Attempt of Microbial Mutagen Screening with Rec-assay; Isolation of Chartreusin

A strong Cotton effect, which practically govern the sign of the optical rotation at 589 nm ([M]_d), was studied in phenyl 1-thio-α (and β)-d-glycopyranosides with our new chiroptical technique. The proposal optical rotatory dispersion (ORD) method, with calculations based on a one term Drude equation, showed the presence of a strong Cotton effect at 200–210 nm. Circular dichroism (CD), with accumulation technique, also gave the same Cotton effect. Agreement in these two methods suggests the usefulness of the proposed ORD calculation method. The rotational strengths and the signs were shown to reflect the anomeric configurations and conformations (α-anomer gave positive and β-anomer gave negative signs; axial gave strong and equatorial gave weak bands). This result is an extension of the ring oxygen helicity rule of alkyl and alkyl thioglycosides to phenyl 1-thioglycopyranosides, and probably to other aromatic glycosides.     

Conformation of Some Hexuronic Acids and d-Galactopyranosiduronic Acid Moiety in the Peracetylated and Permethylated Derivatives of Orange Pectic Acid in Solutions

1. The 1C conformation was estimated for α-d-galactopyranosiduronic acid moiety of pectic acid in the permethylated derivative dissolved in 1 n NaOD-D₂O and in the peracetylated derivative dissolved in dimethyl sulfoxide-d₆, and the C1 conformation was estimated for some derivatives of d-galactopyranuronic acid in chloroform-d by NMR spectroscopy.

2. Random conformation of the whole macromolecule was estimated for pectic acid in water on the basis of no appearance of any induced Cotton effects in the 200 ~ 700 mμ region in the ORD spectra of pectic acid-anionic dye complexes.

3. The conformation was supported by the fact that the rate of periodate oxidation of pectic acid at 5° was slightly decreased in comparison with that of amylase in 7 m urea solution.

     

A Growth Factor for Malo-lactic Fermentation Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium has been isolated from tomato juice, and found to be a β-glucoside. The NMR spectra of TJF and its acetate revealed that the glucosyl residue linked to the hydroxyl group at C-2′ or C-4′ of d- or l-pantothenic acid moiety. Then, 2′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (I), 4′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (II) and 4′-O-(β-d-glucopyranosyl)-d(R)-pantothenic acid (II-a) were synthesized, and Il-a and 4′-O-(β-d-glucopyranosyl)-l-pantothenic acid (II-b) were obtained by the optical resolution of the acetate of II. Among the above compounds, II-a was identical with natural TJF regarding to the biological activity, NMR and ORD spectra, and thin-layer chromatography.     

Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.     

The Mutational Effect of Ile58 at Subsite C in Hen Egg-White Lysozyme on Substrate Binding,Enzymatic Activity,and Protein Stability

Partial acid hydrolysis of Saccharomyces cerevisiae mannan gave 2-O-α-d-Manp-d-Man (1), 3-O-α-d-Manp-d-Man (2), 6-O-α-d-Manp-d-Man (3), O-α-d Manp-(1→2)O-α-d-Manp-(1→2)-d-Man (4), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-d-Man (5), O-α-d Manp-(1→6)-6-O-α-d-Manp-(1→6)-d-Man (6), O-α-d Manp-(1→2)-O-α-d-Manp-(1→2)-6-O-α-d-Manp-(1→6)-d-Man (7), O-α-d-Manp-(1→2)-O-α-d-Manp-(1→6)-O-α-d-Manp-(1→6)-d-Man (8), and O-α-d-Manp-(1→6)-O-[α-d-Manp-(1→2)]-O-α-d-Manp-(1→6)-d-Man (9).     

Crystalline d-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

The crystalline d-mannitol dehyrogenase (d-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of d-fructose to d-mannitol. d-Sorbitol was oxidized only at the rate of 4% of the activity for d-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of d-fructose or the oxidation of d-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035 m for d-fructose and 0.020 m for d-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 × 10^?5 m for NADH₂ and 2.7 × 10^?4 m for NAD.     

Synergistic Antimicrobial Effect of Acetic Acid,Sodium Chloride and Essential Oil Components

Acetic acid, NaCl and essential oil components were examined for their synergistic antimicrobial effect, using air-borne microorganisms and purely cultured fungi. Antimicrobial assays were carried out at 27°C, using 2% glucose Sabouraud agar. In order to completely suppress the growth of all the contaminating air microorganisms over a period of one month, more than 0.2% acetic acid or more than 25% NaCl was required in the medium. Any one of the essential oil components examined, at a concentration of as high as 1 mm or more, permitted considerable growth of various air microorganisms within several days after contamination. However, in combination with both 0.1% acetic acid and 3% NaCl, perillaldehyde, citral (αβ-unsaturated aliphatic aldehydes), citronellol, geraniol, perillalcohol (primary alcohols) or cuminaldehyde, at a concentration of 0.5 mm, completely suppressed the growth of all the contaminating air microorganisms over a period of one month.

Cinnamaldehyde was approximately twice as potent as these compounds in this respect, l-Menthol (secondary alcohol) and d-carvone (α,β-unsaturated ketone), at a concentration of 1 mm but not 0.5 mm, completely suppressed such microbial growth under the same exprimental conditions. Citronellal (α,β-saturated aldehyde) and linalool (tertiary alcohol) were somewhat less effective than l-menthol and d-carvone. Hydrocarbons examined (d-limonene, α-pinene, α-pinene, camphene, β-myrcene, β-caryophyllene and p-cymene), even at 2 mm, were only moderately effective in this respect.

Similar synergistic antimicrobial effects of these substances were observed when using purely cultured fungi.

These results strongly suggest that acetic acid, NaCl and certain essential oils (or their components), when combined together, are applicable at relatively lower concentrations for effective preservation of certain foods without applying synthetic preservatives.     

Enzymatic Assay of d-Xylose Isomerase Activity

The d-xylose isomerase activity was assayed spectrophotometrically as NADH oxidation in a coupled reaction with the d-arabitol dehydrogenase. The assay system is based on the following reactions:

d-Arabitol dehydrogenase was purified from the d-sorbitol-grown cells of Agrobacterium tumefaciens. The standard assay condition is as follows: 5 μmoles of Tris-HCl buffer (pH 7.0), 0.2 μmole of MnCl₂, 2 μl of reduced glutathione (25 mg/ml), 0.05 μmole of NADH, 6 units of d-arabitol dehydrogenase, 5 μmoles of d-xylose and d-xylose isomerase in a total volume of 0.30 ml. The reaction was carried out at 30°C. With the assay system, it was confirmed that d-xylose isomerase did not produce d-xylulose from d-lyxose.     

Syntheses of 14C-Labeled Pyrethrin I,Allethrin, Phthalthrin,and Dimethrin on a Submillimole Scale

Studies on the metabolic fate and degradation chemistry of pyrethroid insecticide chemicals are greatly facilitated by the use of compounds radiolabeled, in separate preparations, in the acid and alcohol moieties. Acid-labeled preparations were made by converting d-trans-chrysanthemic acid-1-¹⁴C (88 mg, 1.3 mCi/mm) into d-trans-d-pyrethrin-1-¹⁴C (68 mg, 1.3 mCi/mm), d-trans-d-allethrin-¹⁴C (43 mg, 1.3 mCi/mm), d-trans-dimethrin-¹⁴C (54 mg, 0.294 mCi/mm), and d-trans-phthalthrin-¹⁴C (47 mg, 0.294 mCi/mm), incorporating approximately 81% of the starting radiocarbon into the four pyrethroid preparations. Alcohol-labeled preparations were made by converting acetone-1,3-¹⁴C into d-trans-dl-ailethrin-¹⁴C (146 mg, 0.162 mCi/mm) and formaldehyde-¹⁴C into d-trans-phthalthrin-¹⁴C (299 mg, 0.276 mCi/mm). Each labeled compound had a high stereochemical purity and a radiochemical purity of greater than 99%. Detailed procedures were worked out for all conversions which took place in high yields except in one case: the synthesis of allethrin labeled in the alcohol moiety.     

Accumulation of S-Adenosylmethionine by Molds

Aspergillus tamani accumulated about 20 μmoles of S-adenosylmethionine (SAM) in 1 g of dry cells when cultured secondarily in a medium containing more than 10 mm of l- methionine. The accumulation was not so high when l-methionine was replaced by d- methionine. Addition of nucleic acid-related substances was not effective for the accumulation. Addition of d, l-ethionine in place of methionine caused accumulation of S-adenosylethionine (SAE) in place of SAM. Among 100 strains of molds tested, a number of strains belonging to the genera Penicillium, Aspergillus, Rhizopus and Mucor could accumulate SAM in their mycelia. Especially Mucor jansseni had the highest ability; it accumulated 45 μmoles of SAM in 1 g of dry cells.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Formation of 2-(5-Hyclroxymethyl-2-formylpyrrol-1-yl)alkyl Acid Lactones on Roasting Alkyl-α-amino Acid with d-Glucose

d-Glucose and several alkyl-α-amino acids (glycine, dl-α-alanine, dl-α-amino-n-butyric acid, l-valine, l-leucine and dl-α-amino-n-caproic acid) were roasted at 200°C or 250°C in a simple two components system. From the roasting products were newly isolated a series of 2-(5-hydroxymethyl-2-formylpyrrol-1-yl)alkyl acid lactones which were characterized by elementary analysis, UV, IR, MS (GC-MS) and NMR spectra.

These lactones have characteristic aroma which may contribute to the flavor produced by sugar-amino acid reaction. The subjective evaluation of aroma of the lactones obtained wrere as follows: 2-(5-hydroxymethyl-2-formyipyrrol-1-yl)propionic acid lactone, caramel and a little scorching; -n-butyric acid lactone, maple and strong sweet; isovaleric acid lactone and isocaproic acid lactone, miso, soy sauce and a little chocolate-like.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

Mechanism of Asymmetric Production of d-Amino Acids from the Corresponding Hydantoins by Pseudomonas sp.

The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).

l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.

It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and directly converted to the corresponding d-amino acids through the successive reactions of d-HYD hydrolase and d-NCA hydrolase in parrallel with the spontaneous racemization of l-5-substituted hydantoins to those of dl-form.     

Isolation and Identification of ( – ) - Quinic Acid as an Unidentified Major Tea-component

A new enzyme, N-acetyl- d-hexosamine dehydrogenase (N-acety 1-α-d-hexosamine: NAD⁺ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12 %. The molecular weight of the enzyme was about 124,000 on gel filtration and 30,000 on SD S-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-d-glucosamine (Km = 5.3mm) and N-acetyl-d-galactosamine (Km = 0.8mm) as the sugar substrate. But it was not so active on N-acetyl-d-mannosamine. NAD⁺ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-d-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-d-glucosaminic acid. The enzyme activity was inhibited by Hg and SDS, but many divalent cations, metal-chelating reagents, and sulfhydryl reagents had no effect.     

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium.

• The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.

• The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.

• The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.

• There was no strain tested so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.

     

Purification and Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.     

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.