Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

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An enzyme, pyridoxamine 5'-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5'-phosphate transaminase did not contain pyridoxal 5'-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5'-phosphate.

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The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio andFlavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.

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Two neutral proteinases (I and II) of Aspergillus sojae were isolated from wheat bran culture by DEAE-cellulose chromatography. Neutral proteinase I was purified by sequential chromatography on columns of CM-cellulose, Sephadex G-100 and hydroxylapatite. Neutral proteinase II was purified by chromatographies on DEAE-Sephadex A-50, hydroxylapatite and Sephadex G-100. Crystalline preparation was obtained from neutral proteinase II, but not from neutral proteinase I. The purified preparation of each enzyme was found to be homogeneous on sedimentation analysis, disc electrophoresis and disc electrofocusing. A notable difference in specific activity was observed between the two neutral proteinases, i.e., 1, 050 and 35 proteinase units per mg of enzyme protein for neutral proteinases I and II, respectively. Determination of the ultraviolet absorption spectra indicated that the E^1%_1cm at 280mμ of neutral proteinases I and II were 16.7 and 9.0, respectively. The molecular weights of neutral proteinases I and II were estimated to be 41, 700 and 19, 800, respectively, by gel filtration, and their isoelectric points were found to be pH 4.7 and pH 4.2, respectively, by disc electrofocusing.

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Some enzymatic properties of purified neutral proteinases I and II from Aspergillus sojae were investigated.
Neutra_??_ proteinase I: The optimum pH for casein digestion was around 7 at 30°C for 10min and the optimum temperature was 55°C at pH 7.3 for 10min in the presence of Ca^2-. The enzyme activity was almost completely lost at 55°C, pH 7.3, within 10min, but was stabilized by Ca²⁺ to some extent. At low temperature, the enzyme was highly stable at the range of pH 3 to 11, and at 49°C the most stable pH was 6 to 7.
Neutral proteinase II: The optimum pH for casein digestion was around 6 at 30°C for 10min and the optimum temperature was 65°C at pH 7.3 for 10min. At low temperature, the enzyme was highly stable at the range of pH 4 to 11 and relatively stable at pH 1 to 2, but unstable at around pH 3. Though the enzyme was remarkably thermostable showing the remaining activity of ca. 80% even by the treatment at 99°C for 10min at pH 7.3, it was very unstable at 75°C. This unstability was considered to be resulted from the autolysis of the enzyme, which was conspicuously observed at pH 3 and pH 9. The presence of Ca²⁺ accelerated the inactivation of the enzyme.
None of metallic salts tested promoted activities of the both enzymes, but Cu²⁺, Cd^2-, Hg^2-, Pb²⁺ and Sn⁴⁺ inhibited markedly neutral proteinase I even at low concentrations. EDTA, iodine and NBS inactivated the both enzymes, whereas sulfhydryl reagents, DFP and potato inhibitor did not affect their activities. Sodium thioglycolate, L-cysteine, KCN, 2-mercaptoethanol and phosphate ions inactivated neutral proteinase I to some extent, but did not neutral proteinase II.

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Metallic mercury-releasing enzyme (MMR-Enz) which catalyzes the reduction of mercury in organic and inorganic mercurials to metallic mercury was purified about 90-fold from the cell-free extract of mercury-resistant Pseudomonas by means of ammonium sulfate precipitation and repeat of column chromatography on Sephadex G-150 and on DEAE-Sephadex. The purified enzyme showed a single band in electrophoresis on polyacrylamide gel, and also _??_ characteristic absorption spectrum indicative of flavoprotein. A prosthetic group of the enzyme was identified as FAD by thin-layer chromatography. The identification was confirmed by the reconstitution of active D-amino acid oxidase from the apoenzyme, and by the reactivation of ultraviolet light-irradiated MMR-Enz with FAD. Organic mercurials (phenyl, methyl and ethyl mercurials) and inorganic mercurials were reductively decomposed forming metallic mercury by the action of the purified MMR-Enz in the presence of reduced NAD (P) generating system and cytochrome c-I.

