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A green bio-separation alternative can be performed with a non-toxic and biodegradable aqueous biphasic system (ABS) composed of short-chain aliphatic alcohol-based top phase (1-propanol and 2-propanol) and carbohydrate-based bottom phase (glucose, sucrose, and maltose). A model protein, bovine serum albumin (BSA) was adopted to determine the effects of types and concentration of phase-forming components; protein concentration; and system pH on the protein partition efficiency in the ABS. Results showed that the 1-propanol/maltose ABS gave an overall better partition efficiency of BSA to the alcohol-rich top phase compared to the 1-propanol/sucrose ABS, 1-propanol/glucose ABS, and 2-propanol/sugar ABS attributed to the lower hydrophilicity of 1-propanol and the stronger sugaring-out effect exerted by the maltose. A maximum partition coefficient (K) of 20.01 ± 0.05 and recovery yield (Y) of 95.42% ± 0.01 of BSA were obtained with the 35% (w/w) 1-propanol/22% (w/w) maltose ABS at pH 5.0 which contained 10% (w/w) BSA. The K and Y of BSA in 1-propanol/maltose ABS was further enhanced with the addition of 3% (w/w) of ionic liquids, 1-butyl-3-methylimidazolium bromide ([Bmim]Br) as the adjuvants which provides the protein stabilizing effect. The Fourier Transform Infrared Spectrum (FTIR) analysis revealed that the protein structure remained unaltered upon the separation process.

Keratinase is widely applied in the bioconversion of feather waste to produce value-added bioproducts. The wide applications of industrial keratinase have urged the advancement in producing keratinase with high productivity and yields. The efficiency of aqueous two-phase electrophoresis system (ATPES) to recover extracellular Kytococcus sedentarius TWHKC01 keratinase was investigated. The operation time of 10 min and voltage of 10 V were applied for the recovery of K. sedentarius TWHKC01 keratinase with the PEG 1000/sulfate ATPES. Keratinase was recovered in the PEG-rich phase of the ATPES consists of 23% (w/w) PEG 1000, 18.5% (w/w) ammonium sulfate of pH 9, 20% (w/w) crude feedstock and with 3% (w/w) of NaCl added. Highest partition coefficient (Ke) of 4.63 ± 0.17, recovery yield (YT) of 78.75% ± 0.61, and purification fold (PFT) of 3.89 ± 0.84 were obtained in the optimal ATPES coupled with cathode electrode in top phase and anode electrode in the bottom phase. The introduction of electric field to the ATPS has enhanced the selectivity (S) of the system towards the extracellular keratinase by two-fold from 2.79 ± 0.46 to 5.68 ± 0.43, demonstrating the efficiency of ATPES in separating the keratinase from crude feedstock when compared to conventional two-phase system.

Ectoine is a zwitterionic amino acid derivative that can be naturally sourced from halophilic microorganisms. The increasing demands of ectoine in various industries have urged the researches on the cost-effective approaches on production of ectoine. Ionic liquids-based aqueous biphasic system (ILABS) was applied to recover Halomonas salina ectoine from cells hydrolysate. The 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 was used in the ILABS and the recovery efficiency of ILABS to recover ectoine from H. salina cells lysate was evaluated by determining the effects of phase composition; pHs; crude loading and additional neutral salt (NaCl). The hydrophilic ectoine was targeted to partition to the hydrophilic salt-rich phase. A total yield (YB) of 96.32% ± 1.08 of ectoine was obtained with ILABS of phase composition of 20% (w/w) (Bmim)BF4 and 30% (w/w) sulfate salts; system pH of 5.5 when the 20% (w/w) of crude feedstock was applied to the ILABS. There was no significant enhancement on the ectoine recovery efficiency using the ILABS when NaCl was added, therefore the ILABS composition without the additional neutral salt was recommended for the primary purification of ectoine. Partition coefficient (KE) of 30.80 ± 0.42, purity (PE) of 95.82% and enrichment factor (Ef) of 1.92 were recorded with the optimum (Bmim)BF4/sulfate ILABS. These findings have provided an insight on the feasibility of recovery of intracellular biomolecules using the green solvent-based aqueous system in one single-step operation.

