Lysozyme extraction from hen egg white in an aqueous two-phase system composed of ethylene oxide–propylene oxide thermoseparating copolymer and potassium phosphate

The recovery and purification of lysozyme from hen egg white has been investigated in an aqueous two-phase systems composed of thermoseparating random copolymers of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate. In the primary extraction step lysozyme was satisfactorily partitioned to the top polymer-rich phase in a system composed of 40% (w/w) EO₅₀PO₅₀, 10% (w/w) potassium phosphate, and 0.85 M sodium chloride at pH 9.0, diluted 3-fold with crude egg white, where contaminating proteins were discarded in the bottom phosphate-rich phase. After the primary phase separation the upper EO₅₀PO₅₀ phase was removed and subjected to temperature-induced (65 °C) phase separation, which resulted in the partitioning of pure lysozyme to the top water phase. The separation system was found to be efficient in achieving the purification of lysozyme in a high yield of 85% and specific activity of 32,300 U/mg of protein, with a purification factor of 16.9 and a concentration of lysozyme in the water phase of 2.3 g/l in two extraction steps.     

Supermacroporous hydrophobic affinity cryogels for protein chromatography

N-Methacryloyl-l-tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacroporous cryogel [PHEMATrp] was prepared for lysozyme purification form chicken egg white. MATrp was synthesized by reacting methacryloyl chloride with l-tryptophan methyl ester and provided hydrophobic functionality to the cryogel. PHEMATrp cryogel with 60–100 μm pore size was obtained by free radical polymerization of HEMA and MATrp having a specific surface area of 50 m²/g. PHEMATrp cryogel was characterized by swelling studies, FTIR and SEM. The equilibrium swelling ratios of the cryogels were 7.18 g H₂O/g for PHEMA and 6.99 g H₂O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concentration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8 mg/g (0.49 and 7.85 mg/mL), respectively. Lysozyme molecules were desorbed with 0.5 M ethylene glycol solution with 91% recovery. It was observed that PHEMATrp cryogel can be used without significant decrease in lysozyme adsorption capacity after five adsorption–desorption cycles. PHEMATrp cryogel was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE. Possible denaturation of purified lysozyme was checked with fluorimetric measurements. Specific activity of the purified lysozyme was found as 43,140 U/mg using Micrococcus lysodeikticus as substrate.     

Large-scale separation of clavine alkaloids from Ipomoea muricata by pH-zone-refining centrifugal partition chromatography

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)–acetonitrile–water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their ¹H, ¹³C NMR and ESI-MS data.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Polymer recycling in aqueous two-phase extractions using thermoseparating ethylene oxide–propylene oxide copolymers

This is a study on the recovery and recycling of copolymer in aqueous two-phase systems containing random copolymers of ethylene oxide (EO) and propylene oxide (PO). The random copolymers separate from water solution when heated above the lower critical solution temperature (LCST). The primary phase systems were composed of EOPO copolymer and hydroxypropyl or hydroxyethyl starch. After phase separation the upper EOPO phase was removed and subjected to temperature induced phase separation. Copolymers with different EO/PO compositions have been investigated, EO50PO50 [50% EO and 50% PO (w/w)], EO30PO70 and EO20PO80. The temperature required for thermoseparation decreases when the PO content of the copolymer is increased. The effect on the recovery of copolymer after addition of salts, a second polymer or protein was investigated. The added components increased the recovery of copolymer after thermoseparation, e.g., increased the amount copolymer separated from the water phase after thermoseparation. Recycling of copolymer and measurements of polymer concentrations in the primary top and bottom phases after repeated recycling steps was performed. The fluctuation in polymer concentration of the phases was very small after recycling up to four times. Partitioning of the proteins BSA and lysozyme was studied in primary phase systems after recycling of copolymer. The partition coefficients of total protein and lysozyme was not significantly changed during recycling of copolymer. More than 90% of the copolymer could be recovered in the thermoseparation step by optimising the temperature and time for thermoseparation. In repeated phase partitionings in EOPO–starch systems the EO50PO50 copolymer could be recovered to 77% including losses in primary system and thermoseparation, which is equivalent to a total copolymer reuse of 4.3 times.     

