The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine m...The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine milk exosomes.The milk exosome partition behaviors and bovine milk separation were investigated,and the ATPSs and bovine milk whey addition was optimized.The optimal separation conditions were identified as 16%(mass)polyethylene glycol 4000,10%(mass)dipotassium phosphate,and 1%(mass)enzymatic hydrolysis bovine milk whey.During the separation process,bovine milk exosomes were predominantly enriched in the interphase,while protein impurities were primarily found in the bottom phase.The process yielded bovine milk exosomes of 2.0×10^(11)particles per ml whey with high purity(staining rate>90%,7.01×10^(10)particles per mg protein)and high uniformity(polydispersity index<0.03).The isolated exosomes were characterized and identified by transmission electron microscopy,zeta potential and size distribution.The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey,presenting a novel approach for the large-scale isolation of exosomes.展开更多
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 ...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.展开更多
This paper presents the evaluation of an aqueous two-phase system （ATPS） for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol （PEG）/salt systems...This paper presents the evaluation of an aqueous two-phase system （ATPS） for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol （PEG）/salt systems was investigated. The suitable system for elastase extraction was PEG/KHEPO4-KEHPO4, in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters （e.g. PEG molecular mass, pH, NaCl addition） on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KHEPO4-KEHPO4, was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% （w/w） PEG 2000 and 11.7% （w/w） KHEPO4-KEHPO4. The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.展开更多
The isothermal solubility data of aqueous two-phase system ethanol+water+K 2HPO 4 were determined with the turbidity titration method at 303.2 K. The binodal curves were described by using the Mistry equation very w...The isothermal solubility data of aqueous two-phase system ethanol+water+K 2HPO 4 were determined with the turbidity titration method at 303.2 K. The binodal curves were described by using the Mistry equation very well. An experimental procedure for measuring the liquid-liquid equilibrium data of the aqueous two-phase system was proposed, in which the concentrations of the coexisting phases were determined with the corresponding densities of the solution. The tie lines were satisfactorily described by using the Othmer-Tobias and Bancroft equations.展开更多
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 s...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.展开更多
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 mode...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%.展开更多
The partition coefficients of baicalin were measured in ethylene oxide and propylene oxide (EOPO)/salt aqueous two-phase systems at 298.15K. It was found that most of baicalin partitioned into EOPO-rich phase. The par...The partition coefficients of baicalin were measured in ethylene oxide and propylene oxide (EOPO)/salt aqueous two-phase systems at 298.15K. It was found that most of baicalin partitioned into EOPO-rich phase. The partition coefficients of baicalin varied from 10 to 120. The effect of various factors, including tie-line length, salt composition, molecular weight of EOPO, and solution pH, on the partition behavior was investigated in EOPO/salt systems. Furthermore the partition coefficients of baicalin were correlated using the modified Diamond-Hsu model. Good agreement with experimental data is obtained. The average relative deviations are less than 5.0%.展开更多
The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene...The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.展开更多
A fast and convenient C―S bond formation reaction was achieved catalyzed by CuI/L-proline in aqueous two-phase system providing a simple method for synthesis of aryl sulfides in good yields in short time.
