Katsuya Kato

Inverting Enantioselectivity of Burkholderia cepacia KWI-56 Lipase by Combinatorial Mutation and High-throughput Screening Using Single-molecule PCR and In Vitro Expression

The enantioselectivity of lipase from Burkhorderia cepacia KWI-56 has been inverted using a novel in vitro technique for construction and screening of a protein library by single-molecule DNA amplification by PCR followed by in vitro coupled transcription/translation system termed single-molecule-PCR-linked in vitro expression (SIMPLEX). Four amino acid residues (L17, F119, L167, and L266) in the hydrophobic substrate-binding pocket of the lipase were selected for mutation based on a structural model of a substrate-enzyme complex, and a combinatorial mutation library was constructed by SIMPLEX and screened for (R) and (S)-configurations of p-nitrophenyl 3-phenylbutyrate. Some combinations of amino acid substitutions in the four positions of the lipase were found as effective for changing the enantiopreference from the (S)-form substrate to the (R)-form. Two variants were expressed in the original host cells and purified to homogeneity, showing completely reversed enantioselectivity for the (R)-form of ethyl 3-phenylbutyrate (selectivity factor E(R)=38 or 33), whereas the wild-type lipase was (S)-selective (selectivity factor E(S)=33). Thus the semi-rational and semi-random combinatorial design of a mutant library followed by a high-throughput screening based on their enzymatic activity should be a powerful tool to engineer the enantioselectivity of enzymes.

TiO2 Synthesis Inspired by Biomineralization: Control of Morphology, Crystal Phase, and Light-Use Efficiency in a Single Process

Hydroxyapatite is mineralized along the long axis of collagen fiber during osteogenesis. Mimicking such biomineralization has great potential to control inorganic structures and is fast becoming an important next-generation inorganic synthesis method. Inorganic matter synthesized by biomineralization can have beautiful and functional structures that cannot be created artificially. In this study, we applied biomineralization to the synthesis of the only photocatalyst in practical use today, titanium dioxide (TiO(2)). The photocatalytic activity of TiO(2) mainly relates to three properties: morphology, crystal phase, and light-use efficiency. To optimize TiO(2) morphology, we used a simple sequential peptide as an organic template. TiO(2) mineralized by a β-sheet peptide nanofiber template forms fiber-like shapes that are not observed for mineralization by peptides in the shape of random coils. To optimize TiO(2) crystal phase, we mineralized TiO(2) with the template at 400 °C to transform it into the rutile phase and at 700 °C to transform it into a mixed phase of anatase and rutile. To optimize light-use efficiency, we introduced nitrogen atoms of the peptide into the TiO(2) structure as doped elemental material during sintering. Thus, this biomineralization method enables control of inorganic morphology, crystal phase, and light-use efficiency in a single process.

Detoxification of Bisphenol A and Nonylphenol by Purified Extracellular Laccase from a Fungus Isolated from Soil

Purified laccase from a fungus (family Chaetomiaceae) was used for the enzymatic oxidation of bisphenol A and nonylphenol, endocrine-disrupting chemicals. It rapidly oxidized both chemicals in the absence of mediators and within 24 h their estrogenic activities were completely removed.

Diastereoselective Friedel–Crafts reaction of α-trifluoromethyl imines derived from chiral amines

Abstract The Friedel–Crafts reactions of chiral N-(2,2,2-trifluoroethylidene)-1-arylethylamines 1a and 1b with various electron-rich aromatic compounds were examined. The reactions proceeded readily at room temperature in the presence of BF3·Et2O. Substituted products 2–12 were obtained in low to very high stereoselectivities (up to 100% d.e.). The absolute configuration of compound 12 was determined by X-ray analysis. Moreover, the chiral auxiliary from compounds 3 and 12 was selectively removed by palladium-catalyzed hydrogenolysis.

Catalytic Properties of Lipases Immobilized on Various Mesoporous Silicates

Lipases SP525, AK, LIP, and PS were immobilized on three kinds of mesoporous silicates (FMS, PESO, and SBA) with diameters of 27 to 92 Å. The amount of lipase activity adsorbed on these supports was related to the pore size of the silicate. Enantioselectivities of immobilized lipases were similar to those of free lipases, and recycling could be done in both aqueous and organic solvents.

Multistep Growth Mechanism of Calcium Phosphate in the Earliest Stage of Morphology-Controlled Biomineralization

We studied the effect of surface-functional-group position on precipitate morphology in the earliest stage of calcium phosphate biomineralization and determined the detailed mechanism of precipitation starting from nucleation to precipitate growth. The biomineralization template was a β-sheet peptide scaffold prepared by adsorption with carboxyl groups arranged at strict 7 Å intervals. Phosphate was then introduced. Within 10 s, highly ordered embryos of calcium phosphate were formed and confined by a peptide nanofiber pattern. They repeatedly nucleated and dissolved, with the larger embryos absorbing the smaller ones in a clear demonstration of an Ostwald-ripening-like phenomenon, then aggregated in a line pattern, and finally formed highly ordered nanofibers of amorphous calcium phosphate. This multistep growth process constitutes the earliest stage of biomineralization.

