Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis

Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC(Re)), have narrow substrate specificity toward (R)-3-hydroxyacyl-coenzyme A with acyl chain length of C3-C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC(Re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[(R)-3-hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB(-)4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB(-)4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli. Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild-type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC(Re). Molecular weight distributions of PHA polymers synthesized by the wild-type enzyme (PhaC(Re)) and its mutants.     

Synthesis of (4R,15R,16R,21S)- and (4R,15S,16S,21S)-rollicosin

A convergent synthesis of (4R,15R,16R,21S)-rollicosin (1) and (4R,15S,16S,21S)-rollicosin (2) was accomplished. Hydroxy lactone 6a and/or 6b were synthesized from 4-pentyn-1-ol, and α,β-unsaturated lactone 7 was synthesized from γ-lactone 8 and 5-hexen-1-ol. Inhibitory activity of these compounds was examined with bovine heart mitochondrial complex I.     

CVD growth of single-walled carbon nanotubes with narrow diameter distribution over Fe/MgO catalyst and their fluorescence spectroscopy

Single-walled carbon nanotubes (SWNTs) with a narrow diameter distribution are synthesized by thermal chemical vapor deposition (CVD) of methane over Fe/MgO catalyst on the basis of parametric study considering Fe loading, reaction temperature and time, methane concentration, and structure of a support material. We found that the porous MgO support gives the SWNTs with a narrow diameter distribution with the mean diameter and standard deviation of 0.93 and 0.06 nm, respectively, only when the Fe loading and reaction temperature are relatively low. The higher Fe loading and/or the higher reaction temperature enlarged the nanotube diameter, forming double-walled carbon nanotubes (DWNTs) in addition to SWNTs. This result indicates that only the diameter of Fe nanoparticles determines the growth of either SWNTs or DWNTs on the MgO support. The fluorescence and absorption spectra of the nanotube dispersion in D(2)O solution with sodium dodecyl sulfate (SDS) were studied to identify their chirality distribution. The fluorescence of the uniform-diameter SWNTs indicates the formation of the near armchair structures. On the other hand, the SWNTs synthesized over the catalyst with a high Fe loading, 3 wt %, showed a wide chirality distribution including the near zigzag structure. The synthesis of the SWNTs with a narrow diameter distribution could be applied to the selection of SWNTs with a specific chirality based on postsynthesis separation.     

Process of destruction of large unilamellar vesicles by a zwitterionic detergent, CHAPS: partition behavior between membrane and water phases

The process of vesicle destruction by zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), was examined to clarify the vesicle-micelle transition mechanism. The physicochemical properties including turbidity, apparent particle size, Cl(-) permeability, electron spin resonance (ESR) spectroscopic parameters, and freeze-fracture electron microscopy were investigated. The concentration of CHAPS was analyzed using HPLC to determine the partition coefficient during the solubilization process. The data obtained revealed that maximum turbidity and apparent particle size were found at the effective ratio (R(e)) of 0.21 and 0.49, respectively. With a further increase in CHAPS concentration, turbidity and particle size abruptly decreased, suggesting the formation of mixed micelles. The partition coefficient changed throughout the solubilization process. In the presence of low concentrations of CHAPS, CHAPS partitioned into vesicles without destruction of membrane bilayers. When the R(e)<0.04, the partition coefficient was independent of the detergent concentration with value of 24 M(-1). At R(e) greater than 0.05, the membrane barrier abruptly decreased. At 0.04相似文献   

Catalytic,asymmetric Mannich-type reactions of N-acylimino esters: reactivity,diastereo- and enantioselectivity,and application to synthesis of N-acylated amino acid derivatives

In the presence of a catalytic amount of Cu(OTf)(2)-chiral diamine 3e complex, N-acylimino esters reacted with silyl enol ethers to afford the corresponding Mannich-type adducts in high yields with high enantioselectivities. A wide variety of silyl enol ethers derived from ketones, as well as esters and thioesters, reacted smoothly. In the reactions of alpha-substituted silyl enol ethers (alpha-methyl or benzyloxy), the desired syn-adducts were obtained in high yields with high diastereo- and enantioselectivities. Several intermediates for the synthesis of biologically important compounds were prepared using this novel catalytic asymmetric Mannich-type reaction, and at the same time, absolute and relative stereochemical assignments were made. In addition, it has been revealed that alkyl vinyl ethers reacted with N-acylimino esters in the presence of a catalytic amount of the Cu(II) catalyst to give the corresponding Mannich-type adducts in high yields with high enantioselectivities. This is the first example of catalytic asymmetric Mannich-type reactions with alkyl vinyl ethers. The reaction mechanism, structure of chiral catalyst-electrophile complexes, and transition states of these catalytic asymmetric reactions were assumed based on X-ray crystallographic analysis of the Cu(II)-chiral amine complex, PM3 calculations, and FT-IR analyses, etc. Finally, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12, 1), a new inhibitor of ceramide trafficking from endoplasmic reticulum to the site of sphingomyerin (SM) synthesis, has been synthesized efficiently using the present Mannich-type reaction as a key step. The synthesis involved three steps (two-pot), and total yield was 82.9%.     

Flow-through micro sensor using immobilized peroxidase with chemiluminometric FIA system for determining hydrogen peroxide.

A micromachined flow cell (overall size; 25 x 25 x 1 mm3) was designed for the fast determination of hydrogen peroxide, based on a luminol-H2O2 chemiluminescence reaction catalyzed by immobilized peroxidase (POD). The flow cell consisted of a sandwich of anisotropically etched silicon and glass chips and contained a spiral channel (20 turns, 50 cm long, 150 microm wide, 20 microm depth, channel volume 1.4 microl) and two holes (1 mm diameter). POD was covalently immobilized with 3-(trimethoxysilyl)propyldietylenetriamine and glutaraldehyde on the inner surface of the channel. The chip was placed in front of a window of a photomultiplier tube and used as a flow cell in a single-line flow-injection analysis system using a luminol solution as a carrier solution. The sample volume for one measurement was 0.2 microl. The maximal sampling rate was 315 h(-1) at a carrier solution flow rate of 10 microl min(-1). A calibration graph for H2O2 was linear for 5 nM - 5 microM; the detection limit (signal-to-noise = 3) was 1 nM (7 fg in 0.2 microl injection). The H2O2 concentration in rainwater was determined using this sensor system.     

Carbonylative cross-coupling reaction of aryl iodides with alkylaluminums by palladium complex catalysis

Secondary and/or tertiary alcohols and unsymmetrical ketones have been obtained in moderate to good yields by the palladium-catalyzed (5 mol%) carbonylative coupling of aryl iodides with alkylaluminum compounds under very mild conditions (20–50°C, 1 atm of carbon monoxide). The type of the reaction product depended on the aluminum reagent employed. While the selective formation of secondary alcohols was observed in the reaction with i-Bu₃Al, the use of Et₃Al led to a mixture of a ketone and two alcoholic products. With Et₂AlCl predominantly unsymmetrical ketones were produced. In all cases, formation of directly cross-coupled products was not observed. DME and benzene can be used as solvents, but THF is unsuitable. Nickel catalysts were found to be ineffective for this reaction.     

Preparation of hydrolyzed pH responsive ion track membrane

The porous copolymer films of N-methacryloyl-L -alanine methyl ester and diethyleneglycol-bis-allylcarbonate (diallyl oxydiethylene dicarbonate) were hydrolyzed with 1 M NaOH solution for 40 min at 25°C. The pores of the films showed pH response in diameter. The pore diameter of 3,7 μm in the buffer solution of pH 3 was closed completely at pH 5.     

PHOTOCHEMISTRY OF ACETYLENIC KETONES IN MICELLAR SOLUTIONS AS STUDIED BY PRODUCT-YIELD-DETECTED ESR AND TRANSIENT ABSORPTION TECHNIQUES

The electron spin resonance (ESR) spectra of the transient radical pairs in the photoreduction of 1,5-diphenyl-1,4-pentadiyn-3-one ( I ) and 1,3-diphenyl-2-propyn-1-one ( II ) in sodium dodecyl sulfate (SDS) micellar solutions have been obtained by using the product-yield-detected ESR (PYESR) technique. The PYESR spectra, detected by tracing the microwave effect on the spin-adduct yield as functions of the magnetic field, show the ESR spectra of the ketyl radical of the ketone and SDS radical as the components of the radical pairs. In addition, the growth and the decay processes of the radical pair were observed through detecting the effect of microwave pulse as functions of the delay period between a laser pulse and the off and on time, respectively, of a microwave pulse. The absorption spectra of transient species have also been obtained by using the laser flash photolysis technique. Through the analysis of these data and molecular orbital calculations, the role of acetylenic groups in the photoreactivity of acetylenic ketones is discussed.