and ≤1.22× K₂SO₄. Based on the CART analysis, a recommendation for improved mesos compounds was developed. Each of the individual trees was analyzed and the cutoff points determined so that all the growth characteristics could be considered in the final concentrations chosen. Using the combined results from the CART analysis, the suggested medium would include WPM with CaCl₂ 2.7×, MgSO₄ 2.7×, K₂SO₄ 0.8×, KH₂PO₄ 0.75×.