Recyclable Aqueous Two-phase Systems Research Articles

Recyclable aqueous two-phase system formed by two temperature-responsive polymers for the chiral resolution of mandelic acid enantiomers

Aqueous two-phase systems (ATPSs) have been used in the chiral resolution of various enantiomers in recent years. However, it is difficult to recycle and reuse the phase-forming components and chiral selectors in these applications. Therefore, a completely recyclable ATPS that is based on two temperature-responsive polymers, viz., poly(MAH-β-CD-co-NIPAAm-DMAEMA-BMA) (PNDB-CD) and poly(NVCL-AM) (PVAm), was employed for the chiral separation of mandelic acid (MA) enantiomers. The two polymers can be recovered by simply adjusting the temperature; meanwhile, the polymer PNDB-CD can be used as a chiral selector. The factors that affect the chiral resolution process were also investigated. A maximum separation factor (α) of 1.49 was achieved via a one-step extraction under the following conditions: PVAm concentration of 14.0 wt%, PNDB-CD concentration of 9.0 wt%, MA concentration of 5.0 mg/g, and system pH of 4.0. Moreover, the two polymers can be easily recycled after the chiral separation process. The recyclable ATPS used for enantioseparation in the current study can provide new ideas for other applications by using enantioselective liquid–liquid extraction (ELLE).

Separation of antibody IgG201 by an aqueous two-phase system with recyclable pH-responsive polymers

Since their clinical application success, therapeutic monoclonal antibodies (mAbs) have been playing important roles in the treatment of many diseases. A significant obstacle to reducing antibody drug costs is reducing the use of expensive materials in the purification manufacturing process. New technologies, such as aqueous two-phase systems (ATPSs), are an option to solve this problem. In this study, a recyclable aqueous two-phase system was developed. Two pH-responsive polymers were synthesized and characterized. The phase-forming polymers and salt concentrations, system pH, and temperature were studied. Finally, under conditions of 6% PMDB3.36 with 20 mmol/L sodium acetate and 30 mmol/L Na2SO4 at pH 5.5, the recovery of target monoclonal antibody IgG201 was 94.4 % and partition coefficient was 8.7. The purity was 92.2 %, as determined bysize exclusion chromatography (SEC). The pH-sensitive polymers in this ATPS can be recycled near their isoelectric point.

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems.

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.

Partition of Tea Saponin with a Novel Recyclable Thermo-pH Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPS) have the advantages of environmentally friendly, high mass transfer efficiency, and mild extraction conditions. However, it is difficult to recycle these polymers, which limits the large-scale application of ATPS. In this study, a novel recyclable ATPS was constructed with thermo-responsive polymer PN and pH-responsive polymer PADB4.78 for the partition of tea saponin. PN represents poly-(N-isopropylacrylamide), and PADB4.78 represents poly-(acrylic acid-dimethylamine ethyl methacrylate-butyl methacrylate), where 4.78 in the subscript indicate the isoelectric point of the polymer. The recoveries of PN and PADB4.78 were 95.36% and 93.48%, respectively, after two cycles. Meanwhile, the phase formation mechanism of ATPS was studied by surface tension and low-field nuclear magnetic resonance (LF-NMR). The effects of polymer concentration, pH, temperature, types and concentrations of salt were investigated on tea saponin partition. In the 1.5% (w/v) PN/3.5% (w/v) PADB4.78 ATPS, the optimal partition coefficient (K) of crude tea saponin were 0.15 in the presence of 1.5 mM KCl at pH 7.6 and 25 °C while the extraction recovery (ERb) reached 92.13%. The K and ERb of tea saponin from tea seeds were 0.12 and 94.50% with 7.5 mM LiBr at pH 8.0 and 25 °C, respectively.