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The accumulation of 5'-IMP or 5'-XMP by adenine-requiring or adenine-guanine-requiring mutants of Bacillus subtilis with weak 5'-nucleotidase was greatly increased by cultivating them at a high temperature (40°C). The accumulation was markedly increased by addition of hypoxanthine or xanthine to the medium.
It is suggested that in these mutants, 5'-IMP and 5'-XMP are secondarily resynthesized through salvage pathways from their respective bases that have been accumulated first in the medium and these processes are activated at this high temperature.

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The effect of overnight fasting on the fatty acid composition and structure of glycerolipids in rat liver and its microsomes was examined. Fasting caused alterations in the fatty acid composition of each glycerolipid in the different ways. Decreased percentage of linoleate and increased percentage of arachidonate were observed in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In contrast, linoleate was increased and oleate was decreased in triglyceride (TG). Increased percentage of tetraenoic PC species was dependent on the increase in stearoyl-arachidonyl species. In TG, palmitoyl-dilinolyl and more polyunsaturated species were increased markedly.
These results suggest that the metabolism of hepatic glycerolipids is completely modified in the fasting rats.

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The action pattern of crystalline ribonuclease of Rhitopus niveus was investigated. The enzyme degraded homopolynucleotides in the following order: poly-U>poly-C>poly-A>poly-I_??_poly-G. The enzyme also attacked yeast RNA and produced G-, A-, U- and C-cyclic p in the decreasing order. A significant weakness of activity of hydrolyzing mononucleoside 2', 3'-cyclic phosphates was found as a characteristic of the enzyme and thus under certain conditions, the cyclic phosphates were accumulated almost quantitatively in the digests of yeast RNA and homopolynucleotides with the enzyme.

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Decomposition of major milk protein fractions by milk protease was investigated by determination of liberated tyrosine and by polyacrylamide get electrophoresis (PAE). The rate of liberation of tyrosine from unfractionated casein, α_s-casein and κ-casein was similar, but that from β-casein was about half of that from other caseins. However, β-casein showed drastic changes in PAE pattern. Two of the decomposed products of β-casein seemed to have the equal mobility to temperature-sensitive and R-(or γ-) casein, respectively. Decomposed products of κ-casein gave the pattern similar but not identical to para-κ-casein. No significant change occurred in PAE patterns when unfractionated whey protein, β-lactoglobulin and α-lactalbumin were incubated with milk protease.

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Comparative studies of temperature-sensitive (TS-) and R-caseins with two decomposed products (F-II and III) of β-casein by milk protease were performed. In polyacrylamide gel electrophoresis at pH 9.1 and 3.0, F-II and III had the mobilities which were the same as TS- and R-caseins, respectively. Amino acid composition, molecular weight and sedimentation coefficient of F-II almost coincided with those of TS-casein. F-III and R-casein also appeared to coincide each other in these properties. It is suggested that TS- and R-caseins are possibly the decomposed products of β-casein by milk protease.

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The purified alkaline and neutral proteinases from Aspergillus oryzae dissolved and hydrolyzed soybean proteins to peptides, but produced only small amounts of free amino acids. Peptidases from A. oryzae were found to play an important role in digestion of the peptides formed by the above proteinases to yield free amino acids. Especially, the contribution rate of leucine aminopeptidase II in glutamic acid liberation from soybean proteins was 79.11% under the conditions of the present experiments with the L₁₆-type orthogonal arrangement. A remarkable increase of glutamic acid in the hydrolyzate of the soybean proteins was also exhibited by the action of the above enzyme in the addition tests. On the basis of these results, determinations of activity of some exopeptidases were proposed for better evaluation of shoyu koji.

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Aerobacter aerogenes No. 505 isolated from soil by Uyeda produced L-valine extracellularly by an aerobic shaking culture. Under anaerobic conditions the production of this amino acid was inhibited while lactic acid as well as a small amount of alanine were produced. The changes in ORP during the incubation under both conditions were investigated. When L-valine was the main product under aerobic conditions the ORP showed a constant value (rH 8.0) from 16 to 40 hr after inoculation. But when lactic acid was the main product and alanine was produced as the only amino acid under anaerobic conditions, the ORP drifted to rH 0 (zero). The phenomenon of the conversion of fermentation was shown clearly by the ORP of the culture broth.

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The endpotential of lactic acid fermentation by Rhizopus G-36 was rH 13 to 14 when measured in the presence of trace amounts of redox dye mixtures. Without dyes, the rH was 18 to 22 and this fungal culture was slower in reaching endpotentials than bacterial cultures. It was postulated that the amount of redox substances exhibiting electromotive activity was not sufficient in this culture.
rH value 13 to 14 was not obtained under such conditions that lactic acid was not produced; that is in a medium with higher concentration of the nitrogen source in the presence of Fe²⁺ and Zn²⁺, or in a medium containing acetate in place of glucose as the carbon source.

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Mycelia of Rhizopus G-36 after 36 hr-culture produced lactic acid even in the absence of oxygen. But unexpectedly, the ORP under anaerobic secondary culture was exactly the same as that in the aerobic shaking culture (rH 13.2).
A method for homogenization of the culture without secondary oxidation was improved. The ORP of anaerobically homogenized cultures was rH 11, and was thought to be due to the activities of all redox systems in the mycelium.
The respiration system of this strain was switched from cytochrome system to flavin system at the point of change in KCN-sensitivity. The ORP of this strain may be influenced by respiration through the flavin system.

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Bacillus No. P-4-N isolated from soil produced an alkaline polygalacturonase (APGase) in alkaline media. The characteristic point of this microorganism was especially good growth in alkaline media, and that very poor growth was detected in neutral media such as nutrient broth. An important factor for the production of APGase was the addition of manganese to the medium containing sodium carbonate, which was also another imporant factor. The APGase of Bacillus No. P-4-N was purified by Sephadex G-100 and DEAE-cellulose columns followed by Sephadex G-200 gel filtration. The enzyme was most active at pH 10_??_10.5 and stable pH was about 6. The enzyme was heat stable: about 80% of the activity remained after heating at 80°C for 5 min. Calcium ion was effective to stimulate the activity and to stabilize the enzyme. The molecular weight estimated by Sephadex gel filtration method was 6_??_7×10⁴. The enzyme was completely inactivated by EDTA, but not by urea, NaCl and PCMB. The enzyme split poly, galacturonic acid at random and yielded galacturonic acid, digalacturonic acid and higher oligogalacturonic acids. If the enzyme is a single entity, it is a type of liquefying APGase.

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The metabolism of an organophosphorus fungicide, Kitazin P^® (S-benzyl O, O-diisopropyl phosphorothioate) by mycelia of Pyricularia oryzae, the rice blast fungus, was studied by using ³²P- and ³⁵S-doubled-labeled, ³⁵S-labeled and non-labeled compounds.
The mycelia incorporated the fungicide from the medium, metabolized most of it to water-soluble metabolites and released them into the medium. The main metabolite from Kitazin P was identefied as O, O-diisopropyl hydrogen phosphorothioate by ion-exchange chromatographic separation of double-labeled metabolites. S-Benzyl O-isopropyl hydrogen phosphorothioate, diisopropyl hydrogen phosphate and/or isopropyl dihydrogen phosphate were suspected to be minor metabolites. From the toluene-soluble fraction several metabolites were separated by thin-layer chromatography. The most eminent metabolite among them was found to be the m-hydroxy derivative of Kitazin P by thin-layer chromatographic identification of the labeled metabolite and gas-liquid chromatographic identification of the metabolite and its methylated and acetylated products with respective authentic samples. The o- and p-hydroxy derivatives were not found in the metabolites.
No significant difference in the rate and extent of metabolism was found between susceptible and resistant clones against the fungicide, when its concentration was below the growth-inhibitory concentration to the both clones.

