EXTRACTION OF INTERFERON-γ USING PEG/K_3PO_4 AQUEOUS TWO-PHASE SYSTEM

The aqueous two-phase system of PEG-4000/K3PO4 is selected to extract interferon-γ（γ-IFN）from the broth of homogenized E.coil cells,which were recombined genetically and werefermented in high expressivity.The effects of pH value and the concentrations of PEG and phos-phate on the partition behavious of γ-IFN and contaminating proteins have been investigated.Theresults show that the pH value is the most influential factor effecting the extraction yield of γ-IFN.Under a suitable condition,an effective separation with high yield of γ-IFN can be achieved.

Simultaneous Cell Disruption and Aqueous Two-Phase Extraction for Isolation of Intracellular Recombinant Proteins

A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins from microbial cells. The process was named as simultaneous cell disruption and aqueous two-phase extraction (SDATE). Advantages, such as high cell disruption efficiency, biochemical activities preservation of proteins, cell debris elimination, and prelimiary purification of the target protein were being claimed. When this technique was employed for isolating recombinant Tumor Necrosis Factor (TNF) from E. coli, overall protein concentration and TNF activity were found to have been increased. More than 95% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficient was greater than 3 and the TNF purification factor was greater than 6. It is shown that less separation steps were being utilized in the new technique, meaning a reduction in separation time and less process extractors required.

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system （ATPS） of PEG/phosphate was proposed for selective separation and enrichment of proteins. The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases. Five proteins （bovine serum albumin, Cytochrome C, lysozyme, myoglobin, and trypsin） were used as model proteins to study the effect of phosphate concentration and pH on proteins partition. The PEG/phosphate system was firstly applied to real human saliva and plasma samples, some proteins showed obviously different partition in two phases. The primary results manifest the selective separation and enrichment of proteins in ATPS provided the potential for high abundance proteins depletion in proteomics. ~ 2009 Feng Qu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Extraction of Theanine from Waste Liquid of Tea Polyphenol Production in Aqueous Two-phase Systems with Cationic and Anionic Surfactants

Extraction of theanine from waste liquid of tea polyphenol production was studied in aqueous surfactanttwo-phase system （ASTP） with cationic surfactant （CTAB） and anionic surfactant （SDS）. Results indicate that the region of ASTP is narrow and there is only a two-phase region of cationic surfactant. The increase in concentrations of NaBr and Na2SO4 are beneficial to the formation of ASTP. Theanine concentration in the bottom phase increases with increasing concentration of theanine, whereas the Partition coefficient and extraction rate only change a littlewhen the concentration of theanine is above 0.2 g.L-＇. With the increase of SDS concentration, the phase ratio and the partition coefficient decrease, while the extraction efficiency of theanine increases and the concentration of theanine changes a little in the range from 2.4/7.5 to 2.8/7.2 for SDS/CTAB ratio. The temperature has a notable ef- fect on the concentration of theanine in the bottom phase, partition coefficient and extraction rate of theanine. The increase of waste liquid decreases the phase ratio, increases the concentration and extraction rate of theanine in the bottom ohase, since the orotein and the saccharide enter the bottom nhase with theanine.

Ionic Liquid-salt Aqueous Two-phase System, a Novel System for theExtraction of Abused Drugs

A 1-butyl-3-methylimidazolium chloride-salt aqueous two-phase system was studied on extraction of abused drugs. The effects of sorts of salts, temperature, concentration of salt and drugs on system were investigated systematically. A satisfactory extraction efficiency of 93% was obtained for papaverine while that of morphine was 65%. The extraction mechanism was primarily discussed.

Measurement of Mass Transfer Coefficients and Their Modelling of Continuous Counter-Current Aqueous Two-Phase System in a Packed Extraction Column

Overall dispersed side volumetric mass transfer coefficients for protein and amino acids were measured in continuous countercurrent PEG4000/KHP aqueous two-phase systems in a 57mm I.D. packed extraction column. A model for overall dispersed side volumetric mass transfer coefficients was derived by describing the motion of the drops based upon Navier-Stokes equation combined with the relationship between mass transfer coefficients and the drop velocity. The model provides good predictions and can be successfully used in aqueous two-phase extraction. The average relative deviation between calculated values and experimental data ranges from 8% to 14%.

Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.