Partition of spiramycin in a recyclable aqueous two-phase system based on pH-responsive and thermosensitive polymers

Aqueous two-phase systems have been widely studied for the separation and recovery of bioproducts. In this study, the thermosensitive polymer EO30PO70 and pH-responsive polymers (PADB4.66 and PADB6.26) were used to develop a recyclable aqueous two-phase system suitable for spiramycin partitioning. EO30PO70 represents poly-(ethylene oxide-propylene oxide), where 30 and 70 indicate the proportions of ethylene oxide and propylene oxide, respectively. PADB4.66 and PADB6.26 represent poly-(acrylic acid-dimethylamine ethyl methacrylate-butyl methacrylate), where the numbers (4.66 and 6.26) in the subscripts indicate the isoelectric point of the polymers. The phase separation mechanism of ATPS was studied through low-field nuclear magnetic resonance (LF-NMR) and surface tension. Moreover, the parameters affecting spiramycin partitioning, such as system components and composition, pH, temperature, and type and concentration of additive salt, were investigated. The best partitioning conditions were determined using 4.70% PADB6.26 and 31.67% EO30PO70 in the presence of 0.05 mol/L NaSCN at 35 °C and pH = 9.2. The highest partition coefficient of spiramycin from fermentation broth was 11.92, with an extraction recovery of 97.69%. In addition, phase-forming polymers PADB6.26 and EO30PO70 could be recovered by regulating the pH or temperature, and their recoveries reached 92.39% and 98.76%, respectively.

Study of Microbial Transglutaminase Partitioning in Thermo-pH-Responsive Aqueous Two-Phase Systems.

The recyclable aqueous two-phase systems (ATPS) responding to environmental stimuli have been widely studied in the purification of biologics. In this study, a thermo-responsive polymer PNE was copolymerized after optimization of monomer ratio. In addition, its lower critical solution temperature (LCST, 31°C) and first recovery (99.43%) were determined. Then, PNE was used to form two recyclable ATPS with another thermo-responsive polymer PNDBN and a pH-responsive polymer PADB4.91, which were polymers already prepared in the lab. Meanwhile, the partition behavior of microbial transglutaminase (MTG) was explored using these two ATPS. The result showed that the PNE/PADB4.91 ATPS was superior to PNDBN/PNE ATPS owing to its faster phase formation and better partition performance. In order to optimize the partition behavior, several parameters were investigated based on PNE/PADB4.91 ATPS. In the ATPS constructed with 2.5% (w/v) PNE and 3.5% (w/v) PADB4.91, the maximal partition coefficient (1/KE) and enzymatic recovery (ERB) of MTG were 12.9 and 95.21% in the presence of 10mM KCl when the temperature, pH, and the addition amount of MTG were 25°C, 7.0, and 2mg/mL, respectively.

Partitioning of tylosin in recyclable aqueous two-phase systems based on two pH-responsive polymers

Abstract Aqueous two-phase systems (ATPS) are a promising biotechnology in bioseparation. However, recycling of the phase-forming polymer is a key issue. In this research, new recyclable ATPS composed of two pH-responsive polymers (PADB4.01 and PMDBH3.27) were prepared. The ATPS could be recycled by adjusting the pH of the solution, with recovery of 93.8%. The ATPS were evaluated for the first time in the partitioning of tylosin. The effects of the main parameters, such as the polymer concentration, pH, temperature, salt species and concentrations, on the partition of crude tylosin and tylosin from fermentation broth were studied. In the 2% PMDBH3.27/3% PADB4.01 ATPS, the partition coefficient (K) and extraction recovery (ERtop) of crude tylosin could reach 5.12 and 93.9%, respectively, in the presence of 90 mM KCl at pH 5.83 and 25 °C. In the 2.5% PMDBH3.27/3% PADB4.01 ATPS, the K and ERtop of tylosin from fermentation broth reached 3.58 and 87.3%, respectively, in the presence of 20 mM KBr at pH 5.69 and 25 °C. Through partition mechanism analysis by Fourier transform infrared spectroscopy (FT-IR) and interphase potential measurements, the appearance of a new absorption peak and interphase potential difference, respectively, may contribute to the partitioning of tylosin in the PADB4.01-enriched top phase.