Background:D-Galactose dehydrogenase(GalDH;EC 1.1.1.48)belongs to the family of oxidoreductases that catalyzes the reaction ofβ-D-galactopyranose in the presence of NAD+to D-galacto-1,5-lactone and NADH.The enzyme ha...Background:D-Galactose dehydrogenase(GalDH;EC 1.1.1.48)belongs to the family of oxidoreductases that catalyzes the reaction ofβ-D-galactopyranose in the presence of NAD+to D-galacto-1,5-lactone and NADH.The enzyme has been used in diagnostic kits to neonatal screen for galactosemia diseases.This article reports the partitioning optimization of recombinant Pseudomonas fluorescens GalDH in aqueous two-phase systems(ATPS).Methods:Preliminary two-phase experiments exhibited that the polyethylene glycol(PEG)concentration,pH value,and concentration of salt had a significant influence on the partitioning efficiency of recombinant enzyme.According to these data,response surface methodology(RSM)with a central composite rotatable design(CCRD)was performed to condition optimization.Results:The optimal partition conditions were found using the 14.33%PEG-4000 and 11.79%ammonium sulfate with pH 7.48 at 25℃.Yield,purity,recovery,and specific activity were achieved 92.8%,58.9,268.75%,and 373.9 U/mg,respectively.PEG and ammonium sulfate concentration as well as pH indicated to have a significant effect on GalDH partitioning.Enzyme activity assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis demonstrated the suitability of predicted optimal ATPS as well.The Km and molecular weight values for the purified GalDH were 0.32 mM and 34 kDa,respectively.Conclusions:Ultimately,our data showed the feasibility of using ATPS for partitioning and recovery of recombinant GalDH enzyme.展开更多
One of the bottlenecks for bioproduction of butyric acid as bulk chemical is the difficulty in separating butyric acid from the fermentation broth,compared with the petroleum-based chemical synthesis method.In the pre...One of the bottlenecks for bioproduction of butyric acid as bulk chemical is the difficulty in separating butyric acid from the fermentation broth,compared with the petroleum-based chemical synthesis method.In the present work,a novel separation methodology was developed based on an aqueous two-phase system with inor-ganic salts.Calcium chloride was screened out for effective separation of butyric acid from butyric acid-water-salt systems.Within appropriate concentration range of butyric acid and salt,butyric acid was enriched in the upper phase and most of calcium ions remained in the lower phase.This"salting out"effect is very efficient to separate butyric acid from the simulated butyrate fermentation broth,which consists of butyric acid and acetic acid with concentration ratio of 4︰1,so that the final ratio of butyric acid/acetic acid in the upper phase is improved to 9.87. The aqueous two-phase system was used to separate butyric acid from the actual fermentation broth with satisfac-tory result.展开更多
The phenomenon of two dilute aqueous phases composed of sodium dodecylsulfate(SDS) and cetyl trimethyl ammonium bromide(CTAB) was investigated under various conditions such as concentrations and molar ratios of the tw...The phenomenon of two dilute aqueous phases composed of sodium dodecylsulfate(SDS) and cetyl trimethyl ammonium bromide(CTAB) was investigated under various conditions such as concentrations and molar ratios of the two surfactants, the addition of sodium chloride and temperature. Vesicles formation was found in the both phases by TEM image.展开更多
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 progr...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.展开更多
The aqueous two-phase system of PEG-4000/K<sub>3</sub>PO<sub>4</sub> is selected to extract interferon-γ（γ-IFN）from the broth of homogenized E.coil cells,which were recombined genetically a...The aqueous two-phase system of PEG-4000/K<sub>3</sub>PO<sub>4</sub> 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.展开更多
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 w...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.展开更多
In the investigation of effect of KSCN on the partitioning of lysozyme in PEG2000/ammonium sulfate aqueous two-phase system, it was found that the KSCN could alter the pH difference between the two phases. and thus af...In the investigation of effect of KSCN on the partitioning of lysozyme in PEG2000/ammonium sulfate aqueous two-phase system, it was found that the KSCN could alter the pH difference between the two phases. and thus affect the partition of lysozyme. The relationship between partition coefficients of lysozyme and pH differences between two phases was discussed.展开更多
A series of ethylene oxide (EO)-propylene oxide (PO) randomco-polymers (EOPO) were used to form aqueous two-phase systems (ATPS)with ammonium sulfate. Effects of EOPO's properties on the phaseseparation behaviors ...A series of ethylene oxide (EO)-propylene oxide (PO) randomco-polymers (EOPO) were used to form aqueous two-phase systems (ATPS)with ammonium sulfate. Effects of EOPO's properties on the phaseseparation behaviors and on the partition of cephalexin and7-aminodesacetoxicephalosporanic acid (7-ADCA) in ATPS wereinvestigated. Both the molar mass and molar ratio of EO to PO of EOPOcould greatly influence partition behaviors of cephalexin and 7-ADCAas well as the binodal curve of ATPS.展开更多
