Simple Determination of Trace Amounts of Anionic Surfactants in River Water by Spectrophotometry Combined with Solid-phase Extraction

A simple and sensitive specrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6×10^?8 M to 5.0×10^?7 M and for SDS from 2.0×10^?9 M to 3.0×10^?7 M. The relative standard deviation (n=5) for 5.0×10^?7 M DBS was 3.1% and for 2.5×10^?7 M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples.     

Studies on Nicotinoids as an Insecticide

In search for the mechanism of insecticidal action of nicotinoids, the kinetics of house fly head cholinesterase inhibition by nicotine were studied to determine the type of inhibition. The pH dependency of inhibition was interpreted in terms of protonation of nitrogen atom in the molecule and the inhibition was shown to be the mixed type closing to competitive type. The Michaelis constants are 3.5 × 10^?4 M and 4.1 × 10^?4 M, while the apparent inhibition constants obtained are 1.0 × 10^?3 M and 2.3 × 10^?3 M at pH 7.4 and 8.4, respectively. The type of the inhibition by nicotine monomethiodide carring univalent cation was competitive and the apparent inhibition constant is 1.5 × 10^?4 M. These data indicated that the cationic head of nicotinium ion interacts with the anionic site in the active center of cholinesterase.     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

Kinetic Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

Effects of the substrate and the coenzyme on the crystalline yeast phosphoglyceric acid mutase activity have been investigated. Lineweaver-Burk plots at different concentrations of the substrate (d-3-phosphoglyceric acid: 3×10^?7 to 8×10^?3m) and the coenzyme (d-2, 3-diphosphoglyceric acid: 8×10^?7 to 10^?5m) change in such a way to indicate the involvement of an enzyme-substrate-coenzyme ternary complex as an active intermediate in the enzymic reaction process. It is concluded that the reaction catalyzed by the yeast enzyme follows the sequential pathway and that a phosphorylated enzyme does not participate as an obligatory intermediate in the reaction mechanism, if it occurs. Kinetic studies indicate Km values of 6×10^?4m for d-3-phosphoglyceric acid and 8×10^?7m for d-2, 3-diphosphoglyceric acid. The substrate is a competitive inhibitor of the coenzyme with a Ksi (inhibition constant) of 3.2×10^?3m. The coenzyme inhibition is not observed at concentration tested. A kinetic treatment to determine the mechanism of the enzyme reaction from the experimental data which are obtaind in the range of inhibitory substrate concentrations is presented.     

Physicochemical and Enzymatic Properties of ATP: Nucleotide Pyrophosphotransferase

The molecular weight determined by the sedimentation equilibrium and SDS Polyacrylamide gel electrophoresis was 29,000 and 28,000, respectively. Isoelectric point of the enzyme was determined as pH 7.7. This enzyme contained large amounts of alanine, aspartic acid, glutamic acid and serine, and no cysteine residue was found. The enzyme was inhibited by SDS, KMnO₄, EDTA and tetracycline. GTP and GDP were the most active as pyrophosphate acceptor to the enzyme. The apparent Km for ATP was 2.2×10^?4 m and that for GTP was 2.1×10^?4m in the reaction of ATP+GTP→AMP+pppGpp. On the other hand, in the reaction of 2ATP→AMP+pppApp, the apparent Km for donor and acceptor ATP was 1.7×10^?3m. Effects of pH and metal ions on the enzymatic synthesis of pppGpp were also studied.     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.     

Depolymerization of Hyaluronic Acid by D-Fructose 6-Phosphate

Depolymerization of hyaluronic acid obtained from Streptococcus zooepidemicus by D-fructose 6-phosphate was investigated for characterization of reducing sugar-mediated degradation of biopolymers under physiological conditions. The extent of depolymerization was monitored by the decrease of viscosity of a reaction mixture containing 1.0% hyaluronic acid, D-fructose 6-phosphate, and 1.0 × 10^?2 mM of Cu²⁺ in phosphate buffer, pH 7.4. It was found that the depolymerization of hyaluronic acid was dependent on the concentration of the reducing sugar and was specifically accelerated by the presence of Cu²⁺. The reaction was found to be significantly inhibited by catalase, superoxide dismutase (SOD), 1,2-dihy­ droxybenzene 3,5-disulfonic acid (Tiron), and chelating agents such as EDTA and diethylene triamine penta­ acetic acid (DETAPAC), although the inhibition by SOD was low. Almost the same depolymerization rates were observed in hyaluronic acid preparations of different molecular weight (1.1 × 10⁶, 8.8 × 10⁵, and 6.8 × 10⁵). The rates, however, were different for hyaluronic acids obtained from S. zooepidemicus, rooster comb, and umbilical cord. It was concluded that depolymerization of the polysaccharide was caused by active oxygen species generated by the autoxidation of D-fructose 6-phosphate in the presence of Cu²⁺, in a mechanism similar to that previously reported for the degradation of DNA and inactivation of virus in vitro.     

