Microbial hydrolysis of pepsidines was studied by using bacteria. A strain of bacteria which has been isolated from soil and identified as Bacillus pumilus, could release successively N-terminal acetyl moiety and valine from pepsidine C. The residual peptides thus obtained were crystallized and identified as desacyl-pepsidine and desacylvalyl-pepsidine. The correlation between their structures and pepsin inhibitory activities was also studied.

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In order to examine the effect of the reduction of individual essential amino acids from either the lysine-deficient diet or the threonine-deficient diet on the liver lipid content, growing rats were fed the 7% amino acid mixture diet for 14 days. The extent of deficiency of individual amino acids was lowered 50% as compared to that in the control diet. In rats fed the diet deficient in lysine or threonine liver lipids were accumulated as reported previously. It was found that the reduction of sulfur (S)-containing amino acids, valine or isoleucine from the lysine-deficient diet, and the reduction of S-containing amino acids from the threonine-deficient diet resulted in preventing the liver lipid accumulation. Whereas, the feeding of the diet deficient in lysine and tryptophan or in threonine and tryptophan showed a decreasing tendency in liver lipid content compared to the lysine-deficient diet or the threonine-deficient diet, respectively. On the other hand, the reduction of individual essential amino acids other than S-containing amino acids, valine, isoleucine and tryptophan from the lysine-deficient diet or other than S-containing amino acids and tryptophan from the threonine-deficient diet did not cause to lower the liver lipid content.

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Three kinds of guanosine polyphosphates (G3P, G4P and G5P) in addition to 5'GMP, 5'GDP and 5'GTP were accumulated by microbial conversion of 5'XMP using a mutant KY 13510 derived from Brevibacterium ammoniagenes. These nucleotides were purified from the culture filtrate by a method including strongly basic anion-exchange column chromatography, charcoal treatment and DEAE-Sephadex A-25 column chromatography. Labilities of the nucleotides in acid and alkali, enzymatic degradation and carbon-13 NMR spectroscopy lead to structural assignments for G3P, G4P and G5P as guanosine 3'-diphosphate, 5'-monophosphate, guanosine 3'-diphosphate, 5'-diphosphate (MSI) and guanosine 3'-diphosphate, 5'-triphosphate (MSII), respectively. More than 3.3g of G3P, 5.2g of G4P and 0.8g of G5P per liter were accumulated in this system. A practical procedure for the production of MSI and MSII was described.

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The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2 KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Ghrconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.
Best solubilization of particulate enzymes was attained with the highest recovery when 10mg of Triton X-100 and 30mg of protein of particulate fractions in 1ml of 0.01M phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.
By comparison of the total, enzyme activity of particulate enzymes with that of NAD (P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD (P)-linked enzymes.

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A shoyu flavor concentrate was prepared with as much precaution as possible not to damage the original. The procedure consisted of distillation under reduced pressure, extraction with dichloromethane, and then concentration. The odor of this concentrate seemed to be almost the same as that of original shoyu. Another concentrate containing minor components was fractionated from the above concentrate by a gas chromatographic method. These two concentrates were analyzed by use of combined gas chromatography-mass spectrometry.
Fifty components were identified, fifteen of which have not been reported previously in shoyu. They are 2-hexanone, 2-methyl-3-tetrahydrofuranone, 4-pentanolide, furfuryl acetate, ethyl phenylacetate, 2-phenylethyl acetate, 2-methylpyrazine, 2, 6-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 3-ethyl-2, 5-dimethylpyrazine, borneol, bornyl acetate, benzyl alcohol and 2, 6-dimethoxyphenol.

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Unstable compounds with intense sweet odor were isolated from shoyu and identified as 4-hydroxy-2-ethyl-5-methyl-3 (2 H) -furanone and 4-hydroxy-5-ethyl-2-methyl-3 (2 H) -furanone (tautomer ratio, about 3:2) on the basis of spectrometrical investigation. This may be the rare case of isolation of the tautomeric compounds from a natural product.
They were concluded to be major and important flavor components in shoyu.