Green separation of bromelain in food sample with high retention of enzyme activity using recyclable aqueous two-phase system containing a new synthesized thermo-responsive copolymer and salt

High cost and enzyme deactivation in purification process are the two main obstacles for the use of enzyme as green catalyst. The objective of this work was to overcome these limitations by developing a cost-effective aqueous two-phase system (ATPS) for efficient purification of enzymes with remarkable separation efficiency and high retention of enzyme activity. The ATPS was formed by thermo-responsive block copolymer PEG113-b-PNIPAM149 and salt as phase-forming components combining economy, recovery and sustainability. This strategy fabricated block copolymer with specified molecular weight and low LCST, which not only achieved better phase splitting but also ensured easy recycling for block copolymer. The developed ATPS demonstrated excellent extraction and biocompatibility for bromelain in real sample with 94.87% separation efficiency and 77.06% activity, which were remarkably higher than those obtained in EOPOEO-based ATPS. The recycling of copolymer was introduced to minimize cost, with recovery efficiency of 90% in the five cycles.

Polymerization of a new thermo-responsive copolymer with N-vinylcaprolactam and its application in recyclable aqueous two-phase systems with another thermo-responsive polymer

BackgroundThe recovery characteristics of phase-forming polymers are essential for aqueous two-phase systems (ATPS) to recycle in bioseparation engineering.ResultsA new thermo-responsive copolymer (PVBAm) is suggested based on N-vinylcaprolactam, acrylamide, and butyl methacrylate. Together with another thermo-responsive polymer, poly (N-isopropyl acrylamide) (PN), it has been applied to form a recyclable ATPS. PVBAm and PN were designed to obtain structures and molecular weights allowing a lower critical solution temperature (LCST). By polymerization optimization, both PN and PVBAm were obtained with recoveries 98.5% and 95% above their LCST (i.e., PN 32.5 °C and PVBAm 40.5 °C), respectively, which allows each ATPS phase to be effectively recycled. The recycled ATPS based on PVBAm and PN was applied to the partitioning of vitamin B12. Under optimized conditions (5% PVBAm/3.5 %PN ATPS, in the presence of 0.8 M KCl, pH 4.0), the partition coefficient of vitamin B12 reached a value of 5.81.ConclusionThe new ATPS based on the thermo-responsive copolymer PVBAm/PN possessed appropriate recycling characteristics regarding LCST, as well as recovery and phase separation characteristics.

Synthesis of pH-responsive polymers forming recyclable aqueous two-phase systems and application to the extraction of demeclocycline

The application of aqueous two-phase systems (ATPS) to bioseparation and biocatalyst engineering has attracted interest. However, despite the distinct advantages of the technique, the scaling up of ATPS is limited by the cost of the system components. In this study, two novel recyclable pH-responsive polymers were synthesized and tested for the partition of demeclocycline. The recoveries of two recyclable pH-responsive polymers could reach over 95.0% by only adjusting the solution pH to the isoelectric points of the polymers. The control variate method was used to investigate the effects of polymerization conditions on polymer synthesis. Polymer PADB4.6 was firstly synthesized in 500 l reactor with two-stage heating method (25–40 ℃ and 40–62 ℃), as the heating rate is an important factor in the scale-up process. The polymer characteristics (molecular weight, viscosity, surface tension, and zeta potential) were studied to understand their influences on the synthetic process. Furthermore, the phase formation mechanism was studied with low-field nuclear magnetic resonance (LF-NMR). The optimal partition coefficient and recovery of demeclocycline using the recyclable ATPS were 0.24 and 82.9% at pH 6.30 in presence of 20 mmol/L MgSO4.