Formation of Adenosine Triphosphate in Isolated Aleurone Particles of Rice Grains

Using isolated aleurone particles of rice grains it was found that ADP was easily transformed to ATP. This reaction is likely explained by either a transphosphorylation of a phosphate group from phytic acid to ADP or a mechanism involving adenylate kinase. The optimum temperature and pH for this reaction were 30°C and 7.8 respectively. The apparent Km for ADP was 4.0 × 10^?4 m. Cyanide arsenate and azide at 10^?3 to 10^?2 m had no effect on the ATP formation, indicating that the formation was not due to oxidative phosphorylation.     

Enzymatic Conversion of Dethiobiotin to Biotin in Cell-free Extracts of a Bacillus sphaericus bioB Transformant

The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant. Among the sulfur compounds tested, only S-adenosyl-l-methionine was active, while l-methionine and l-cysteine had no significant effect. Protein concentrations higher than 15mg/ml in the reaction mixture were needed to detect biotin synthase activity. When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe²⁺, Mn²⁺, and Ca²⁺ among the metal ions tested also had some effects.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Inhibitory Activities of 3-Thiophenecarboxylic Acid and Related Compounds on Plant Growth

3-Thiophenecarboxylic acid (1) showed strong growth-inhibitory activity toward the following plants but not Glycine max Merrill; Brassica campestris subsp. rapa Hook. fil. et Anders, Sesamum indicum L., Lactuca sativa L. var. longifolia Lam, Echinochloa utilis Ohwi et Yabuno and Allium tuberosum Rottler. Compound 1 strongly inhibited the growth of roots of S. indicum and L. sativa even at the low concentration of 5.0 × 10^?5 m. The growth-inhibitory activity of 1-related compounds (2–6) on S. indicum was also studied. Among the compounds, 3-thiopheneacetic acid (6) showed the strongest inhibitory activity, but 3-thiophenecarboxaldehyde (2), 3-thiophenemethanol (3), and 3-thiophenecarboxamide (5) showed no activity. The radicles of plants treated with these active compounds showed negative geotropism.     

Production of l-aspartic acid by biotransformation and recovery using reverse micelle and gas hydrate methods

l-Aspartic acid (l-Asp) was produced using Escherichia coli (ATCC 11303), and its recovery from the reaction mixture was studied using reverse micelle and gas hydrate methods. The effect of initial substrate concentration on l-Asp production was also investigated, and inhibition was shown to occur above 0.75 mol L^?1. The values of the kinetic constants were determined as r_max=2.33×10^?4 mol L^?1 min^?1, K_M=0.19 mol L^?1, and K_ss=3.98 mol L^?1. The reverse micelle phase used for extraction contained Aliquat-336, 1-decanol and isooctane, and a micro-injection technique was used for extraction of l-Asp. The reverse micelle system is a useful technique for obtaining small particle sizes, which can be used for the synthesis of nanoparticle biomolecules. Recovery of l-Asp from reverse micelles using CO₂ hydrates was carried out, giving a recovery of 55%. The formation of CO₂ hydrate from the reverse micelle solution breaks the micelle by reducing the amount of water in the micelle structure, thus precipitating the l-Asp.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Synthesis of (1→2)-β-d-Glucan by Cell-free Extracts ofAgrobacterium radiobacter IFO 12665b 1 andRhizobium phaseoli AHU 1133

Particulate preparations from Agrobacterium radiobacter IFO 12665b 1 and Rhizobium phaseoli AHU 1133 have been shown to catalyze the synthesis of (1→2)-β-d-glucan from UDP-d-[¹⁴C]glucose. The (1→2)-β-d-glucans synthesized are suggested to be in a cyclic form without other glycosidic linkages and to consist of a mixture of several components with degrees of polymerization of 17 and more. The enzyme systems from A. radiobacter IFO 12665b 1 and R. phaseoli AHU 1133 both required Mn²⁺ and had optimum activities at pH 7.5 ~ 8, and their Km values for UDP-d-[¹⁴C]glucose were 5 × 10~⁵ m and 3.3 × 10^?5 m, respectively.     

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-d-glucitol

Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-d-glucitol (1,5-AG) and l-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (k_cat/K_m) for l-sorbose and 1,5-AG is 1.8 × 10² and 1.5 × 10² s^?1·M^?1, respectively.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.