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Purification was conducted on polyvinyl alcohol (PVA) degrading enzyme produced and secreted into the culture medium by Pseudomonas O-3 strain. The enzyme was found to separate into several fractions by ion-exchange chromatography and gel filtration. Among these fractions, a fraction adsorbed to SP-Sephadex C-50 at pH 6.0 was purified to homogeneity by polyacrylamide gel electrophoresis. Some properties of this purified enzyme were examined. Optimum pH and temperature were 9.0 and 40°C, respectively. The enzyme was stable up to 50°C and in a pH range between 5 and 11 at 5°C. The enzyme activity was inhibited by Co²⁺, Ni²⁺, EDTA, hydroxylamine and salicylaldoxime. In substrate specificity, this enzyme oxidized several kinds of modified PVA, as well as normal PVA, but did not oxidize other synthetic polymers, such as vinylon, polyacrylamide and polyvinyl acetate. The effect of oxygen on this enzyme was examined, and without oxygen, PVA was not broken down by this enzyme. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 26, 000.

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Microbial 16 β-hydroxylation of some steroids with Wojnowicia graminis, Corticium centrifugwn and Bacillus inegaterium has been reported, but not 16 β-hydroxylation of normal 17-oxo steroids with Aspergillus niger. This time, we tried microbial transformation of dehydroepiandrosterone with this fungus, and obtained 4-androstene-3, 17-dione, 17 β-hydroxy-4-androstene-3, 16-dione, 16 β, 17 β-dihydroxy-4-androsten-3-one and a new compound, 16 β-hydroxy-4-androstene-3, 17-dione. This new compound was also obtained by the fermentation of 4-androstene-3, 17-dione and testosterone.

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Two types of L-isoleucine producing mutants were derived from L-threonine producers by the supplement of the resistance to ethionine.
Main control site in L-isoleucine biosynthetic pathway after threonine is threonine dehydratase. In case of Brevibacterium flavum No. 14083, L-isoleucine production was based on the insensitiveness of this key enzyme to feedback inhibition by L-isoleucine. As regards Brevibacterium flavum No. 168, it was based on the increase in the specific activity of this enzyme.
The former produced 11.3g/liter of L-isoleucine and the latter produced 9.92g/liter from glucose. The former showed a vigorous ability of acetic acid assimilation, but the latter did not.

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Culture conditions were studied for L-isoleucine production from acetic acid. Acetate and ammonium concentration in culture liquid exerted a great influence on the fermentation, and optimum concentration was 2_??_5g/liter and 2_??_3g/liter respectively. To maintain these conditions throughout the culture, it was necessary to supply intermittently a small amount of feeding solution which consisted of ammonium acetate and acetic acid. Molecular ratio of the former to the latter was 0.175, and total concentration of acetic acid was 700g/liter.
Carbon dioxide showed an inhibitory influence on L-isoleucine production and adequate ventilation was necessary for satisfactory result. Maximum amount of L-isoleucine was 33.5g/liter after 77-hr cultivation at 28°C and at pH 7.7. Production yield of L-isoleucine was 10% by weight from acetic acid.

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Screening of protein-producing bacteria was conducted to systematically study the ubiquitous nature of the extracellular production of proteins and their excretion mechanisms. A very simple and efficient test revealed that about 15% of bacteria tested (total 1200 strains) accumulated some protein under the cultural conditions employed. Among protein-excretors, five strains produced a large amount of protein in the liquid shake culture.
Many good protein producers including five excellent ones were found to be gram-positive rod and probably belong to Bacillus species. An acid-insoluble product by one of the hyperprotein producers was identified as a protein mixture. Good producer was not found among the known 15 species of bacteria. The implication of these findings is discussed.

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While a continuous ingestion of lead acetate added in drinking water suppressed the rat growth, depressing in some degree the level of hepatic δ-aninolevulinate (ALA) dehydratase, a very small amount of sclerin (SCL) added simultaneously in the water restored the growth and dehydratase level. Moreover, subcutaneous injection of SCL to the rat not only maintained the ALA dehydratase level, but prevented a marked depression of the level of mitochondrial ALA synthetase in liver caused by intraperitoneal injection of lead acetate. Injection of SCL alone increased tolerably (about 1.8 times) the mitochondrial ALA synthetase, but little the extramitochondrial synthetase. The treatment by SCL was attended by a initial decrease, then a gradual increase in the activity of microsomal drug metabolizing enzyme.

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The relative excess of some catabolites of sulfur-containing amino acids in the liver of rats fed a low protein diet might be one of the factors which cause the liver fat accumulation. To investigate the possibility were studied relationships between changes in concentrations of some metabolites of sulfur-containing amino acids and those in fat contents of rats fed a low protein diet consisting of heated soybean flour, casein or wheat flour with or without added methionine, threonine or lysine. The addition of 0.6% methionine to the 25% heated soybean flour diet increased the nonprotein-sulfhydryl (NP-SH) concentration and fat content in the liver. These changes were prevented by the further addition of 0.5% threonine to the diet, although the NP-SH concentration was remarkably higher than that of rats fed the unsupplemented diet. The addition of 0.6% cystine HCI to the 25%. heated soybean flour diet containing sufficient choline elevated the NP-SH concentration and fat content in the liver, which were not affected by the further addition of 0.5% threonine. The addition of 0.6% cystine HCl to the 10% casein diet significantly increased the fat content, and NP-SH and taurine concentrations in the liver. The further addition of 0.5%. threonine completely decreased the fat content, and partially reduced the NP-SH and taurine concentrations. Effects of supplementation of 0.4% lysine HCl to the 70% wheat flour diet on the fat content and NP-SH concentration in the liver demonstrated the trends similar to those of supplementation of cystine to the 10% casein diet. The further addition of threonine remarkably decreased the fat content, NP-SH and taurine concentrations in the liver.

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Some of the kinetic properties of purified cytosine deaminase from Serratia marcescens were studied.
The enzyme was activated by L-amino acids, especially remarkably by histidine and cysteine. The enzyme was also activated by phosphate and pyrophosphate: 90% by 1mM pyrophosphate and 50% by 10mM phosphate. The enzyme activity was affected by various nucleotides: strongly activated by IDP, ITP, GTP and ATP, and inhibited by deoxyguanosine and deoxyGMP.
Many kinds of metal ions inhibited the enzyme reaction: especially Co²⁺ and Cd²⁺ were observed to be effective inhibitors. The apparent Michaelis constant Km for cytosine was found to be 3.4×10^-3M, and Km value was not changed and only maximum velocity V_max was changed in the presence of activators and inhibitors.

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Some of the kinetic properties of purified cytosine deaminase from Pseudomonas aureofaciens were studied.
The enzyme was activated by L-amino acids, especially by asparagine, aspartic acid and histidine, at the optimum pH of 10.0.
The enzyme was also activated by phosphate and pyrophosphate: 50% by 50 mm phosphate and 50% by 20mM pyrophosphate, while the enzyme was not affected by nucleotides and nucleosides. Some metal ions inhibited the enzyme reaction; especially Mn²⁺ and Cd²⁺ were observed to be effective inhibitors.
The apparent Michaelis constants Km for cytosine and 5-methylcytosine were found to be 4.5×10^-3M and 5.7×10^-4M, respectively. This enzyme was inhibited strongly by 1mM p-chloromercuribenzoate and seemed to be a thiol enzyme.

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Glyoxalase I from rat liver was purified about 25-fold by acetone fractionation and ionexchange chromatography on CM-Sephadex and DEAE-cellulose columns. The kinetic study of the enzymatic reaction supported the one-substrate mechanism: the hemimercaptal adduct produced nonenzymatically from methylglyoxal and glutathione is the substrate. The Km value determined was 0.1mM and similar to that of porcine erythrocytes enzyme but differed significantly from that of yeast enzyme. It was inhibited by free glutathione competitively (Ki 1.2mM). Kinetic studies on inhibition of glyoxalase I by MS-3 which was obtained from a cultured mushroom, Stereum hirsutum, indicated the inhibition type was competitive with the hemimercaptal adduct (Ki 4.6×10^-6M). By the graphical study of the multiple inhibition kinetics free glutathione and MS-3 were shown to bind at the same sites of the enzyme.

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Eight fructo-oligosaccharides were isolated from purified oligosaccharide fractions of the roots of Asparagus officinalis L. (Liliaceae). By examination of constituent sugars, gasliquid-chromatographic analysis of methyl derivatives, and investigation of partial acid hydrolyzates and products of β-fructofuranosidase action, they were confirmed to be 1^F(1-β-fructofuranosyl)_n sucrose [n=1 (1-kestose), 2 (nystose), and 3], 6^G (1-β-fructofuranosyl) _n sucrose [n=1 (neokestose), 2, and 3], 1^F, 6^G-di-β-fructofuranosyl sucrose, and a new pentasaccharide 1^F (1-β-fructofuranosyI)₂-6^G-β-fructofuranosyl sucrose.

