1. Several bacteria were isolated from soil which grew on both D- and L-aminolactam and whose cells had an activity to racemize them. They were identified as Achromobacter obae nov. sp., Achr. cycloclastes, Alcaligenes faecalis and Flavobacterium arborescens.
2. Racemization of D- and L-aminolactam was investigated using the lyophilized cells of Achr. obae nov. sp. The optimum pH value of the reaction was about 8.0. The racemizing activity was completely inhibited by 10^-4M hydroxylamine, and the inhibition was removed by 10^-4M pyridoxal phosphate. Five percent D- and L-aminolactam solutions were completely racemized with a concomitant slight formation of L-lysine.

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An enzymatic process has been proposed for producing L-lysine from DL-aminolactam by the author. Several yeasts, including Cryptococcus laurentii, have been isolated from soil and found to catalyze the selective hydrolysis of aminolactam. As well, several bacteria, including Achrornobacter obae nov. sp., have been isolated which catalyze the racemization of aminolactam. This paper reports an investigation of the conversion reaction of D- and DL-aminolactam into L-lysine using both cells of Crypt. laurentii and Achr. obae nov. sp. The optimum pH of the reaction was between 8.0 and 9.0, but most often near 8.0. An inhibitory effect of the substrates on the reaction was observed when the substrate concentration was higher than 10%, but the inhibition was slight at 5% substrate. The optimum temperature of the reaction for 3_??_6hr was around 39°C. A complete conversion reaction of 10% DL-aminolactam was carried out at 40°C for 24 hr, producing L-lysine in a conversion rate of 99.8 %. L-Lysine•HCl isolated from the reaction mixture was 99.5% optically pure.

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The interactions between ovalbumin and κ- or β-casein due to heat-treatment of the mixtures were examined.
The interaction between ovalbumin and κ-casein was confirmed by the viscosity change of the mixture, the change in denaturation level of ovalbumin and the change in the ability of κ-casein to stabilize Ca-α_s-caseinate. Contribution of electrostatic bindings to the interaction was partly confirmed by acetylation of the proteins. The reaction through SH groups of denatured ovalbumin and κ-casein was not responsible for the interaction.
The interaction between ovalbumin and β-casein was recognizable even at 25°C. This interaction was evidenced not by the viscosity change but by the change of sound velosity in the mixture and the partial inhibition by β-casein of the exposure of SH groups of ovalbumin when heated.

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An α-1, 3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-1, 3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-1, 3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.
The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P (phospho)-cellulose. To eliminate the concomitant β-1, 3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-1, 3-glucanase was eluted with a solution of α-1, 3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78, 000 by SDS-polyacrylamide gel electrophoresis.

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Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7 S or 11 S soybean proteins with alcohol. The 7 S component showed the ability to form a stronger gel than the 11 S. This phenomenon depended on pH and alcohol concentration. In 66% ethanol, the viscosity of the 7 S and 11 S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol<tert-butanol<n-propanol<iso-propanol<ethanol<methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7 S.

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Two bacterial strains 26.1 B (I) and 22.39 B (II) were isolated from soil as alkaline lipase producing microorganisms. Strain 26.1 B (I) was identified as Pseudomonas nitroreducens nov. var. thermotolerans, and strain 22.39 B (II) was identified as Ps. fragi. When they were cultivated aerobically in a 20-liter jar fermentor in medium containing 2.0% soybean meal, they secreted a large amount of alkaline lipases. The enzymes were recovered efficiently as precipitates by adjusting the pH of the culture fluids to 4.0 with HCl. The enzymatic characteristics included: optimum pH, 9.5 (I, II); stable pH range, 5_??_11 (I, II); optimum temperatures, 50°C (I) and 75_??_80°C (II); and more than 95% of the enzyme activity remained after incubated at 70°C for 20min (I, II). Lipase activities were inhibited remarkably in the presence of anionic surfactants (I, II) and bile salts (I, II).

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Extracellular asparaginase from Candida utilis was partially purified by precipitation with acetone and by column chromatography on DEAE Sephadex A-50 and Sephadex G-200. The specific activity of the enzyme preparation was 3900 units per mg of protein. Candida asparaginase characteristically had deaminating activity for D-asparagine as well as for L-asparagine. But this enzyme was not able to hydrolyzed L- or D-glutamine. SH inhibitor, chelating agents and metal ions did not show any inhibition or activation of L-asparaginase activity. Optimum pH was about 6 for both L- and D-asparagine. This asparaginase was stable between pH 4 and pH 10 in heating for 10min at 50°C.

