Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114

The detergent Triton X-114, because of its convenient cloud point temperature (22 °C), has been used extensively to extract membrane proteins and to separate them in two phases according to their hydropathy. The upper detergent-poor phase contains mostly hydrophilic proteins, whereas hydrophobic ones are found mainly in the lower detergent-rich phase. In this work, we developed a method to fractionate membrane proteins and estimate their hydropathy based on a series of cloud point partitions with Triton X-114. With this method, beetroot plasma membrane proteins were separated in different fractions according to their hydropathy, following the binomial distribution law as expected. This method revealed the presence of both hydrophilic and hydrophobic Ca²⁺-dependent protein kinases in those membranes. At least five distinct Ca²⁺-dependent kinases were observed in in-gel kinase activity assays. This separation procedure was also used as the first step in the purification of a hydrophobic 60-kDa kinase.     

Assessing bioavailability of the solubilization of organic compound in nonionic surfactant micelles by dose–response analysis

It is uncertain in some extent that organic compounds solubilized in micelles of a nonionic surfactant aqueous solution are bioavailable directly by the microbes in an extractive microbial transformation or biodegradation process. In this work, a dose–response method, where a bioequivalence concept is introduced to evaluate the synergic toxicity of the nonionic surfactants and the organic compounds, was applied to analyze the inhibition effect of organic compounds (naphthalene, phenyl ether, 2-phenylethanol, and 1-butanol) in nonionic surfactant Triton X-100 micelle aqueous solutions and Triton X-114 in aqueous solutions forming cloud point systems. Based on the result, a mole solubilization ratio of organic compounds in micelle was also determined, which consisted very well with those of classic semi-equilibrium dialysis experiments. The results exhibit that bioavailability of organic compounds solubilized in micelles to microbial cells is negligible, which provides a guideline for application of nonionic surfactant micelle aqueous solutions or cloud point systems as novel media for microbial transformations or biodegradations.     

Speciation of platinum in blood plasma and urine by micelle-mediated extraction and graphite furnace atomic absorption spectrometry

A highly sensitive and selective technique for the speciation of platinum by cloud point extraction prior to determination by graphite furnace atomic absorption spectrometry (GFAAS) was described. The separation of Pt(II) from Pt(IV) was performed in the presence of 4-(p-chlorophenyl)-1-(pyridin-2-yl)thiosemicarbazide (HCPTS) as chelating agent and Triton X-114 as a non-ionic surfactant. The extraction of Pt(II)–HCPTS complex needs temperature higher than the cloud point temperature of Triton X-114 and pH = 7, while Pt(IV) remains in the aqueous phase. The Pt(II) in the surfactant phase was analyzed by GFAAS, and the concentration of Pt(IV) was calculated by subtraction of Pt(II) from total platinum which was directly determined by GFAAS. The effect of pH, concentration of chelating agent, surfactant, and equilibration temperature were investigated. An enrichment factor of 42 was obtained for the preconcentration of Pt(II) with 50 mL solution. Under the optimum experimental conditions, the calibration curve was linear up to 30 μg L^?1 with detection limit of 0.08 μg L^?1 and the relative standard deviation was 1.8%. No considerable interference was observed due to the presence of coexisting anions and cations. The accuracy of the results was verified by analyzing different spiked samples (tap water, blood plasma and urine). The proposed method was applied to the speciation analysis of Pt in blood plasma and urine with satisfactory results.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I_core–hydrophobin I (EGI_core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI_core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI_core–HFBI was quantitatively back-extracted (K_{EGIcore–HFBI}=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI_core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI_core–HFBI into a water phase.     

Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10 mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.     

Studies on the cloud point and micellar phases of triton X-100 using 220 MHz proton magnetic resonance techniques

High-resolution proton magnetic resonance techniques at 220 MHz were employed to follow the transformation of Triton X-100 between its micellar and cloud point phases as a function of temperature. The results obtained suggest that while a phase separation occurs rather sharply above the cloud point, the increase in temperature below the cloud point is accompanied by the gradual formation of very large structures suspended in the aqueous phase. The proton magnetic resonance studies show that the separation of phases, which occurs above the cloud point, appears to be accompanied by a fractionation of the polydisperse detergent. In addition, a lowering of the cloud point of Triton X-100 by dipalmitoyl phosphatidylcholine was observed by visual means and the results are reported here.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

Phase separation temperatures of mixtures of Triton X-114 and Triton X-45: application to protein separation.

