Faculty Profiles - ONO Tsutomu

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Factors affecting protein transfer into surfactant-isooctane solution: A case study of extraction behavior of chemically modified cytochrome c

T Ono, M Goto

BIOTECHNOLOGY PROGRESS 14 ( 6 ) 903 - 908 1998.11

Language：English Publisher：AMER CHEMICAL SOC

The extraction mechanism of proteins by surfactant molecules in an organic solvent has been investigated using a chemically modified protein. We conducted guanidylation on lysine residues of cytochrome c by replacing their amino groups with homoarginine to enhance the protein-surfactant interaction. Results have shown that guanidylated cytochrome c readily forms a hydrophobic complex with dioleyl phosphoric acid (DOLPA) through hydrogen bonding between the phosphate moiety and the guanidinium groups. Although improved protein-surfactant interaction activated the formation of a hydrophobic complex at the interface, it could not improve the protein transfer in isooctane. It has been established that the protein extraction mechanism using surfactant molecules is mainly governed by two processes: formation of an interfacial complex at the oil-water interface and the subsequent solubilization of the complex into the organic phase. In addition, a kinetic study demonstrated that guanidylation of lysine accelerated the initial extraction rate of cytochrome c. This fact implies that the protein transferability from aqueous phase into organic phase depends on the protein-surfactant interaction which can be modified by protein surface engineering.

DOI： 10.1021/bp9800844

Extraction and activity of chymotrypsin using AOT-DOLPA mixed reversed micellar systems

Masahiro Goto, Yoshio Ishikawa, Tsutomu Ono, Fumiyuki Nakashio, T. Alan Hatton

Biotechnology Progress 14 ( 5 ) 729 - 734 1998.9

Language：English Publisher：AIChE

Novel reversed micellar solutions formulated with a mixture of AOT (dioctyl sulfosuccinate) and DOLPA (dioleyl phosphoric acid) show good potential for use in reversed micellar protein extraction operations. Chymotrypsin is easily extracted from an aqueous phase into organic isooctane containing 10 mM AOT and DOLPA in a 4:1 ratio. The extraction ability of the mixed reversed micelles of 10 mM was higher than that of 200 mM AOT alone. The results of extraction indicated that the AOT-DOLPA mixed reversed micelles are very useful for separating and enriching chymotrypsin. Back-extraction of chymotrypsin from the organic phase to a fresh aqueous phase is also accomplished by adding an alcohol to the organic phase. Although the backtransfer of chymotrypsin from the reversed micelles formed by AOT alone is very slow and difficult, in the AOT-DOLPA mixed reversed micelles, the back-extraction can be achieved completely by addition of 10% (v/v) isobutyl alcohol to the reversed micellar phase. The time to attain to the equilibrium of back-extraction was reduced from more than 24 to 2 h by adding the alcohol. On the basis of the activity data, the best composition of AOT and DOLPA was a 4:1 ratio and the total surfactant concentration was 10 mM. The activity of chymotrypsin recovered from the mixed reversed micelles was higher than that of the initial protein before the forward-transfer. This result means that the novel mixed reversed micellar solutions are useful not only in separation but also in purification of proteins.

DOI： 10.1021/bp9800790

Protein refolding by reversed micelles utilizing solid-liquid extraction technique

Y Hashimoto, T Ono, M Goto, TA Hatton

BIOTECHNOLOGY AND BIOENGINEERING 57 ( 5 ) 620 - 623 1998.3

Language：English Publisher：JOHN WILEY & SONS INC

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. (C) 1998 John Wiley & Sons, Inc.

DOI： 10.1002/(SICI)1097-0290(19980305)57:5<620::AID-BIT14>3.0.CO;2-F

Catalytic properties of novel reversed micellar system on trans-esterification by α-chymotrypsin in organic media

Ly Iskandar, Tsutomu Ono, Noriho Kamiya, Masahiro Goto, Fumiyuki Nakashio, Shintaro Furusaki

Biochemical Engineering Journal 2 ( 1 ) 29 - 33 1998

Language：English Publisher：Elsevier

Novel reversed micelles formulated by sodium dioleylphosphate (SDOLP) has been developed for enzymatic reaction in organic media. Comparison study between sodium bis-(2-ethylhexyl)-sulfosuccinate (Aerosol-OT or AOT) and SDOLP, which was carried out by investigating the reactivity of each reversed micellar system as reaction media for enzymatic transesterification reaction of N-acetyl-1-phenylalanine ethyl ester (APEE) and 1-propanol, leads to the upshot that α-chymotrypsin immobilized within SDOLP reversed micelles can persevere its high catalytic activity in isooctane. The initial reaction rate of the SDOLP reversed micellar system is almost 60 times higher than that in the AOT system. Additional kinetic results, including the Michaelis constant and the maximum reaction rate, reveal that the inner core of the SDOLP reversed micelles provides a better environment for enzymatic reaction than that of the conventional AOT reversed micelles. © 1998 Elsevier Science S.A. All rights reserved.