Synthesis of amino-functionalized mesoporous silica sheets and their application for metal ion capture

Abstract Novel mesoporous silica sheets with surface amino-functional groups (sheet-NH2) were prepared for the efficient adsorption of aqueous metal ions. The sheet-NH2 were synthesized by a dual-templating process using Pluronic P123 and N-palmitoyl-l-alanine as templates. Two silicone regents were used to form the silica framework: tetraethoxysilane and 3-aminopropyltriethoxysilane. In the synthetic process, the as-prepared silicate was refluxed in ethanol to remove the organic templates remaining from the surface amino groups derived from APTES. The prepared sheet-NH2 showed widths of several micrometres and small thicknesses of approximately 50 nm. The pore diameter, pore volume and BET surface area of sheet-NH2 were determined by nitrogen adsorption–desorption isotherms to be 3.1 nm, 0.73 cm3 g−1 and 189.9 m2 g−1, respectively. TEM, XRD, FT-IR and TG–DTA analysis demonstrated that the sheet material had a disordered mesoporous structure and contained organic chains. The adsorptions of aqueous zinc(II) and copper(II) metal ions were examined and compared with amino-functionalized conventional mesoporous silica (MCM-NH2), calcined mesoporous silica sheets and silica beads with no porous structures. Notably, the sheet-NH2 exhibited the highest adsorption of both zinc and copper ions among the examined materials. In addition, the metal ion sorption equilibrium data of sheet-NH2 were fitted to the Langmuir isotherm model.

Interparticle mesoporous silica as an effective support for enzyme immobilisation

Mesoporous silica materials with cylindrical pores (MPSC) and interparticle pore structures (IPMPS) were synthesized by typical sol–gel methods, and their structural properties were characterized. The enzyme immobilised on IPMPS exhibited higher specific reactivity because of an improvement in the substrate affinity of the enzyme immobilised on the pore spaces of the IPMPS support. Fourier transform infrared and circular dichroism spectroscopy indicated that the highly ordered structure of formaldehyde dehydrogenase (FDH) is not altered by binding to IPMPS and MPSC surfaces. Interestingly, after 10 repeated reactions, FDH immobilised on IPMPS exhibited a residual activity higher than that of FDH immobilised on MPSC. The cycle performance of the enzyme immobilised on MPSC decreased because of support aggregation by the released enzyme. Meanwhile, IPMPS has a high-intensity surface electrical charge that is highly dispersible in the presence of enzymes. In addition, enzymes that were inactive because of being buried inside mesopores are quantitatively determined. The dependency of the activity of FDH immobilised on IPMPS and MPSC as a function of substrate (formaldehyde) concentration was also evaluated to determine the potential application of these materials as biosensors. Formaldehyde concentrations of 3.0–500 μM could be detected using FDH immobilised on the IPMPS support.

Enzyme structure and catalytic properties affected by the surface functional groups of mesoporous silica

The enzyme subtilisin from Bacillus licheniformis (4.1 nm × 7.8 nm × 3.7 nm) was easily immobilized onto a mesoporous silica (MPS) surface by a direct one-step method and the amount of subtilisin immobilized on each functionalized MPS surface was similar (approximately 0.30 mg of enzyme/mg of MPS support). The catalytic performance (hydrolytic activity and enantioselectivity) of the immobilized subtilisin was found to depend on the properties of the organofunctional group on the MPS surface. In particular, the hydrolytic activity of enzyme immobilized on ethyl-group-modified MPS increased relative to the behavior of free subtilisin (relative activity 143%). The activity of subtilisin immobilized on the modified MPS was improved by facilitation of contact between enzyme and hydrophobic substrate by increase in hydrophobicity with an immobilized carrier. On the other hand, the enantioselectivity of subtilisin immobilized on 3-mercaptopropyl-group-modified MPS significantly decreased (enantioselectivity of 2.6 compared to 4.3 for free subtilisin). This decrease in enantioselectivity indicated that the mercapto group on the MPS surface was changed in the secondary structure of enzyme by interacting between enzyme and immobilized support. The denaturation temperature of subtilisin immobilized on no-substituted MPS increased (65 °C compared with 57 °C for free subtilisin). The denaturation temperature of immobilized subtilisin was dependent on the absorbed fraction of thermal energy by functional groups on the MPS surface.

Regulation of cellular responses to macroporous inorganic films prepared by the inverse-opal method.

Regenerative medicine for repairing damaged body tissues has recently become critically important. Cell culture scaffolds are required for the control of cell attachment, proliferation, and differentiation in in vitro cell cultures. A new strategy to control cell adhesion, morphology, and proliferation was developed by culturing mouse osteoblast-like MC3T3-E1 cells on novel cell culture scaffolds fabricated using ordered nanometer-sized pores (100, 300, 500, and 1000 nm). Results of this study indicate that after 72 h of incubation, the number of cells cultured on a silica film with a pore size of 1000 nm was similar to or slightly lower than that cultured on a non-porous control silica film. Films with 100-500 nm pore sizes, however, resulted in the cell growth inhibition. Morphology of the cultured cells revealed increased elongation and the formation of actin stress fibers was virtually absent on macroporous silica films with 100-500 nm pore size. Vinculin molecules expressed in cells cultured on the non-porous silica films showed many clear focal adhesions, whereas focal contacts were insufficiently formed in cells cultured on macroporous films. The influence of hydroxyapatite (HAp) and alumina scaffolds on the behavior of MC3T3-E1 cells was also evaluated. The proliferation rate of MC3T3-E1 cells cultured on HAp films with 1000 nm pore size was increased to approximately 20% above than that obtained of cells cultured on non-porous HAp films. These results demonstrate that the pore size and constituents of films play a role in controlling the morphology and proliferation rate of MC3T3-E1 cells.