Crude Loading Research Articles

Application of Alcohol-Salt Aqueous Biphasic System for the Recovery of Ectoine

Ectoine is an osmoregulatory compound synthesized by halophiles which has attracted attention in the biotechnology, pharmaceuticals, and cosmetics industries due to its stabilizing and protective properties. Conventional methods for ectoine recovery are complex, costly, and often result in low yields. Therefore, there is a growing interest in exploring simple and cost-effective strategies for ectoine recovery. The aqueous biphasic system (ABS) has been employed for the recovery and purification of numerous biocompounds, but the study of low-molecular weight compounds partitioning in ABS remains limited. This study aimed to investigate the feasibility of alcohol–salt ABS for ectoine recovery from Halomonas salina DSM5928T cells. The influences of types and compositions of phase-forming components, crude load concentration, pH, and adjuvants on ectoine recovery were evaluated. Results revealed that ectoine favoured partitioning into the salt-rich bottom phase of alcohol–salt ABS owing to its inherent hydrophilic characteristic. ABS consisting of 16% (w/w) 1-propanol, 20% (w/w) sulphate at pH 6.0, 30% (w/w) crude load, and 1% (w/w) sodium chloride resulted in a partition coefficient (KE) of 9.61 ± 0.05 and a yield (YB) of 97.50% ± 0.21. A purity (PE) of 86.73% was achieved with the 1-propanol-sulphate ABS. Alcohol–salt ABS proved to be an effective approach for ectoine recovery, meeting the raising market demand for industrial applications.

Influence of crude oil pollution on the content and electrophoretic spectrum of proteins in Carex hirta plants at the initial stages of vegetative development

The role of proteins in the general adaptive response of Carex hirta plants to soil pollution by crude oil has been studied. It was established that a possible element of the process of adaptation of C. hirta plants to combined stress – conditions of soil polluted by crude oil – may be the synthesis of stress proteins – high molecular weight of more than 60 kD and low molecular weight, not exceeding 22–45 kD. The synthesis of all 5 HSP families was detected in the leaves and rhizomes, and only sHSP (starting from Mr 32 kD), Hsp 60 and Hsp 100 proteins were synthesized in the roots under the influence of crude oil pollution. The development of C. hirta adaptation syndrome under the influence of crude oil pollution of the soil was promoted by enhanced synthesis of proteins with Mr 85, 77, 64, 60 and 27 kD in the leaves, 118 and 41 kD in the rhizomes and proteins with Mr 105, 53, 50 and 43 kD in the roots of the plants. The decrease in the amount of proteins with Mr 91, 45, 28 kD in the leaves, proteins with Mr 85, 76 and 23 kD in rhizomes and proteins with Mr 64 and 39 in the roots of C. hirta plants under conditions of crude oil polluted soil could be a consequence of inhibition of synthesis or degradation of protein molecules providing the required level of low molecular weight protective compounds in cells. The root system and rhizomes of C. hirta plants undergo a greater crude oil load, which leads to increased protein synthesis in these organs and decreased in the leaves, correspondingly. However, a decrease in protein content in the leaves may indicate their outflow in the roots and rhizomes. Сrude oil contaminated soil as a polycomponent stressor accelerated the aging of leaves of C. hirta plants, which could be caused by increased synthesis of ABA. ABA in its turn induced the synthesis of leaf-specific protein with Mr 27 kD. These proteins bind significant amounts of water with their hydrate shells maintaining the high water holding capacity of the cytoplasm under drought conditions. ABA inhibits the mRNA synthesis and their corresponding proteins, which are characteristic under normal conditions, and induces the expression of genes and, consequently, the synthesis of specific proteins including 27 kD protein. By stimulating the expression of individual genes and the synthesis of new polypeptides, ABA promotes the formation of protective reactions and increases the resistance of plants to crude oil pollution.

Bioseparation of phycocyanin from Phormidium tergestinum using an aqueous two-phase system

This study aimed at purification of phycocyanin (PC) from Phormidium tergestinum using an aqueous two-phase system (ATPS) comprised of polyethylene glycol (PEG) and salts. The partitioning efficiency of PC in ATPS and the effect of phase composition, pH, crude loading, and neutral salts on purification factor and yield were investigated. Results showed that PC was selectively partitioned toward bottom phase of the system containing potassium phosphate. Under optimum conditions of 20% (w/w) PEG 4000, 10% (w/w) potassium phosphate, 20% (v/v) crude load at pH 7, with addition of 0.5% (w/w) NaCl, PC from P. tergestinum was partially purified up to 5.34-fold with a yield of 87.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of PC was ∼19 kDa. Results from this study demonstrated ATPS could be used as a potential approach for the purification of PC from P. tergestinum.