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Purified Candida asparaginase was proved to be homogeneous by gel filtration, ultracentrifugation and disc electrophoresis. The enzyme was found to have properties as glycoprotein containing mannose. The ratio of mannose to protein was 1 to 2 in purified enzyme. Specific activity was 5500 units per mg of protein. Isoelectric point was pH 4 to 4.5 and sedimentation coefficient was found to be about 8.2 S. Antitumor activity of Candida asparaginase was inferior to E. coli enzyme. It was thought as the reason why the Candida asparaginase had less affinity to L-asparagine and it was cleared faster from the blood than E. coli asparaginase.

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Bacillus No. C-11 which utilized rayon waste was isolated. This strain belongs to the genus Bacillus from its morphological and biochemical characteristics but grew better in alkaline media than in neutral media. Residual sugars of rayon waste were 34.7% after 2 days cultivation, 25.5% after 4 days and 7.0% after 9 days. Yeast extract and N-source, such as polypeptone or urea stimulated the utilization of rayon waste. The long period cultivation optimum pH was about 11, and the short period cultivation optimum pH was about 9. Partially purified hemicellulase from Bacillus No. C-11 was most active at pH 7, but still active at pH 10. The stable pH for this enzyme action was in the range of 5.5 to 9, and from the hemicellulose enzymatic digest, xylose, xylobiose, xylotriose and oligosaccharides were detected.

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The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50mM sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35%. and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (-15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.

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From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI=9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5_??_10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41, 000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA>native DNA_??_RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercaptoethanol and glutathione had no effect. Polyamines showed no activation of the nuclease for DNA degradation.

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A nuclease from N. crassa mycelia was found to attack both heat-denatured and native DNA in endonucleolytic manner. The products of exhaustive degradation of heat-denatured DNA were mainly di- to pentanucleotides bearing 5'-phosphoryl groups. 5'-Mononucleotides amounted to 4.4% of the total products and the base distribution was in the following order: dTMP_??_dCMP>dGMP>dAMP. Analysis of the residues at 5'- and 3'-termini of the oligonucleotides showed that thymidine was predominant at both termini, especially at 3'-termini. Also the analysis of terminal residues produced by limited digestion (27% and 55.5% of the substrate were rendered acid soluble, respectively) gave the same results as above. Therefore, it was suggested that N. crassa nuclease has some preference for thymidine residue to hydrolyze the sequence of -T↓pT- or -T↓pX- predominantly. The activity toward synthetic polymers was in the following order; poly d(A-T)_??_poly dA•poly dT>poly d(G-C)>poly dG•poly dC. The correlation between GC-contents and the activity was also investigated.

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In electron microscopic observation, neither wax nor cuticle was observed on the outermost layers of callus tissues. Chemical estimation of wax in the callus surface was attempted by thin-layer chromatography of solvent extracts of callus tissues in comparison with those of barley and rice leaves. Hydrocarbons and free alcohols were detected in lyophilized callus tissues, but no wax esters or ketones were detected. Germination test indicated that germination of spores of Aspergillus oryzae was less favored on hydrophobic membranes than that of spores of Alternaria sp. and Botrytis cinerea.
From these results, we inferred that the lack of cuticle and wax in the outermost layer of callus tissues facilitated spore germination and penetration, and A. oryzae, a saprophytic fungus, could also readily penetrate into callus tissues.

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A new antibiotic K-52 B, different from K-52 A, was isolated from the culture broth of Streptoverticillium roseoverticillatum subsp. albosporum, strain No. K-52. The antibiotic K-52 B was thought to be a similar saccharide to K-52 A from its physicochemical properties but differed from K-52 A in the presence of nitrogen content. Antibiotic K-52 B inhibited the growth of Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa on a chemically defined medium. The growth inhibition was, however, reversed by L-glutamine, L-glutamic acid, L-asparagine and L-aspartic acid.

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1-Aminoproline-U-¹⁴C was administered to rats by intraperitoneal injection. The radioactivity was distributed in all the tissues examined. Among them, kidney, lung, liver and spleen had high specific activity. The radioactivity in the tissues and blood decreased rapidly as a function of time, except in brain. About 80% of the radioactivity administered was excreted in urine within 24 hr. Besides intact 1-aminoproline, several radioactive compounds were detected in the urine sample, and one of them was identified as 1-aminopropyl hydrazone of pyridoxal.

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The larval homogenate of sweet potato weevil, Cylas formicarius, induced terpene formation in sweet potato root tissue. We indicated that the terpene-inducing factor of the larvae consisted of high molecular weight component(s) and low molecular weight component(s). The high molecular weight component(s) was heat-unstable and partially inactivated by pronase, indicating proteinacious properties. The low molecular weight component(s) was heat-stable.

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The quenching effect of α-, γ- and δ-tocopherols on the methylene blue sensitized photooxidation of methyl linoleate was investigated, and the ¹O₂ quenching ability of tocopherols was determined, The ¹O₂ quenching rate constants for α-, γ- and δ-tocopherols in ethanol were estimated to be 2.6×10⁸M^-1 sec^-1, 1.8×10⁸M^-1 sec^-1 and 1.0×10⁸M^-1 sec^-1, respectively. And the rate constants for the chemical reaction between each tocopherol and ¹O₂ were 6.6×10⁶M^-1 sec^-1, 2.6×10⁶M^-1 sec^-1 and 0.7×10⁶M^-1 sec^-1 for α-, γ- and δ-tocopherols, respectively. The results show that α-tocopherol is the most effective compound toward ¹O₂ among the three tocopherols. The photooxidation of each tocopherol produced two peroxides which, after chemical reduction, were identified to be tocopherol hydroquinone by gas chromatography-mass spectrometry analysis. The photooxidation mechanism of these tocopherols was assumed to be different from that of autoxidation.

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Pyridoxal-5'-phosphate was synthesized in excellent yield by phosphorylation of 1-secondaryamino-1, 3-dihydro-7-hydroxy-6-methyl-furo (3, 4-c) pyridine which was readily obtained by a condensation reaction between pyridoxal and a secondary amine.

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Livers of growing rats fed a 5 or 20 protein calories percent (PC%) diet containing purified whole egg protein for three weeks were perfused in situ and the release of triglycerides (TG) and the oxidation of fatty acid by the liver alone were estimated by infusing palmitic acid-1-¹⁴C to the perfusion medium.
The release of TG from the liver of the 5 PC% group was significantly lower in both unfractionated perfusate plasma and perfusate plasma very low density lipoprotein (VLDL) than that of the 20 PC% group, whereas the content of liver TG of the 5 PC% group was higher than that of the 20 PC% group. Significantly lower radioactivity appeared in TG of both unfractionated perfusate plasma and perfusate plasma VLDL of the 5 PC% group than that of the 20 PC% group, while total radioactivity of liver TG was higher in the 5 PC% group than in the 20 PC% group. The ¹⁴CO₂ production in the perfused liver of the 5 PC% group increased gradually with time rather than decreased in comparison with that of the 20 PC% group.
These findings suggest that a major factor responsible for the liver lipid accumulation in rats fed the low protein diet is not an impaired fatty acid oxidation in the liver but an impaired secretion of TG from the liver.

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Substrate specificity of the two forms of glucoamylase (glucoamylases I and II) from the mycelia of Penicillium oxalicum has been investigated. Both glucoamylases hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but not isomaltose and isomaltotriose. Glucose was the sole hydrolysis product found in the digests of these substrates. Both enzymes hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. Maltose was hydrolyzed about one-sixth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen and rice starch in the yields of almost complete hydrolysis, and also hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylases I and II were glycoproteins containing 3.93% and 3.22% neutral sugar, respectively. Glucoamylase II contained mannose, galactose, and glucose as neutral sugar constituent.

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Penicillium oxalicum produced two forms of glucoamylase and an α-glucosidase. The α-glucosidase has been isolated from the mycelia in an electrophoretically homogeneous form, and its properties have been investigated. The enzyme had a pH optimum at 3.5 with maltose as substrate. The enzyme hydrolyzed maltose, maltotriose, phenyl α-maltoside, isomaltose, panose, isomaltotriose, amylose, and soluble starch liberating glucose, but not sucrose. The Km value for maltose was 1.232mM. Phenyl α-maltoside was hydrolyzed to glucose and phenyl α-glucoside by the enzyme. Isomaltose was the main transglucosylation product from maltose and soluble starch. It was found that the enzyme liberated α-glucose from amylose.