Ionic liquid-based aqueous biphasic system (ILABS) has emerged as an attractive green approach for the extraction and separation of various biomolecules. The growing market demands of cytochrome c (cyt c) due to its vast uses in medical applications have urged the search for cost-efficient approaches for the production and purification of cyt c. In this study, the feasibility of ILABS to recover cyt c from simulated Saccharomyces cerevisiae cell cultures was investigated by evaluating the effects of phase composition, pH, and additives concentration on the recovery efficiency of cyt c. The ILABS was developed using the hydrophilic ionic liquids, 1-hexyl-3-methylimidazolium bromide ((C6mim)Br) and potassium citrate. The optimal separation conditions for recovery of cyt c from simulated cells cultures were attained with ILABS of pH 9 comprising of 28% (w/w) of (C6mim)Br and 24% (w/w) of potassium citrate and the addition of 0.2% (w/w) NaCl at room temperature (25 °C). Cyt c was recovered in the IL-rich phase with partition coefficient (Kc) of 364.00 ± 1.09, recovery yield (Y) of 99.76% ± 0.03 and selectivity (S) of 223.31 ± 0.56. The results suggest that ILABS can efficiently recover cyt c from microbial fermentation broth with high recovery yields and separation efficiency.

Microbial carotenoids are gaining attention because of the unsustainable supplies of natural plant sources of carotenoids. The downstream processes of the microbial carotenoids are often limited by high cost of production and multi-steps unit operation. Aqueous biphasic system (ABS) comprised of alcohol and salt is suggested as a potential approach to overcome these limitations for recovery of Gordonia terrae carotenoids in this study. Aliphatic alcohols inclusive of 1-propanol and 2-propanol and sulphate, citrate and phosphate salts were used to develop the alcohol/salt ABS to recover the microbial carotenoids. The optimum ABS for the integration of G. terrae cells disruption and simultaneous recovery of carotenoids was developed by evaluating the effects of concentration of phase-forming components, pH, amount of crude loads, concentration of additives, sonication time on the recovery yield of carotenoids. Repetitive batch-extraction of carotenoids using the optimum alcohol/salt ABS was performed to investigate the feasibility of ABS as the sustainable and consistent method for recovery of microbial carotenoids. High recovery yield of carotenoids (90.86 ± 2.97%) was obtained with 20% (w/w) 1-propanol and 20% (w/w) trisodium citrate salt of pH 9. A total of 10% (w/w) of G. terrae cells was loaded into the optimized system with the addition of 4% (w/w) of NaCl. The optimized system is suggested to perform without sonication because degradation of the carotenoids was observed with sonication applied. A total of 4 batches of ABS extraction were conducted until all carotenoids were completely extracted from the G. terrae cells. The high-water content and cost-effective one-step ABS allows the process integration of extraction and separation of carotenoids from G. terrae fermentation.

Microbial astaxanthin with strong antioxidant activity is greatly demanded for diverse applications. Extractive disruption in aqueous biphasic system (ABS) integrates the cells disruption and biomolecules recovery processes in one-step operation, allowing the direct recovery of intracellular biomolecules with biphasic system upon released from cells. In this study, astaxanthin was recovered from recombinant Kluyveromyces marxianus yeast cells via extractive disruption using alcohol/salt ABS. Recombinant K. marxianus yeast is engineered to produce high concentration of free form astaxanthin. Highest partition coefficient (K = 90.02 ± 2.25) and yield (Y = 96.80% ± 0.05) of astaxanthin were obtained with ABS composed of 20% (w/w) 1-propanol and 20% (w/w) sodium citrate of pH 5, 0.5% (w/w) yeast cells loading and additional of 1% (w/w) 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 to improve the migration of astaxanthin to alcohol-rich top phase. The incorporation of 2.5 h of ultrasonication to the biphasic system further enhanced the astaxanthin recovery in ABS. The direct recovery of astaxanthin from recombinant K. marxianus cells was demonstrated with the ultrasonication-assisted alcohol/salt ABS which integrates the extraction and concentration of astaxanthin in a single-step operation.