DOI： 10.1016/S1369-703X(98)00013-8

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Application of reversed micelles in bioengineering

T Ono, M Goto

CURRENT OPINION IN COLLOID & INTERFACE SCIENCE 2 ( 4 ) 397 - 401 1997.8

Language：English Publishing type：Book review, literature introduction, etc. Publisher：CURRENT SCIENCE LTD

Reverse micelles have found wider biological applications recently in terms of providing an enabling hydrophilic environment for solubilized functional biomaterials in organic media. Further, separation and purification of target proteins from aqueous solution using micellar aggregates created in hydrophobic media as well as efficient refolding of proteins in micellar solutions have become possible.

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Design of surfactants suitable for protein extraction by reversed micelles

Masahiro Goto, Tsutomu Ono, Fumiyuki Nakashio, T. Alan Hatton

Biotechnology and Bioengineering 54 ( 1 ) 26 - 32 1997.4

Language：English Publisher：John Wiley & Sons Inc

New surfactants have been synthesized for potential use in reversed micellar protein extraction operations. Preferential solubility of the surfactant in an aliphatic solvent such as hexane, heptane, or isooctane and the formation of reversed micelles accompanied with solubilization of significant quantities of water can be achieved by using strongly hydrophobic, twin alkyl chains as the hydrophobic moiety. Different surfactants having identical water-solubilizing capacities can have significantly different behavior in protein extractions, where extraction efficiency appears to be governed by the nature of the interfacial complex that forms between surfactants and proteins. Bulky surfactant chains provide a steric hindrance to the adsorption of the surfactant to the protein surface, thus inhibiting solvation of the protein/surfactant complex, and hence protein extraction. Under these conditions, a precipitate forms either in the bulk aqueous phase or at the interface. Surfactants that can form a close-packed complex with the protein are excellent protein-solubilizing agents. Dioleyl phosphoric acid (DOLPA) appears to be the best surfactant currently available for protein extraction.

DOI： 10.1002/(SICI)1097-0290(19970405)54:1<26::AID-BIT3>3.0.CO;2-#

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分子集合体を利用した抽出分離技術 (特集分離技術シンポジウム)

後藤雅宏, 小野努

分離技術 27 ( 3 ) 193 - 198 1997.4

Effect of organic solvents on extraction behaviour of cytochrome c using reversed micellar solutions

Tsutomu Ono, Takayuki Miyazaki, Masahiro Goto, Fumiyuki Nakashio

Solvent Extraction Research and Development 1996 1 - 9 1996.12

The effect of organic solvents on the extraction behaviour of cytochrome c has been investigated with reversed micellar solutions. An aliphatic solvent such as hexane, octane or isooctane was suitable for protein extraction operations using the reversed micelles. Organic solvents having a different hydrophobicity show significantly different behaviour in protein extraction, where extraction efficiency appears to be governed by the nature of the interfacial complex that is formed by surfactants and proteins. Solvent effects on the protein extraction are more dominant in the AOT system rather than that in the DOLPA system. Addition of alcohol appears to decrease the activity of proteins in the back-extraction step, although it is effective for recovering proteins from the reversed micellar solutions.

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Extraction behavior of hemoglobin using reversed micelles by dioleyl phosphoric acid

T Ono, M Goto, F Nakashio, TA Hatton

BIOTECHNOLOGY PROGRESS 12 ( 6 ) 793 - 800 1996.11

Language：English Publisher：AMER CHEMICAL SOC

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.

DOI： 10.1021/bp960055y

Novel immobilized enzymes utilizing reversed micellar solutions as an optical resolution tool

T Ono, M Goto, F Nakashio, TA Hatton

JOURNAL OF CHEMICAL ENGINEERING OF JAPAN 29 ( 4 ) 695 - 697 1996.8

Reversed micelles recognize an active protein

Masahiro Goto, Tsutomu Ono, Fumiyuki Nakashio, T. Alan Hatton

Biotechnology Techniques 10 ( 3 ) 141 - 144 1996

Language：English Publisher：Springer Netherlands

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.

DOI： 10.1007/BF00158935

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Protein Extraction by New Reversed Micelles with Di(Tridecyl) Phosphoric Acid

Masahiro Goto, Motohiro Kuroki, Tsutomu Ono, Fumiy uki Nakashio

Separation Science and Technology 30 ( 1 ) 89 - 99 1995

Language：English

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 sulfosucci-nate) 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. © 1995, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1080/01496399508012215

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有機媒体中における分子集合系のタンパク質の分離場としての利用--逆相ミセルによるタンパク質の抽出とその可溶化機構

小野努, 後藤雅宏

表面 32 ( 9 ) p601 - 611 1994.9

Protein extraction and extraction mechanism by reversed micelles

32 ( 9 ) 601 1994

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