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A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the glucan. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27, 000 in molecular weight, was found to cleave a β-(1→4) linkage adjacent to a β-(1→3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and Iaminaranases.

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Acidic aroma constituents of Turkish tobacco have been studied. Three new compounds, 4-isopropyl-7-oxo-2E-octenoic acid (I), 6-isopropyl-3-methyl-9-oxo-2Z, 4E-decadienoic acid (II) and 6-isopropyl-3-methyl-9-oxo-2E, 7E-decadienoic acid (III) were isolated from cured Turkish tobacco and their structures were elucidated on the base of the spectroscopic properties. These new constituents are considered to be derived from tobacco thunberganoids.

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To investigate the effects of catabolites of sulfur-containing amino acids on the liver fat content, rats fed a protein-free or 10% casein diet were orally or intraperitoneally administered sulfur-containing amino acids and their catabolites. Force-feeding of the proteinfree diet supplemented with 0.01 and 0.05% methionine reduced the excretion of urinary nitrogen, but it did not affect the liver fat content. Supplementation with 0.2% methionine induced significant liver fat accumulation accompanied with remarkable increases in the concentrations of nonprotein sulfhydryl (NP-SH) and taurine in the liver. The effects of the addition of 0.2% cystine HCI to the protein-free diet were similar to those of 0.2% methionine. Forcefeeding of the protein-free diet supplemented with 0.2% cysteine sulfinic acid or 0.2% thiopyruvate did not bring about any changes in the concentrations of sulfur-containing metabolites and fat content in the liver and the excretion of urinary nitrogen. On the contrary supplementation with 0.2% hypotaurine remarkably increased the liver fat content and taurine concentration without any changes in NP-SH concentration. The effects of injection of methionine, cysteine and hypotaurine to rats fed 10% casein diet on the fat content and sulfurcontaining metabolites in the liver were similar to those of oral administration of these compounds. These findings supported the possibility that the excess of catabolites of sulfurcontaining amino acids might be one of the factors which cause the liver fat accumulation.

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The thermal rearrangement of 3-O-allyl-α⁴-norpyridoxol derivatives (II) resulted in the formation of the ortho-Claisen rearrangement products and the para-Claisen rearrangement product. The former compounds (IIIa, IVa and Va) were subsequently transformed into 2, 3-dihydrofuro (2, 3-c) pyridine (VIa). Hydrolysis of the benzyl ether and the t-butyl ether of the rearrangement products (III, IV and V) with hydrochloric acid produced 4-propenyl-α⁴-norpyridoxols (XII, XIII and XIV). No significant anticoccidial activity was shown by these compounds.

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Three quinolone alkaloids were isolated from fresh leaves and fruits of Evodia rutaecarpa Hoox. f. et THOMS. and were structurally elucidated as 1-methyl-2-pentadecyl-4 (1H) -quinolone (I), 1-methyl-2-tridecyl-4 (1H) -quinolone (dihydroevocarpine, II) and 1-methyl-2-undecyl-4 (1H) -quinolone (III) on the basis of spectral measurement and synthesis.

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Destomycin B, a member of destomycin family antibiotics having anthelmintic activity, was shown to be 5-O-[2, 3-O-(6-amino-6-deoxy-L-glycero-D-gtaco-heptopyranosylidene)-β-D-mannopyranosyl]-1, 3-di-(methylamino)-1, 2, 3-trideoxy-myo-inositol based on chemical and spectroscopic studies.

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A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.

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Isopropanol-utilizing microorganisms were newly isolated from soils and several of them accumulated two acids in the culture broth, α-ketoglutaric acid being a major one and succinic acid a minor one. Two strains (N-79 and S-1), classified as the genus Mycobacterium, were examined for the cultural conditions with respect to the accumulation of the acids. The accumulation of α-ketoglutaric acid depended greatly on the pH value in the broth, which is required to be kept at around 4 for the maximum accumulation. By means of the pH-controlled culture (at 3.5) with a jar fermentor, strain N-79 accumulated a-ketoglutaric acid at a rate of 0.015g/liter/hr. The data obtained in this work indicate that the metabolism of isopropanol by strain N-79 probably proceeds via the acetone pathway without the interconversion between isopropanol and n-propanol.

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