Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids.

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase.

Enhanced accumulation of anthocyanin in cultured strawberry cells by repetitive feeding of L-phenylalanine into the medium.

L-Phenylalanine (Phe), biosynthetic precursor of anthocyanin, was repetitively added into a suspension culture of strawberry cells, constantly producing anthocyanins. In the repetitive feeding culture, the maximum anthocyanin accumulation per culture was 30% and 81% higher than those in a single Phe-feeding culture and non-feeding culture, respectively. This means that the inhibitory effect of Phe on cell growth was successfully avoided and its intracellular free-state concentration was maintained at a higher level by the repetitive feeding of L-phenylalanine at relatively lower intra-medium concentration.

Catalytic activity of laccase hosted in reversed micelles.

Nanostructured reversed micelles induce a high laccase activity in organic solvents, because enzymes can maintain their highly dimensional structure in water pools of reversed micelles [RMs]. Laccase attracts considerable attention as a novel industrial enzyme due to its high capability to catalyze the oxidation of aromatic compounds. The catalytic activities of lyophilized laccase and laccase entrapped in RMs were compared using an oxidative reaction.

Refolding of cytochrome c using reversed micelles.

A novel protein refolding method using reversed micelles has been developed, which could replace the conventional dilution method using a buffer solution. The novel refolding method enables efficient refolding at a high protein concentration. In the present study, denatured bovine heart cytochrome c was directly solubilized in AOT reversed micelles using the solid-liquid extraction technique.

Lanthanoid element recognition on surface-imprinted polymers containing dioleylphosphoric acid as a functional host.

Surface-imprinted polymers have been newly developed for the separation of lanthanoid elements: i.e. La(III), Ce(III), and Dy(III).

Activation of lignin peroxidase in organic media by reversed micelles.

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution.

Inhibitory effects of extracts from peels of Citrus natsudaidai encapsulated in hybrid liposomes on the growth of tumor cells in vitro.

Inhibitory effects of extracts from peels of Citrus natsudaidai (natsumikan) encapsulated in hybrid liposomes (HL) composed of L-alpha-dimyristoylphosphatidylcholine and polyoxyethylene (20) sorbitan monolaurate on the growth of tumor cells were examined. The extracts with lower polar solvents inhibited the growth of B-16 mouse melanoma and human lung carcinoma cells, although the extracts with higher polar solvents showed no antitumor activity. In particular, the inhibitory effects of extracts with lower polar solvents encapsulated in HL were enhanced as compared with those of free extracts.

Surface imprinting polymers for the recognition of nucleotides.

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition.

Complex formation of cytochrome C with a calixarene carboxylic acid derivative: a novel solubilization method for biomolecules in organic media.

A calixarene carboxylic acid derivative has been found to form a complex with the cationic protein cytochrome c. The solubilized cytochrome c was stable and showed peroxidase activity in chloroform. The calix[6]arene and the calix[8]arene achieved quantitative extraction of the protein.

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells.

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28).

Formation and Characterization of Reversed Micelles Composed of Phospholipids and Fatty Acids.

The formation of reversed micellar systems composed of phosphatidylcholine (PC) and fatty acid was newly demonstrated by a significant increase in water content in the organic ethyl oleate phase when the micelles were prepared by the contact method. The solubilized water concentration in the reversed micellar organic phase reached 3 wt%. The new systems are expected to be used as highly biocompatible reversed micellar systems.

Small-angle X-ray scattering analysis of stearic acid modified lipase.

Stearic acid modified lipase (from Rhizopus japonicus) exhibited remarkable interesterification activity in n-hexane, but crude native lipase did not. The structure of the fatty acid modified lipase had not been analyzed until now. We analyzed the modified lipase by small-angle X-ray scattering (SAXS) measurements in order to clarify the structure.

Preparation and catalytic performance of surfactant-manganese peroxidase-Mn(II) ternary complex in organic media.

A novel preparation method for surfactant-MnP-Mn(II) ternary complex utilizing water-in-oil emulsions has been developed. The surfactant-MnP complex was spectroscopically characterized, strongly suggesting that the heme environment of the surfactant-MnP complex in benzene is identical to that of native MnP in the aqueous buffer. o-Phenylenediamine oxidation catalyzed by the surfactant-MnP-Mn(II) ternary complex was performed in benzene.

Important parameters affecting efficiency of protein refolding by reversed micelles.

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution.

Formation of biocompatible reversed micellar systems using phospholipids.

Formation of reversed micellar systems using biocompatible components was revealed by a significant increase of water content in the organic phase. Soybean lecithin (SL), which is a mixture of different phospholipids, and phosphatidylcholine (PC) purified from soybean were used as the amphiphilic molecule. Fatty acid and fatty acid ethyl esters were used as the organic solvent.

Surfactant-lactoperoxidase complex catalytically active in organic media.

A surfactant-lactoperoxidase (LPO) complex catalytically active in organic solvents was developed by the emulsion coating method. The oxidation of 2,6-dimethoxyphenol (2,6-DMP) was conducted by the surfactant-LPO complex in organic media. The LPO complex efficiently catalyzed the oxidation of 2,6-DMP in various organic solvents, although lyophilized LPO did not display the catalytic activity at all.

Characterization and catalytic property of surfactant-laccase complex in organic media.

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media.

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation.

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium.

Intermittent light irradiation with second- or hour-scale periods controls anthocyanin production by strawberry cells*.

Anthocyanin production by strawberry cells depends not only on light intensity but also on the light/dark cycle operation with hour- or second-scale periods. These findings are useful for designing and operating photobioreactors for enhanced anthocyanin production. Intermittent illumination with a second-scale period produces the same amount of anthocyanin as continuous light, suggesting that the light intensity distribution within a photobioreactor does not cause suppressed production.

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*.

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution.

Analysis of pigment accumulation heterogeneity in plant cell population by image-processing system.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity.

Surfactant-histidine-heme ternary complex as a simple artificial heme enzyme in organic media.

A surfactant-heme complex which shows peroxidase activity in organic media has been prepared by a method utilizing water-in-oil (W/O) emulsions. Both the aqueous phase pH and the type of surfactant appeared to have prominent effect on the catalytic activity of the heme complex in benzene. The catalytic efficiency of the heme complex was enhanced more than ten times by adding histidine to the aqueous phase of W/O emulsions in the preparation process.

Protein separation using affinity-based reversed micelles.

Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction.

Affinity extraction of proteins with a reversed micellar system composed of Cibacron Blue-modified lecithin.

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions.