Biomedical subjects
Publications and source records attributed to Tatsuo Maruyama.
DNA was site-specifically conjugated to a substrate peptide of microbial transglutaminase fused to the N- or C-terminus of target proteins without the loss of the proteins' functions of interest.
Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A.
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.
Guanine quenching of a fluorescence-labeled DNA probe is a powerful tool for detecting a mutation in a targeted site of a DNA strand. However, a different guanine adjacent to a targeted site can interfere with detection of a point mutation, resulting in unsatisfactory sensitivity. In the current study, we developed a simple method to improve sensitivity of the guanine quenching method using a masking DNA oligonucleotide. The simple addition of a masking DNA oligonucleotide was found to mask the interference of a different guanine in a target oligonucleotide on fluorescence and to enhance difference in the quenching ratio between wild-type and mutant oligonucleotides. Based on this strategy, we succeeded in discriminating various mutations from the wild-type YMDD motif of the hepatitis B virus DNA polymerase gene using guanine quenching with a masking oligonucleotide.
The ribonuclease protection assay is a generally applicable technique for the detection of known mutations. We have developed a simple and rapid method for mutation detection based on the ribonuclease protection assay using fluorescently labeled oligodeoxyribonucleotide probes. The fluorogenic ribonuclease protection (FRAP) assay uses two differently labeled oligodeoxyribonucleotides, a donor probe and an acceptor probe, to obtain a fluorescence resonance energy transfer (FRET) signal. We have utilized the FRAP assay for the detection of a single-base mutation in the YMDD motif of the hepatic B virus DNA polymerase gene. The occurrence of mismatch-selective RNA cleavage was successfully discriminated by measuring the FRET signal between the donor and acceptor probes. Moreover, mutation sensing was successfully visualized by a UV transillumination. This simple and rapid mutation sensing method should facilitate a high-throughput mutation analysis.
Subtilisin Carlsberg conjugated with comb-shaped poly(ethylene glycol) was solubilized in common ionic liquids without adding water, and exhibited higher transesterification activity in an ionic liquid, [Emim][Tf2N], than in organic solvents commonly used for enzymatic biotransformation.
We have encapsulated a surfactant-protease complex (the main protease used being alpha-chymotrypsin) in an organic phase of a supported liquid membrane (SLM) for the optical resolution of various amino acids. L-Isomers of amino acids were enantioselectively permeated through the SLM. The mechanism of the amino acid permeation through the SLM was considered to be as follows; an L-amino acid was enantioselectively esterified with ethanol by a surfactant-protease complex encapsulated in the SLM, and the resulting L-amino acid ethyl ester dissolved into the organic phase of the SLM and diffused across the SLM. Another surfactant-alpha-chymotrypsin complex in the receiving phase catalyzed ester hydrolysis to produce the initial L-amino acid and ethanol, which are water-soluble. Thus, the L-amino acid was selectively transported to the receiving phase through the SLM on the basis of the molecular recognition of the surfactant-protease complex in the SLM. It was found that the catalytic activity and enantioselectivity of the surfactant-protease complex governed the permeate flux of amino acids and the enantiomeric excess in the membrane separation.
Surface molecular imprinting was applied to the preparation of an ion-selective polymeric membrane for the first time. The use of acrylonitrile-butadiene rubber and a porous solid support in the polymer matrix resulted in improved flexibility and mechanical strength of the imprinted membrane. The asymmetric porous structure of the membrane was observed by scanning electron microscopy. The selectivity of the zinc(II)-imprinted membrane was evaluated by competitive adsorption and permeation studies. The imprinted membrane showed higher adsorption affinity and permeation selectivity towards the imprinted zinc ion than the non-imprinted counterpart. On the basis of the results obtained, the permeation mechanism of the metal ions was considered to be hopping of metal ions on the binding sites in the membranes.
We have developed a novel, lipase-facilitated, supported liquid membrane (SLM) for the selective separation of organic acids by encapsulating a surfactant-lipase complex in the liquid membrane phase. This system exhibited a high transport efficiency for 3-phenoxypropionic acid and enabled the selective separation of organic acids due to the different solubilities of the acids in the organic phase and the variable substrate specificity of the surfactant-lipase complex in the liquid membrane phase. We found that various parameters, such as the amount of surfactant-lipase complex in the SLM, the lipase concentration in the receiving phase, and the ethanol concentration in the feed phase, affected the transport behavior of organic acids. The optimum conditions were 5 g L(-1) of the surfactant-CRL complex in the SLM (CRL=lipase from Candida rugosa), 8 g L(-1) of PPL in the receiving phase (PPL=lipase from porcine pancreas), and an ethanol concentration of 50 vol %. Furthermore, we achieved high enantioselective transport of (S)-ibuprofen attributable to the enantioselectivity of the surfactant-CRL complex.
Fluorescence quenching by guanine allows DNA hybridization to be monitored and any point mutations in oligonucleotides to be detected. However, fluorescence quenching is often affected by untargeted guanine located in a protruding end (single-strand DNA) of the probe-target DNA duplex resulting in an unsatisfactory sensitivity. In the present study, we used enzymatic digestion of the protruding end of a probe-target DNA duplex to avoid interference by untargeted guanine on fluorescence quenching for detection of a nucleobase mutation. Enzymatic digestion of the protruding end of the DNA duplex fully prevented interference by untargeted guanine, and produced a marked difference in the quenching ratios (36% for wild-type, and 0% for mutant).