Environmentally Benign and Recyclable Aqueous Two-Phase System Composed of Distillable CO2-Based Alkyl Carbamate Ionic Liquids

Ionic liquid-based aqueous two-phase systems (IL-ATPSs) have been studied comprehensively as a potential alternative method for protein purification. One of the distinct features of IL-ATPS is that the polarity of the IL can be adjusted by different combinations of cation and anion, providing flexibility in governing the partition of proteins between phases. However, the conventional IL-ATPS usually has poor environmental footprint and low recyclability, thereby hampering the systems for practical use in protein separation. To address these shortcomings, here we explored the distillable CO2-based alkyl carbamate ILs as phase-forming components. N,N-dimethylammonium N′,N′-dimethylcarbamate (DIMCARB) was able to form ATPS with polypropylene glycol (PPG). The liquid–liquid equilibrium data of ATPSs composed of PPG with different molecular mass and DIMCARB were determined. As both phase-forming components are thermosensitive, the effect of temperature on the phase diagram of the PPG 400 + DIMCARB system was also evaluated. The partition behavior of model proteins (i.e., bovine serum albumin and lysozyme) in the PPG + DIMCARB systems were assessed. Both model proteins tend to migrate to the more hydrophilic IL-rich phase, when a higher temperature or a higher molecular mass of PPG was used. The DIMCARB was recovered via rotary evaporation, while the PPG 400 was recovered from a secondary two-phase system thermally induced from the primary ATPS. The recovered components were subsequently used to prepare a new batch of ATPS. The phase composition of the new system as well as the partition coefficients of both model proteins in the recycled system were maintained, thereby proving the feasibility of reusing both phase-forming components. Overall, the recyclable and biodegradable phase-forming components used in the newly developed PPG + DIMCARB system make the ATPS a sustainable and eco-friendly system for protein purification.

Recyclable aqueous two-phase system based on two pH-responsive copolymers and its application to porcine circovirus type 2 Cap protein purification

Aqueous two-phase system (ATPS) has great potential in industrial applications of bio-separations and bio-reactions. However, its large-scale application is limited by recovery difficulty of phase systems. In this paper, a recyclable ATPS was prepared by two pH-responsive copolymers (PADB4.99 and PMDM7.08) and applied for purification of porcine circovirus type 2 Cap protein fermentation broth (PCV2 Cap protein). Phase separation mechanism was studied by using low-field nuclear magnetic resonance (LF-NMR). The results showed that relatively desirable ATPS were formed successfully when two copolymer concentrations were 4%–6% (w/w) with pH 7.5–8.6. The main parameters, such as copolymer concentration, temperature, pH, type and concentration of salts, were investigated. The results demonstrated that the best ATPS consisted of 4% (w/w) PADB4.99 and 6% (w/w) PMDM7.08, the optimal partition coefficient (K) and extraction recovery (ER) of PCV2 cap protein were 0.25 and 94.2% in the presence of 50 mM Li2SO4, 3.53 and 93.4% in the presence of 40 mM KCl, respectively, at 30 °C, pH 8.1. The maximum purity of PCV2 Cap protein was 88.4%. In addition, the two phase-forming copolymers could be recycled by adjusting solution pH to their isoelectric points (pI), with recoveries of over 97.5%.

Synthesis of thermo-responsive polymers recycling aqueous two-phase systems and phase formation mechanism with partition of ε-polylysine

Aqueous two-phase systems (ATPS) have the potential application in bioseparation and biocatalysis engineering. In this paper, a recyclable ATPS was developed by two thermo-responsive copolymers, PVBAm and PN. Copolymer PVBAm was copolymerized using N-vinylcaprolactam, Butyl methacrylate and Acrylamide as monomers, and PN was synthesized by N-isopropylacrylamide. The lower critical solution temperature (LCST) of PVBAm and PN were 45.0°C and 33.5°C, respectively. The recoveries of both polymers could achieve over 95.0%. The phase behavior and formation mechanism of PVBAm/PN ATPS was studied. Low-field nuclear magnetic resonance (LF-NMR) was applied in the phase-forming mechanism study in ATPS. In addition, combining the analysis results of surface tension, transmission electron microscopy and dynamic light scattering, the phase-forming of the PVBAm/PN ATPS was proved. The application was performed by partition of ε-polylysine in the 2% PVBAm/2% PN (w/w) ATPS. The results demonstrated that ε-polylysine was extracted into the PN-rich phase, the maximal partition coefficient (1/K) and extraction recovery of pure ε-polylysine were 6.87 and 96.36%, respectively, and 7.41 partition coefficient and 97.85% extraction recovery for ε-polylysine fermentation broth were obtained in the presence of 50mM (NH4)2SO4 at room temperature. And this method can effectively remove the most impurities from fermentation broth when (NH4)2SO4 exists in the ATPS. It is believed that the thermo-responsive recycling ATPS has a good application prospect in the field of bio-separation.

