A Novel Synthetic Procedure for “Reverse” C-Nucleosides

The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible α-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN.     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

The volatile sulfur components produced by boiling soybean meal hydrolyzates (AMINOSAN-EKI) have been identified as dimethyl sulfide and hydrogen sulfide. No mercaptan or disulfides were detected.

The main precursor of dimethyl sulfide is supposed to be methionine methylsulfonium compound derived from methionine and pectin substances (–COOCH₃) during the hydrolysis of soybean meal by hydrochloric acid.     

Application of Nitrate Reductase for the Determination of Nitrate in Meat and Fishery Products

A rapid and specific method is described for the determination of nitrate in meat and fishery products.

Nitrate separated from foods by extraction with 1/50Ν sodium hydroxide and ultrafiltration was readily reduced to nitrite by the use of respiratory nitrate reductase (NR) from Escherichia coli K-12. The nitrite so obtained can be determined by the specific diazotation-coupling reaction method.

The use of an enzymatic reaction resulted in quantitative reduction of nitrate, and the method was relatively free of interferences. Recoveries of 10 and 100 ppm of nitrate from 5 samples of meat and fishery products ranged from 92.8 to 97.8% for 10 ppm and 97.8 to 99.4% for 100 ppm with a detection limit of 0.5 ppm.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

“Nojirimycin” as a Potent Inhibitor of Glucosidase

Glucose-6-phosphate dehydrogenase in a yeast, Hansenula mrakii IFO 0895 is induced when the cells are cultured in a medium containing lipid hydroperoxide. The enzyme was purified from H. mrakii to the homogeneous state on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 52kDa by SDS-PAGE and 130 kDa by Sephadex G-150column chromatography, respectively. The enzyme was specific to glucose-6-phosphate and NADP⁺, and K_mvalues for glucose-6-phosphate and NADP⁺ were 293µM and 24.1 µM, respectively. The enzyme activity was inhibited by diethylpyrocarbonate and 2, 4, 6-trinitrobenzene sulfonate, and by metal ions such as Zn^{2 +}, Cd^{2 +}, Cu^{2 +}, and Al^{3 +} . tert-Butyl hydroperoxide, a kind of lipid hydroperoxide, slightly(approximately 20%) increased the enzyme activity.     

Role of Acetyl CoA: Deacetylcephalosporin C Acetyltransferase in Cephalosporin C Biosynthesis by Cephalosporium acremonium

Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg²⁺ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase.     

Genetic Interaction between the ero1-1 and leu2 Mutations in Saccharomyces cerevisiae

The conditional ero1-1 mutant, deficient in the ER-localized PDI oxidase Ero1p, is blocked in disulfide bond formation under restrictive conditions, such as high temperature, lack of oxygen, or high concentrations of membrane-permeant thiols. Previous studies of the physiological consequences of the ero1-1 mutation were carried out in a leu2 mutant. The ero1-1 leu2 strain does not grow in standard synthetic complete medium at 30 °C, a defect that can be remedied by increasing the L-leucine concentration in the medium or by transforming the ero1-1 leu2 strain with the LEU2 wild-type allele. In addition, the LEU2 gene can partially complement the growth impairment at 37 °C of the ero1-1 leu2 mutant. The leucine transporter Bap2p exhibits a dramatic decrease in stability in an ero1-1 strain, which may account for the pronounced leucine demand observed in the ero1-1 leu2 mutant.     

Conversion of the Extracellular Dextransucrase Isoenzymes from Leuconostoc mesenteroides NRRL B-1299

Effect of oxygen tension on l-lysine, l-threonine and l-isoleucine accumulation was investigated. Sufficient supply of oxygen to satisfy the cell’s oxygen demand was essential for the maximum production in each fermentation. The dissolved oxygen level must be controlled at greater than 0.01 atm in every fermentation, and the optimum redox potentials of culture media were above ?170 mV in l-lysine and l-threonine and above ?180 mV in l-isoleucine fermentations. The maximum concentrations of the products were 45.5 mg/ml for l-lysine, 10.3 mg/ml for l-threonine and 15.1 mg/ml for l-isoleucine. The degree of the inhibition due to oxygen limitation was slight in the fermentative production of l-lysine, l-threonine and l-isoleucine, whose biosynthesis is initiated with l-aspartic acid, in contrast to the accumulation of l-proline, l-glutamine and l-arginine, which is biosynthesized by way of l-glutamic acid.     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

Sedimentation Analysis of the Interacting Mixture of Dextran Sulfate and Low Density Lipoprotein

The reversible interaction between dextran sulfate (D) and the low density lipoprotein of human serum (P) was investigated by sedimentation velocity. Analysis of the velocity patterns of dextran sulfate—lipoprotein mixtures revealed that the maximum number of binding sites on dextran sulfate molecule is approximately 6. It was also shown that the species of the complex formed is affected by the mixing ratio of the two constituents: at the molar ratio (P/D) 0.69, the complex exists in average as DP_1.6 and at 0.98 as DP_2.2. The linear increment of sedimentation coefficient of the complex due to the binding of one lipoprotein molecule was 7.8S. Finally, the mechanism of precipitation of the complexes was discussed.