Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose.

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16-2.5 mM glucose, with a correlation coefficient, r, of 0.998.

A Rapid Enrichment Technique for the Ultratrace Determination of Nickel in Water Samples Using a Nanofiber-composite Membrane Filter.

A new method for the rapid enrichment and highly sensitive determination of nickel ion has been developed by using a nanofiber-composite membrane filter, which was fabricated by stacking a nanofibrous material made of nylon 6 over a water-permeable membrane filter. The noncharged nickel-α-furil dioxime complex was adsorbed on a nanofibrous layer of the membrane filter under significantly higher flow rates than those used for conventional solid-phase extraction techniques. Highly sensitive determinations with detection limits at sub-parts per billion levels were achieved by enrichment from 50 mL of the complex solution, and the enrichment was completed within 3 min. The color that was developed on the membrane filter was successfully subjected to visual colorimetric analysis and quantitative determination by solid-phase spectrophotometry. In addition, colorimetric determination was feasible with a handheld spectrometer after elution of the colored agent with 50 µL of acetone. This combination of rapid enrichment and spectrometric measurement in a small-volume sample provides a useful analytical method suitable for on-site analysis, which requires neither expensive instruments nor high laboratory skills.

Role of Cys73 in the thermostability of farnesyl diphosphate synthase from Geobacillus stearothermophilus.

Farnesyl diphosphate synthase (FPPase) is an enzyme that catalyzes the condensation between one molecule of dimethylallyl diphosphate (DMAPP) and two molecules of isopentenyl diphosphate (IPP) to produce farnesyl diphosphate (FPP). FPP is an important precursor in the isoprenoid synthesis pathway. In this study, the crystal structure of FPPase from Geobacillus stearothermophilus (GsFPPase) was determined at 2.31 Å resolution. The structure of GsFPPase shows a three-layered all α-helical fold and conserved functional domains similar to other prenyltransferases. We have analyzed the structural features of GsFPPase related to thermostability and compared it with those of human and avian mesophilic FPPases. "Semi-conserved" regions which appear to be possible features contributing to the thermostability of FPPase were found.

Lubricant and Bactericidal Properties of Calcium Salts of Fatty Acids: Effect of Degree of Unsaturation.

Fatty acids containing a C18 alkyl chain such as stearic acid (C18:0 fatty acid), oleic acid (C18:1 fatty acid) and linoleic acid (C18:2 fatty acid) are common emulsifiers in skin-care products and cosmetics and are also used in skin cleansers. In this study, we prepared calcium salts (Ca salts) of the above fatty acids to determine the effect of the degree of unsaturation of the alkyl chain. Scanning electron microscopy images and X-ray diffraction patterns show that C18:0 and C18:1 fatty acid Ca salts are plate-shaped, lamellar-crystalline powder, while C18:2 fatty acid Ca salt is amorphous powder. Therefore, C18:2 fatty acid Ca salt exhibits a lower lubrication ability than do C18:0 and C18:1 fatty acid Ca salts. In addition, the bactericidal ability against Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes improved with increasing degree of unsaturation. These findings suggest that Ca salts of unsaturated fatty acids have potential applications in cleansing and cosmetic products.

Palmitoleic acid calcium salt: a lubricant and bactericidal powder from natural lipids.

Palmitoleic acid is a promising bactericidal agent for cleansing products with alternative bactericidal abilities. In this study, we focus on the physical and biological activity of palmitoleic acid calcium salt (C16:1 fatty acid Ca salt) because it forms via an ion-exchange reaction between palmitoleic acid and Ca ions in tap water, and remains on the skin surface during the cleansing process. Here, we prepared C16:1 fatty acid Ca salt to investigate its crystal structure and physical and bactericidal properties. The Ca salt was a plate-shaped lamellar crystalline powder with a particle diameter of several micrometers to several tens of micrometers; it exhibited significant lubricity and alternative bactericidal activity against Staphylococcus aureus (S. aureus) and Propionibacterium acnes (P. acnes). We also examined other fatty acid Ca salts prepared from lauric acid (C12:0 fatty acid), palmitic acid (C16:0 fatty acid), and oleic acid (C18:1 fatty acid). The bactericidal activities and lubricity of the fatty acid Ca salts changed with the alkyl chain length and the degree of unsaturation. The C16:1 fatty acid Ca salt exhibited the strongest selective bactericidal ability among the four investigated fatty acid Ca salts. These findings suggest that C16:1 fatty acid and its Ca salt have potential applications in cleansing and cosmetic products.