Preparation of novel alkaline pH-responsive copolymers for the formation of recyclable aqueous two-phase systems and their application in the extraction of lincomycin.

Aqueous two-phase systems have potential industrial application in bioseparation and biocatalysis engineering; however, their practical application is limited primarily because the copolymers involved in the formation of aqueous two-phase systems cannot be recovered. In this study, two novel alkaline pH-responsive copolymers were synthesized and examined for the extraction of lincomycin. The two copolymers could form a novel alkaline aqueous two-phase systems when their concentrations were both 6% w/w and the pH was 8.4(±0.1)-8.7(±0.1). One copolymer was synthesized using acrylic acid, 2-(dimethylamino)ethyl methacrylate, and butyl methacrylate as monomers. Moreover, 98.8% of the copolymer could be recovered by adjusting the solution pH to its isoelectric point (pH 6.29). The other copolymer was synthesized using the monomers methacrylic acid, 2-(dimethylamino)ethyl methacrylate, and methyl methacrylate. In this case, 96.7% of the copolymer could be recovered by adjusting the solution pH to 7.19. The optimal partition coefficient of lincomycin was 0.17 at 30°C in the presence of 10 mM KBr and 5.5 at 40°C in the presence of 80 mM Ti(SO4)2 using the novel alkaline aqueous two-phase systems.

Poly(N,N-diallylaspartic acid-alt-sulfur dioxide): its synthesis and application

Monomer pairs, dimethyl N,N-diallylaspartate hydrochloride [(CH2=CH–CH2)2NH+CH(CO2Me)CH2CO2Me Cl−] (I)/SO2 and N,N-diallylaspartic acid hydrochloride [(CH2=CH-CH2)2NH+CH(CO2H)CH2CO2H Cl−] (II)/SO2, underwent alternate cyclocopolymerization to give cationic polyelecyrolytes (CPEs), poly(I-alt-SO2) (III) and poly(II-alt-SO2) (IV), respectively, in very good yields. (+) III upon acid hydrolysis was converted to cationic (+) IV, bearing in each repeating unit the triprotic acid residues [(NH+…(CO2H)2] of aspartic acid hydrochloride. Under the influence of pH, (+) IV has been equilibrated to water-insoluble diprotic polyzwitterionic acid (±) V, water-soluble monoprotic poly(zwitterion-anion) (± −) VI and finally its conjugate base polydianion (=) VII. Basicity constants of CO2− and amine group have were determined. The pH-responsive polymer was demonstrated to be an efficient antiscalant against CaSO4 scaling. For potential separation and purification of biomolecules, a recyclable aqueous two-phase system (ATPS) was constructed using water-soluble (=) VII and urethanized polyvinyl alcohol; the ionic polymer can be recycled by precipitating it as (±) V at a lower pH.

Alternate cyclopolymer of diallylglutamic acid and sulfur dioxide

The alternate cyclocopolymerization of a glutamic acid-based monomer and SO2led to a pH-responsive polydianionic electrolyte (PDE) which is used to construct a recyclable aqueous two-phase system (ATPS) and found to be an effective antiscalant.