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The nuclear magnetic resonance spectra of erythrose, threose and the 5-O-methyl derivatives of arabinose, xylose and lyxose were examined. In certain cases α, β-anomeric ratios in deuterium oxide might be measurable. And with erythrose evidence was obtained which indicated that 55% of it existed in a form other than furanose form. There was a correlation between the C-1 proton signals of the 5-O-methyl-pentoses and those of the stereochemically similar isopropylidene hexoses, obtained from the corresponding isopropylidene diethyl thioacetal. The evidence indicated 5, 6-substitution in the diethyl thioacetals of mannose, glucose and altrose.

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The mechanism of dispersion of solute in gel chromatography using various Sephadex gels was quantitatively studied. In order to simplify the mathematical treatment, non-adsorptive low-molecular weight substances such as NaCl and glucose were chosen as samples. A pulse response experiment was carried out in a column. The longitudinal dispersion coefficient and the diffusion coefficient in gel phase were determined separately by applying the moment method to the elution curve. Then, their contribution to the column efficiency characterized by HETP was studied. Particularly, the effect of gel phase diffusion was examined in detail. The gel phase diffusion coefficient was apparently much smaller than the molecular diffusion coefficient. Consequently, it was revealed that gel phase diffusion played a much more important role in gel chromatography than what was expected by other investigators.

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Neither bacteriophage øX174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ø X 174 DNA synthesis started.

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3 Z, 6 Z-Dienoic acids (C₈-C₁₂ and C₁₈) were for the first time synthesized by coupling 2-acetylenic bromides and 2-(3'-butynyloxy)-tetrahydropyrane followed by stereoselective hydrogenation and oxidation.

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About one hundred of unsaturated long straight chain compounds (C₁₀-C₁₈) containing a terminal hydroxyl or acetoxyl group were synthesized and their attractiveness to male Lepidoptera were tested in various environments. In this screening test, male insects of ninetythree species which belong to fifteen families were attracted specifically. It was indicated that monoene alcohols and acetates constitute a main group of sex pheromones of the Lepidoptera being native in Japan. Discussions are made on the structure-activity relationship in the attractants and on the chemical structural features of components, which are expected to be present commonly in the certain family, namely, Torticidae, Noctuidae etc.

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A specific nicotinamide adenine dinucleotide (NAD)-linked D-xylose dehydrogenase was first demonstrated in the D-xylose-grown cells of Arthrobacter sp. This enzyme was specifically induced by D-xylose. No other effective sugar was found for the production of this enzyme by growing culture. This enzyme catalyzes the following reaction:
D-Xylose+NAD⁺-D-xylonolactone+NADH+H⁺
This enzyme was purified 186-fold in specific activity from the extracts of D-xylose-grown cells by the procedures including chromatographies on DEAE-cellulose, DEAE-Sephadex and Sephadex G-100. The enzyme is specific on the dehydrogenation of D-xylose with NAD. Seven pentoses, 5 hexoses, and 7 polyols were not substrates with NAD or NADP. NAD was active coenzyme of this enzyme. The Km values were 17.4mM for D-xylose and 0.27mM for NAD. The pH optimum was at pH 10.4, but the enzyme activity was most stable at neutral pH, 7.0 to 7.5. The molecular weight of the enzyme was calculated as 62, 000±2000 and isoelectric point was pH 5.58. These evidences clearly indicated the presence of a novel dehydrogenase specific on D-xylose, D-xylose dehydrogenase in Arthrobacter sp.

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5-Methyltubercidin and its α-anomer were synthesized from 4-methoxy-5-methyl-2-metbylthio-7-(2, 3, 5-tri-O-benzyl-α- and β-D-ribofuranosyl) pyrrolo [2, 3-d] pyrimidines, which in turn prepared by condensation of anion of 4-methoxy-5-methyl-2-methylthiopyrrolo [2, 3-d] pyrimidine with 2, 3, 5-tri-O-benzyl-D-ribofuranosyl bromide. The anomeric configuration was rigorously established by deriving 5-methyltubercidin to the quaternary cyclonucleoside.

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Grayanotoxin (G)-II (1), C₂₀H₃₂O₅, mp 196_??_8°C, is one of the toxic diterpene alcohols obtained from Leucothoe grayana Max (Ericaceae).¹⁾ These alcohols are known for their novel carbon skeleton. We elucidated the biological oxidation of G-II (1) by a microorganism to give 3-dehydro G-II (2), C₂₀H₃₀O₅, mp 233°C.

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