Selective recovery of lactate dehydrogenase using affinity foam

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.     

Effect of invertase on the batch foam fractionation of bromelain

Foam fractionation can be used to enrich a hydrophobic protein such as bromelain from an aerated dilute protein solution because the protein foams. On the other hand, a protein such as invertase, which is hydrophilic, is not likely to foam under similar aerated conditions. While a foam fractionation process may not be approapriate for recovering a hydrophilic protein alone, it is of interest to see how that non-foaming protein affects the foaming protein when the two are together in a mixture. The bromelain enrichment, activity and mass recovery were observed as a function of the solution pH in order to explore how invertase can affect the recovery of bromelain in a foam fractionation process.     

Foam fractionation applications

Biotechnological downstream processing faces several challenges, such as dilute product streams and contained target products which are sensitive to heat, oxidation, other chemicals, etc. State-of-the-art separation methods, e.g. chromatography, are not always the best option due to variable yield losses and high costs. Foam fractionation appears as a promising alternative unit operation in biotechnological downstream processing. From its applications in metal industry and on fish farms, it was developed further towards the recovery of phytonutrients, metabolites and proteins. However, no large scale applications of foam fractionation in biotechnological downstream processing exist yet. This is due to the complexity of various biotechnological media, which makes a universalized approach for systematic process design of protein separations difficult. Ongoing research in the fields of process engineering, surface chemistry and protein chemistry can help to close this gap. Although many different substances, such as detergents, have been separated or recovered using foam fractionation, this review focuses mainly on biotechnological applications, more specifically on protein separation.     

A downstream process with microemulsion extraction for microbial transformation in cloud point system

A downstream process strategy for a whole microbial transformation to produce l-phenylacetylcarbinol (PAC) in a nonionic surfactant Triton X mediated cloud point system was developed. By application of a Winsor I microemulsion, the product and the nonionic surfactant in the microbial transformation broth was separated successfully. Then the nonionic surfactant was recovered with a Winsor II microemulsion. In a single stage Winsor I microemulsion extraction process, the product recovery ratio 76.9% and the nonionic surfactant recovery ratio 66.5% were achieved. A discrete countercurrent extraction operation was also carried out to improve the separation efficiency. Finally, character of the recovery nonionic surfactant was also examined.     

泡沫分离技术研究进展

本综述了泡沫分离技术的研究进展,介绍了分离过程中操作参数（气流速度,泡沫区高度,液相高度,温度）,溶液体系性质（进料浓度,pH值,离子强度,表面活性剂种类）,分离设备等因素对分离效果的影响,并介绍了泡沫分离在固体粒子,溶液中的离子分子,废水处理以及生物产品的分离过程中的应用,指出了泡沫分离技术目前存在的问题及发展方向。     

Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions

The lipopeptide, surfactin, is produced by Bacillus subtilis. A study has been made on large-scale production of this surfactant. A good yield was obtained from a glucose substrate fermentation by continuously removing the product by foam fractionation. The surfactin could be easily recovered from the collapsed foam by acid precipitation. The yield was also improved by the addition of either iron or manganese salts. Hydrocarbon addition to the medium, which normally increases biosurfactant production, completely inhibited surfactin production by B. subtilis.     

Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.     

Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents

The effects of foaming on the production of the hydrophobin protein HFBII by fermentation have been investigated at two different scales. The foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed-batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without the antifoam Struktol J647. In the presence of antifoam, HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam, a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment, averaged over all overflowed material throughout the whole fermentation, of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L⁻¹ in the fermenter. It is also shown that uncontrolled foaming resulted in reduced concentration of biomass in the fermenter vessel, affecting total production. This study illustrates the potential of foam fractionation for efficient recovery of HFBII through simultaneous high enrichment and recovery which are greater than those reported for similar systems.     

Carbon dioxide induced soybean protein precipitation: protein fractionation, particle aggregation, and continuous operation

A novel protein fractionation technique using a volatile electrolyte has been developed. Carbon dioxide was used to isoelectrically precipitate 80% and 95% pure glycinin and beta-conglycinin fractions from soybean isolate. The protein fractions precipitated as primary particles 0.2-0.3 microm in diameter, which under optimum conditions may be recovered as aggregates up to 500 microm in diameter. The dependency of protein fractionation efficiency on aggregate settling rates has been demonstrated. The isoelectric points of the two main soybean fractions, glycinin and beta-conglycinin, were calculated to be pH 5.2 and 4.95, respectively. Solution pH was accurately controlled by pressure in the isoelectric pH range of the different soybean protein fractions, and a pH "overshoot" was eliminated. Volatile electrolyte technology was also applied to a continuous process in order to eliminate the particle recovery concerns associated with batch precipitation and to demonstrate the potential for scale-up. Glycinin was effectively recovered on-line (94% glycinin recovery) with a purity approaching that of the batch process (95%).     

Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.

A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.     

Purification and partial characterization of the extracellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602

The gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase, or endopeptidase I, from Bacillus sphaericus 9602 was purified to apparent protein homogeneity. The purification was achieved by a six-step procedure: ammonium sulfate fractionation, phenyl-Sepharose chromatography, two consecutive DEAE-Trisacryl chromatographies, chromatofocusing and Sephacryl S-200 permeation chromatography. The enzyme was purified 5000-fold with a 38% recovery of lytic activity. It is an acidic protein (pI 5.4) of hydrophobic nature. Kinetic studies have shown a Km value of 0.57 mM and an apparent Vmax of 8.3 mumol min-1 (mg enzyme)-1 with N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)meso-diaminopimelyl (L)-D-[14C]alanine as substrate. The enzyme was inhibited by o-phenanthroline and EDTA and was reactivated by zinc, cobalt and manganese ions; thus endopeptidase I is a metallo enzyme, probably a zinc enzyme. Moreover it is a heat-stable protein with an apparent inactivation temperature of 80 degrees C.     

Isolation and purification of protease from human placenta by foam fractionation

The effect of operating parameters like pH, protein concentration, column geometry, and gas flow rate on the separation efficiency of proteolytic enzymes from crude human placental homogenate has been studied in a batch foam column. Purification has been found to be optimum at pH 8.0, close to the isoelectric pH, at which the surface adsorption of the protein on the foam bubbles is maximum. Both purification and recovery varied significantly with total protein concentration. Stable bubble formation was hindered at lower protein concentrations, while extraneous proteins rather than the protease were preferentially adsorbed at higher protein concentrations, decreasing the purification efficiency. Column diameter and column height should be optimized for any specific feed protein concentration and gas flow rate. However, the enrichment ratio was found to decrease with the increase in flow rate. The results indicate that foam fractionation is an effective separation process for recovering valuable biochemicals from biological materials.     

Protein recovery from surfactant precipitation

The recovery of lysozyme from an aqueous solution containing precipitated lysozyme-AOT complexes formed by the direct addition of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) to a lysozyme solution was studied using both solvents, and a counterionic surfactant. Ethanol,methanol and solvent mixtures dissolved the surfactant precipitate and recovered lysozyme as a solid. Recovery efficiency and protein stability varied with the type of solvent used. An entirely different method of recovery was also evaluated using a counterionic surfactant: tri-octylmethylammonium chloride (TOMAC) which bound to AOT releasing lysozyme into solution.Complete recovery (100%) of lysozyme was achieved at a molar ratio of 2:1(TOMAC:AOT), and the original protein activity was maintained in the final aqueous phase.The recovered lysozyme retained its secondary structure as observed in circular dichroism(CD) spectra. Specific activity studies show that counterionic surfactant extraction does not alter the biological activity of the enzyme.     

Cytochemical location of urease in the cell wall of two different lichen phycobionts

The enzyme urease has been located in the cell wall of recently isolated phycobionts from Evernia prunastri and Xanthoria parietina lichens. Cytochemical detection is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea in the presence of cobalt chloride. Cellular fractionation reveals that about 80% of total urease activity was associated to the cell wall on both phycobionts whereas only 20% was recovered as soluble protein.     

Protein recovery using gas–liquid dispersions

Two separation techniques, foam separation and colloidal gas aphrons (CGAs), both of which are based on gas–liquid dispersions, are compared as potential applications for protein recovery in downstream processing. The potential advantages of each method are described and the concentration and selectivity achieved with each method, for a range of proteins is discussed. The physical basis of foam separation is the preferential adsorption of surface active species at a gas–liquid interface, with surface inactive species remaining in bulk solution. When a solution containing surface active species is sparged with gas, a foam is produced at the surface: this foam can be collected, and upon collapse contains surface active species in a concentrated form. CGAs are microbubble dispersions (bubble diameters 10–100 μm) with high gas hold ups (>50%) and relatively high stability, which are formed by stirring a surfactant solution at speeds above a critical value (typically around 5000 rpm). It is expected that when proteins are brought into contact with aphrons, protein adsorbs to the surfactant through electrostatic and/or hydrophobic forces. The aphron phase can be separated easily from the bulk solution due to its buoyancy, thus allowing separation of protein in a concentrated form.     

