A collagenolytic enzyme specific for native collagen and gelatin was isolated from Pseudomonas marinoglutinosa by DEAE-cellulose column chromatography, Sephadex G-150 gel filtration and by disc electrophoresis on polyacrylamide gel.
The molecular weight of the enzyme was approximately 74, 000 and its isoelectric point was found to be around 4.5. The optimum pH and temperature for Z-GPLGP hydrolysis were around 7.6 and 38°C, respectively. The enzyme was rather stable up to 50°C and in the range between pH 5.0 and 10.0, and was stabilized by Ca²⁺ to some extent. Some chelating agents and metal ions such as Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ inactivated the enzyme, but diisopropyl phosphofluoridate, sulfhydryl agents and some trypsin inhibitors did not affect the activity.
The EDTA-inactivated enzyme was restored its activity by added Ca-salt to almost completely and very slightly by Co-, Mn- and Sr-salt.
Metal analysis showed the enzyme contained 1g atom of zinc and 4g atoms of calcium per mole.

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For the purpose of studying the mechanism of conidiogenesis in Piricularia oryzae, methods were developed to separate the phase of mycelial growth from that of conidiation and also to evaluate them quantitatively.
When P. oryzae was grown on media with low carbon: nitrogen ratio and high nitrogen concentration, conidiation did not take place, in spite of its vegetative growth. Conidiation occurred in a very short period of time when the above mycelia were replaced on nutritionally poor media. Cellophane membrane was overlaid on solid medium and conidia were spread uniformly. Evenly grown mycelial mat which could be easily transferred onto the post-culture medium was obtained. As the preculture medium, MSA medium with carbon: nitrogen ratio of 6.3 and nitrogen concentration of 1.5g/liter was used. The evenly grown mycelial mat was cut into small square mats of 1.44cm² each and the small square mycelial mats were transferred onto the post-culture medium together with the cellophane membrane. The conidiation took place in the post-culture and the vegetative growth in the preculture and the conidiation in the post-culture could be observed separately and quantitatively.
Conidiation did not occur at all in the preculture and the degree of conidiation which took place in the post-culture varied according to the precultural conditions. This means that it is a certain state of physiological condition in the preculture which determines the degree of conidiation in the post-culture. The anthers designated this state of physiological condition as the “latent activity of conidiation” (LAC). For the purpose of the quantitative estimation of this activity, we expressed LAC in terms of the degree of conidiation in the post-culture under a defined cultural condition. The LAC was subject to change very easily, declined rapidly and disappeared upon prolonged preculture. Only young mycelia showed to have this activity.
The influences of the precultural condition on the development of the LAC and vegetative growth were generally parallel. However, the LAC was generally more sensitive to the environmental condition than the vegetative growth, especially to the temperature change.
The conidia formed were uniform in size and had high rate of germination. Several strains of P. oryzae tested showed very similar behavior.

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Effects of phospholipids on the bacterial protoplasts or membranes were investigated. Phosphatidylinositol (PI) has, most of all, active bursting action on the protoplasts of Bacillus megaterium which was found to contain a very small amount of inositol, about 0.006% of dry cell weight. This action of PI was less active on the spheroplasts or protoplasts of Escherichia coli or Bacillus subtilis than Bac. megaterium. The bursting action of PI was dependent on temperature, but not on pH or osmotic pressure(concentration of sucrose). This action of PI on the protoplasts of Bac. megaterium was more marked when the incubation was carried out in phosphate buffer than in Tris buffer. High concentration of Mg ion inhibited this PI action in the phosphate buffer, but accerelated that in the Tris buffer.
Phospholipids, especially PI, elevated the activity of succinate dehydrogenase of membrane fraction of Bac. megaterium, but sodium laurylsulfate (SLS) inhibited this enzyme.
These actions of PI were compared with those of other phospholipids and detergenic substances.

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The π-electron distribution of various aromatic compounds has been calculated by a molecular orbital method.
The reaction of hydroxylation was assumed to be radical type.
Relation between electronic structures and mono-hydroxylation of aromatic compounds by microorganisms was investigated.
A distinct parallelism was ruled out between the electronic structure and hydroxylation of aromatic compounds.
Hydroxylation of aromatic compounds occurred where the superdelocalizability Sr (R) showed large value.

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Some enzymatic properties and substrate specificities of the two peptidoglutaminases (peptidoglutaminase-I (PGase-I) and II (PGase-II) isolated from Bacillus circulans were investigated.
The both resembled each other with respect to optimum pH and temperature for their activities, susceptibility to various reagents and effects of many metal ions. But, the pH-rate profile of PGase-II was more broad than that of PGase-I. Thermal stability of PGase-I was better than that of PGase-II at a degree of about 5°C.
However, they were definitively different each other with respect to their substrate specificities. PGase-I specifically deamidated the γ-amide of L-glutamine residing at the carboxyl terminal on peptides, and was inactive to glutamine derivative substituted at both α-amino and α-carboxyl groups. On the other hand, the best substrate for PGase-II was tri-peptides, X-Gln-Y, where X was carbobenzoxy-, t-amyloxycarbonxy- or amino acid residues, and Y was amino acids. Though L-glutamine presented in polypeptide chains composed of more than four amino acid residues was a poor substrate, two L-glutamines in oxidized insulin A chain were well attacked by the enzyme.
The both PGase were inactive to asparagine and asparaginyl peptides.

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α-Glucosidase was purified from Saccharomyces logos by precipitation with ethanol, and chromatographies on Sephadex G-200, DEAE-Sephadex, DEAE-cellulose and Duolite A-2. The purified α-glucosidase was homogeneous on ultracentrifugation and zone electrophoresis using cellulose acetate membrane. The sedimentation coefficient was calculated to be 9.6S. The molecular weight was estimated to be approximately 2.7×10⁵ by gel-filtration technique.
The optimum pH was found to be in the range of 4.6_??_5.0, and the optimum temperature was 40°C. The enzyme exhibited higher hydrolytic activity toward maltose rather than toward phenyl-α-glucoside and turanose, and no activity toward sucrose.
The enzyme was a glycoprotein containing carbohydrate of about 50%.

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The substrate specificity of Saccharomyces logos α-glucosidase has been investigated. The enzyme was active especially on maltose and phenyl-α-maltoside. The ratio of hydrolysis for maltose: phenyl-α-maltoside: phenyl-α-glucoside was estimated to be 100:110:5.5. Therefore, the substrate specificity of the enzyme was quite different from those of other Saccharomyces species, though similar to those of mold α-glucosidases.
Km values for maltose, phenyl-α-maltoside and phenyl-α-glucoside were calculated to be 7.7mM, 3.6mM and 8.7mM, respectively. Of the substrates tested, the enzyme showed a preference for phenyl-α-maltoside.

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Cell-free extracts of Brevibacterium thiogenitalis culture grown in the presence of copper catalyzed the oxidation of NADH₂ and succinate through an electron transport chain which contained menaquinones and cytochromes a, b and c. On the other hand, extracts of cells grown in the absence of copper lacked cytochromes a and c, and contained cytochrome d. These findings, as well as the results obtained in inhibition experiments, suggest that in copperdeficient cells the major part of NADH₂ was oxidized via a bypass in which the electrons were transferred directly from fiavoprotein or cytochrome b to molecular oxygen.
Electron transport from these substrates to molecular oxygen resulted in ATP synthesis. The average P/O ratios in extracts of the copper-sufficient cells were 0.33 for generated NADH₂, 0.20 for added NADH₂, and 0.34 for succinate, and those in extracts of the copperdeficient cells were 0.15, 0.13 and 0.21, respectively. In addition, a linear relationship was found between the yield of L-glutamate from acetate and the P/O ratios with both NADH₂ and succinate as substrates.
From these results, it is reasonable to consider that the poor yield of L-glutamate from acetate in copper-deficient cells was due to a reduction in energy supply, which was caused by the low efficiency of oxidative phosphorylation.

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Crystalline D-glucose-isomerizing enzyme from Bacillus coagulans, strain NH-68 has been shown to consist of subunits by the method of electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels.
The dissociation behavior of the enzyme has been characterized. The enzyme dissociates into inactive subunits by the preincubation with 0.05% SDS in the presence of 5×10^-3M MnCl₂ or CoCl₂, but not in the absence of these metal salts. In 8M urea, however, the enzyme does not dissociate into subunits and the activity is completely recovered by dilution of the urea. Metal salts, such as MnCl₂ and CoC₂, also do not affect activity in the presence of urea.

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Relationships between callus origin and the nicotine contents as well as conditions of nicotine production in tobacco tissue cultures were investigated. Nicotine contents of callus tissues were remarkably affected by plant growth regulators in the culture medium. Thus, nicotine production was promoted by the regulators at lower concentrations, but gradually inhibited when the concentrations increased over an optimal region which was different among several kinds of the regulators. The nicotine contents also considerably depended on conditions of the callus induction as well as organ from which they were derived, at least just after callus induction. The differences due to the induction conditions were considered to be gradually lost during successive cultures. Thus, the nicotine contents appeared gradually to change to a certain level which mainly depended on the concentration of the regulators added to the culture medium. When such stabilized callus tissues were transferred to a culture medium containing another regulator or different concentration of the regulator, their nicotine contents rapidly changed to a new level depending on the culture conditions during a few successive cultures. The stabilized callus tissues grown on a medium containing 0.1ppm α-NAA contained 0.5% of nicotine or more, which was almost the same level in root of the intact plant.

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Corn steep liquor markedly inhibited the growth of the grass, alfalfa, as previously reported. The active principles from corn steep liquor were isolated. From the acidic fraction of corn steep liquor, white needles and yellow needles were isolated. The compound of white needles, which inhibited the germination of alfalfa seeds at the concentrations above 500ppm, was found to be identical with ferulic acid known as germination inhibitor of plant seeds. The compound of yellow needles did not inhibit the germination of alfalfa seeds, but stimulated the growth of the plant. It has a structure related to β-acid which has been isolated from rice-bran and has been identified as 2, 6-dihydroxycinchoninic acid. This active substance is a new quinoline, 2, 8-dihydroxycinchoninic acid and was named zeanic acid.

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Increase in fruit set of grape was observed when vines were sprayed with zeanic acid before blossom, but when sprayed after blossom the effect was not clear.
Promotion of growth of radish cotyledons by zeanic acid was observed in the presence and absence of kinetin. Zeanic acid also promoted growth of dwarf mutant of rice seedlings (cv. Tanginbozu), but inhibited that of normal seedlings (cv. Tonewase). Zeanic acid showed promotive effect on proliferation of rice callus tissue in the absence of kinetin and auxin.
Zeanic acid showed low toxic activity against mice, earthworm and killifish, and no antimicrobial activity.

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The biosynthetic pathway of vitamin B₆ (abbreviated as B₆) has been studied with the cell-suspension of B₆-producing bacteria, Achronrobacter cycloclastes A. M. S. 6201. The distribution of ¹⁴C in the B₆ molecule prepared with the cell-suspensions containing glycerol-1, 3-¹⁴C, glycerol-2-¹⁴C or γ-aminobutyric acid-U-¹⁴C was investigated by using three novel degradation methods. The results showed that carbon skeletons of glycerol and γ-aminobutyric acid were used for the formation of the major part of B₆ carbon skeleton respectively. The implication of these compounds as precursors of B₆ was discussed.

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Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A-50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.
The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.

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Feeding tests were carried out on rats to clarify the mechanisms of fatty liver formation induced by autoxidized methyl linoleate. Lipid peroxides prepared by autoxidation of highly purified methyl linoleate were given orally to rats. Triglyceride and glycogen contents in liver were determined and enzyme activities including triglyceride synthetase and α-glycerophosphate dehydrogenase were also examined. The following results were obtained. 1. Triglyceride accumulation in rat liver fed autoxidized methyl linoleate was observed. 2. Increase in triglyceride content in rat liver was soon followed by the decrease of hepatic glycogen. 3. When rats were starved prior to introduction of autoxidized methyl linoleate, hepatic triglyceride accumulation did not occur. 4. The activities of α-glycerophosphate dehydrogenase and triglyceride synthetase in liver, and those of glutamic oxalacetic transaminase and leucine aminopeptidase in plasma were practically similar among the rats of test groups fed fresh or autoxidized methyl linoleate and the control fed diet without methyl linoleate. 5. The addition of l-carnitine which is a stimulator of fatty acid oxidation retarded the accumulation of the hepatic triglyceride mentioned above.

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To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of ¹⁴C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C₄-dicarboxylic acids, indicated that C₄-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, n-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or n-lactate. From these results it was concluded that the uptake of the C₄-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specific dehydrogenase system.

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A major polyphenol from P. japonicus, named fukinolic acid (XII), was isolated and its structure was investigated. Fukinolic acid afforded a new polyphenol named fukiic acid (I) and caffeic acid on hydrolysis in molar ratio 1:1. The structure of fukiic acid was established as 2, 3-dihydroxy-4-(3', 4'-dihydroxyphenyl)-3-carboxybutyric acid (I), and its methyl derivatives were described.
Based on evidences presented, the structure for fukinolic acid was proposed as XII. It was further demonstrated that fukinolic acid caused the brown discoloration on action of a polyphenoloxidase from the plant tissues.