The functionalization of the cytochrome P450cam monooxygenase system, which requires electron transfer among three different proteins, was investigated in the micro-scale aqueous compartments of stable water-in-oil (W/O) emulsions formed with the nonionic surfactant tetraethylene glycol dodecyl ether. Neither an organic-aqueous biphasic system nor a non-emulsified organic-aqueous solution containing the same amount of surfactant showed substantial hydroxylation of camphor, a natural substrate of P450cam, whereas substantial monooxygenation activity was detected when stable aqueous compartments were provided by the formation of W/O emulsions. Since the camphor hydroxylation in W/O emulsions was modest, we explored the integration of an enzymatic NADH regeneration system in order to effectively provide a reducing equivalent. Two different dehydrogenases, bacterial glycerol dehydrogenase (GLD) and yeast alcohol dehydrogenase (ADH), were selected, and each of these was coupled with the P450cam catalytic cycle in W/O emulsions. As a result, the camphor hydroxylation rate was successfully improved by approximately 5-fold when GLD was employed under optimized conditions. These results reveal the potential utility of the micro-scale cell-like aqueous compartments of W/O emulsions for multicomponent enzymatic reactions especially for substrates with low aqueous solubility.
Oxidative degradation of phenolic environmental pollutants in organic media was investigated using a laccase complexed with surfactants. The catalytic activity of the surfactant-laccase complex in isooctane was markedly enhanced by appropriately adjusting the water content of the reaction medium using reverse micelles. The surfactant-laccase complex showed little activity towards the oxidative reaction of bisphenol A in water-saturated isooctane (i.e., 0.0055% [v/v] water) while effectively catalyzed the same reaction in isooctane containing 4% (v/v) water, which is over the maximum water solubility. The latter system was homogeneous and was only achieved by the aid of reverse micelles. With respect to the oxidation of bisphenol A, two products, 4-isopropylphenol and 4-isopropenylphenol, were identified by gas chromatography-mass spectrometry (GC-MS) analyses, indicating the oxidative degradation of the bis-phenolic structure of bisphenol A. We also found that the surfactant-laccase complex turned out to handle other environmental pollutants, chlorophenols, by the simultaneous addition of water and a redox mediator into the reaction medium using reverse micelles.
A three-phase flow, water/n-heptane/water, was constructed in a microchannel (100-microm width, 25-microm depth) on a glass microchip (3 cm x 7 cm) and was used as a liquid membrane for separation of metal ions. Surface modification of the microchannel by octadecylsilane groups induced spontaneous phase separation of the three-phase flow in the microfluidic device, which allows control of interfacial contact time and off-chip analysis using conventional analytical apparatus. Prior to the selective transport of a metal ion through the liquid membrane in the microchannel, the forward and backward extraction of yttrium and zinc ions was investigated in a two-phase flow on a microfluidic device using 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (commercial name, PC-88A) as an extractant. The extraction conditions (contact time of the two phases, pH, extractant concentration) in the microfluidic device were examined. These investigations demonstrated that the conventional methodology for solvent extraction of metal ions is applicable to solvent extraction in a microchannel. Finally, we employed the three-phase flow in the microchannel as a liquid membrane and observed the selective transport of Y ion through the liquid membrane. In the present study, we succeeded, for the first time, in the selective separation of a targeted metal ion from an aqueous feed solution to a receiving phase within a few seconds by employing a liquid membrane formed in a microfluidic device.
We developed a highly enantioselective separation system for the optically active compounds, (S)-ibuprofen and l-phenylalanine, from their racemic mixtures by employing a supported liquid membrane (SLM) encapsulating a surfactant-lipase complex (or a surfactant-alpha-chymotrypsin complex). In the present system, enzymes encapsulated in the liquid-membrane phase effectively drove the enantioselective transport of optically active compounds through the SLM. This novel SLM allowed high enantioselectivity (ee over 91%) in the optical resolution of racemic ibuprofen and phenylalanine.
Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media.
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.
Lipase-catalyzed alcoholysis between vinyl cinnamate and benzyl alcohol in fluorous solvents was investigated. This is the first report of a lipase-catalyzed reaction in a fluorous solvent. Forming the poly(ethylene glycol)(PEG)-lipase PL complex enhanced lipase activity over 16-fold over that of native lipase powder. The PEG-lipase PL complex exhibited markedly higher alcoholysis activities in fluorous solvents than in conventional organic solvents such as isooctane and n-hexane. The optimum reaction temperature for FC-77 (perfluorooctane) was 55 [degree]C and the optimum pH for the preparation of the PEG-lipase complex was 9.0; similar to the conditions for lipase PL-catalyzed reaction in aqueous solution. The alcoholysis reaction in fluorous solvent requires the addition of a FC77-miscible organic solvent (isooctane) in order to dissolve non-fluorinated substrates. Lipase activity in the fluorous solvent was significantly influenced by the volume ratio of isooctane in the reaction medium. Vinyl cinnamate inhibition of the lipase-catalyzed reaction occurred at a much lower concentration in the fluorous solvent than in isooctane. These results can be explained by the localization of substrates around lipase molecules, induced by adsorption of the substrates to the PEG layer of the PEG-lipase complex.
Catalytic oxidation of biphenyl derivatives was investigated using laccase in a homogeneous aqueous-organic system. A thermostable laccase from Trametes sp. showed the highest catalytic activity for the oxidation of 4-hydroxybiphenyl (4-HB) at a reaction temperature of 60 degrees C when dimethylsulfoxide (DMSO) was employed as a co-solvent. Furthermore, the catalytic performance was successfully enhanced by the incorporation of a laccase mediator system (LMS) into the aqueous-DMSO media. The catalytic performance strongly depended on the type of mediator, and the highest activity was observed with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator, suggesting the importance of the selection of a suitable mediator. It was verified that this mediator system is applicable to the oxidation of several biphenyl derivatives with hydroxyl groups.