Partitioning of yeast RNA in aqueous two-phase system

Summary The partition behaviour of RNA extracted from yeast was examined using three types of aqueous two-phase systems: potassium phosphate—polyethyleneglycol (PEG) system, ammonium sulphate—PEG system and dextran sulphate—PEG—NaCl system. The molecular weight of PEG showed rather remarkable effects of RNA partition than did the electrolyte. High molecular weight RNA was concentrated at the interface in these systems. This suggests the possibility of its simple concentration or extraction.     

Partitioning of caseinomacropeptide in aqueous two-phase systems

This study evaluates the influence of type of salt and temperature on the partition coefficient of caseinomacropetide (CMP) to determine the best conditions for the recovery of CMP in aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG) 1500 and an inorganic salt (potassium phosphate, sodium citrate, lithium sulfate or sodium sulfate). In all systems, CMP presented affinity for the PEG-rich phase. The PEG1500+lithium sulfate showed the highest values of partitioning coefficient. In addition, thermodynamic parameters (DeltaH degrees , DeltaS degrees , DeltaG degrees) as a function of temperature, were calculated for the system PEG1500-sodium citrate at different PEG concentrations and the results imply thermodynamic differences between partitioning of CMP in this system.     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Novel serine penicillocarboxypeptidase CPD-S3 from Penicillium janthinellum IBT 3991: Purification, characterization, and uses in peptide synthesis and modification

A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of -configuration with basic amino acid residues in either P₁ or P_1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P₁ could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.     

Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.     

Partitioning of protease from stomach of albacore tuna (Thunnus alalunga) by aqueous two-phase systems

Partitioning of protease from stomach of albacore tuna using an aqueous two-phase system (ATPS) was investigated. The best ATPS conditions for protease partitioning from stomach extract (SE) and acidified counterpart (ASE) were 25% PEG1000–20% MgSO₄ and 15% PEG2000–15% MgSO₄, which increased the purity by 7.2-fold and 2.4-fold with the recovered activity of 85.7% and 89.1%, respectively. Electrophoretic study revealed that SE had a major protein with a molecular weight (MW) of 40.6 kDa, while protein with MW of 32.7 kDa was predominant in ASE and ATPS fractions. Pepsinogen in SE might be activated to pepsin by acidification and partitioning process. SE was quite stable at 0 and 4 °C up to 14 days. The loss in protease activity in ASE and selected ATPS fractions was more pronounced when storage time and temperature increased. Therefore, ATPS can be effectively used to recover and purify protease from albacore tuna stomach.     

Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetyl esterase

Trichoderma reesei RUT C-30 acetyl esterase, known to catalyze transacetylation reactions in water/vinyl acetate two-phase mixtures, was studied with respect to regioselectivity of acetylation of oligosaccharides in aqueous environment. Using series of oligosaccharides and their methyl glycosides, it was found that the enzyme catalyzes an efficient acetylation at O-3 position of the non-reducing terminal units of gluco-, xylo- and manno-oligosaccharides and a less efficient acetylation of O-2 position of the reducing end units of gluco- and xylo-oligosaccharides. The axial hydroxyl group at O-2 position of the reducing end mannose in mannooligosaccharides was not recognized by the enzyme and its acetylation was not observed. The structure of isolated transacetylation products was established by NMR, ESI-MS analysis and on the basis on their resistance towards action of glycosidases acting from the non-reducing end of oligosaccharides. The position of acetylation allowed deduce on some of the structural requirements of the enzyme for the acetyl group acceptors. T. reesei RUT C-30 acetyl esterase was also found to be capable of liberation of acetyl groups from terminal units of oligosaccharides, which speaks for its classification as an exo-acting acetyl esterase.     

Separation of presumptive plasma membranes from mitochondria by partition in an aqueous polymer two-phase system

Light-induced absorbance changes (LIAC), indicating the reversible reduction of a b-type cytochrome, and with a possible connection to blue light photomorphogenesis, have been found in a presumptive plasma membrane rich centrifuge fraction from LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results technique membrane particles are separated according to differences in surface properties rather than size and density. LIAC could be separated into two fractions: one partitioning into the polyethylene glycol rich upper phase and another preferring the dextram rich lower phase. Mitochondria (cytochrome c oxidase) were recovered in the lower phase. A dual distribution of LIAC was found with all materials tested: corn coleoptiles, corn shoots, barley shoots and cauliflower inflorescences. About 80–90% of the cytochromes in the upper phase were related to LIAC, whereas only 10–15% of those in the lower phase were of this kind. The LIAC preferring the upper phase was probably bound to the plasma membrane, since plasma membrane vesicles are known to have a high partition in these phase systems. The lower phase LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results within one hour after the initial pelleting) for purification of presumptive plasma membranes, yielding a preparation which contained five times less mitochondrial contamination than the preparation obtained with sucrose gradient centrifugation (the 33/45% w/w sucrose interface fraction).     

Separation of yeast protoplasts from membrane ghosts using an aqueous two-phase system

Protoplasts, prepared from Saccharomyces cerevisiae, were separated from membrane ghosts using an aqueous two-phase system of Dextran T 500, 5% (w/w) and polyethylene glycol (PEG) 8000, 5% (w/w). The protoplast preparation was prewashed in the top phase. After resuspension in fresh top phase an equal volume of bottom phase was added. A protoplast preparation almost free from membrane ghosts was obtained as a precipitate in the bottom phase. The membrane ghosts partitioned to the interphase.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Characterisation of anaerobic mixed cultures by partition in an aqueous two-phase system

A method to characterise a dynamic microbial consortium is described. By exploiting differences in surface properties between different cells and between cells of different physiological status, it was possible to develop a partition pattern for a mixed culture under different conditions. The separation method used was partition in aqueous two-phase systems and when using a counter current extraction process one could clearly differentiate the partition profile between resting, active and overloaded biomethanation cultures.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.     