Triton X-114 solutions separate above 22 degrees C into two immiscible aqueous phases. The more dense phase is enriched in detergent, and the less dense phase is depleted of detergent, relative to the original single phase. This phenomenon has been used to partition proteins according to hydrophobicity. The phase separation temperature is sensitive to the length of the polyoxyethylene headgroup. When Triton X-45, with a shorter headgroup, is mixed with Triton X-114 in various proportions, the phase transition temperature can be adjusted anywhere between 0 and 22 degrees C. Partitioning properties of the resulting mixtures are similar to those of Triton X-114 alone.     

Phase separation of integral membrane proteins in Triton X-114 solution

A solution of the nonionic detergent Triton X-114 is homogeneous at 0 degrees C but separates in an aqueous phase and a detergent phase above 20 degrees C. The extent of this detergent phase separation increases with the temperature and is sensitive to the presence of other surfactants. The partition of proteins during phase separation in solutions of Triton X-114 is investigated. Hydrophilic proteins are found exclusively in the aqueous phase, and integral membrane proteins with an amphiphilic nature are recovered in the detergent phase. Triton X-114 is used to solubilize membranes and whole cells, and the soluble material is submitted to phase separation. Integral membrane proteins can thus be separated from hydrophilic proteins and identified as such in crude membrane or cellular detergent extracts.     

The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.     

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.     

Cloud point formation based on mixed micelle in the presence of electrolyte for extraction, preconcentration, and spectrophotometric determination of trace amounts of hydrazine in water and biological samples

A cloud point extraction process using mixed micelle of the anionic surfactant sodium dodecyl sulfate and the nonionic surfactant Triton X-114 to extract hydrazine from aqueous solutions was investigated. The method is based on the condensation reaction of hydrazine with p-(dimethylamino)benzaldehyde, azine formation, and mixed micelle-mediated extraction of azine in the presence of NaCl electrolyte as an inducing phase separation. An azine product was concentrated in surfactant-rich phase after separation. The optimal extraction and reaction conditions (e.g., surfactant, reagent and electrolyte concentrations, and centrifuge time) were studied and the analytical characteristics of the method (e.g., limit of detection, linear range, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 0.50-110ngml(-1) of hydrazine and the detection limit of the method is 0.08ngml(-1). The interference effect of some cations, anions, and organic compounds was also tested. The method was successfully applied to the determination of hydrazine in water and biological samples.     

Novel procedure for extraction of a latent grape polyphenoloxidase using temperature-induced phase separation in triton x-114

Polyphenoloxidase from grape berries is extracted only by nonionic detergents with a hydrophilic-lipophilic balance between 12.4 and 13.5. The enzyme was partially purified in latent form, free of phenolics and chlorophylls, by using temperature phase partitioning in a solution of Triton X-114. This method permits the purification of the enzyme with the same fold purification as the commonly used method, but with a yield three times higher and a 90% reduction in time needed. The latent enzyme can be activated by different treatments, including trypsin and cationic and anionic detergents. Cetyltrimethylamonium bromide was found to be the most effective detergent activator, followed by sodium dodecyl sulfate. Polyphenoloxidase in grape berries, in spite of being an integral membrane protein, had an anomalous interaction with Triton X-114, remaining in the detergent-poor phase after phase separation. This could be explained by its having a short hydrophobic tail that anchors it to the membrane.     

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.     

Separation/Preconcentration Methods for the Determination of Aluminum in Dialysate Solution and Scalp Hair Samples of Kidney Failure Patients

A new method is reported for the separation of aluminum ions (Al(3+)) from interfering cations in pharmaceutical and biological samples through solid-phase extraction (SPE) using 2-methyl-8-hydroxyquinoline (8-hydroxyquinaldine) on activated silica. While separated Al(3+) was preconcentrated by cloud point extraction (CPE) using 3,5,7,2'-4'-pentahydroxyflavone (morin) as complexing reagent, the resulting complex was entrapped in nonionic surfactant (Triton X-114) as prior step to its determination by spectrofluorimetry (SPF). The validity of separation/preconcentration of Al(3+) was checked by certified reference material of human hair and standard addition method. The chemical variables affecting the analytical performance of the separation/preconcentration methods were studied and optimized. The enrichment factor and detection limit of Al(3+) for the preconcentration of 10?ml of dialysate solution and acid-digested samples of scalp hair samples were found to be 25 and 0.34?μg/L, respectively. The relative standard deviation for six replicates of standard containing 20?μg/L of Al(3+) was <10%. In all DS, the concentration of Al was >10?μg/L. The level of Al in scalp hair samples of kidney failure patients was higher than healthy controls.     