Crude palm oil (CPO) and palm-pressed mesocarp fibre oil (PPMFO) consist of minor components such as Vitamin E, phytosterols, squalene, and carotene that play numerous health function in human. However, limitations are present in current separation processes to separate or enrich minor components from plant sources. In this study, interest is focusing on the enrichment efficiency of the minor components from CPO and PPMFO through proposed sequential adsorption-desorption strategy. The strategy started with the adsorption-desorption properties evaluation of six adsorbents. Both Diaion HP20 and Sepabeads SP850 showed better adsorption-desorption properties than silica gel, Florisil, Diaion HP2MG and Amberlite XAD-7HP. Diaion HP20 was selected as the suitable adsorbent to perform a series of selective desorption process using three different solvents: methanol, IPA, and n-hexane in Soxhlet extraction. Vitamin E, phytosterols, and squalene from CPO were obtained in the 1st fraction using methanol solution and their concentration increased from the initial concentration with the enrichment factor (EF) of 3.4, 3.9, and 1.8, respectively, which slightly higher than those minor components obtained from PPMFO, 1.2, 1.8, and 1.4, respectively. The carotene from both CPO and PPMFO was enriched in the 3rd fraction by using n-hexane solution with an enrichment factor of 1.1 and 1.5, respectively. In conclusion, the obtained result revealed the efficiency of the proposed sequential adsorption-desorption strategy to enrich the minor components from CPO and PPMFO.

Recent advances in genetic engineering technology have provided various methods in recombinant proteins production for pharmaceutical purposes. However, the development of the effective purification strategy remains the major challenge for large-scale production of therapeutic proteins because high purity of the proteins is often demanded. In this study, recombinant Oryza sativa L. cv Tainung 67 human serum albumin (OsrHSA) was expressed and purified with a single-step polyethylene glycol (PEG)/phosphate aqueous biphasic system (ABS). The optimum condition of PEG/phosphate ABS for the purification of OsrHSA was determined by investigating the effects of PEG molecular weight; pH; tie-line length (TLL); and volume ratio (VR) of ABS on the OsrHSA purification efficiency. The purification efficiency of OsrHSA was evaluated based on the partition coefficient (KHSA) and recovery yield (RB) of the OsrHSA in the PEG/phosphate ABS. An RB of 69.8% of OsrHSA was recovered in the optimum PEG/phosphate ABS consists of TLL of 26.0% (w/w) of pH 7.5. In addition, large-scale ABS were attempted and results showed that the recovery efficiency of 3000 g ABS was consistent with a 10 g ABS. This novel finding has demonstrated that the feasibility of ABS as a potential tool to purify HSA protein in one-step operation.

Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (Ke = 7.53 ± 0.35) and yield (YT = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO4], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.

Bromelain is an important industrial proteolytic enzyme due to the wide application in food, cosmetic and therapeutic industries. There is a great demand in developing a cost-efficient method to recover bromelain from plant sources. Low interfacial tension and biocompatible aqueous biphasic system (ABS) is widely applied in various separation processes of bioproducts. Thus, recovery of bromelain from Ananas comosus (pineapple) pulp using polymer/salt ABS was attempted in this study. The ABS operating factors including polyethylene glycol (PEG) molecular weight, phase composition, pH, centrifugal force and additional of neutral salt were studied. The optimal condition for ABS recovery of bromelain from crude extract was achieved with ABS consisting of 17% (w/w) PEG 4000 and 14% (w/w) phosphate salt; centrifugal force of 6000 × g; pH of 8.0 and at 25 °C. Bromelain exhibited a high specific activity (SA) of 2.7 U and purification factor ( P F ) of 16.3 in the top phase of PEG 4000/phosphate ABS with a yield ( Y T ) of 55.6%. The findings demonstrated that ABS is a potential one-step operation in replacement to the existing methods in recovery of bromelain from plant sources.

Tea is receiving great interests due to potential health benefits associated with long term consumption. Theaflavins, thearubigins and catechins presence in black tea possess high antioxidant capacity and biological activities. Concentration and extraction of these potent antioxidants from Camellia sinensis var. assamica leaves with an aqueous biphasic system (ABS) composed of thermo-sensitive ethylene oxide–propylene oxide (EOPO) random copolymer and salt was demonstrated. The thermo-sensitive EOPO can be recovered upon heating above a lower critical solution temperature (LCST). The partition coefficients of polyphenols based on phenolic content in the EOPO/salt ABS were determined to understand the partition preference of the polyphenols in the extraction system. Effects of different types of salts, tie-line length (TLL), volume ratio ( V R ), sample loading, pH and equilibration time on the extraction efficiency of polyphenols using ABS were evaluated. The EOPO polymer recovery was performed after the primary aqueous biphasic extraction. The highest recovery of polyphenols in EOPO-rich phase was obtained in EOPO 2500/sulphate ABS at pH of 7.0, TLL of 41.4% (w/w), V R of 1.0, sample loading of 0.3% (w/w) and equilibration time of 12.5 min. A total of 96.54 ± 0.09% of polyphenols was recovered in the EOPO-rich phase of the primary EOPO 2500/sulphate ABS with a partition coefficient of polyphenols ( K pc ) of 26.93 ± 0.76. Later, 95.2% of the EOPO 2500 polymer was recovered after the thermo-induction of the polymer-rich phase. This study has successfully demonstrated the feasibility of thermo-sensitive ABS in the extraction and enrichment of antioxidants from plant sources.