Production of phosphomonoesterases by Nicotiana tabacum 1507 in an aqueous two-phase system

Studies were conducted on Nicotiana tabacum 1507 cultivation in an aqueous two-phase system (PD(5)) formed by adding 4% PEG (MW 20,000) and 7.5% dextran (MW 70,000) to the medium. The time course of growth and changes in the phase volumes of the PD(5) system, as well as the biosynthesis, secretion, and partitioning of phosphomonoesterases during 11 days of cultivation, were followed. In comparison with N. tabacum 1507 cultivation as a free suspension, on day 8 of cultivation in the PD(5) system the yields of acid and alkaline extracellular phosphomonoesterases were 18 and 10 times higher, respectively. Partitioning took place mainly in the bottom phase with specific activity being 4.5 and 3.5 times higher, respectively. (c) 1996 John Wiley & Sons, Inc.     

两相法分离纯化橡胶树树皮质膜

橡胶树树皮质膜H~+-ATPase在橡胶树产排胶过程中扮演着重要角色,制备高纯度及高活性的质膜是研究质膜H~+-ATPase特性和功能的必要条件。该研究以一年生巴西橡胶树(Hevea brasiliensis)树皮为材料,利用差速离心法获得粗膜微粒体,通过两相分配法分离纯化质膜,并研究两相体系中不同浓度聚合物(5.9%、6.1%、6.3%、6.5%、6.7%,W/W)和KCl(2、5、8、11、14 mmol·L~(-1))对质膜蛋白得率和纯化效率的影响。通过Bradford法对质膜蛋白得率进行检测,同时采用酶活性检测法对质膜纯度进行检测,分析结果表明选用6.4%(W/W)聚合物浓度和5mmol·L~(-1)KCl组成的两相体系可获得较高纯度和得率的橡胶树树皮质膜。通过电镜观察法在形态学上对质膜纯度进一步评价,利用铅铀能侵染全部膜组分使其染色,而磷钨酸只能专一性地侵染质膜并使其染色这一特性,分别使用铅铀和磷钨酸对切片进行染色,并通过透射电镜对切片染色程度进行直接观察,结果表明提取的粗膜微粒体中质膜组分较少,存在大量的细胞器膜污染,而纯化后的质膜膜组分较单一,其他膜组分污染较少,而且质膜大小较均一,可以用于进行后续橡胶树树皮质膜H~+-ATPase特性和功能的研究。     

Production of alkaline protease by Bacillus licheniformis in an aqueous two-phase system

Alkaline protease production by Bacillus licheniformis was studied in an aqueous two-phase system composed of 5% (w/w) polyethylene glycol 6000 (PEG 6000) and 5% (w/w) dextran T500. The top phase was continuous and rich in PEG while the bottom phase was dispersed and rich in dextran. The cells were retained in the bottom phase and at the interface. The two-phase system produced less enzyme in total amount than the control in the early phase, but after 50 h the enzyme produced in the control system decreased while the aqueous two-phase system continued its production and finally the total enzyme activity reached 1.3 times that of the control culture. In order to improve the productivity of protease, repeated batch cultivation were successfully carried out four times by optimizing the top phas composition of freshly added media, which resulted in 13.8, 35.9, 27.8 and 34.7 units ml⁻¹ h⁻¹ of protease based on the amounts of replaced top phases, respectively.     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-based aqueous two-phase systems

Alcohol/salt-based aqueous two-phase systems (ATPSs) were used to recover lipase derived from Burkholderia pseudomallei (B. pseudomallei). Nine biphasic systems, comprised of an alcohol-based top phase (ethanol, 2-propanol and 1-propanol) and a salt-based bottom phase (ammonium sulfate, potassium phosphate and sodium citrate), were evaluated for their effectiveness in lipase recovery. The stability of lipase in each of the solutions was tested, and phase diagrams were constructed for each system. The optimum partition efficiency for the purification of lipase was obtained in an ATPS of 16% (w/w) 2-propanol and 16% (w/w) phosphate in the presence of 4.5% (w/v) NaCl. The purified lipase had a purification factor of 13.5 and a yield of 99%.     

Partitioning of host and recombinant cells in aqueous two-phase polymer systems

The separation of host and recombinant Escherichia coli bacterial cells has been studied using the surface-sensitive technique of partitioning in aqueous two-phase polymer systems. Experiments were designed to probe charge-and hydrophobicity-related property differences of antibiotic-resistant recombinant cells and their antibiotic-sensitive hosts. Differential partitioning was observed in both charge-sensitive and non-charge-sensitive phase systems for three host-recombinant cell systems, but the non-charge-related effects appear to have a greater impact on partitioning behavior. This result suggests that plasmid-encoded products related to antibiotic resistance modify the surface hydrophobicity of the E. coli bacterial cell and that these differences can be exploited for cell separation.     

Chromatographic resolution of nucleic acids extracted from Penicillium chrysogenum

Summary High molecular weight DNA extracted from Penicillium chrysogenum has been fractionated using RPC-5 Analog, into three distinct types designated 1, 2 and 3. Types 1 and 2 have the same buoyant density of 1.710 g/cm³ and together appear to comprise the nuclear DNA. Type 1 is enriched for repeated sequences which are normally observed in restriction digests of P. chrysogenum total DNA. Conversely, type 2 appears to be composed entirely of non-repetitive sequences. Type 3 has been identified as mitochondrial DNA, having a buoyant density of 1.695 g/cm³ and an estimated molecular weight of 31.6×10⁶ Daltons.