Enantioresolution of 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl oxide using inclusion complex with chiral 2,2'-dihydroxy-1,1'-binaphtyl.

An enantioresolution of 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl oxide (BINAPO) into its enantiomers was achieved using the inclusion complex with a commerciallyavailable chiral 2,2'-dihydroxy-1,1'-binaphthyl ((R)-BINOL), giving the two enantiomers with 99% ee and 72% ee, respectively.

Conformation and absolute configuration of (1S,2S)-2-(phenyl-selan-yl)cyclo-hexyl (R)-2-meth-oxy-2-(1-naphth-yl)propionate.

The relative and absolute configurations of the title compound, C(26)H(28)O(3)Se, were assigned from the known configuration of (R)-(-)-2-meth-oxy-2-(1-naphth-yl)propionic acid used as starting material, and by examination of the Bijvoet (Friedel) pairs, using the anomalous dispersion data collected with Mo Kα radiation at low temperature. The geometry around the carbonyl group exists in the syn conformation, as reflected in torsion angles involving this group, and the stability of the structure is affected by weak bifurcated intra-molecular C-H⋯O hydrogen bonds.

Zinc-mediated allylation and alkylation of aminals in the presence of TMSCl and diisopropylamine.

An alkylation of aminals with organozinc reagents derived from allyl bromide, benzyl bromide, alpha-bromoacetate, and alpha-bromonitrile proceeded efficiently in the presence of TMSCl and diisopropylamine. This reaction system was applied to the synthesis of an antispasmodic: butaverine.

Lipase-catalyzed enantioselective esterification of mono-aza-benzo-15-crown-5-ether derivatives in organic media.

For the purpose of developing a new chiral crown ether unit as a chiral synthon, three racemic mono azabenzo-15-crown-5-ethers, i.e. (R,S)-1-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-propan-2-ol, (R,S)-2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-1-phenyl-ethanol and (R , S)-1-[2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-ylmethyl)-phenyl]-ethanol were esterified with vinyl acetate using a lipase from Candida antarctica. The enzymatic acylation of alcohols produced monoacylated products. Two optically active azacrown ethers, (R)-propionic acid 1-methyl-2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-ethyl ester and (R)-acetic acid 1-[2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-ylmethyl)-phenyl]-ethyl ester were obtained within 48% and 36% yields, respectively and, at an enantiometric excess of over 99% in each case.

Fluorescence spectroscopic study of alpha-chymotrypsin relevant to the enantioselectivity for optical resolution of amino acid esters in organic solvents.

The fluorescence emission wavelength of alpha-chymotrypsin (CT) correlated with its enantioselectivity (E value) for the resolution of DL-tyrosine ethyl ester. The changes in the E value of the CT due to the changes in the solvent composition were closely related to its fluorescence properties (delta lambda(em)), which were most probably associated with the structural modification of the enzyme. A linear relationship was established between E value and delta lambda(em) in aqueous acetonitrile with high correlation coefficients (r = 0.94).

Electrical communication between NAD-dependent enzyme and metal electrode using ferrocene-labeled NAD derivatives.

The target N6-ferrocene substituted NAD derivatives (ferrocene-labeled NAD, Fc-NAD) were synthesized as an electron-relay NAD cofactor. The synthesized NAD derivatives possessed some biological activities as substrates in the alcohol dehydrogenase systems. The oxidation current depended on both addition of ethanol and spacer length of Fc-NAD.