Partitioning of bio-active compounds from rinds of garcinia indica using aqueous two-phase system: Process evaluation and optimization

The simultaneous extraction of anthocyanins (ACNs), garcinol (GL), isogarcinol (IGL) and hydroxycitric acid (HCA) from the rinds of Garcinia indica (GI) fruit and their partition behaviour in the aqueous two-phase system (ATPS) formed by 1-propanol-(NH4)2SO4 was investigated. The effect of different variables that govern the partition characteristics were studied and the amount of crude loaded into the ATPS, the concentration of 1-propanol and (NH4)2SO4 were found to be the significant variables. The Response Surface Methodology (RSM) was adopted to optimize the process variables through desirability based multi-response optimization for the partitioning of GL and IGL to the alcohol rich top phase and ACNs and HCA to the salt-rich bottom phase by considering the partitioning coefficients (K) and extraction efficiency of all the four bioactive components as responses. A ATPS consisting of 15.202% (w/w) 1-propanol, 10.242% (w/w) (NH4)2SO4 having the TLL of 28.505% (w/w) at a crude load of 25% (w/w) able to partition 97.39% GL (K = 370.770) and 92.38% IGL (K = 120.581) in the top phase and 99.19% ACNs (K = 0.080) and 99.83% HCA (K = 0.016) in the bottom phase with a purity higher than 99% by implementing secondary ATPS.

English

The aim of this paper is to present the first record of ctenophore species Pleurobrachia pileus (O. F. Muller, 1776) in the coral reef as was recently found in Iraqi marine waters. The specimens were collected from two sites, the first was in Khor Abdullah during May 2015, and the second site was located in the pelagic water of the coral reef area, near the Al-Basrah deep sea crude oil marine loading terminal. Three samples were collected at this site during May 2015, February and March 2018 which showed that P. pileus were present at a densities of 3.0, 2.2 and 0.55 ind./ m3 respectively. The species can affect on the abundance of other zooplankton community through predation. The results of examining the stomach contents revealed that they are important zooplanktivorous species; their diets comprised large number of zooplankton as well as egg and fish larvae. The calanoid copepods formed the highest percentage of the diet, reaching 47%, followed by cyclopoid copepods 30%, and then the fish larvae formed 20% of the diet. The current investigation showed that the density of zooplankton decreased significantly in the second site of all the groups, but the highest was in Calanoida group (324 ind./ m3) coincident with the presence of noticeable numbers of P. pileus (0.55-3.0 ind./ m3), in the same site compared to the density of Calanoida in the first site, which amounted to 991ind./ m3; as well, the increase was noticeable in relation to the rest of the other groups of zooplankton during the same period; such a state confirms the effect of predation by P. pileus on the zooplankton community. The study recommended that more attention should be given to investigate this group of zooplankton, and specific care should be exerted in preserving the specimens collected from the sea.

Primary purification of intracellular Halomonas salina ectoine using ionic liquids-based aqueous biphasic 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.

Optimization of aqueous two-phase partitioning for purification of recombinant Eupenicillium terrenum fructosyl peptide oxidase

Abstract Fructosyl peptide oxidase (FPOX), a flavoenzyme belonging to the class of oxidoreductases, has been used as a diagnostic enzyme for HbA1c measurement test. Design of experiments methodology was applied to evaluate the partition behavior of recombinant Eupenicillium terrenum FPOX in aqueous two-phase systems (ATPS). Different ATPS parameters including polyethylene glycol (PEG) molecular weight, PEG concentration, salt type, salt concentration, pH, system buffer type, system buffer concentration, temperature, neutral salt type, neutral salt concentration, and crude enzyme loading were investigated using a Plackett–Burman design (PBD). Taking into consideration an increase in partition coefficient (KE), PEG concentration, pH, system buffer concentration, and NaCl concentration, which played significant roles in enzyme partition, were chosen for further optimization by Box–Behnken design (BBD) in response surface methodology (RSM). The optimized condition led to maximum recovery (106.8%), yield (95.1%) and purification factor (42.4) values which were predicted to be achieved in ATPS formed by 14.0% (w/w) PEG-4000, 15.0% (w/w) Na2CO3, 5.0% (w/w) NaCl and 55.0 mM potassium phosphate buffer with pH 8.0. The partitioned recombinant FPOX was obtained as a single band into the upper PEG-rich phase and its specific activity was calculated to be 201.23 ± 2.1 U/mg. In summary, our data showed that the ATPS optimization using design of experiments can be an appropriate method for recovery of recombinant FPOX.

Aqueous two phase based selective extraction of mannose/glucose specific lectin from Indian cultivar of Pisum sativum seed

Pisum sativum lectin (Psl) being a high-value protein has marked its application in the biomedical and therapeutic field. Aqueous two phase extraction (ATPE) was implemented as a selective partitioning technique for the partial purification of Psl from its seeds. PEG/citrate based biodegradable aqueous two phase system (ATPS) was screened and the factors such as the type and concentration of citrate salts, molar mass and concentration of polyethylene glycol (PEG), tie line length (TLL) and additive (NaCl) concentration, pH, crude load and volume ratio were studied for the selective partition of Psl. The Psl was successfully extracted to the top phase in the ATPS formed with 18% PEG 6000/16% sodium citrate at 41.01% TLL, 2% NaCl and pH of 7.5. A volume ratio of 0.76 and a crude load of 20% showed maximum activity yield of 122.12% with the purification factor of 16.26. The subunits of Psl namely α and β were identified with a molecular weight of 6 and 18 kDa respectively during the purity analysis using SDS PAGE and HPLC.

Evaluating the possibility of utilizing hollow fiber membranes for recovering VOC generated by oil tankers

ABSTRACT In this research, we evaluated the possibility of using hollow fibre membranes combined with a VOC recovery system to recover high-capacity of VOCs in crude oil loading conditions. We initially investigated the performance of a basic membrane module under various conditions, and then developed and tested a large-scale membrane system. The ranges of conditions were determined based on practical and operational environments in terminals and tankers. The results of the basic membrane module experiment indicated that the recovery ratio of a multi-hydrocarbon mixture gas, in contrast to that of single hydrocarbon gases, increases proportionally with an increase in flow rate up to a point and then decreases in a curvilinear manner. Our findings demonstrate the strong dependence of VOC recovery ratio on the pressure and flow rate, while the influence of temperature is less significant. A large-scale membrane test was then conducted based on the optimised conditions of the basic membrane module, and the total recovery ratio between flow rates of 500 (N m3)/h and 1000 (N m3)/h were compared. This study demonstrates the possibility of utilising membranes for large-scale VOC separation and the necessity for further research to minimise recovery losses due to membrane module enlargement and moisture intrusion.

Purification of a Bacteriocin-Like Inhibitory Substance Derived from Pediococcus acidilactici Kp10 by an Aqueous Micellar Two-Phase System.

Aqueous micellar two-phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behavior of biomolecule between a micelle-rich and a micelle-poor phase. In this study, an AMTPS composed of a nonionic surfactant, Triton X-100 (TX-100) was used for purifying a bacteriocin-like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behavior and activity yield of the BLIS were investigated. The obtained coexistence curves showed that the mixtures of solutions composed of different surfactant concentrations (5-30% w/w) and 50% w/w crude load were able to separate into two phases at temperatures of above 60 °C. The optimum conditions for BLIS partitioning using the TX-100-based AMTPS were: TX-100 concentration of 22.5% w/w, CFCS load of 50% w/w, incubation time of 30 min at 75 °C, and back-extraction using acetone precipitation. This optimal partitioning resulted in an activity yield of 64.3% and a purification factor of 5.8. Moreover, the addition of several additives, such as sorbitol, KCl, dioctyl sulfosuccinate sodium salt, and Coomassie® Brilliant Blue, demonstrated no improvement in the BLIS separation, except for Amberlite® resin XAD-4, where the activity yield was improved to 70.3% but the purification factor was reduced to 2.3. Results from this study have demonstrated the potential and applicability of TX-100-based AMTPS as a primary recovery method for the BLIS from a complex fermentation broth of P. acidilactici Kp10. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2719, 2019.