Micell-mediated extraction for the spectrophotometric determination of nitrite in water and biological samples based on its reaction with p-nitroaniline in the presence of diphenylamine

A cloud point extraction process using the nonionic surfactant Triton X-100 to extract nitrite from aqueous solution was investigated. The method is based on the color reaction of nitrite with p-nitroaniline in the presence of diphenylamine in acid media and micell-mediated extraction of an azo product. The optimal extraction and reaction conditions (e.g., acid concentration, reagent concentration, effect of time) were studied, and the analytical characteristics of the method (e.g., limit of detection, linear range, molar absorptivity, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 2-40 ng ml(-1) of nitrite ion. The detection limit of the method is 0.87 ng ml(-1) of nitrite ion. The interference effect of some anions and cations was also tested. The method was applied to the determination of nitrite in tap water, waste water, and human urine samples.     

Extractive biodegradation of diphenyl ethers in a cloud point system: Pollutant bioavailability enhancement and surfactant recycling

The biodegradation of diphenyl ethers (DEs) in the environment is limited by their high hydrophobicity. The enhancement of DE bioavailability by a cloud point system (CPS) was investigated in this study. Three CPSs (i.e., Triton X-114, Triton X-114 + Triton X-45, and Brij30 + TMN-3) were tested to promote DE biodegradation. Biocompatibility tests showed that the biodegradation of DE and 4-bromodiphenyl ether (4-BDE) was inhibited by TX-114, unaffected by TX-114 + TX-45, and promoted by Brij30 + TMN-3 over 48 h of cultivation with Cupriavidus basilensis and 4% (w/v) nonionic surfactants. Further optimization with 2% (w/v) Brij30 + TMN-3 yielded residual DE and 4-BDE quantities of 143 and 154 mg/L, respectively, lower than quantities in the control. During degradation, DE content did not decrease in the dilute phase, but sharply decreased in the coacervate phase, indicating that the DEs gradually diffused and transferred from the coacervate phase to the dilute phase for degradation by microbial cells. This behavior also enhanced the bioavailability of DEs in the CPS. By removing the cell-rich dilute phase and adding fresh degradation medium and DE to the coacervate phase, surfactants were successfully recovered and reused twice without affecting DE biodegradation. Results demonstrated that a CPS with 2% (w/v) Brij30 + TMN-3 not only enhanced the bioavailability of DEs, but also decreased the treatment cost through surfactant recycling, which is beneficial for large-scale applications.     

Choline acetyltransferase in mammalian synaptosomes: Evidence for an integral membrane-bound form

Choline acetyltransferase activity was detected in extensively washed membranes prepared from rat and guinea pig synaptosomes. When these preparations were treated with the non-ionic detergent Triton X-114 and heated to 37°C to cause phase separation, a significant percentage was found to associate with the detergent-rich phase, indicating that the enzyme might be an integral membrane-bound protein. In guinea pigs receiving septal lesions, a large reduction in both total and in Triton X-114-extractable choline acetyltransferase in hippocampal synaptosomes was observed indicating that the detergent-extracted form is associated with cholinergic nerve terminals. When membrane-bound choline acetyltransferase from lysed, washed synaptosomes was incubated in Triton X-114, 30% of the membrane-associated enzyme could be extracted into the detergent-rich phase. This extraction could be improved by reducing the chloride content of the extraction medium. When the chloride content of synaptosomes, prepared from rat cerebral cortex, was manipulated, by either exposure to γ-aminobutyric acid, muscimol or to a medium containing reduced levels of chloride, the ability of antibodies against choline acetyltransferase to specifically immunolyse (in the presence of complement) the cholinergic synaptosome population was enhanced. These results suggest that the choline acetyltransferase found in the nerve terminal region exists in at least two forms (a soluble and a lipophilic form) which are partially interconvertible. The conversion between the two forms can be influenced by chloride ions.