Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350–sodium citrate aqueous two-phase systems (ATPS) was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, sodium citrate and NaCl concentrations of 15.1, 11.0 and 8.52% respectively. However, under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.     

The Aqueous Two-Phase System Polyethylene Glycol/Dextran as a Reaction Medium for the Microsomal Monooxygenase System

An aqueous two-phase system of dextran and polyethylene glycol was investigated as a reaction medium for pig liver microsomes in order to find out if the partition of the microsomes, of the substrate 7-ethoxycoumarin and of the product 7-hydroxycoumarin favoured any biotechnological perspectives. Cytochrome-P-450 and NADPH-cytochrome P-450 reductase concentrations and the monooxygenase 7-ethoxycoumarin deethylation activity were measured under a variety of the system parameters. Microsomes totally partition into the bottom phase whereas the concentration ratio of substrate to product is higher in the microsome free top phase. An unfavourable effect is the specific partial deactivation of the cytochrome P-450 by polyethylene glycol.     

用蛋白质印迹转移技术分析正常及硒性白内障大鼠晶状体及房水中蛋白质的性质

本文用蛋白质印迹转移技术分析了正常及硒性白内障大鼠晶状体及房水中蛋白质的性质。结果表明,晶状体中的脲溶性蛋白质可被抗α及抗γ晶体蛋白血清识别,提示α及γ晶体蛋白均为脲溶性蛋白质的主要成份。患白内障时房水中的蛋白质含量明显增加,且主要被抗γ血清识别,而被抗α血清识别的成份很少,表明在大鼠硒性白内障形成过程中,有较多低分子量蛋白质漏出到房水中,且其主要成份为γ晶体蛋白。此外,我们还发现正常及硒性白内障大鼠晶状体膜蛋白质与抗α及抗γ血清起反应的程度及分布有所不同,提示晶状体细胞膜与晶体蛋白之间存在着相互作用。     

PEG／（NH4）2SO4双水相体系在加杨叶总黄酮萃取分离中的应用

目的：寻找加杨叶粗提液中的总黄酮的有效方法。方法：利用双水相体系萃取分离、紫外分光光度法直接测定。结果：萃取分离加杨叶总黄酮的最佳双水相体系是25％PEG400与12％（NH4）2SO4,最佳萃取条件为：pH=9,NaCl的添加量为3％,粗提液3mL,温度25℃。结论：该方法的相对标准偏差（RSD）≤0．28％（n=5）,具有良好的精密度和选择性,为黄酮类化合物萃取分离的一种有效方法。     

Speciation in aqueous vanadate-ligand and peroxovanadate-ligand systems

In the present focused review, the speciation studies of aqueous vanadate-ligand (L) and peroxovanadate-L systems are addressed. The paper focuses solely on the systems studied at our department in the context of potential insulin-enhancing effects, including the following ligands: imidazole, alanylhistidine, alanylserine, lactate, picolinate, citrate, phosphate, maltol, and uridine. We summarise the results of detailed and thorough potentiometric (glass electrode) and ⁵¹V NMR (Bruker AMX-500 MHz) spectroscopic studies, performed at 25 °C in 0.150 M Na(Cl), a medium representing human blood. The importance of experimental conditions is discussed and illustrated. A detailed overview of our methodology, based on combining potentiometric and ⁵¹V integral and chemical shift data by means of the computer program LAKE, is also given. We list the important steps of equilibrium analysis and the kinds of information available from different sets of NMR spectra. The ligand picolinate is chosen to exemplify our working method, but conclusions are drawn from all systems, reviewing trends and common features. An overview of all systems is given in two tables, including e.g. types and number of species formed. Previously unpublished modelling results at physiological conditions are also shown for all peroxovanadate-ligand systems.     

Maximizing EPS production from Pseudomonas aeruginosa and its application in Cr and Ni sequestration

Heavy metal contamination of water bodies has been a cause of grave concern around the globe. Analysis of various industrial effluents has revealed a perilous level of Cr (VI) and Ni (II). Pseudomonas aeruginosa is an extracellular polymeric substances (EPSs) producing bacterium. EPS has a great potential in the sequestration of heavy metal ions. In the present study efforts have been made to understand the effect of time, pH, and temperature on production of EPS by P. aeruginosa (MTCC 1688). The extracted EPS has been applied for removal of Ni (II) and Cr (VI) ions from aqueous system. The results revealed that highest EPS yield (26 mg/50 mL) can be obtained after 96 h of incubation at pH 6 and 32 °C temperature in 50 mL of culture. Treatment of 10 mg/L Cr (VI) and Ni (II) with 30 mg/L EPS resulted in the removal of 26% and 9% of Cr (VI) and Ni (II), respectively. Fourier-transform infrared spectral analysis revealed the involvement of –OH, –NH, C–O, diketone, and ester functional groups of EPS in the attachment of Cr (VI) ion while involvement of amide and –CO groups in Ni (II) binding with EPS. Scaling-up the production of EPS using bioreactor may further help in developing an efficient process for treatment of water polluted with Cr and Ni.     

Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase

Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated P_ADB3.8) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB). In the systems, cellulase was immobilized on pH-response copolymer P_MDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH₄)₂SO₄. Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS.     

Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds

Conventional treatments for breast cancer are costly and have serious side effects. Non-conventional natural treatments have gained wide acceptance due to their promise of a cure with minimal or no side effects, but little scientific evidence exists. One such common remedy is the seed of the Lepidium sativum plant. Presented here is the first reported use of the aqueous extract of Lepidium sativum seeds on breast cancer cells. The ability of the extract to induce apoptosis and necrosis in the human breast cancer cell line MCF-7, compared to normal human skin fibroblasts (HFS), was determined by morphological changes in the cells using light microscopy, DNA fragmentation assay, and florescent stains (Annexin V and propidium iodide) using flow cytometry and fluorescent microscopy. Apoptosis was induced in both cells, and more in MCF-7, when they were treated with 25% and 50% extract, while necrosis was observed mainly after exposure to elevated extract concentrations (75%). DNA fragmentation resulted for both cells, in a time and dose-dependent manner. Both cells, at all extract concentrations, showed no significant differences in the number of living, dead, apoptotic, and necrotic cells. Finally, the results may indicate that apoptotic changes in MCF-7 may be independent of caspase-3, which is involved in apoptosis and is lacking in MCF-7 cells.     

Composition of the aqueous phase of chromaffin granules

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 ± 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine: ATP molar ratio (4.41 ± 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine: protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.     

Separation of bovine X and Y sperm based on surface differences

Aqueous two-phase partition involving thin-layer counter current distribution (TLCCD) has been used to assess surface heterogeneity of ejaculated bovine sperm. When partitioned in charge-insensitive aqueous two-phase systems, which detect non-charge associated surface properties, the sperm fractionates into two distinct populations. Using a Y-chromosome-specific DNA marker, it has been shown that one of these populations is enriched in Y chromosome bearing sperm. However, this population is not pure—it consists of 80% Y sperm, with the other 20% being X sperm. All the sperm in the original population that had begun to undergo the acrosome reaction were separated into this same peak; the sex chromosome composition of these sperm is unknown. Since the aqueous partition of sperm is based on surface properties these results suggest that two populations of Y sperm exist that have different surface characteristics. © 1993 Wiley-Liss, Inc.