When a solution of soybean acid-precipitated or IIS protein was frozen and stored at -1 to -5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble com-ponents revealed that new components which may be aggregates or associates of the I1S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 1IS component. NEM-IIS was not insolubilized by frozen storage at -5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at -1 to -3°C.

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Protease from a strain of Serratia contained one gram atom of zinc ion per mole and the zinc ion was essential for the activity. Also zinc-free apoenzyme was isolated as a crystalline form from the native-enzyme. Several metalloenzymes were prepared by the addition of corresponding metal ions to the apoenzyme. Studies on activities toward the hydrolysis of casein showed that relative activities of native- (zinc), cobalt- and manganese-enzyme were 1.0, 1.2 and 0.8, respectively. Toward the hydrolysis of hippurylleucinamide, however, specific activity of cobalt-enzyme was about 10 times that of the native- (zinc-) enzyme. Spectroscopic studies did not reveal any significant differences in conformations among native-enzyme, apoenzyme and the other metalloenzymes.

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A new enzyme has been obtained in a crystalline state from the muscle of blue white dolphin. This enzyme resembles to methemoglobin reductase from erythrocyte with respect to (a) elution pattern of DEAE-Sephadex column chromatography, (b) absorption spectra, (c) molecular weight and (d) activity of reducing methemoglobin, metmyoglobin and ferric cytochrome c. However, distinct differences can be observed between two enzymes with regard to (a) sedimentation coefficient, (b) diffusion coefficient, (c) frictional ratio, (d) pH-mobility curve and (e) specific activity of reducing the above three substrates. It is advocated that enzyme is termed metmyoglobin reductase.

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Chemical screening of some profusely growing wild leguminous seeds of Acacia, Albizzia and Leucaena species, for their nutritional constituents suggested the possibility of their inclusion in animal nutrition. However, since the whole seeds proved to be unpalatable as well as toxic in some cases, it was thought of utility to employ their isolated proteins instead. Accordingly, with a view to explore newer and effective sources of dietary pro-teins, simple and effective solubility method was employed for their extraction, precipita-tion, fractionation and isolation from the undesirable wild seed constituents to obtain the protein fractions in the pure form which could be incorporated in animal nutrition. Dis-tribution of total nitrogen and dispersion of nitrogenous constituents in the seeds have also been studied. Extraction of proteins of considerable purity is best effected with sodium chloride solution (5%, w/v) and subsequent dialysis of the extract. Proteins were isolated from seven varieties of leguminous seeds by acid-alkali extraction method and their protein efficiency ratio and the biological value have been evaluated by animal experiments with and without supplementing the isolated protein diets with the missing essential amino acids.

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The accumulation of vitamin B₂ by Pichia guilliermondii Wickerham grown on hydro-carbon was investigated. Addition of the following materials stimulated vitamin B₂ pro-duction: metal ions such as ferrous, cobalt, manganese, and calcium ions; organic nutrients such as yeast extract and casamino acid; amino acids such as proline and arginine; vitamins such as B₁, nicotinic acid, inositol, and p-aminobenzoic acid. Optimal aeration rate for vitamin B₂ production was obtained in a 500-ml shaking flask containing 75ml of the medium.

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Sulfite reductase using reduced methyl viologen as an electron donor was purified about 94-fold from a red alga, Porphyra yezoensis. The enzyme was ultracentrifugically homo-genous and could reduce sulfite to sulfide quantitatively with an uptake of six electrons. The enzyme had a pH optimum in the vicinity of 7.5. The Km for sulfite was determined to be 6.5×10^-4M. The purified preparation of the algal reductase showed its absorption maximum at 385mμ and slight shoulders at 408, 456, 485, 600 and 664mμ in addition to an intense peak at 278mμ. Metal analysis of the purified enzyme suggested the presence of iron and copper in the molecule. NADPH, NADH or the reduced form of spinach ferredoxin could not be a direct electron donor for the purified algal sulfite reductase.