A novel method for the separation/analysis rhodamine B has been described. The ionic liquid (1-tetradecyl-3-methylimidazolium bromide)/anion surfactant (sodium dodecyl sulfate)/NaCl two-phase systems (ATPSs) is presen...A novel method for the separation/analysis rhodamine B has been described. The ionic liquid (1-tetradecyl-3-methylimidazolium bromide)/anion surfactant (sodium dodecyl sulfate)/NaCl two-phase systems (ATPSs) is presented as a simple, rapid and effective sample pretreatment technique coupled with ultraviolet spectrometry for analysis rhodamine B in soft drink. The effects of parameters on the ATPSs extraction of rhodamine B such as amount of surfactant, ionic liquid and salt, pH, temperature, stabilization and centrifugal time have been studied in details. Under the optimized conditions, the linear range of calibration curve for rhodamine B was 0.05 - 7.0 μg·mL-1 and the detection limit was 3.2 ng·mL-1. The phase equilibrium and the mechanism of phase separation for ATPSs have been discussed. This method has been applied to the determination of rhodamine B in soft drink.展开更多
Temperature sensitivity of waxy crude oils makes it difficult to study their flow behaviour in the presence of water especially near their wax appearance temperature （WAT）. In this study a method was proposed and im...Temperature sensitivity of waxy crude oils makes it difficult to study their flow behaviour in the presence of water especially near their wax appearance temperature （WAT）. In this study a method was proposed and implemented to mitigate such difficulties which was applied in predicting mixture temperatures （Tin） of a typical Malaysian waxy crude oil and water flow in a horizontal pipe. To this end, two analytical models were derived firstly from calorimetry equation which based on developed two correlations for defining crude oil heat capacity actualized from the existed specific heat capacities of crude oils. The models were then applied for a set of experiments to reach the defined three predetermined Tm （26 ℃, 28 ℃ and 30 ℃）. The comparison between the predicted mixture temperatures （Tin,1 and Tin,2） from the two models and the experimental results displayed acceptable absolute average errors （0.80% 0.62%, 0.53% for model 1; 0,74%, 0.54%, 0.52% for model 2）. Moreover, the average errors for both models are in the range of standard error limits （4-0,75Z） according to ASTM E230. Conclusively, the proposed model showed the ease of obtaining mixture temperatures close to WAT as predetermined with accuracy of ± 0.5 ℃approximately for over 84% of the examined cases. The method is seen as a practical reference point to further study the flow behaviour of waxy crudes in oil-water two-phase flow system near sensitive temperatures.展开更多
基金supported by the National Natural Science Foundation of China(22378350).
文摘The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine milk exosomes.The milk exosome partition behaviors and bovine milk separation were investigated,and the ATPSs and bovine milk whey addition was optimized.The optimal separation conditions were identified as 16%(mass)polyethylene glycol 4000,10%(mass)dipotassium phosphate,and 1%(mass)enzymatic hydrolysis bovine milk whey.During the separation process,bovine milk exosomes were predominantly enriched in the interphase,while protein impurities were primarily found in the bottom phase.The process yielded bovine milk exosomes of 2.0×10^(11)particles per ml whey with high purity(staining rate>90%,7.01×10^(10)particles per mg protein)and high uniformity(polydispersity index<0.03).The isolated exosomes were characterized and identified by transmission electron microscopy,zeta potential and size distribution.The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey,presenting a novel approach for the large-scale isolation of exosomes.
基金Supported by the Fundamental Research Funds for the Central Universities(JUSRP11205)
文摘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.
基金Project (No. 20276064) supported by the National Natural ScienceFoundation of China
文摘This paper presents the evaluation of an aqueous two-phase system （ATPS） for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol （PEG）/salt systems was investigated. The suitable system for elastase extraction was PEG/KHEPO4-KEHPO4, in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters （e.g. PEG molecular mass, pH, NaCl addition） on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KHEPO4-KEHPO4, was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% （w/w） PEG 2000 and 11.7% （w/w） KHEPO4-KEHPO4. The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.