Carboxymethyl cellulase (CMCase) hydrolyses cellulose into glucose and is useful in various industrial applications. Conventional CMCase purification methods are rather complicated and time-consuming; thus, a cost-effective strategy for CMCase recovery is on demand. Polyethylene-glycol (PEG)/sodium citrate aqueous biphasic system (ABS) was adopted in this study to investigate the effectiveness of the ABS in the recovery of extracellular Bacillus subtilis CMCase from fermentation broth. Comprehensive optimization steps were executed that took into consideration the ABS variables of PEG molecular weight, tie-line length (TLL), volume ratio (VR), crude loading, pH and the addition of sodium chloride (NaCl). A CMCase recovery yield (YB) of 88.82% ± 0.69, a purification fold (PF) of 4.8 and a partition coefficient (K) of 0.44 ± 0.03 were achieved from the bottom phase of the PEG 6000/citrate ABS with TLL of 42.16% (w/w), VR of 0.29, 1% of (w/w) NaCl, pH 7.0, and 20% (w/w) crude loading. CMCase was mainly segregated to the salt-rich bottom phase because of the hydrophilicity of the enzyme surface. The highly effective recovery technique was further confirmed by SDS-PAGE analysis. Overall, the present study suggests that the ABS is a potential purification strategy for extracellular CMCase.

Methods of extraction and separation of antioxidants from plant sources by chromatography and solvent extraction are often time and solvent consuming, and result in low yields. A simple and mild thermo-induced aqueous micellar biphasic system (AMBS) was used to recover α- and γ-mangostins from Garcinia managostana peels. AMBS composed of non-ionic surfactants such as Triton X-114, Triton X-45, and Pluronic triblock copolymers were developed. Effects of the types of the non-ionic surfactant, its concentration, sample load, pH of the AMBS and the equilibration temperature on recovery of mangostins were studied. The optimum condition for recovery of α- and γ-mangostins was achieved with an AMBS composed of 3% (w/w) Pluronic L-81 and a sample loading of 0.5% (w/w) crude peels. At equilibration temperature of 40 °C and pH 8, 2646.6 ± 1.2 μg/mL of α-mangostin and 379.4 ± 1.2 μg/mL of γ-mangostin were recovered in the micelle-rich phase of the AMBS with a log partition coefficient of α- (log K α ) and γ-mangostin (log K γ ) of 4.00 ± 0.02 and 3.36 ± 0.01, respectively. α- and γ-mangostins could be recovered simultaneously with a yield of 52.93% and 7.59%, respectively in a single-step. This study demonstrated that AMBS can serve as a tool to efficiently recover and separate various bioactive compounds from plant sources.

Liquid biphasic flotation (LBF) offers advanced downstream processing of bioproducts by floating of gas bubbles within the biphasic system to enhance the separation efficiency of target bioproducts. In this study, the feasibility of alcohol/salt LBF for primary capture of extracellular Bacillus subtilis xylanase from crude feedstock was investigated. The effects of types of phase-forming components; phase composition; concentration of crude feedstock load; and flotation time on the recovery performance of xylanase in LBF were evaluated. The recovery of xylanase was achieved in LBF comprised of 28 % (w/w) 2-propanol, 22 % (w/w) ammonium sulfate and 10 % (w/w) B. subtilis crude feedstock load. A maximum partition coefficient (Ke) of 13.29 ± 0.14, selectivity (S) of 24.71 ± 0.96 and recovery yield (YT) of 90.03 % ± 0.10 were recorded in the optimum 2-propanol/sulfate LBF with flotation time of 20 min. SDS-PAGE analysis revealed that B. subtilis xylanase was successfully recovered in the 2-propanol-rich top phase with high purity. The findings demonstrated that LBF is a practicable technology for the large-scale production of microbial xylanase in a one-step operation.