The application of foaming for the recovery of Surfactin from B. subtilis ATCC 21332 cultures

Foaming, a proficient method for the recovery of surface active solutes from dilute solutions, was successfully applied for the concentration of the lipopeptide biosurfactant Surfactin from B. subtilis ATCC 21332 cell culture broths. Foaming was only partially successful in concentrating Surfactin when applied as a separate semi-batch unit downstream of the cell culture stage. Surfactin partitioned strongly into the foam during the latter stages of the semi-batch process, where enrichments of over 50 could be obtained. However, simultaneous high enrichments and recoveries of Surfactin could not be obtained as the majority of Surfactin (around 70% of the total recovered) was produced at a low concentration during the early stages of foaming. Foam fractionation was considered for both cell free and cell containing broths; the presence of cells increased the foamability of the solution and therefore yielded more dilute Surfactin preparations. More favourable recovery and enrichment of Surfactin occurred when foaming was integrated with the cell culture stage. The use of low stirrer speeds was essential in producing foam at a controlled rate. By collecting fractions of the foam produced between 10 and 30 hours, from systems stirred at 166 and 146 rpm, a highly concentrated Surfactin extract could be obtained. The Surfactin concentration in the foam was 1.22 and 1.67 g l(-1) respectively, which represented enrichments and percent recoveries of over 60. This study points to the utility of foaming as a method for the recovery of surface-active fermentation products, particularly when used in an integrated production/recovery system.     

Colloidal gas aphrons: A novel approach to protein recovery

Sebba (1987) defined colloidal gas aphrons (CGA) as microbubbles stabilized by surfactant layers, which are created by stirring surfactant solutions at speeds greater than a critical value. A high shear impeller is used for stirring and critical values for the impeller speed must be exceeded to create these stable gas liquid dispersions (typically >5000 rpm). Although there have been no previous reports of direct protein recovery using CGA, it is likely that, with appropriate choice of surfactant, proteins should adsorb to these surfactant bubbles by means of electrostatic and/or hydrophobic interactions. This is the basis of this study, in which the use of CGA for protein recovery from aqueous solution is considered. A surfactant which has been characterized previously for generation of CGA was chosen (Jauregi et al., 1997), i.e., the anionic surfactant sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT). Lysozyme, a well-characterized protein, was chosen as the protein to be recovered. Lysozyme was recovered successfully from aqueous solution using CGA generated from AOT. At optimum conditions, lysozyme recovery, enrichment ratio, and separation ratio were 95%, 19 and 302 respectively, with enzyme activity maintained. These results indicate the exciting potential of this technique. A wide range of process conditions including initial concentration of protein and surfactant, surfactant/protein molar ratio, pH, and ionic strength were considered. High recoveries and enrichments were generally obtained at protein concentrations 0.11 mg/mL. However, at high ionic strength (0.29M) poor separation and recoveries were obtained at low protein concentrations (counter-ions diminishing electrostatic interactions between protein and aphrons at this condition). In general, (ns/np)a was determined to be between 10 and 16 for experiments in which high levels of recovery/separation parameters were found. For most conditions, protein precipitation was observed; however, this precipitate could be resolubilized without loss of enzyme activity.     

Separation of soybean saponins from soybean meal by a technology of foam fractionation and resin adsorption

Foam fractionation and resin adsorption were used to recover soybean saponins from the industrial residue of soybean meal. First, a two-stage foam fractionation technology was studied for concentrating soybean saponins from the leaching liquor. Subsequently, resin adsorption was used to purify soybean saponins from the foamate in foam fractionation. The results showed that the enrichment ratio, the recovery percentage, and the purity of soybean saponins by using the two-stage foam fractionation technology could reach 4.45, 74%, and 67%, respectively. After resin adsorption and desorption, the purity of soybean saponins in the freeze-dried powder from the desorption solution was 88.4%.     

Efficient recovery of secretory recombinant proteins from protease negative mutant Escherichia coli strains

Osmotic shock and lysozyme/EDTA methods were used to recover secreted recombinant proteins from protease negative mutant strains of E. coli. Up to 80% of protein A--lactamase fusion protein was recovered from protease negative mutants by simple osmotic shock. Fractionation by lysozyme/EDTA treatment, increased the recovery of protein A--lactamase fusion protein from the mutant strain up to 93%. Mild fractionation condition allowed efficient recovery of secreted protein from protease negative mutant strains, but not from the parent strain possessing proteases. © Rapid Science Ltd. 1998     

Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.