Tatsuo Maruyama

FT-IR analysis of BSA fouled on ultrafiltration and microfiltration membranes

Abstract Protein fouling is a critical problem for ultrafiltration (UF) and microfiltration (MF). In the latest decade, a Fourier-transform infrared (FT-IR) spectroscopic method has been developed to quantify protein secondary structure by employing the amide I spectral region. The most attractive feature of FT-IR analysis is its ability to analyze proteins in various conditions. In this study, we employed FT-IR to quantify the conformational change of protein fouled on polysulfone (PS) UF membrane and polytetrafluoroethylene (PTFE) MF membrane. Bovine serum albumin (BSA) was adopted as a model protein. BSA adsorption onto the membranes was performed at 4°C and gel-like BSA deposits on the membranes were prepared by filtration at room temperature. FT-IR analysis revealed that the BSA adsorbed onto PS UF membrane had little change in the secondary structure, whereas the BSA adsorbed onto PTFE MF membrane had remarkable changes in the secondary structure, which were a decrease in α-helix content from 66 to 50% and an increase in β-sheet content from 21 to 36%. In addition, gel-like BSA deposits on both of the membranes had marked changes in secondary structure, which were similar to the changes in the BSA adsorbed onto the PTFE MF membrane. And the BSA concentration did not significantly affect the changes in the secondary structure of BSA fouled on both the UF and MF membranes.

Cancer Cell Death Induced by the Intracellular Self-Assembly of an Enzyme-Responsive Supramolecular Gelator

We report cancer cell death initiated by the intracellular molecular self-assembly of a peptide lipid, which was derived from a gelator precursor. The gelator precursor was designed to form nanofibers via molecular self-assembly, after cleavage by a cancer-related enzyme (matrix metalloproteinase-7, MMP-7), leading to hydrogelation. The gelator precursor exhibited remarkable cytotoxicity to five different cancer cell lines, while the precursor exhibited low cytotoxicity to normal cells. Cancer cells secrete excessive amounts of MMP-7, which converted the precursor into a supramolecular gelator prior to its uptake by the cells. Once inside the cells, the supramolecular gelator formed a gel via molecular self-assembly, exerting vital stress on the cancer cells. The present study thus describes a new drug where molecular self-assembly acts as the mechanism of cytotoxicity.

Poly(ethylene glycol)-lipase complex that is catalytically active for alcoholysis reactions in ionic liquids

Lipase-catalyzed alcoholysis was investigated in three different ionic liquids. Lyophilized native lipase had a low activity in all the ionic liquids but a poly(ethylene glycol) (PEG)-lipase complex (with a molar ratio of the polymer/enzyme of 10:1) had an increased activity of over 14-fold. Of several lipases tested, PEG-lipase PS (from Pseudomonas cepacia) exhibited the highest activity (1.07 mmol/(h g−1 protein)) in 1-octyl-3-methylimidazolium hexafluorophosphate.

Versatile Supramolecular Gelators That Can Harden Water, Organic Solvents and Ionic Liquids

We developed novel supramolecular gelators with simple molecular structures that could harden a broad range of solvents: aqueous solutions of a wide pH range, organic solvents, edible oil, biodiesel, and ionic liquids at gelation concentrations of 0.1-2 wt %. The supramolecular gelators were composed of a long hydrophobic tail, amino acids and gluconic acid, which were prepared by liquid-phase synthesis. Among seven types of the gelators synthesized, the gelators containing L-Val, L-Leu, and L-Ile exhibited high gelation ability to various solvents. These gelators were soluble in aqueous and organic solvents, and also in ionic liquids at high temperature. The gelation of these solvents was thermally reversible. The microscopic observations (TEM, SEM, and CLSM) and small-angle X-ray scattering (SAXS) measurements suggested that the gelator molecules self-assembled to form entangled nanofibers in a large variety of solvents, resulting in the gelation of these solvents. Molecular mechanics and density functional theory (DFT) calculations indicated the possible molecular packing of the gelator in the nanofibers. Interestingly, the gelation of an ionic liquid by our gelator did not affect the ionic conductivity of the ionic liquid, which would provide an advantage to electrochemical applications.

Poly(ethylene glycol)-lipase complexes that are highly active and enantioselective in ionic liquids

Lipase-catalyzed alcoholysis between vinyl acetate and 2-phenyl-1-propanol was investigated in dialkylimidazolium-based ionic liquids. Although native lipase powder exhibited very low activity in an ionic liquid, forming a poly(ethylene glycol)(PEG)-lipase complex improved the lipase activity in the ionic liquid. The activity of the PEG-lipase complex was higher in ionic liquids than in common organic solvents (n-hexane, isooctane and dimethylsulfoxide). Fluorescence measurements using 4-aminophthalimide revealed that the ionic liquids were more hydrophilic than the organic solvents used for non-aqueous enzymology. A kinetic study of lipase-catalyzed alcoholysis in an ionic liquid ([Bmim][PF6]) revealed that the Michaelis constant (Km) for 2-phenyl-1-propanol in the ionic liquid was half that in n-hexane, suggesting that the ionic liquid stabilized the enzyme-substrate complex. Finally, we carried out enantioselective alcoholysis of 1-phenylethanol in ionic liquids employing the PEG-lipase complex, and obtained high enantioselectivity, comparable to that in n-hexane.