There has been significant interest in ionic liquid aqueous two-phase systems (ILATPSs) with properties such as rapid phase segregation which can lead to a reduction in time taken for protein recovery. Evaluations of ILATPSs were performed with various types of ionic liquid ((Emim)BF4, (Emim)Br, (Bmim)BF4, and (Bmim)Br) and salts (potassium-, sodium-, and magnesium-based) as phase components to figure out their competencies in the recovery of lipase from a fermentation broth of E. coli using banana waste as a substrate. The results of this study revealed that an ILATPS comprising (Emim)Br/potassium phosphate significantly enhanced lipase recovery upon partitioning lipase. Optimization of the composition of the ILATPS using response surface methodology (RSM) significantly improved the purification factor (PF) and partition coefficient (Ke). The influences of crude loading (CL), pH changes, and the presence of NaCl on the recovery performance were also studied. Recovery of E. coli BL21 lipase was accomplished in an (Emim)Br/potassium phosphate ILATPS using 26.5% (w/w) (Emim)Br, 19% (w/w) potassium phosphate at pH 7.6, 3% NaCl and a crude loading of 7% (w/w). Using this ILATPS, lipase was successfully recovered in a single purification step, which gave a yield, PF and Ke of 93.75%, 3.394 and 1.352, respectively. A high PF value indicates that (Emim)Br/potassium phosphate is capable of attaining an excellent degree of lipase purity, suggesting that the proposed ILATPS is suitable for implementation in a large scale process for lipase purification.

This study aimed at recovery of thermostable lipase from Escherichia coli BL21 using porous glass beads grafted with polyethylene glycol (PEG) in aqueous impregnated resins system (AIRS). The influencing parameters such as concentration and pH of extraction solution, concentration of NaCl, size of the beads, and pH of the desorption solution on the partition behaviour of lipase were evaluated. Smaller adsorbent (4 mm) had a 65.5% of recovery yield with approximately two-fold higher purification factor compared to that obtained with the larger adsorbent. Recombinant lipase was purified successfully using AIRS with a purification factor of 7.6 and yield of 78.4% under optimum conditions of 18% (w/w) PEG 4000, 10% (w/w) of potassium citrate at pH 9 with 3% (w/w) of NaCl. Optimum desorption was obtained with 4.0 mm of porous glass beads at pH 9.

The α- and γ-mangostins from Garcinia mangostana pericarps (GMP) exhibit antioxidant, anti-bacterial, anti-inflammatory and anti-tumor properties. The extraction yields α- and γ-mangostins are often limited by the presence of the GMP cell walls. Therefore, the extraction and recovery of mangostins from GMP with an Aspergillus niger cellulase-assisted aqueous micellar biphasic system (CA-AMBS) was developed for enhanced yield of mangostins. Effects of the concentration of cellulase, the incubation time and the temperature of the system on the recovery of mangostins were investigated. The optimum condition for the recovery of α- and γ-mangostins was obtained with the addition of 0.5% (w/w) cellulase incubated at 40°C for 2 h. High log partition coefficients of α-mangostins (log Kα 4.79 ± 0.02) and γ-mangostins (log Kγ 4.02 ± 0.02) were achieved. High yields of α-mangostins (73.4%) and γ-mangostins (14.0%) were obtained from the micelle-rich bottom phase with final concentrations of 3.67 mg/mL and 0.70 mg/mL, respectively. The back-extraction of mangostins was performed with the addition of 30% (w/w) of isopropanol and 0.05 M of KCl at pH 9 to the bottom phase of the CA-AMBS. The yields of the α- and γ-mangostins from GMP were considerably enhanced with the CA-AMBS and the direct recovery of mangostins was demonstrated without additional downstream processing steps.

Ionic liquids-based aqueous two-phase system (ILATPS) offers immediate phase separation and thus reduces the overall processing time, is significantly advantageous as compared to conventional ATPS such as polymer/polymer ATPS and polymer/salt ATPS. In this study, ILATPSs composed of imidazolium-based ionic liquid (IL) and salt were experimentally evaluated for their efficiencies in recovering Bacillus cereus cyclodextrin glycosyltransferase (CGTase) from fermentation broth. The phase-forming behavior of 1ethyl-3-methylimidazolium tetrafluroborate, (Emim)BF4/sodium citrate ILATPS and (Emim)BF4/sodium carbonate ILATPS were first studied by constructing the binodal curves. Effects of the ILs concentration, pH value, feedstock loading, and addition of sodium chloride (NaCl) on the recovery of CGTase in ILATPS were investigated. The optimum conditions for the recovery of CGTase were obtained in an ILATPS consisting of 35% (w/w) (Emim)BF4, 18% (w/w) sodium carbonate and 3% (w/w) NaCl. Experimental results showed that 78% of CGTase could be recovered in the IL-rich phase in single-step purification with a purification fold (PF) of 15.4. The high PF indicates that this ILATPS is feasible to be applied in the recovery and separation of CGTase from the fermentation broth.