基金Supported by Naturd Science Foundation of Fujian Province(No. E0 2 10 0 2 2 ),Japan Science and Technology Corpora-tion(JST)
文摘The isothermal solubility data of aqueous two-phase system ethanol+water+K 2HPO 4 were determined with the turbidity titration method at 303.2 K. The binodal curves were described by using the Mistry equation very well. An experimental procedure for measuring the liquid-liquid equilibrium data of the aqueous two-phase system was proposed, in which the concentrations of the coexisting phases were determined with the corresponding densities of the solution. The tie lines were satisfactorily described by using the Othmer-Tobias and Bancroft equations.
基金This study was jointly supported by the National Natural Science Foundation of China(20275003 and 20335010).
文摘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.
基金Supported by the National Natural Science Foundation of China.
文摘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%.
基金Supported by the National Natural Science Foundation of China (No. 29736180).
文摘The partition coefficients of baicalin were measured in ethylene oxide and propylene oxide (EOPO)/salt aqueous two-phase systems at 298.15K. It was found that most of baicalin partitioned into EOPO-rich phase. The partition coefficients of baicalin varied from 10 to 120. The effect of various factors, including tie-line length, salt composition, molecular weight of EOPO, and solution pH, on the partition behavior was investigated in EOPO/salt systems. Furthermore the partition coefficients of baicalin were correlated using the modified Diamond-Hsu model. Good agreement with experimental data is obtained. The average relative deviations are less than 5.0%.
基金Project (No. 20276064) supported by the National Nature ScienceFoundation of China
文摘The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.
基金Supported by the Zhejiang Province Natural Science Foundation, China(No.407240)
文摘A fast and convenient C―S bond formation reaction was achieved catalyzed by CuI/L-proline in aqueous two-phase system providing a simple method for synthesis of aryl sulfides in good yields in short time.
文摘Background:D-Galactose dehydrogenase(GalDH;EC 1.1.1.48)belongs to the family of oxidoreductases that catalyzes the reaction ofβ-D-galactopyranose in the presence of NAD+to D-galacto-1,5-lactone and NADH.The enzyme has been used in diagnostic kits to neonatal screen for galactosemia diseases.This article reports the partitioning optimization of recombinant Pseudomonas fluorescens GalDH in aqueous two-phase systems(ATPS).Methods:Preliminary two-phase experiments exhibited that the polyethylene glycol(PEG)concentration,pH value,and concentration of salt had a significant influence on the partitioning efficiency of recombinant enzyme.According to these data,response surface methodology(RSM)with a central composite rotatable design(CCRD)was performed to condition optimization.Results:The optimal partition conditions were found using the 14.33%PEG-4000 and 11.79%ammonium sulfate with pH 7.48 at 25℃.Yield,purity,recovery,and specific activity were achieved 92.8%,58.9,268.75%,and 373.9 U/mg,respectively.PEG and ammonium sulfate concentration as well as pH indicated to have a significant effect on GalDH partitioning.Enzyme activity assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis demonstrated the suitability of predicted optimal ATPS as well.The Km and molecular weight values for the purified GalDH were 0.32 mM and 34 kDa,respectively.Conclusions:Ultimately,our data showed the feasibility of using ATPS for partitioning and recovery of recombinant GalDH enzyme.
基金Supported by the National High Technology Research and Development Program of China(2009AA02Z206,2006AA02Z239)the National Basic Research Program of China(2007CB707805)the Ministry of Science and Technology,China
文摘One of the bottlenecks for bioproduction of butyric acid as bulk chemical is the difficulty in separating butyric acid from the fermentation broth,compared with the petroleum-based chemical synthesis method.In the present work,a novel separation methodology was developed based on an aqueous two-phase system with inor-ganic salts.Calcium chloride was screened out for effective separation of butyric acid from butyric acid-water-salt systems.Within appropriate concentration range of butyric acid and salt,butyric acid was enriched in the upper phase and most of calcium ions remained in the lower phase.This"salting out"effect is very efficient to separate butyric acid from the simulated butyrate fermentation broth,which consists of butyric acid and acetic acid with concentration ratio of 4︰1,so that the final ratio of butyric acid/acetic acid in the upper phase is improved to 9.87. The aqueous two-phase system was used to separate butyric acid from the actual fermentation broth with satisfac-tory result.
文摘The phenomenon of two dilute aqueous phases composed of sodium dodecylsulfate(SDS) and cetyl trimethyl ammonium bromide(CTAB) was investigated under various conditions such as concentrations and molar ratios of the two surfactants, the addition of sodium chloride and temperature. Vesicles formation was found in the both phases by TEM image.
基金Zhejiang University and TalentIntroduction Program of China for Postdoctoral Researcher for the financial supportfinancially supported by the National Key Research&Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286)。
文摘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.
文摘The aqueous two-phase system of PEG-4000/K<sub>3</sub>PO<sub>4</sub> 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.
基金Supported by the National Natural Science Foundation of China(No.20777029)Department of Education Project of Jilin Province,China(No.20070156)
文摘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.
基金This work is supported by State Key PrO-iect of Fundamental Research !(G1998061302) the National Natural Science Foundation
文摘In the investigation of effect of KSCN on the partitioning of lysozyme in PEG2000/ammonium sulfate aqueous two-phase system, it was found that the KSCN could alter the pH difference between the two phases. and thus affect the partition of lysozyme. The relationship between partition coefficients of lysozyme and pH differences between two phases was discussed.
基金Supported by the National Natural Science Foundation of China (No. 29873014) and the Key Disciplinary Foundation of Shanghai.
文摘A series of ethylene oxide (EO)-propylene oxide (PO) randomco-polymers (EOPO) were used to form aqueous two-phase systems (ATPS)with ammonium sulfate. Effects of EOPO's properties on the phaseseparation behaviors and on the partition of cephalexin and7-aminodesacetoxicephalosporanic acid (7-ADCA) in ATPS wereinvestigated. Both the molar mass and molar ratio of EO to PO of EOPOcould greatly influence partition behaviors of cephalexin and 7-ADCAas well as the binodal curve of ATPS.
文摘A novel method for the separation/analysis rhodamine B has been described. The ionic liquid (1-tetradecyl-3-methylimidazolium bromide)/anion surfactant (sodium dodecyl sulfate)/NaCl two-phase systems (ATPSs) is presented as a simple, rapid and effective sample pretreatment technique coupled with ultraviolet spectrometry for analysis rhodamine B in soft drink. The effects of parameters on the ATPSs extraction of rhodamine B such as amount of surfactant, ionic liquid and salt, pH, temperature, stabilization and centrifugal time have been studied in details. Under the optimized conditions, the linear range of calibration curve for rhodamine B was 0.05 - 7.0 μg·mL-1 and the detection limit was 3.2 ng·mL-1. The phase equilibrium and the mechanism of phase separation for ATPSs have been discussed. This method has been applied to the determination of rhodamine B in soft drink.
基金Supported by the Malaysia's Ministry of Higher Education(FRGS/4F136)the University Teknologi Malaysia(RUG/01H68)
文摘Temperature sensitivity of waxy crude oils makes it difficult to study their flow behaviour in the presence of water especially near their wax appearance temperature （WAT）. In this study a method was proposed and implemented to mitigate such difficulties which was applied in predicting mixture temperatures （Tin） of a typical Malaysian waxy crude oil and water flow in a horizontal pipe. To this end, two analytical models were derived firstly from calorimetry equation which based on developed two correlations for defining crude oil heat capacity actualized from the existed specific heat capacities of crude oils. The models were then applied for a set of experiments to reach the defined three predetermined Tm （26 ℃, 28 ℃ and 30 ℃）. The comparison between the predicted mixture temperatures （Tin,1 and Tin,2） from the two models and the experimental results displayed acceptable absolute average errors （0.80% 0.62%, 0.53% for model 1; 0,74%, 0.54%, 0.52% for model 2）. Moreover, the average errors for both models are in the range of standard error limits （4-0,75Z） according to ASTM E230. Conclusively, the proposed model showed the ease of obtaining mixture temperatures close to WAT as predetermined with accuracy of ± 0.5 ℃approximately for over 84% of the examined cases. The method is seen as a practical reference point to further study the flow behaviour of waxy crudes in oil-water two-phase flow system near sensitive temperatures.