Extraction of Tetracycline via Ionic Liquid Two-phase System

A new method to extract and separate tetracycline（TC） in a new aqueous tyro-phase system（ATPS） consisting of hydrophilic [Bmim]BF4 and NaH2PO4 was investigated. The effects of the amount of salt, ionic liquid as well as tetracycline on extraction efficiency were studied systematically. The results show that ionic liquid-ATPS exhibited the highest extraction efficiency higher than 90%. When the concentration of NaH2PO4 was 33.3%--38.5% （mass fraction）, the amounts of ionic liquid and TC were 1.0--2.0 mL and 1.8 mL, respectively, and ionic liquid could be re-used.

The phase behavior of n-ethylpyridinium tetrafluoroborate and sodium-based salts ATPS and its application in 2-chlorophenol extraction

In this paper,the aqueous two-phase systems(ATPS)containing n-ethylpyridinium tetrafluoroborate([EPy]BF_(4)),sodium-based salts,and water were studied and the extraction efficiency of 2-chlorophenol was measured to study the ATPS performance in extracting phenolic compounds.The binodal curves of[EPy]BF_(4)+sodium carbonate(Na_(2)CO_(3))ATPS and[EPy]BF_(4)+sodium dihydrogen phosphate(NaH_(2)PO_(4))ATPS have been determined at 308.15 K,318.15 K,and 328.15 K and atmospheric pressure.After getting good correlation with Merchuk equation,the binodal curves together with gravimetric method were used to calculate the tie-lines data.Furthermore,the reliability of tie-lines data was verified using Othmer-Tobias and Bancroft equations.Then,the salt influence and temperature influence on the phase behavior were discussed and the results show the salt-outing ability of Na_(2)CO_(3) is better than NaH_(2)PO_(4).With the aim of studying the ATPS performance in extracting phenolic compounds,extraction efficiency for 2-chlorophenol at different temperatures were studied and the results show that[EPy]BF_(4)+Na_(2)CO_(3) ATPS is preferred than[EPy]BF_(4)+NaH_(2)PO_(4) ATPS in applications.

Aqueous Two-phase Systems with Ultrasonic Extraction Used for Extracting Phenolic Compounds from Inonotus obliquus

Objective To optimize the extracting technology of assessing the maximum yield of phenolic compounds (PC) from Inonotus obliquus by single factor experiments and orthogonal array design methods through aqueous two-phase systems combined with ultrasonic extraction. Methods The range of the independent variables, namely levels of acetone and ammonium sulfate, and ultrasonic time were identified by a first set of single factor experiments. The actual values of the independent variables coded at four levels and three factors were selected based on the results of the single factor experiments. Subsequently, the levels of acetone and ammonium sulfate, and ultrasonic time were optimized using the orthogonal array method. Results The optimum conditions for the extraction of PC were found to use 7.0 mL acetone, 5.5 mg ammonium sulfate, with ultrasonic time for 5 min. Under these optimized conditions, the experimental maximum yield of PC was 37.8 mg/g, much higher than that of the traditional ultrasonic extraction (UE, 29.0 mg/g). And the PC obtained by this method had stronger anti-oxidative activities than those by traditional UE method. Conclusion These results indicate the suitability of the models developed and the success in optimizing the extraction conditions. This is an economical and efficient method for extracting polyphenols from I. obliquus.

Separation/Extraction/Detection of Chloramphenicol Using Binary Small Molecule Alcohol-Salt Aqueous Two-phase System Coupled with High-performance Liquid Chromatography

A green and sensitive sample separation and purification method coupled with high-perfomiance liquid chromatography(HPLC)was developed for the analysis of chioramphenicol(CAP).One element small molecule alcohol-salt aqueous two-phase system(ATPS)can't effectively adjust the polarity of the system,but binary small molecule alcohol-salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics.A binary aqueous two-phase system based on 1-propanol+2-propanol and NaH2PO4 system was formed and applied to the separation and purification of trace CAP in real samples.The influence factors on partition behaviors of CAP were discussed,including the types and mass of salts,the volume ratio of alcohol,the pH,temperature and the standing time.The optimal condition was found at pH=5.0,2.5 g of NaH2PO4,3.0 mL of 1-propanol and 2-propanol(volume ratio 1:1)and 30℃by using response surface methodology.Under this optimal condition,the extraction efficiency of CAP reached 98.91%,and partition coefficient of CAP was 17.31.