The partitioning of β-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of β-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of β-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of β-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the β-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of β-mannanase from fermentation broth in one single-step strategy.

An aqueous two-phase system (ATPS) composed of thermo-separating polymer and salt was deployed in this study for the recovery of Bacillus cereus cyclodextrin glycosyltransferase (CGTase) and recycling of phase components simultaneously. CGTase was recovered using ATPS over two successive extractions and the recycled phase-forming polymers were utilized for subsequent extraction. ATPSs constructed by different molecular weight of the thermo-separating copolymers [ethylene oxide–propylene oxide (EOPO)] and potassium phosphate salt were tested for their effects on partitioning of the enzyme CGTase into top phase. Besides, the effects of phase composition, tie-line length (TLL), crude load, volume ratio (VR) and pH of the ATPS on the partitioning behavior of CGTase were studied to achieve optimum purification condition. Copolymer recovery was measured in each successive ATPS. The optimum purification of enzyme CGTase was obtained in the EOPO 3900/phosphate ATPS comprising TLL of 41.2% (w/w), VR of 1.25, and crude load of 20% (w/w) and pH of 7.0. CGTase from B. cereus was successfully purified by the ATPS up to 13.1-fold with a yield of 87% and the EOPO 3900 could be recovered up to 80% after the first enzyme extraction from ATPS.

In this works, the polymer–salt aqueous two phase system (ATPS) which is polyethylene–glycol (PEG) with sodium citrate was constructed to purify the enzyme CGTase from fermentation broth. Impacts of parameters such as phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and the addition of sodium chloride (NaCl) on the partition behavior of cyclodextrin glycosyltransferase (CGTase) were investigated. The study exhibited that the optimum system for the purification of the enzyme CGTase was achieved on the 19.0% PEG and 11.5% citrate system with TLL of 38.89% (w/w), VR of 2.0, with additional 4% (w/w) NaCl and 20% crude load at pH 7.0. CGTase from Bacillus sp. was partially purified by the ATPS up to 16.3-fold with a yield of 70%.

A polymer–salt aqueous two-phase system (ATPS) consisting of polyethylene–glycol (PEG) with sodium citrate was developed for direct recovery of a bacteriocin-like inhibitory substance (BLIS) from a culture of Pediococcus acidilactici Kp10. The influences of phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and sodium chloride (NaCl) on the partition behaviour of BLIS was investigated. Under optimum conditions of ATPS, the purification of BLIS was achieved at 26.5% PEG (8000)/11% sodium citrate with a TLL of 46.38% (w/w), VR of 1.8, and 1.8% crude load at pH 7 without the presence of NaCl. BLIS from P. acidilactici Kp10 was successfully purified by the ATPS up to 8.43-fold with a yield of 81.18%. Given that the operation of ATPS is simple, environmentally friendly and cost-effective, as it requires only salts and PEG, it may have potential for industrial applications in the recovery of BLIS from fermentation broth.

An extractive bioconversion with Bacillus cereus cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) in aqueous two-phase system (ATPS) was investigated for the synthesis and recovery of cyclodextrins (CDs). Optimum condition for the extractive bioconversion of CDs was achieved in ATPS consisted of 7.7% (w/w) polyethylene glycol (PEG) 20,000 and 10.3% (w/w) dextran T500 with volume ratio (VR) of 4.0. Enzymatic conversion of starch occurred mainly in dextran-rich bottom phase whereas the product, CDs was transferred to top phase and a higher partition coefficient of CDs was achieved. Repetitive batch of CDs synthesis was employed by replenishment of the top phase components and addition of starch every 8 h. An average total CDs concentration of 13.7 mg/mL, (4.77 mg/mLa-CD, 5.02 mg/mLb-CD and 3.91 mg/ mLc-CD) was recovered in the top phase of PEG 20,000/dextran T500 ATPS. This study showed the effectiveness of ATPS application in extractive bioconversion of CDs synthesis with B. cereus CGTase.