Intermittent partition walls promote solvent extraction of metal ions in a microfluidic device

Liquid–liquid extraction of metal ions was carried out in a microfluidic device which had intermittent partition walls in the center of the confluent microchannel 100 µm wide, 20 µm deep and 3 cm long. The intermittent partition walls (50 µm long) stabilized a two-phase (n-heptane–water) flow and allowed clear phase separation at the end-junction of the microchannel. Using this two-phase flow in the microchannel, yttrium ions were successfully extracted in a complex form with an extractant PC-88A (2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester) from a feed aqueous phase to a n-heptane phase within a contact time of 1.5 s. Although the apparent interfacial area in the microchannel was reduced by introducing the partition walls, the presence of the partition walls improved the extraction efficiency 2–3 fold at a contact time of 0.12–0.24 s. Flow analyses using fluorescent beads and a computational fluidic dynamics simulation revealed that the partition walls induced a slight turbulence in the two-phase flow in the microchannel. This slight turbulence would result in the mixing of the aqueous phase and promote the transport of yttrium ions from the aqueous feed phase to the organic extractant phase.

Homogeneous enzymatic reactions in ionic liquids with poly(ethylene glycol)-modified subtilisin

Subtilisin Carlsberg was covalently modified with comb-shaped poly(ethylene glycol) (PM13). PM13-modified subtilisin (PM13-Sub) was readily solubilized in three different ionic liquids (ILs), i.e., [Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]. Analysis of homogeneous enzymatic reactions in the ILs revealed that PM13-Sub exhibited excellent catalytic performance while the native enzyme suspended in ILs showed no activity. Hydrophobicity of ILs slightly affected enzyme activity, and the relatively hydrophobic IL [Emim][Tf2N] was the preferred medium for enzymatic reactions, similar to enzymatic reactions in conventional organic solvents. Enzyme activity was much higher in [Emim][Tf2N] than in conventional organic solvents, and excellent activity was associated with unique properties of ILs such as hydrophobicity and high polarity. Furthermore, PM13-Sub showed good stability in [Emim][Tf2N], and maintained 80% of its initial activity after 60 h.

Enzyme-facilitated enantioselective transport of (S)-ibuprofen through a supported liquid membrane based on ionic liquids

Coupling lipase reactions with a supported liquid membrane (SLM) based on ionic liquids showed facilitative and selective permeation of (S)-ibuprofen through the SLM, indicating successful optical resolution of a racemic mixture using the enzyme-facilitative SLM.

Synthesis of gold nanoparticles using various amino acids.

Gold nanoparticles (4-7nm) were synthesized from tetraauric acid using various amino acids as reducing and capping agents. The gold nanoparticles were produced from the incubation of a AuCl4(-) solution with an amino acid at 80°C for 20min. Among the twenty amino acids tested, several amino acids produced gold nanoparticles. The color of the nanoparticle solutions varied with the amino acids used for the reduction. We adopted l-histidine as a reducing agent and investigated the effects of the synthesis conditions on the gold nanoparticles. The His and AuCl4(-) concentrations affected the size of the gold nanoparticles and their aggregates. The pH of the reaction solution also affected the reaction yields and the shape of the gold nanoparticles.

Activation of lipase in ionic liquids by modification with comb-shaped poly(ethylene glycol)

Abstract Outstanding activation of an enzyme in ionic liquids (ILs) has been demonstrated by covalent modification with comb-shaped poly(ethylene glycol) (PEG) (PM13). Candida rugosa lipase modified with PM13 (PM13–CRL) was readily solubilized in all the ILs tested ([Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]) containing 0.5% (v/v) of water, whereas native lipase did not dissolve in any of the ILs. The results for transesterification of 2-phenyl-1-propanol with vinyl acetate using lipase in ILs revealed that the PM13–CRL conjugate exhibits a high catalytic activity while suspended native lipase shows little activity. The hydrophobicity of ILs somewhat affected the enzyme activity and a more hydrophobic IL such as [Emim][Tf2N] was preferable for the lipase reaction, as was also observed in enzymatic reaction in conventional organic solvents. The enzyme activities in ILs were much higher than those in organic solvents, the excellent activity being associated with unique properties such as the hydrophobicity and the high polarity of ILs. Furthermore, the PM13-–CRL conjugate exhibited a high storage stability in [Emim][Tf2N].