Physical characterisations of a single-stage Kühni-type aqueous two-phase extraction column

The main parameters which influence the behaviour of phase separation in a single-stage Kühni-type aqueous two-phase extraction column containing polyethylene (PEG) and di-potassium hydrogen phosphate were characterised. Two aqueous two-phase system (ATPS) composed of 12% (w/w) PEG 1450 and 12% (w/w) di-potassium hydrogen phosphate (designated as 12/12) and 12% (w/w) PEG 1450 and 11% (w/w) di-potassium hydrogen phosphate (designated as 12/11) were chosen in this study. The hold-up ɛ_D increased with increasing impeller speeds and mobile phase flow rates. Phase separation for the 12/11 system was slower than that for the 12/12 system, which resulted in higher dispersed phase hold-up values for the 12/11 system. For 12/12 system, mass transfer of plasmid DNA (pDNA) from the dispersed mobile phase to the stationary phase increased rapidly with increasing impeller speeds of 130, 160 and 200 rpm which was reflected in the decreased values for C_T/C_To. The degree of back-mixing quantified by the axial dispersion coefficient D_ax was estimated to be 2.7 × 10⁻⁶ m² s⁻¹.     

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture,model of the Escherichia coli cytoplasmic membrane

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters

Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25°C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350 g h⁻¹. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.     

Improving the lactic acid production of Actinobacillus succinogenes by using a novel fermentation and separation integration system

This work describes the development of a novel integrated system for lactic acid production by Actinobacillus succinogenes. Fermentation and separation were integrated with the use of a microfiltration (MF) membrane, and lactic acid was recovered by resin adsorption following MF. The fermentation broth containing residual sugar and nutrients was then recycled back into the fermenter after lactic acid adsorption. This novel approach overcame the problem of product inhibition and extended the cell growth period from 41 h to 120 h. Production of lactic acid was improved by 23% to 183.4 g L⁻¹. The overall yield and productivity for glucose were 0.97 g g⁻¹ and 1.53 g L⁻¹ h⁻¹, respectively. These experimental results indicate that the integrated system could benefit continuous production of lactic acid at high levels.     

Enhancement of pilot scale production of l(+)-lactic acid by fermentation coupled with separation using membrane bioreactor

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l(+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l(+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m²/h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l(+)-lactic acid in an industrial scale.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

高速逆流双水相色谱法纯化卵白蛋白

生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%.     

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A₅₄₅/A₂₈₀ > 4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900 rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5 mm glass beads and 22 min of residence time with a yield of 1.35 mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17 USD/mg, which is underneath the commercial price of a BPE standard (>$30 USD/mg).     

Bio-resolution of a chiral epoxide using whole cells of Bacillus megaterium ECU1001 in a biphasic system

Optically active epoxides can be prepared by kinetic resolution of racemic mixtures using stereospecific epoxide hydrolases. To increase the bio-resolution efficiency of a sparingly water-soluble epoxide (glycidyl phenyl ether, GPE), we investigated the use of organic/aqueous two-phase system. Various conditions were systematically examined and optimized in shake flasks. Isooctane was found to be the most suitable solvent as the organic phase. The phase volume ratio (ϕ_o/w) and biocatalyst concentration were shown to be sensitive parameters affecting both the reaction rate and the enzyme enantiospecificity in the biphase system. An isooctane/aqueous system was developed to overcome the low solubility and instability of GPE in the aqueous phase, resulting in a significant improvement of enatiomeric ratio (E-value) from 39.5 to 94.0 and an average productivity of 18.8 mg GPE/(h g) biocatalyst to 48.9 mg GPE/(h g) biocatalyst, respectively. Resolution of a 90.1 g/l solution of racemic glycidyl phenyl ether in isooctane phase was successfully carried out in a mechanically stirred reactor (120 ml), affording (S)-glycidyl phenyl ether in high (100%) enantiomeric excess with a yield of 44.5%.     

Influence of pH and salt concentration on the cross-flow microfiltration of BSA through a ceramic membrane

In this paper, the influence of pH in the 4–8 interval and NaCl concentration up to 25 mM on the cross-flow microfiltration of BSA was investigated. A tubular ceramic membrane with a pore size of 0.14 μm was employed and its point of zero charge was calculated. The evolution of permeate flow and BSA transmission with time was determined at 45 °C, a cross-flow velocity of 3.5 m/s and a transmembrane pressure of 100 kPa. The curves of permeate flow were explained according to the resistances in series model. Maximum protein transmission was obtained at the isoelectric point of BSA (4.9), with significant transmission also at the point of zero charge of the membrane and null transmission at pH 4 and 8. The highest permeate flow was observed at pH 7 and the lowest at 4.9. Finally, the addition of salt resulted to some extent in an improvement of both protein transmission and permeate flow.     

Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH₄)₂SO₄. 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH₄)₂SO₄ at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH₄)₂SO₄ system and the control, respectively.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.