Efficient Purification of Camptothecin via Aqueous Two-phase System Composed of 1,1,3,3-Tetramethylguanidinium Benzoate Ionic Liquid and K2CO3

After being hydrolyzed into corresponding hydrosoluble carboxylate by 1,1,3,3,-tetramethylguanidine （TMG）, camptothecin was extracted via the aqueous two-phase systems composed of ionic liquid 1,1,3,3- tetramethylguanidinium（TMGM） carboxylate and K2CO3, and was regenerated at the final by dilute aqueous hy- drochloric acid. Among the ionic liquids, TMGM benzoate showed the best effect, TMGM formate the worst, this results were consistent with the liposolubilities of different carboxylic acid components in the ionic liquids. Through two-step extraction of TMGM benzoate ionic liquid/K2CO3 aqueous two-phase under a favorable condition, camptothecin was obtained in a purity of 98.3% and a total yield of 85.1%, from the crude camptothecin extract with a purity of 14.2%.

Biological evaluations of decellularized extracellular matrix collagen microparticles prepared based on plant enzymes and aqueous two-phase method

For patients with extensive full-thickness burns who do not have sufficient autologous splitthickness skin for skin grafts,the application of biological skin substitutes may be considered.The aim of this study was to find an optimal new type method for the production of a biovital skin substitute based on acellular dermal matrix(ADM)and preclinical evaluations.In this work,25 methods of ADM production were assessed.The proposed methods are based on the use of the following enzymes:papain,Carica papaya lipase(CPL),and purification using a polymer/salt aqueous two-phase system.The obtained ADM samples were characterized via scanning electron microscopy(SEM),porosity measurement and water vapor transmission test.Results showed that the collagen bundles of ADM microparticles were intact and orderly.Through differential scanning calorimetry(DSC),thermo gravimetric analysis(TGA)and biocompatibility tests,the results indicated that the proportion of papain and CPL was the same and 5 h processing time are the optimum conditions for ADM preparation and the material showed good biocompatibility.Our results suggested that the potential of developing this kind of decellularization process to manufacture ADM scaffolds for clinical application.

Recovery of natural active molecules using aqueous two-phase systems comprising of ionic liquids/deep eutectic solvents

Due to environmental protection requirements,extraction of bioactive compounds from plant materials using environment-friendly green solvents has always been a research hotspot.And great efforts of scholars have been made in this direction,as well as environment-friendly solvents have been used to develop many innovative extraction techniques.Ionic liquids(ILs)and deep eutectic solvents(DESs)are two kinds of typical designable green solvents,which are potential replacements for traditional volatile organic solvents used for extracting.Under the substances action of inorganic salts or polymers,ILs/DESs can form an aqueous two-phase system(ATPS),which has obvious advantages for separating natural products.This paper discussed the phase separation principle of ILs/DESs-based ATPSs and reviewed the applications in the extraction of natural active molecules in recent years,as well as to promote the development of separation of the active constituents in Chinese materia medica.

Study on PEG-(NH_4)_2SO_4 Aqueous Two-Phase System and Distribution Behavior of Drugs

The distribution behavior of chlorpromazine hydrochloride (CPZ), procaine hydrochloride (PCN) and procaine amide hydrochloride (PCNA) in polyethylene glycol (PEG800 or PEG1500)-(NH4)2SO4 aqueous two-phase sys-tems has been investigated. The result shows that the PEG-(NH4)2SO4 aqueous two-phase system has potential ex-traction capability in small molecular drug separation. In PEG800-(NH4)2SO4 system, the extraction efficiencies (E) of CPZ, PCN and PCNA amount to 92.8%, 74.5% and 74.4%, respectively, with the distribution coefficients (KD) being 25.7, 5.9 and 5.8, correspondingly. In PEG1500-(NH4)2SO4 system, the extraction efficiencies (E) of CPZ, PCN and PCNA are 93.7%, 71.3% and 63.2%, respectively, with distribution coefficients (KD) of 39.6, 6.6 and 5.0, correspondingly. Based on the study on ultraviolet and fluorescence spectra and also distribution behavior of the drugs in PEG-(NH4)2SO4 aqueous two-phase system, extraction mechanism was further proposed that both hydro-gen bond and hydrophobic interaction are involved in extraction.

Purification Processes and Market Potential of Bromelain in Brazil

Bromelain is the denomination given to the group of endoproteases obtained from members of Bromeliacea family. These enzymes have a wide range of proven applications and have been an object of study for worldwide researchers for decades. Over the years, several different downstream processes were studied in order to determine which technique would be worthwhile to be implemented in Brazil and provide the national market with such product. The objective of the present study is to relate the main studies in Brazil that has proven that bromelain purification can be cost-effective, in addition to the well-known benefits owned by such enzymes, and highlight the applications that create their market potential in the Brazilian market.