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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.

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Experiments were carried out on the effects of substrate or competitive inhibitor on the rate of appearance of N-terminal isoleucine residue of pepsin and peptides released from pepsinogen in its conversion to pepsin. Assumptions were made from these experiments, that an active site is initially formed in pepsinogen by acidification of its solution, and that peptide bond between 41-glutamyl and 42-isoleucyl residues locates in the juxtaposition to the active site forming an intramolecular enzyme-substrate complex. Thus, N-terminal tail of pepsinogen is released by _??_ hydrolysis catalyzed by its own active site.
It was indeed ascertained in this study that neither _??_ small amount of pepsin which could be accompanied by pepsinogen preparation used contributes to the initial step of hydrolysis of pepsinogen nor pepsin formed accelerates the following activation process.
Therefore, it was concluded that the conversion of pepsinogen to pepsin is self-degradation process.

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Gas-liquid chromatographic study revealed that organophosphorus fungicide Inezin^®, (S-benzvl O-ethyl phenylphosphonothioate) was metabolized to O-ethyl hydrogen phenylphosphonothioate or ethyl hydrogen phenylphosphonate by mycelial cells of Pyricularia oryzae. Metabolic fate of the removed benzyl or benzylthio group was further studied by labeling benzene ring of benzyl radical of the fungicide with ¹⁴C, and dibenzyl disulfide, benzvl alcohol, toluene-α-sulfonic acid and benzoic acid were found as metabolites by ion exchange chromatography and thin-layer chromatography. Gas-liquid chromatographic study also ascertained that a part of Inezin was hydroxylated at m-position of the benzene ring of benzvl radical, but o- or p-hydroxylation of benzyl radical was not seemed to occur.
No significant difference was found in metabolism of the fungicide between susceptible and resistant clones of the fungi.

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The structure-activity relationships of 1-phenylpyrrolidine-2, 5-dione derivatives were investigated on Sclerotinia sclerotiorum by the agar dilution method. In addition, several representative compounds were tested for antimicrobial spectra in vitro with 15 pathogenic microbes and for foliage protection activity in green house tests with rice blast, rice brown spot, rice sheath blight and kidney bean stem rot. It was found that 3, 5-dihalo-substituents on the benzene moiety are essential to high antifungal activity against Sclerotinia sclerotiorum. Generally, 1-(3', 5'-dihalophenyl) pyrrolidine-2, 5-diones are active against Corticiaceae, Dematiaceae, Pleosporaceae and Sclerotiniaceae, especially active against Sclerotinia sclerotiorum and Botrytis cinerea (the conidia form of Sclerotinia fuckeliana). N-(3, 5-Dichlorophenyl) itaeonimide showed _??_ peculiarly broad antimicrobial spectrum. In green house tests, these compounds showed high activity against rice brown spot, rice sheath blight and kidney bean stem rot. Results of green house tests on the above-mentioned diseases correlate fairly well with those of in vitro tests.

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The pathway of the leaf alcohol reaction, in which n-hexen-1-ols were converted to 2-propyl-5-ethylbenzylalcohol by refluxing with sodium at 160°C has been confirmed by the tracer technique. First, 2-trans-hexen-1-ol-1-¹⁴C and -5-¹⁴C were synthesized, then the labeled alcohols were subjected to the leaf alcohol reaction. The 2-propyl-5-ethylbenzyl alcohol-¹⁴C obtained was led to suitable degradation compounds.
By the radioassay of the starting, condensed and degradative compounds, the ratios of radioactivity among these compounds were determined. Results demonstrated that the C-1 and C-3 of one molecule of 2-hexen-1-ol respectively combined with the C-4 and C-2 of another molecule of the compound to be converted to 2-propyl-5-ethylbenzyl alcohol.

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