T_??_inactivation of the enzyme protein by the autoxidized products of linoleic acid was stuied with special reference to the incorporation of the autoxidized products into the protein and the consequent damage to the amino acid residues of the protein. Inactivation of ribonuclease (RNase) was due to the incorporation of linoleic acid hydroperoxides (LAHPO) or their secondary products (SP). Pepsin showed a lesser degree of inactivation and incorporation with respect to LAHPO, but SP activated the pepsin activity. Trypsin was affected only by SP. In RNase lysine, histidine, tyrosine, methinoine and cystine were the most labile amino acids to LAHPO attack, while methionine was the only amino acid which suffered damage in trypsin and pepsin. The SP caused damage of methionine, lysine and histidine in RNase, methionine, cystine and histidine in trypsin and methionine and aspartic acid in pepsin. The reactions of LAHPO were enhanced by a prooxidant, ascorbic acid (AsA), while antioxidants acted otherwise. The incorporation of SP into RNase and pepsin was enhanced by AsA while antioxidants were ineffective.

View full abstract

A crude enzyme preparation hydrolyzing konjac mannan was extracted from germinating konjac tubers, and purified by chromatography with DEAE-cellulose and alkali-swollen cellulose, and by gel-filtration on Sephadex G-100. The purified enzyme preparation showed optimal activity at pH 4.7, optimum temperature at 40°C. It was considerably stable at pH's between 4.0 and 8.0, but inactivated rapidly by temperaters above 50°C. Hydrolysis of the mannan by this enzyme proceeded by typical random mechanism, and the rate was in agreement with an empirical equation, p=0.43 E^0.77 t^0.5. As the Km and V_max Values for mannan, 7.14×10^-2 (%) and 23.8×10^-3 (_??_OD500_nm) were obtained, respectively.

View full abstract

The growth of a threonine-sensitive and-requiring strain of Brevibacterium flavum, QL-5, depended on the threonine concentration in a minimal medium, but it was much slower than that of a simple threonine-requiring mutant, T-36, at a low concentration of threonine. This slow growth of the former was stimulated by the addition of isoleucine plus serine or NaCl.
Since these effectors also stimulated the threonine-sensitivity, isoleucine plus serine at a relatively higher concentration of threonine or an excess amount of NaCl inhibited the growth.
The effect of NaCl was ascribed to that of sodium ions. Lithium chloride as well as BeCl₂ showed a similar effect but KCl did not.
Similar promoting effects were also observed with all other threonine-sensitive mutants and a simple threonine-requiring mutant tested in various culture media.
The lysine production by QL-5 was also stimulated by isoleucine plus serine.

View full abstract

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE- and CM-cellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10, 000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohem-agglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.

View full abstract

Nitrite reductase (EC 1. 6. 6. 4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5, ×10^-4M inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61, 000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.

View full abstract

One of the microbial products produced from dibenzothiophene by Pseudomonas abikonensis or P. jianii was identified as a new substance, trans-4 [2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid, and the other as its hemiacetal form. From the structures of the products, an oxidation pathway of dibenzothiophene was suggested. Moreover, the culture broth and the isolated products were examined for plant growth activities on rice plants.

View full abstract

N-Acetate-methyl signals were examined in the NMR spectra of a series of mucopolysaccharides and some glycopeptides in reference with those of the component monosaccharides involving axial or equatorial acetamide group. The signals showed no significant change on the chemical shifts and shaps in 8M urea, 1 N NaOH, 1 N HCl and D₂O. The signals appeared in δ 2.00 to 2.06 ppm which fall in equatorial region. These results indicate that hexosamine moieties in these polymers keep the C l conformation in solutions. A deshielding effect of N-methyl group on the N-acetate-methyl signals was evident with N-methylated mucopolysaccharides.

View full abstract

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5 per cent protein levels to rats for six weeks on their plasma lipids has been studied. The results have been compared with rats fed casein at 10 per cent level. The plasma lipid metabolism in experimental and control rats has been studied by using acetate-1-¹⁴C, palmitate-1-¹⁴C, glucose-U-¹⁴C and NaH₂³²PO₄. Plasma total lipids and glycerides (mg/ml) were significantly high in all millet protein fed rats. Plasma total and esterified cholesterol was lower in M-5% groups and unaffected in M-10% and M-15% groups. Plasma total phospholipids and LPC and PC were reduced in M-10% and M-15% groups. The incorporation of palmitate-1-¹⁴C, acetate-1-¹⁴C and glucose-U-¹⁴C into plasma triglycerides showed that the secretion of hepatic TG into plasma was not impaired. The labelling pattern of plasma cholesterol and phospholipids suggest that the availability of these in liver is not the limiting factor for the pathogenesis of fatty liver observed in rats fed millet.

View full abstract

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3_??_2.6 times in both enzymes at the concentration of 50mM of potassium chloride. Such activation was also caused by monoand di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-gluco-sidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.

View full abstract

GDP-glucose pyrophosphorylase activity was found to be widely distributed in Streptomyces. Streptomyces olivaceus IFO 3409 showed the highest activity among the strains tested. When air-dried cells of Streptomyces olivaceus (100mg/ml) were incubated with GTP•(NH₄)₃ (20μmoles/ml), D-glucose-l-phosphate (100μmoles/ml), potassium phosphate buffer, pH 7.0 (270μmoles/ml) and MgSO₄ (20μmoles/ml), GDP-glucose was synthesized in 25% yield against GTP. GDP-glucose was isolated and identified. The strains capable of synthesizing GDP-glucose in higher yield were searched. Streptomyces sp. AKU 2801 seems to be the most suitable strain for the preparation of GDP-glucose.

View full abstract

The known B. subtilis phages PBSI, M2, SP 01, SP 10 and newly isolated B. natto phage NP-38 were examined for the induction of γ-polyglutamic acid (PGA) depolymerase synthesis. These phages, except M2, were capable of inducing the synthesis of the depolymerase in the infected bacteria. A direct evidence that the synthesis of depolymerase lay under the phage genome was obtained by the isolation of a depolymerase-deficient mutant of phage. A measurable amount of depolymerase was not detected in either intact or disrupted particles of phages. Thus, it seemed that the phage particles did not involve a bound enzyme.
The depolymerase was purified about 960 folds from the lysate of phage NP-38. The purified enzyme did not have a hydrolytic action on known protein substrate. The molecular weight was estimated to be about 20, 000 from the result of a gel filtration.

View full abstract

The effects of oxygen and carbon dioxide in inosine fermentation were investigated from the industrial viewpoint. Oxygen supply at the rate of more than 5×10^-7mole/ml•min was indispensable for maintaining the high productivity of inosine in jar fermentors as well as in shaking flasks. Oxygen deficiency due to insufficient oxygen supply, on the other hand, resulted in the inhibition of inosine production, even though glucose added to the medium was entirely assimilated. In addition to sufficient oxygen supply ventilation was also indispensable since an increased tension of carbon dioxide reduced the inosine-producing capability of the cells.

View full abstract

The concentration of dissolved carbon dioxide in the liquid phase was in proportion to the partial pressure of carbon dioxide in the gas phase, and that of bicarbonate ions was in proportion to the dissolved carbon dioxide tension and pH of the liquid. Although the partial pressure of carbon dioxide was maintained at the same level, the bicarbonate ion concentration varied depending upon the difference in pH values. Thus, the influence of the carbon dioxide tension and bicarbonate ion concentration on the product formation was investigated independently by a dissolved carbon dioxide controlled culture at a constant pH level.
The production of inosine was highly affected by the partial pressure of carbon dioxide. The yield of inosine, however, was shown to be independent of the bicarbonate ion concentration in the culturing liquid.

View full abstract

In inosine fermentation, the yield of the product was closely related to the partial pressure of carbon dioxide in the culture system rather than the bicarbonate ion concentration in the culture liquid. The inhibitory effect of carbon dioxide was restored by the methods of reducing the partial pressure of carbon dioxide lower than a certain level. Both ventilation and chemical absorption were applicable for the restoration of the carbon dioxide inhibition, but ventilation had great advantages over the other method from an industrial view-point. In the estimation of aeration-agitation conditions for the scale-up of submerged fermentation in which carbon dioxide was inhibitory, the rate of aeration was to be established to make sufficient ventilation and overcome this inhibition. A scheme for the procedure for the estimation of aeration-agitation conditions under the consideration of influences of carbon dioxide on submerged fermentation was proposed in this paper.

View full abstract

A new phosphorylating agent, 2-methylthio-4H-1, 3, 2-benzodioxaphosphorin-2-oxide (MTBO), prepared from its thiono isomer, 2-methoxy-4H-1, 3, 2-benzodioxaphosphorin-2-sulfide (MOBS), by means of the Pistschimuka reaction, reacted with several aliphatic alcohols in the presence of cyclohexylamine to give the corresponding O-alkyl S-methyl phosphorothiolates in 60 to 45% yield.
The phosphorothiolates could be converted to phosphate diesters as well as monoesters by the action of an oxidizing agent such as isoamyl nitrite or iodine in dry alcohol or in water.

