Optimization of the surfactant (AOT) concentration in a reverse micellar extraction process

Hentsch, Marc; Menoud, P.; Steiner, L.; Flaschel, Erwin; Renken, A.

doi:10.1007/bf02439326

Cited by 17 publications

(13 citation statements)

References 10 publications

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“…At surfactant concentrations above 25 mM, solubilization increased only slightly. A similar protein solubilization-AOT concentration profile was achieved for the PT of CHY; however, the AOT concentration at which the protein solubilization plateau was reached was about 3 mM (Hentsch et al, 1992). Hentsch et al employed a much smaller w/o-ME/protein ratio than we employed here.…”

Section: Effect Of the Aot Concentrationmentioning

confidence: 64%

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

Hayes

1997

Biotechnol. Bioeng.

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The extraction of solid‐phase α‐chymotrypsin, bovine serum albumin (BSA), and lysozyme by water‐in‐oil microemulsion (w/o‐ME) solution containing Aerosol‐OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein‐containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid‐phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein‐AOT‐water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein‐containing w/o‐MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT‐protein interactions or the protein's affinity for microemulsion‐encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270–282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid α‐chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583–593, 1997.

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Section: Effect Of the Aot Concentrationmentioning

confidence: 64%

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

Hayes

1997

Biotechnol. Bioeng.

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“…In the forward extraction step, selection of sur-factants and pH play significant roles in protein stabilization. Sodium di-2-ethylhexyl sulfosuccinate (AOT) is the most common surfactant used in chymotrypsin purification [8][9][10][11]. pH influences ionic molecular interactions in solution and therefore, influences the efficiency of extraction by reverse micelles [8,10].…”

Section: Introductionmentioning

confidence: 99%

“…This step is usually very slow and salt is added into the aqueous phase to assist the process [9]. However, increasing chloride ion concentration will decrease chymotrypsin yield by competing with chymotrypsin in the extraction process and the effect is particularly significant at low ionic strength [8,11]. According to Goto et al [12] and Hu and Gulari [9], the limitations of the backward extraction step are due to the difficulty in separating proteins from AOT reverse micellar phase and the excessive time involved in the process.…”

Section: Introductionmentioning

confidence: 99%

Extraction of Chymotrypsin from Red Perch (Sebastes marinus) Intestine Using Reverse Micelles: Optimization of the Backward Extraction Step

Zhou¹,

Budge²,

Ghaly³

2012

J Bioproces Biotechniq

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ObjectivesThe aim of this study was to optimize the backward extraction step of the reverse micelles method while purifying chymotrypsin from fish processing waste. The specific objectives were: (a) to study the effect of pH (6.5, 7.0, 7.5, 8.0 and 8.5) and salt concentration (0.5, 1.0, 1.5 and 2.0 M) and alcohol addition in the backward extraction step of the reverse micelles method on the enzyme activity (A E ), protein concentration (Cp), specific activity (SA), purification fold (PF) and recov- AbstractFish processing waste can be used to produce valuable by-products such as chymotrypsin which has applications in the food, leather, chemical and clinical industries. In this study, a reverse micelles system of AOT/isooctane was used to extract chymotrypsin from crude aqueous extract of red perch intestine. The effects of pH and KCl concentration of the backward extraction step on the total volume (TV), volume ratio (VR), total activity (TA), enzyme activity (A E ), specific activity (SA), purification fold (PF), protein concentration (Cp) and recovery yield (RY) were studied. Changing the pH from 6.5 to 8.5 and the KCl concentration from 0.5 to 2.0 M during the backward extraction step had no effects on the TV or VR. Increasing the pH from 6.5 to 7.5 increased A E , SA, Cp, PF and RY by up to 47.06%, 30.0%, 27.0%, 26.9% and 18.47%, respectively but they all then declined with further increases in the pH. Similar trends were observed when the KCl concentration was increased from 0.5 to 1.5 M. The decreases in these parameters were due to the denaturation of protein under high pH. The highest A E , Cp and RY were achieved with pH 7.5 and 1.0 M KCl concentration while the highest SA and PF were achieved with pH 7.5 and 1.5 M KCl concentration. Addition of isobutyl alcohol in the backward extraction step increased the TV, A E , TA, Cp, SA, PF and RY by 13.6%, 336.4%, 342.6%, 81.1%, 146.4%, 146.2% and 345.8%, respectively. Alcohol reduced the interfacial resistance for the reverse micelles and, thus, destroyed the reverse micelles structure. The values of A E , TA, SA, PF and RY obtained with reverse micelles methods were much higher (2.3 fold) than those obtained with the ammonium sulphate method.

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“…Sodium di-2-ethylhexyl sulfosuccinate (AOT) is the most common surfactant used in chymotrypsin purification. It can form reverse micelles without adding co-surfactant (Jolivalt et al, 1990;Hu and Gulari, 1996;Hentsch et al, 1992). pH influences ionic molecular interactions in solution and, therefore, influences the efficiency of extraction by reverse micelles (Jolivalt et al, 1990).…”

Section: Reverse Micelles Methodmentioning

confidence: 99%

“…However, the pH of protein aqueous solutions should be held near a pH of 3 because at that pH the autohydrolysis rate is minimized and chymotrypsin is most stable (Hu and Gulari, 1996). Jolivalt et al (1990) and Hentsch et al (1992) reported that increasing chloride ion concentration will decrease chymotrypsin yield by competing with chymotrypsin in the extraction process and the effect is particularly significant at low ionic strength.…”

Section: Reverse Micelles Methodmentioning

confidence: 99%

Extraction, Purification and Characterization of Fish Chymotrypsin: A Review

K.¹

2011

American Journal of Biochemistry and Biotechnology

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Problem statement: Solid fish waste is generated from the unwanted parts of fish including heads, tails, fins, frames, offal (guts, kidney and liver) and skin. It accounts for up to 80% of material from production of surimi, 66% from production of fillet and 27% from production of headed and gutted fish. Currently, fish wastes are disposed off in land-based waste disposal systems or at sea generating toxic byproducts during the decomposition process. However, fish processing waste can be used to produce commercially valuable by-products, such as chymotrypsin. Approach: A comperehensive review of the literature on the extraction, purification and characeterization of fish chymotrypsin was performed. Results: Chymotrypsin is an endopeptidase secreted by the pancreatic tissues of vertebrates and invertebrates. It has 3 different structures (chymotrypsin A, B and C) varying slightly in solubility, electrophoretic mobility, isoelectric point and cleavage specificity. Only chymotrypsin A and B are found in fish. Compared with mammal chymotrypsin, fish chymotrypsins have similar amino acid composition and molecular weights. Fish chymotrypsins have higher specific activity, especially those from cold-water fish, and low pH and temperature tolerance. The factors affecting the concentration and activity of chymotrypsin in fish are water temperature, fish species, fish age, fish weight and starvation. Chymotrypsin has application in various industries including the food industry, leather production industry chemical industry and medical industry. Conclusion: Extraction techniques for chymotrypsin include: ultra-filtration, ammonium sulphate fractionation precipitation or water-in-oil microemulsions. Purification can be carried out using recrystallization and gel-filtration, ion-exchange and hydrophobic interaction chromatography. Further studies should focus on the optimization of purifiying chymotrypsin from fish processing wastes.

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Optimization of the surfactant (AOT) concentration in a reverse micellar extraction process

Cited by 17 publications

References 10 publications

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

Extraction of Chymotrypsin from Red Perch (Sebastes marinus) Intestine Using Reverse Micelles: Optimization of the Backward Extraction Step

Extraction, Purification and Characterization of Fish Chymotrypsin: A Review

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