PH recycling aqueous two-phase systems applied in extraction of Maitake β-Glucan and mechanism analysis using low-field nuclear magnetic resonance

In this paper, a recycling aqueous two-phase systems (ATPS) based on two pH-response copolymers PADB and PMDM were used in purification of β-Glucan from Grifola frondosa. The main parameters, such as polymer concentration, type and concentration of salt, extraction temperature and pH, were investigated to optimize partition conditions. The results demonstrated that β-Glucan was extracted into PADB-rich phase, while impurities were extracted into PMDM-rich phase. In this 2.5% PADB/2.5% PMDM ATPS, 7.489 partition coefficient and 96.92% extraction recovery for β-Glucan were obtained in the presence of 30mmol/L KBr, at pH 8.20, 30°C. The phase-forming copolymers could be recycled by adjusting pH, with recoveries of over 96.0%. Furthermore, the partition mechanism of Maitake β-Glucan in PADB/PMDM aqueous two-phase systems was studied. Fourier transform infrared spectra, ForteBio Octet system and low-field nuclear magnetic resonance (LF-NMR) were introduced for elucidating the partition mechanism of β-Glucan. Especially, LF-NMR was firstly used in the mechanism analysis in partition of aqueous two-phase systems. The change of transverse relaxation time (T2) in ATPS could reflect the interaction between polymers and β-Glucan.

Synthesis of cefprozil using penicillin G acylase in recyclable aqueous two-phase systems

Cefprozil is an important semi-synthetic cephalosporin antibiotic. In this study, immobilized penicillin G acylase (PGA) is used to catalyze the acylation of 7- amino-3-(1-propenyl)-4-cephalosporanic acid (7-APRA) and 4-hydroxyphenylglycine methyl ester (HPGME) and a recyclable thermo-pH responsive PNB/PADB aqueous twophase system (ATPS) is used to synthesize cefprozil. In this system, the partition coefficient of cefprozil was 2.24 with 60 mmol/L (NH4)2SO4. In addition, the optimal enzymatic reaction conditions were found to be pH 6.5, 20°C, 78 u/mL immobilized PGA, 30 mmol/L 7-APRA and 90 mmol/L HPGME. In the PNB/PADB ATPS, the maximal yield of cefprozil was 75.81% with 60 mmol/L (NH4)2SO4 and in the single aqueous system the yield was 56.02%. The yields are thought to improve because there is a reduction in product inhibition.

Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer

Biodegradation of cellulose involves synergistic action of the endoglucanases, exoglucanases and β-glucosidases in cellulase. However, the yield of glucose is limited by the lack of β-glucosidase to hydrolyze cellobiose into glucose. In this study, β-glucosidase as a supplemental enzyme along with cellulase are co-immobilized on a pHresponsive copolymer, poly (MAA-co-DMAEMA-co-BMA) (abbreviated PMDB, where MAA is α-methacrylic acid, DMAEMA is 2-dimethylaminoethyl methacrylate and BMA is butyl methacrylate). The thermal and storage stabilities of PMDB with immobilized enzymes are improved greatly, compared with those of free cellulase. Biodegradation of cellulose is carried out in a pH-responsive recyclable aqueous two-phase system composed of poly (AA-co- DMAEMA-co-BMA) (abbreviated PADB 3.8, where AA is acrylic acid) and PMDB. Insoluble substrate and PMDB with immobilized cellulase and β-glucosidase (Celluclast 1.5L FG and Novozyme 188, respectively) were biased to the bottom phase, while the product was partitioned to the top phase in the presence of 40 mM (NH4)2SO4. When the degradation reaction of cellulose is carried out with PMDB containing immobilized cellulase and β-glucosidase, the concentration of glucose reaches 4.331 mg/mL after 108 h. The yield of glucose is 50.25% after PMDB containing the immobilized enzymes is recycled five times.

Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers

The synthesis of Cephalexin in novel recyclable aqueous two-phase systems was studied, using immobilized penicillin acylase as biocatalyst, 7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS) was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50mM 7-ADCA and 150mM PGME at pH 5.8, 20°C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.