View full abstract

Methionine auxotrophs were derived by the treatment with ultraviolet ray or N-methylN'-nitro-N-nitrosoguanidine from five strains of Escherichia coli. One of the methionine auxotrophs of E. coli C-6, strain No. 15, produced maximum amount of L-threonine (4.3mg/ml) with the medium containing 5% cane-molasses (as sugars). Double auxotrophs were derived with further mutational treatment from strain No. 15. It was found that L-threonine production was greatly enhanced by cultivating methionine-valine auxotrophs in the presence of L-valine and methionine. One of the methionine-valine auxotroph, strain No. 234, produced maximum amount of L-threonine (10.5mg/ml) from cane-molasses.
The requirement of L-valine for the growth of the strain No. 234 was found to be leaky, and it was suggested that some enzymes relating to L-valine metabolism were mutationally altered to temperature-sensitive.

View full abstract

α-Glucosidase was purified and crystallized from the mycelia of Mucor javanius, by procedures including extraction with urea, fractionations with acetone and polyethylene glycol 6000, successive separation on columns of Sephadex G-200 and DEAF-cellulose, and crystallization by the addition of ampholine reagent. The crystalline enzyme was homogeneous in ultracentrifugal analysis and gel electrophoresis. The purified enzyme showed a sedimentation constant of 5.6S and isoelectric point of pH 8.6. Some properties of the purified enzyme were also investigated. It was recognized that the synthesis of riboflavin α-glucoside was catalyzed by the transglucosylation activity of this α-glucosidase.

View full abstract

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain F11. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.

View full abstract

Homotectoridin (II), C₂₃H₂₄O₁₂- H₂O, mp 186°C, a new isoflavone isolated from ethanolic extracts of the rhizomes of Iris germanica Linnaeus, was formulated as 5, 4'-dihydroxy-8, 3'-dimethoxy-isoflavone-7-O-mono-D-glucoside. Tectoridin (I) was also isolated from the same extract.

View full abstract

Twelve L-glutamyl dipeptides were prepared and on the basis of their tastes were classified into three groups: I brothy taste group (Glu-Asp, Glu-Thr, Glu-Ser and Glu-Glu); II flat taste group (Glu-Gly, Glu-Ala, Glu-Pro and Glu-Val); and III bitter taste group (Glu-Ile, Glu-Leu, Glu-Tyr and Glu-Phe). Examination with ion-exchange, thin layer and paper partition chromatographies showed that the dipeptides in I were more acidic, polar and hydrophilic than those in II, the bitter dipeptides (III) being rather hydrophobic. Similar tests for a brothy taste tripeptide, Glu-Gly-Ser, indicated that this possessed properties resembling I. O-Acetylation of the serine residue of Glu-Gly-Ser lessened its hydrophilicity and the taste became flat. The O-butyrylation resulted in a marked increase in hydrophobicity and the product showed a bitter taste.

View full abstract

The crude lipase powder has been purified 216-fold in specific activity by means of pH adjustment, DEAE-Cellulose, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 column chromatography and the recovery of the activity was 30%. The purified lipase was confirmed to be homogeneous with disc electrophoresis and ultracentrifugal analyses. The purified lipase was stable in the pH range from 7.0 to 10.0. Optimal pH for the lipolysis of polyvinyl alcohol-emulsified olive oil at 45°C was 8.0 and optimal temperature was 60°C. The purified lipase was stable up to 60°C and retained 55% of full activity after heating at 70°C for 20min.

View full abstract

Benthiocarb labeled at benzyl methylene group with carbon-14 was synthesized and studied on the distribution, excretion and metabolism in white mice. Benthiocarb was rapidly translocated into organs after oral administration. Radioactive substances were also rapidly eliminated mainly into urine, slightly into feces and little into expiration. Major metabolites in urine were identified as 4-chlorohippuric and 4-chlorobenzoic acids, and small amounts of glucuronides of the latter acid and 4-chlorobenzyl alcohol were detected. Benthiocarb was degraded in liver homogenates, in which the microsomal fraction showed the largest activity, and the degradation was accelerated by reduced NADP as the cofactor for the reaction. N-Desethylbenthiocarb, bis(4-chlorobenzyl) mono- and di-sulfides, and 4-chlorobenzoic acid were identified in the incubation mixture of the liver homogenates. The main metabolic pathway in mice seemed to be as follows; parent benthiocarb and/or the N-desethylbentiocarb were hydrolyzed, and the produced 4-chlorobenzylmercaptan presumed to be oxidized finally to 4-chlorobenzoic acid, which then conjugated with glycine to produce 4-chlorohippuric acid.

View full abstract