Studies on the Metabolites of Aspergillus versicolor (Vuillemin) Tiraboschi

Three new pigments, named versicolorins A, Band C, as metabolites from the mycelium of Aspergillus versicolor have been isolated. Versicolorin A, C₁₈H₁₀O₇, is fine orange yellow needles, m.p. 289°C (decomp.), [α]_D^-354°. It is an anthraquinoid pigment having three hydroxyl groups and a vinyl ether system contained in a five-membered ring. Versicolorin A trimethyl ether was hydrogenated to a dihydro-derivative, and by oxidation gave 3,5-dimethoxyphthalic acid and a hydroxy acid which may be 1,6,8-trirnethoxy-3-hydroxy anthraquinone-2-carboxylic acid. These chemical behavior and NMR data show that versicolorin A probably has the structure of (I). Versicolorin B, C₁₈H₁₂O₇, is fine orange yellow needles, m.p., 298°C (decomp.), [α]_D^-223° Its trimethyl ether is identical with that of dihydroversicolorin A. Therefore, the structure (II) could be assigned to versicolorin B. Versicolorin C, C₁₈H₁₂O₇, is orange red needles, m.p.>310°C, [α]_D O° Comparison of optical properties, IR and NMR spectra of versicolorin B and its methyl ether with those of versicolorin C and its methyl ether indicates that versicolorin C is very probably a racemate of versicolorin B.     

Studies on Growth Inhibition of Hiochi-bacteria,Specific Saprophytes of Sake

A new growth inhibitant against hiochi-bacteria, C₁₁H₁₈N₂O, m.p. 173~4°C, named muta-aspergillic acid, has been isolated from the crystals of crude hydroxyaspergillic acid, obtained from culture filtrate of Asp. oryzae. Successful separation of these two compounds from each other was accomplished by counter current distribution method. The physical and chemical properties of muta-aspergillic acid as well as its physiological properties are described.     

Solubilities Studies of Basic Amino Acids

The solbilities of l-basic amino acids in the type of free, monohydro-chloride and dihydrochloride in water were determined, and the results were formulated as follows.

l-Arginine: log S = 0.9770+0.01345t (t is from 0°~70°C)

l-Histidine: log S = 0.3627+0.00905t (t is from 0°~70°C)

l-Arginine monohydrochloride: log S = 1.6532+0.01301t (t is from 0°~70°C)

l-Lysine monohydrochloride dihydrate: log S = 1.6990+0.01294t (t is from 0°~55°C)

l-Lysine monohydrochloride monohydrate: log S = 1.7404+0.01256t (t is from 55°~70°C)

l-Lysine dihydrochloride: log S = 2.2138+0.00409t (t is from 0°~70°C)

l-Histidine dihydrochloride: log S = 1.9085+0.00265t (t is from 0°~70°C)

Three component systems (basic amino acid, hydrochloric acid, water) were studied and the solubilities in mixed solution system of alcohol-water were also investigated.     

Studies on the Metabolic Products of Oospora sp.

A microorganism was isolated from the air of a patient-room and classified in the genus Oospora. This microorganism was cultured on a malt extract medium, and the mycellium was separated from the culture filtrate. A new compound (O-1), m.p. 129°C, C₁₁H₁₀O₃, and eburicoic acid, m.p. 290°C, C₃₁H₅₀O₃ were obtained from the dried mycellium. Another new compound (O-2), m.p. 176°C, C₁₁H₈O₅ was obtained from the culture filtrate.     

Isosclerone,a New Metabolite of Sclerotinia sclerotiorum (Lib.) De Bary

Isosclerone, mp 74~76°C, , a new metabolite of Sclerotinia sclerotiorum, was isolated and its absolute structure was determined as (4S)-3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone. It stimulated the root elongation of rice seedlings by ca. 30% at concentrations of 1~10 ppm, and inhibited the growth of shoots and roots at high concentrations above 50 ppm.     

Studies on the Metabolic Products of Rosellinia necatrix Berlese

A new inhibitor of plant growth, named rosellinic acid, C₁₁H₁₀O₅, m.p. 206~208°C, has been isolated from the culture filtrate of Rosellinia necatrix Berlese. Rosellinic acid has one C-methyl, one phenolic hydroxyl and one aryl carboxylic acid group. Fusion with alkali gave 4,5-dihydroxyisophthalic acid, and the results of physical and chemical tests on the rosellinic acid led to the conclusion that the formula of 6-carboxy-8-hydroxy-2-methylchromanone seems the most possible structure.     

Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.     

Catalytic Properties of Tryptophanase from Proteus rettgeri

Formation of sclerotia in a strain of Sclerotinia libertiana Fuckel using Czapek-Dox agar medium was highest at pH 4.0~6.0 and at 22~25°C. The response was better under darkness than under light. However, when a potato-extract medium was used the optimum temperature range extended, and even at 15~27°C mature sclerotia formed. The addition of indole-3-acetic acid (IAA) to the Czapek-Dox medium containing riboflavine stimulated the formation of sclerotia under fluorescent light. Though iodoacetic acid, a ?SH reagent, also had a stimulatory effect, cysteine had little inhibitory effect on sclerotial formation. Formation was markedly inhibited by p-aminobenzoic acid (PABA), especially in the presence of tyrosine or tryptophan, and excess ammonium salts in the medium also produced an inhibitory effect. It was assumed that accumulation of an intermediary metabolite with high reactivity with ?SH groups was necessary for sclerotial formation, but PABA and excess ammonium salts inhibited the accumulation.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

ASSESSING PHYSIOLOGICAL STRESS IN THALASSIOSIRA FLUVIATILIS (BACILLARIOPHYTA) AND DUNALIELLA TERTIOLECTA (CHLOROPHYTA) WITH DCMU-ENHANCED FLUORESCENCE1

Exponentially growing cultures of Thalassiosira fluviatilis Hustedt and Dunaliella tertiolecta Butcher were exposed to 4 min temperature shocks of 5° to 20°C above ambient (20°C). Photosynthetic carbon fixation, changes in in vivo fluorescence and fluorescence on the addition of the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) were measured over the subsequent 24 h. The fluorescence ratio (R, DCMU-enhanced fluorescence/in vivo fluorescence) paralleled changes in photosynthesis over this period; both were significantly reduced (P < 0.05) by temperature shocks of +15° and +20° C, but +5° and +10° C treatments had no inhibitory effect on either relative to the control. The instantaneous response obtained with the fluorescence ratio indicates that the technique might be applicable to routine bioassay procedures and thus replace the time consuming methods now used for the estimation of ¹⁴C-incorporation and growth rates.     

Effects of nitric oxide scavengers on thermoinhibition of seed germination in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Plant seeds sometimes do not germinate at elevated temperature. The thermoinhibition mechanisms of seed germination have yet not revealed. Here we describe a chemical approach to improve seed germination at high temperature. We compared the temperature response of germination between wild-type Arabidopsis thaliana and its T-DNA insertion mutant ΔAtGLB3 that lacks a functional gene encoding GLB3, a homologue of bacterial truncated Hb (trHb). Under optimal temperature conditions (e.g. 22°C), the seeds of ΔAtGLB3 and the wild type germinated at a frequency near 100%. In contrast, at 32°C the seeds of ΔAtGLB3 did not germinate while wild-type seeds retained the same high germination frequency. The germination of ΔAtGLB3 at 32°C was partially restored by supplementation with the nitric oxide-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; cPTIO), 3-(3,4-dihydroxycinnamoyl)quinic acid, bovine serum Hb, or isoprene. The results presented in this study suggest that chemical scavengers for reactive nitrogen species potentially improve seed germination at high temperature.     

Studies on Growth Inhibition of Hiochi-bacteria,Specific Saprophytes of Saké

The authors have succeeded in isolating an antibiotic, from the cultured filtrate of Asp. oryzae, responsible for, at least, the majority of the active substances against hiochi-bacteria, as pale yellow crystals of m.p. 152~153°C. It has been proved that this antibiotic is identical with hydroxyaspergillic acid. The minimum concentration for complete inhibition in diluted Saké-peptone medium against true hiochi-bacilli, Lactobacillus homohiochii H-42 and Lactobacillus heterohiochii H-l, was 10 γ/ml, and that against hiochi-lactobacilli H-7 and H-34 20 γ/ml.     

Studies on Pectolytic Enzymes of Molds

Exopolygalacturonase from Coniothyrium diplodiella has been purified by ammonium sulfate fractionation, chromatography on DEAE-cellulose and column zone electrophoresis. The enzyme was concentrated about 5-fold with a yield of 0.24% on the basis of polygalacturonase activity per weight of total nitrogen. The purified enzyme was homogenous On free-boundary electrophoresis. The enzyme was most active in the pH range 4.0~4.5. The enzyme was stable at 50°C and pH range of 3.5~6.0, but inactivated at higher than 55°C. Hydrolysis of pectic acid by the enzyme went to completion via galacturonic acid liberation from the end of the chain, but pectin was little affected by the enzyme.     

Regioselective carboxylation of catechol by 3,4-dihydroxybenzoate decarboxylase of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> P

3,4-Dihydroxybenzoate decarboxylase in Enterobacter cloacae P241 was induced by adding 3,4-dihydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or 4-acetamidobenzoic acid to the culture medium. After stabilizing the enzyme activity by adding 5 mM dithiothreitol and 20 mM Na₂S₂O₃ to a cell-free extract, catechol at 50 mM was carboxylated in the presence of 3 M KHCO₃ to 3,4-dihydroxybenzoic acid with a molar conversion ratio of 28% after 14 h at 30°C.     

Isolation and Identification of α-Spinasterol and α-Spinasteryl D-Glucoside from Sugar Beet Pulp

A sterol and a steryl glucoside were isolated from dried beet pulp. The sterol was identified with α-spinasterol, the glucoside possessed chemical and physical properties such as follows: The molecular formula C₃₅H₅₈O₆, m.p. 292°, [ α]¹⁹_D-34.1°, acetate; m.p. 168°, benzoate; m.p. 175-177°, and positive for Molish and Lieber-mann-Burchard reactions. When it was hydrolyzed with 1% sulfuric acid, the crystal of α-spinasterol and D-glucose detectable by paper chromatography were obtained. These results gave evidence that the glucoside was in question to be α-spinasteryl D-glucoside.     

Post-harvest age-induced seed dormancy of Acer ginnala and its alleviation by growth regulator and low temperature treatments

One-month-old fruits of Acer ginnala with winged pericarp attached gave 44% germination and this was not increased by cold treatment at 4°C for 0, 10, 20, or 30 days, gibberellic acid treatment at 0, 1, 10, 100 or 1000 mg litre^-1, or ethephon treatment at 0, 2, 20, 200 or 2000 mg litre^-1. After 6 months of storage at 20–25 °C, germination of untreated fruits fell to 5% but could be restored to that of 1-month-old fruits by incubation at 4 °C for 30 days. After 9 months storage, no germination occurred in untreated fruits. Cold treatment (30 days at 4 °C partially restored germination (26%). Treatment with either gibberellic acid (1000 mg litre^-1) and 30 days at 4 °C (40%) or ethephon (100 mg litre^-] and 30 days at 4 °C improved germination (69%). The combination of all three treatments, i.e. 100 mg litre^-1 gibberellic acid, 100 mg litre^-1 ethephon and 30 days at 4 °C, optimised germination (86%). Thus, dormancy of A. ginnala developed during storage but could be reversed by a combination of treatment with low temperature and growth regulators. The highest germination (86%) was achieved after low temperature and growth regulator treatment of stored fruit.     

Purification and Characterization of Lactobacillus bulgaricus Diaphorase

γ-Isomer of 1,2,3,4,5,6-hexachlorocyclohexane (BHC) showed greater decomposition on γ or UV irradiation of five isomers of BHC in crystalline state or in 2-propanol solution. The α- and δ-isomer of BHC and known la, 2a, 3e, 4e, 5e-pentachlorocyclohexane were separated from the irradiation product of crystalline γ-BHC. Four compounds were isolated from the irradiation product of γ-BHC in 2-propanol. Two compounds were tetrachloro-cyclohexenes (C₆H₆C1₄): γ-isomer (mp 86 ~87°C) and ?-isomer (mp 99 ~ 100°C). The other two were isomers of pentachlorocyclohexane (C₆H₇C1₅). One of them (mp 78 ~ 8.5°C) was consistent with known meso-1e,2a,3a,4a,5e isomer. The molecular structure of the other (mp 75°C) established by X-ray crystal structure analysis was 1α, 2α, 3α, 4β, 5α configuration or le 2a 3e 4e 5e conformation of CI atoms. A reaction mechanism was proposed that included a radical chain reaction and chlorine atom migration.     

The House Fly Attractants in Mushrooms

Several active concentrates responsible for the long known house fly attracting property of Amanita muscaria (L.) Fr. were obtained through steam distillation and exhaustive solvent extraction of the filtered juice of the fruiting body. Among the active concentrates, one fraction referred to as D₃ was isolated as colorless crystals, m.p. 22~23°C, with a chromatographycally proved homogeneity and exhibited a marked attractiveness to house flies, especially to mature females.     

Improvements of the bioavailability of straw: acid and enzyme hydrolysis of wheat straw pretreated with saturated lithium chloride in hydrochloric acid

Wheat straw was pretreated with saturated lithium chloride in 4 m hydrochloric acid at 50°C for 1 h, then hydrolysed at 100°C for 1 min, to give 84% conversion to monosaccharides. Particle sizes, 150–355 mesh, were easily hydrolysed. Samples pretreated with saturated lithium chloride in 1 m hydrochloric acid at 27°C for 24 h were hydrolysed by Trichoderma viride cellulase (MVA 1284) [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] to give 20–23% monosaccharides for particle sizes of 150–250 mesh, and 82–95.4% for particle sizes of 250–355 mesh.