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By selected microorganisms the acetates of the racemic sec-alcohols were asymmetrically hydrolyzed to optically active sec-alcohols and the acetates of their antipodes. Microbial hydrolysis of the acetates of the racemic prim-alcohols having an asymmetrical carbon atom at the β-position gave lower optically active alcohols and the acetates of their antipodes, but the acetates having an asymmetrical carbon atom at the γ-position were hydrolyzed only to give racemic alcohols.

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Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.

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3-O-Demethyl and 2, 3-O, O-didemethyl derivatives of natural rotenone (5' β-rotenone), 5' α-rotenone, d-epirotenone (5' β-epirotenone) and 5' α-epirotenone are obtained upon reacting 5' β-rotenone or 5' β-epirotenone with two or three molar equivalents of boron tribromide followed by recyclization of the E-ring using sodium bicarbonate. 3-Methoxy-¹⁴C-5' β-rotenone is prepared in 16% yield by treating 3-O-demethyl-5' β-rotenone with methyl-¹⁴C iodide in the presence of alkali followed by epimerization of the ¹⁴C-5' β-epirotenone byproduct for increased yield of ¹⁴C-5' β-rotenone. 3-O-Demethylation is established as a detoxification mechanism for 5' β-rotenone or for one of its metabolites based on the expiration by mice and rats of 27% and 13%, respectively, of the administered radiocarbon as ¹⁴carbon dioxide.

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Some physicochemical properties of neutral proteinases I and II, zinc-containing metalloenzymes, from Aspergillus sojae were investigated.
Neutral proteinase I: The enzyme protein had a sedimentation coefficient of 3.90S, an intrinsic viscosity of 0.0315dl/g, and a partial specific volume, calculated from the amino acid and carbonhydrate composition, of 0.715cm³/g. The molecular weight was 42, 200 from the Yphantis' procedure, and was 42, 500 from the calculation according to the Scheraga-Mandelkern's formula. The integral numbers of amino acid residues per molecule calculated on the basis of 42, 200 as molecular weight were as follows; Lys₁₆, His₆, Arg₁₃, Trp₈, Asp₅₆, Thr₂₅, Ser₂₃, Glu₃₁, Pro₁₈, Gly₄₀, Ala₃₃, 1/2Cys₄, Val₁₁, Met₆, Ile₁₅, Leu₂₅, Tyr₂₀, Phe₁₀, (amide-ammonia)₂₉, in addition to mannose₆, galactose, , hexosamine₃.
Neutral proteinase II: The enzyme protein had a sedimentation coefficient of 2.32S, an intrinsic viscosity of 0.0270dl/g, and a calculated partial specific volume of 0.714cm³/g. The molecular weight was 16, 800 from the Yphantis' procedure, and was 18, 000 from the sedimentation and intrinsic viscosity. The following amino acid compositions was calculated on the basis of 16, 800 as molecular weight; Lys₈, His₃, Arg₃, Asp₁₉, Thr₁₇, Ser₁₁, Glu₂₃, Pro₅, Gly₉, Ala₂₄, 1/2Cys₄, Val₅, Ile₃, Leu₁₃, Tyr₁₀ Phe₃, (amide-ammonia)₁₅. In the enzyme preparation, neither methionine nor tryptophan was detected and carbohydrate was also absent.
In both neutral proteinases I and II, no free SH group was detected by the PCMB-titration in the presence of 8M urea.

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Further selection for a better strain capable of producing D(-)-a-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) was carried out. Pseudomonas melanogenum KY 3987 was consequently selected as a new strain possessing an APc-specific penicillin acylase.
The acylase could synthesize APc in good yields from 6-APA and phenylglycine ester and form 6-APA only from APc, not from other common penicillins. Since the Pseudomonas acylase was found incapable of forming penicillin G (Pc-G) from 6-APA and phenylacetic acid, in contrast with E. coli and Kluyvera citrophila enzymes, the enzymatic hydrolysate of Pc-G, for example by K. citrophila cells, which contained 6-APA and phenylacetate, became employed as a source of 6-APA instead of purified 6-APA to synthesize APc by the cells of P. melanogenum.

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