Masahiro Goto

Extraction Behavior of Hemoglobin Using Reversed Micelles by Dioleyl Phosphoric Acid

A new surfactant, dioleyl phosphoric acid (DOLPA), has been applied to the extraction of hemoglobin using reversed micelles. The reversed micelles formed by DOLPA can easily extract hemoglobin from aqueous to reversed micellar solutions. DOLPA is the first surfactant to extract hemoglobin completely without using any cosurfactants. On the basis of the difference between DOLPA and AOT reversed micelles in the forward extraction behavior of hemoglobin, the nature of the interfacial complex that would be formed between surfactants and hemoglobin at the oil−water interface was found to be the dominant factor in determining the extraction efficiency of hemoglobin by reversed micelles. In addition, back‐transfer studies of hemoglobin from the DOLPA reversed micelles were also carried out by the phase transfer method. It was found that hemoglobin, once dissolved into the DOLPA reversed micelles, is not transferred to a fresh aqueous solution even when the conditions are adjusted to not allow the forward transfer of hemoglobin. However, the addition of several kinds of alcohol drastically improved the yield in the back‐transfer of hemoglobin. The efficiency in the back‐transfer of hemoglobin strongly depends on the aqueous conditions that are in contact with the reversed micelles, such as pH, ionic strength, and alcohol concentration. A pH higher than the pI of hemoglobin, a salt concentration lower than that of the water pool, and the proper concentration of alcohol are required for the recovery aqueous phase to ensure the back‐transfer of hemoglobin from the DOLPA reversed micelles.

Efficient glucoside extraction mediated by a boronic acid with an intramolecular quaternary ammonium ion

Abstract To develop an efficient sugar extractant on the basis of the mechanistic view a phenylboronic acid bearing a trioctylammonium group at the ortho position ( 4 ) was synthesized. To avoid the complexity we employed a simple two-phase solvent-extraction system which corresponds to the first step in membrane transport, i.e. , extraction from a donating aqueous phase to an organic liquid membrane phase. The extraction rates and equilibria were estimated using α - p -nitrophenyl-D-glucopyranoside as a sugar and 1,2-dichloroethane as an organic phase and compared with those of a 2-methylphenylboronic acid ( 5 ) / trioctylmethylammonium chloride (TOMAC) 1:1 binary system. The extraction rates for 4 were faster by 2.5 – 29 fold than those for 5 + TOMAC. The distribution coefficients were also enhanced by 5 – 8 fold. The results indicate that the intramolecular quaternary ammonium group is very effective to neutralize the anionic charge developed in the boron atom upon sugar-binding and create extractable zwitterionic sugar complexes.

Enzymatic resolution of racemic ibuprofen by surfactant-coated lipases in organic media

SummarySurfactant-coated lipases have been utilized as a biocatalyst for the resolution of racemic ibuprofen. S-(+)-ibuprofen was selectively transferred to the ester form by Mucor javanicus or Candida rugosa lipase. The enzymatic activity of upases in organic media was remarkably enhanced by coating with a nonionic surfactant. The reaction rates of the coated lipases were increased around 100-fold that of the powder lipases.

Effect of using a co-solvent in the preparation of surfactant-coated lipases on catalytic activity in organic media

Surfactant-coated lipases (from Candida cylindracea) were prepared in phosphate buffer (pH 6.9) containing a 2% (v/v) water-miscible organic solvent. The molecular structure of the surfactants used to coat the lipase strongly affected the esterification activity of the modified lipases in isooctane, while both the yield and the enzymatic activity were significantly influenced by the nature of the co-solvent used during the preparation. Although, in general, the most suitable co-solvents were found to be hydrophilic, especially ethanol and N,N-dimethylformamide (DMF), no definite correlation between solvent hydrophobicity (log P) and the properties of the surfactant-coated lipases was revealed. However, when a co-solvent which can dissolve a surfactant well was employed, the surfactant number attached to one lipase molecule tended to decrease and the yield was also lowered. Addition of dimethylsulfoxide (DMSO), formamide, or ethylene glycol to the lipase buffer solution during preparation led to substantial decreases in catalytic activity, even though the modified lipases were obtained in a good yield. This results suggests that these co-solvents affect the three-dimensional structure of the lipase, that is, the catalytic activity of surfactant-coated lipases in organic media may be chiefly determined during the preparation process in an aqueous solution.

Protein extraction by new reversed micelles with di(tridecyl) phosphoric acid

Abstract Di(tridecyl) phosphoric acid (DTDPA) was synthesized as a new surfactant for protein extraction by reversed micelles. DTDPA can form stable reversed micelles in the concentration range above 1 mM. The size of the DTDPA reversed micelles was smaller than that of conventional AOT (sodium-di-2-ethylhexyl sulfosuccinate) reversed micelles under the same conditions. The formation of reversed micelles using DTDPA can be controlled by adjusting the aqueous pH. The extraction behavior of some proteins from an aqueous phase into the reversed micellar phase of DTDPA has been investigated by varying the experimental conditions (pH, ionic strength, surfactant concentration). It was found that reversed micelles with DTDPA can extract many proteins. In particular, hemoglobin, which is difficult to extract with an AOT reversed micelle, can be extracted in the DTDPA reversed micelles. In the new reversed micellar system, phase separation was very fast even at a high concentration of DTDPA.

Reversed micelles recognize an active protein

The extraction behavior of native and heated-denatured α-chymotrypsin has been investigated with two different reversed micellar systems. A large difference in the degree of extraction was observed for the native relative to the denatured α-chymotrypsin. In particular, mixed reversed micelles formulated with DOLPA (dioleyl phosphoric acid) and AOT show a high selectivity for the active α-chymotrypsin.

Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules

Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules have been developed for the re-use of an oil-soluble enzyme in organic media. The esterification rate of the surfactant-coated lipase immobilized in the microcapsules was thirty times that of the powder lipase. More than 90% of the enzymatic activity of the capsulated lipases has been maintained after recycling six times.