Micellar Solubilization of Proteins in Aprotic Solvents and their Spectroscopic Characterisation

Luisi, Pier Luigi; Bonner, Francis J.; Pellegrini, Antonio; Wiget, Peter; Wolf, Romain M.

doi:10.1002/hlca.19790620312

Cited by 133 publications

(35 citation statements)

References 37 publications

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“…Table 1 During the re-extraction of cytohrome c and rubredoxin, small red particles appeared at the interface indicative of some loss of haem. For a-chymotrypsin Luisi et al [5] observed an optimal transfer at pH 10 -11. The percentage uptake was dependent on protein concentration and type of organic solvent used.…”

Section: Resultsmentioning

confidence: 98%

“…Protein transfer from an aqueous into a reversed micellar phase has been shown to depend not only on the composition of both phases but also on the properties of the protein under investigation [13, 141. Moreover from studies on the transfer of amino acids [23] and proteins [5,12,131 into organic solvents with the aid of surface active compounds, it became clear that electrostatic interactions play an important role. Solubilization is only observed when the surfactant and protein are oppositely charged, and in most cases an increase in ionic strength leads to reduced solubilization [7,13,.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Protein transfer from an aqueous phase into reversed micelles

Wolbert¹,

Hilhorst²,

Voskuilen³

et al. 1989

European Journal of Biochemistry

113

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Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum = isoelectric point f0.11 x 1O-j M , -0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di-(2-ethylhexyl)sulphosuccinate Several fields of application for reversed micelles in biotechnology have been suggested (for reviews see [l -41). One of the potential applications is the extraction of proteins from an aqueous or solid phase via a reversed micellar phase into a second aqueous phase. This double transfer process is based on the ability of reversed micelles to extract proteins from an aqueous solution into their aqueous core. Most proteins retain their native conformation during the transfer process and remain in an active form when incorporated into reversed micelles [ 1 -61.Hatton and coworkers have shown that this technique is feasible for the extraction of proteins from a fermentation broth [7] and Leser et al. [8] demonstrated its applicability for the extraction of proteins from the solid phase. Previously [9] we reported that a-amylase could be extracted from an aqueous solution into a reversed micellar medium and re-extracted into a second aqueous phase in a continuous process with a yield of 45% and a concentration factor of eight with respect to enzyme activity. Addition of the nonionic surfactant nonylphenol pentaethoxylate (Rewopal HV5) to the organic phase, led to an increase both in the degree of solubilization of the enzyme and in the pH range in which solubilization occurs [lo]. By increasing the rate of mass transfer and the distribution coefficient of the enzyme during forward extraction, the yield has been improved to 85% and the concentration factor to 17 [ll].From the results published to date, it becomes evident that protein partitioning depends on the pH, ionic strength and type of ions present in the aqueous phase, on the type of surfactant, cosurfactant and organic solvent used, on micellar size, on the temperature and on properties of the protein [ 5 , 12 -161. However, no information is available on the mechanisms by which these variables affect protein partitioning. In this study the partitioning behaviour is related to such protein properties as size, isoelectric point and distribution of charged groups over th...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Protein transfer from an aqueous phase into reversed micelles

Wolbert¹,

Hilhorst²,

Voskuilen³

et al. 1989

European Journal of Biochemistry

113

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“…Luisi et al, 17 using trioctylmethylammonium chloride (TOMAC), showed that electrostatic interactions are determinant in tryptophan solubilization, when comparing the 90% degree of transfer in the high pH range with the 10% of transfer for the zwitterionic species. The decrease in amino acid solubilization at high electrolyte concentrations has been partly explained by a decrease of the electrostatic potential at the solubilization site, due to a more effective screening of the double layer.…”

Section: Introductionmentioning

confidence: 99%

Amino acid solubilization in cationic reversed micelles: factors affecting amino acid and water transfer

Cardoso

Barradas

Kroner

et al. 1999

J. Chem. Technol. Biotechnol.

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The aim of this work is to investigate the driving forces involved in amino acid solubilization in cationic reversed micelles, and to determine in which way different parameters affect the reversed micellar structure and amino acid solubilization, in order to select the best conditions to optimize amino acid extraction. To this end, extraction equilibrium experiments were performed using different experimental conditions and three amino acids with different structures: aspartic acid ± a hydrophilic amino acid, phenylalanine ± a slightly hydrophobic amino acid, and tryptophan ± a hydrophobic amino acid. The study of the effect of amino acid related parameters, such as pH and the initial amino acid concentration in the aqueous phase, and the effect of parameters that in¯uence the reversed micellar structure, such as surfactant concentration, ionic strength and co-surfactant concentration, provides useful information about the driving forces involved, solute±micelle interfacial interactions and solute location in the cationic system trioctylmethylammonium chloride (TOMAC)/ hexanol/n-heptane. These parameters can be adjusted to optimize amino acid extraction. It is shown that amino acids with the same isoelectric point can be selectively separated by exploring the different interactions they establish with the reversed micellar interface.

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“…Luisi et al (1979) first investigated the use of the quaternary ammonium salt trioctylmethyl ammonium chloride (TOMAC) in cyclohexane to solubilize various proteins, including ␣-chymotrypsin and pepsin. Since then, much research work has been conducted to understand the solubilization and the stability of proteins extracted into a reverse micellar phase.…”

Section: Introductionmentioning

confidence: 99%

Solubilization limit of lysozyme into DODMAC reverse micelles

Shin

Vera²

2002

Biotech & Bioengineering

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Dioctyldimethyl ammonium chloride (DODMAC) was used to form reverse micelles and to extract lysozyme from an aqueous solution into an organic phase. The solubilization behavior of lysozyme into a DODMAC reverse micellar phase was examined in terms of the temperature, the type of cations in the aqueous phase, and the surfactant concentration in the organic phase. Complete removal of lysozyme from the aqueous phase was obtained when the pH was set one unit higher than the pI of the protein. However, it was found that there is a solubilization limit of lysozyme in the organic phase. Not all the lysozyme extracted out of the initial aqueous phase was solubilized into the DODMAC reverse micellar phase, resulting in the formation of white precipitate at the aqueous-organic interface. Temperature has a negligible effect on the solubilization limit of lysozyme. The value of the solubilization limit is a strong function of the type of cations present in the aqueous phase, indicating an important role of lysozyme-cation interactions on the extraction process. An increase in the DODMAC concentration from 100-200 mM resulted in little change in the highest concentration of lysozyme obtained in the organic phase.

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Micellar Solubilization of Proteins in Aprotic Solvents and their Spectroscopic Characterisation

Cited by 133 publications

References 37 publications

Protein transfer from an aqueous phase into reversed micelles

Protein transfer from an aqueous phase into reversed micelles

Amino acid solubilization in cationic reversed micelles: factors affecting amino acid and water transfer

Solubilization limit of lysozyme into DODMAC reverse micelles

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