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The crude enzyme preparation obtained by isopropanol-precipitation of a water-extract of wheat bran culture of Trichoderma viride was first fractionated with ammonium sulfate precipitation, followed by DEAE-Sephadex A-25 column chromatography. Two active fractions, F-1 and F-4, were obtained. Fraction F-4 was futher purified by column chromatography with CM-Sephadex C-50 and was finally crystallized by the addition of ammoniun sulfate to 35% saturation. This purified xylanase A was homogeneous as judged by ultracentrifugation and discelectrophoresis. Maximum conditions for the activity of crystalline xylanase were pH 3.5 and 50°C. The enzyme was stable in a concentrated solution below 40°C and between pH 2 and 7, but at low enzyme concentration, i. e. 9.7μg per ml, it was easily inactivated under these same conditions.

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During the course of studies on the effects of mutation in carbohydrate metabolism on the synthesis of purine derivatives, it was found that three mutants of a Bacillus species, which lacked transketolase or D-ribulose 5-phosphate 3-epimerase, accumulated a large amount of D-ribose in the culture medium. The amount of D-ribose was about 35mg per ml of the broth incubated for 6 days. D-Ribose in the broth was purified in crystalline form and was identified from its chemical and physical properties.

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During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5'-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8mg/ml) from uracil and 10000 (4.3mg/ml) from orotic acid when each substrate was added at the concentration of 2mg/ml.
Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5'-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C.4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumu-lation (3.6mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.
UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.

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The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.
The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphos-phates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the in-hibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.
N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloro-mercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.
The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.

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Complexes of amylose with several kinds of n-aliphatic ketones having different chain lengths, different positions and numbers of carbonyl groups in the molecules were prepared. The unit cell dimensions of the complexes were calculated in both the wet and dried states by means of X-ray diffraction analysis. Both the 6₁- and 7₁-helix amyloses were presented in these complexes. It was found that the helix packing diameter of the amylose-ketone complex changes depending upon the linear chain length of the ketone molecule complexed.

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To study the influence of cultural conditions on higher plant cells in suspension culture, the effects of nutritional conditions on the growth of suspended cells were investigated. Calluses were induced from 39 species of Nicotiana plants and 6 species of Populus plants on agar slant media, then these were transferred to suspension cultures. Concentrations of 2, 4-D and kinetin suitable for incubation of callus from each plant were investigated and species having high growth rates in the appropriate medium were selected.
The effects of concentrations of auxins and kinetin, a variety of carbon and nitrogen sources, thiamin and myo-inositol on growth of the selected calluses were also studied. Of these calluses studied, N. glutinosa, N. tabacum var. Xanthi ova and P. hybrids were selected as calluses having high growth rates. Myo-inositol had no effect on any callus growth, and thiamin gave a distinct effect on Populus callus only. Nitrate as a nitrogen and sucrose as a carbon sources, and 2, 4-D as an auxin were most effective in all calluses studied. Kinetin was essential for N. glutinosa among the calluses studied. Although high sugar concentrations tended to lengthen the lag period in the growth curve, there was no differ-ence in the growth rates of the logarithmic phase among the concentrations.

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An indole derivative having blue fluorescence was produced in cooked soybean digest-ed at 37°C for 24 hr with an acid proteinase Molsin (optimum pH: 2.8) from Aspergillus saitoi or a usual acid proteinase pepsin (optimum pH: 1.6) from beef stomach. This in-dole derivative was identical with a condensation product from L-tryptophan and n-hexa-nal. Based on MS, NMR, IR and UV spectrometry, the condensation product was identified as 1-pentyl-2, 3, 4, 9-tetrahydro-lH-pyrido [3, 4-b]-indole-3-carboxylic acid [trivial name: 1-pentyl-1, 2, 3, 4-tetrahydro-2-carboline carboxylic acid-(3)].
Data were presented of the formation of the above indole derivative and of the re-sulting consumption of L-tryptophan and n-hexanal.
The possible ocurrence of the formation of Harmala alkaloids, i.e. 2-carboline deriva-tives, through in vitro digestion of soybean with acid proteinases was discussed.
A carbonyl-trapping ability of L-tryptophan was suggested.