Optimization of recovery of esterase from Serratia marcescens using combination of the solvent impregnated resin and aqueous two-phase extraction techniques

ABSTRACT The performance of tunable aqueous polymer phase impregnated resins (TAPPIR) which is the combination of the solvent impregnated resin principle and an aqueous two-phase system for the separation of esterase from Serratia marcescens was evaluated in this study. Different molecular weight of polyethylene glycol (PEG) (2000, 4000 and 6000) at concentration ranging from 5% to 20% (w/w) and potassium citrate were used to construct the aqueous phase in TAPPIR technology. Optimum composition of PEG and salt for esterase partitioning was determined using response surface methodology. The optimum condition for the purification of esterase was impregnation of 25% (w/w) of PEG 2000 into 4 mm porous glass beads and extraction of esterase using 15% (w/w) potassium citrate at pH 8 containing 12% (w/w) crude loading with the addition of 4% (w/w) NaCl. Esterase from S. marcescens was successfully purified by the TAPPIR technology up to 5.32 of purification factor with a yield of 75.98%.

Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol.

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

Direct recovery of Bacillus subtilis xylanase from fermentation broth with an alcohol/salt aqueous biphasic system

Xylanase enzyme degrades linear polysaccharide β-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (KE), selectivity (S) and yield (YT) of xylanase in top phase of the ABS were measured. Highest KE (6.58±0.05) and selectivity (4.84±0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High YT of 71.88%±0.15 and a purification fold (PFT) of 5.74±0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanol/sulphate ABS with a distinct single band.

Recovery of intracellular ectoine from Halomonas salina cells with poly(propylene) glycol/salt aqueous biphasic system

Abstract Ectoine is a compatible cell protectant sourced from halophilic and halotolerant microorganisms. Various biotechnological applications of ectoine have urged the development of cost-effective downstream processing of ectoine. Aqueous biphasic system (ABS) composed of poly(propylene) glycol (PPG) 425 and salt was adopted for the recovery of intracellular ectoine from Halomonas salina DSM5928T fermentation. Phosphate, citrate and sulfate salts were evaluated for their effects on the recovery of intracellular ectoine using the PPG/salt ABS. In this study, ectoine exhibited a salt-rich bottom phase preference in the PPG/salt ABS because of the hydrophilic surface properties of ectoine. The effects of concentrations of PPG 425 and salts, crude load, pH and concentrations of neutral salts (NaCl and KCl) on the recovery of ectoine were investigated. Optimum recovery of intracellular ectoine was obtained with ABS (pH 5.5) composed of 30% (w/w) PPG 425 and 20% (w/w) sulfate; 15% (w/w) crude load; and 1.5% (w/w) of KCl. High yield of 94.7% of intracellular ectoine was successfully recovered from H. salina cells with an enrichment factor (EF) of 1.7 and a purity of 87.03%.

Aqueous biphasic system for the partial purification of Bacillus subtilis carboxymethyl cellulase

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.

Purification of β-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system.

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.

Recovery and partial purification of thermophilic β-xylosidase derived from recombinant Bacillus megaterium MS941 by aqueous two-phase system

ABSTRACTA polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinant Bacillus megaterium MS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (VR), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, VR of 2.3, crude loading (CL) of 30% (w/w) at pH 6.

Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10.

An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.

A novel aqueous micellar two-phase system composed of surfactant and mannitol for purification of polygalacturonase enzyme from Durio zibethinus Murray and recycling phase components

ABSTRACTA novel aqueous micellar two-phase system composed of surfactant and mannitol was employed for the first time to purify polygalacturonase from Durio zibethinus Murray. The influences of different parameters such as type of surfactants, composition of surfactant/mannitol, pH and crude load on polygalacturonase partitioning were investigated. The results indicated that the polygalacturonase was recovered in 25% Tween 80 and 22% mannitol, at 68.2% of Tie line length, crude load of 30% at pH 5.0. The enzyme was successfully recovered with a high purification factor of 16.1 and yield of 97.3% while phase components were also recovered and recycled above 95%.

Direct recovery of cyclodextringlycosyltransferase from Bacillus cereus using aqueous two-phase flotation

Purification of cyclodextrin glycosyl transferase (CGTase) from Bacillus cereus using polyethylene glycol (PEG)-potassium phosphates aqueous two-phase flotation (ATPF) system was studied in this paper. The effects of varying PEG molecular weight, tie-line length (TLL) value, volume ratio (VR), pH value, crude concentration and gas nitrogen flotation time were investigated. The optimal condition for purification of CGTase was attained at 18.0% (w/w) PEG 8000, 7.0% (w/w) potassium phosphates, VR of 3.0, 20% (w/w) crude load at pH 7, and 80 min nitrogen flotation time at a flow rate of 5 L/min. With this optimal condition, purification factor (PFT) of 21.8 and a yield (YT) of 97.1% were attained. CGTase was successfully purified in a single downstream processing step using the ATPF.