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The reduction of p-mentha-1, 3-dien-7-al (II) with sodium in aqueous ammonia afforded trans- and cis-p-menthan-7-ol via 1-p-menthen-7-al (phellandral) and trans- and cis-p-menthan-7-al, respectively, in which the trans forms were obtained predominantly due to the stereo-electronic requirement.

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The mechanism of activation of pepsinogen was studied. It was found that no peptide bond cleavage occurred in the molecule of denatured pepsinogen at pH 2. It was inferred from this that a specific secondary and tertiary structure is formed in the molecule of pepsino-gen in acid and that it might be necessary for the hydrolysis of the peptide bond. From the circular dichroism studies on pepsinogen and pepsin, it was found that there is a con-formational change in the molecule of pepsinogen at pH 4.3_??_4.5 and that this change is followed by a gradual formation of pepsin.

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Two new gibberellins, gibberellins A₂₆ and A₂₇ were isolated from immature seeds of Japanese morning-glory (Pharbitis nil) and their structures were elucidated as I and IX.

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Seven gibberellin glucosides (F-I_??_F-VII) were isolated from immature seeds of Japanese morning-glory (Pharbitis nil). The structures were elucidated as 2-O-β-glucosyl-gibberellin A₃ (F-I), 2-O-β-glucosyl-gibberellenic acid (F-II), 2-O-β-glucosyl-isogibberellin A₃ (F-III), 3-O-β-glucosyl-gibberellin A₂₆ (F-IV), 3-O-β-glucosyl-gibberellin A₂₇ (F-VI) and 3-O-β-glucosyl-gibberellin A₂₉ (F-VII). The one of the glucosides (F-V) was identified as 3-O-β-glucosyl-gibberellin As isolated from Phaseolus multiflorus.

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Various organoleptic characteristics of cigarette smoke, such as aroma, taste, harshness, offensive odor and taste, strength and dryness, were measured by sensory test using more than fifty kinds of flue-cured tobacco leaves. The results were compared with those of chemical and gas chromatographic analyses of the particulate matter in the smoke. The correlations between each evaluation characteristic and each analytical value were computed and simple equations for tobacco evaluation were set up. The equations obtained were considered to be available for the evaluation of tobacco quality instead of the sensory test, although their applications to lowgrade samples seemed to need further modifications.

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The enzymatic decomposition of phenyl mercuric acetate (PMA) to metallic mercury by a mercury-resistant Pseudomonas has been studied. The formation of the system involved in the decomposition was found to be inducible. Glucose dehydrogenase (D-glucose: NAD oxidoreductase), arabinose dehydrogenase (L-arabinose: NADP oxidoreductase), cytochrome c and the “decomposing enzyme” which catalyzes the splitting of the C-Hg linkage, were separated from cell free extract by gel filtration on a column of Sephadex G-150. The cytochrome fraction was further separated into two types, c-I and c-II, by chromatography on a column of CM-Sephadex. Each of these cytochromes showed absorption peaks at 416, 519 and 547mμ fe in the reduced form, but they were different in the molecular weight; cytochrome c-I was estimated to be about 26000, and cytochrome c-II about 14000. The reconstruction of these enzymes demonstrated that a reduced NAD(P) generating system, glucose dehydrogenase or arabinose dehydrogenase, cytochrome c-I and the “decomposing enzyme” were required for the decomposition of PMA. A hypothetical scheme for the decomposition of PMA was proposed and the decomposition mechanism was discussed.

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Tenuazonic acid has been isolated as a halo-inducing toxin from the culture filtrate of Alternaria longipes, a fungus causative of the tobacco brown-spot disease. Further, its occurrence in leaves of diseased plants was confirmed by a spectrometric method. The role of the acid in the host-parasite interaction was elucidated from the phytopathological standpoint.

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