Bacteria degrading nicotine-N'-oxide were isolated from tobacco leaves and soil of tobacco field. The bacterial metabolites from nicotine-N'-oxide were identified to be N'-methylmyosmine and 4'-keto-4'-(3-pyridyl)butylic acid, and four unidentified compounds were present. Accumulation of N'-methylmyosmine in the cultured medium reached 43%, in quantity of the initial substrate on the third day of culture. On the other hand, nicotine was far more slowly degraded by the same bacteria, and the first metabolite was identified to be 6-hydroxynicotine and the second to be 6-hydroxy-N'-methylmyosmine, suggesting that nicotine and its oxide are metabolized in different pathways.

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The purified red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 has a potent saccharifying activity against cell walls, but the living cell lytic activity of it is considerably lower than that of the culture filtrate. Therefore, the living cell lytic factors in the culture filtrate were examined. The alkaline protease of Pen. lilacinum played an important role for living cell lysis. The synergistic effect on living cell lysis was also detected, when acid proteases from various origins were combined with the cell wall lytic enzyme. These results indicated that the protein layers of red yeast cell surface inhibited the action of a glycanase, cell wall lytic enzyme, and the protein molecule contributed to retain the rigid structure of the wall.

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The removal of phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) was efficiently effected by inoculating and retaining viable cells of Nocardia erythropolis, a bacterium known capable of rapidly degrading phthalate esters, in soil column. When an influent containing 3000 ppm of DEHP was passed through a column inoculated with Nocardia erythropolis, the eluent from the column was gas-chromatographically free of DEHP after 1 day. Residual DEHP on the support after 32 days in the column inoculated with Nocardia erythropolis was only 0.14% against the total amount of DEHP fed, whereas it was 5.2% in the uninoculated column. Microorganisms capable of utilizing DEHP were isolated from the inoculated and uninoculated columns after 32 days operation and identified. The DEHP utilizing microorganisms in the inoculated column were found to belong to Nocardia erythropolis, Nocardia restricta and Pseudomonas putida (Biotype B), and those in the uninoculated column to Nocardia erythropolis, Pseudomonas putida (Biotype A and B) and Pseudomonas acidovorans. In particular, strain I-1 of Nocardia erythropolis isolated from the inoculated column was morphologically and biochemically identical with the inoculated Nocardia erythropolis S-1. Ratio of all Nocardia erythropolis to total cells recovered increased from 10.8% immediately after inoculation to 27.2% after 32 days in inoculated column.

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Cytoplasmic and wall-bound saccharases were prepared from the seedlings of sugar beet, and adsorption-release characteristics of the enzymes were compared by the use of a partially purified wall. Adsorption of the bound enzyme occurred below 0.25 M in NaCl concentration and was unaffected by changes in pH, while the cytoplasmic enzyme was adsorbed only in acidic media of pH 3_??_4. Release of re-adsorbed enzymes with NaCl depended on its concentration, and the cytoplasmic enzyme was liberated more readily than the bound type, giving their maximum values at 0.2M and 0.8M NaCl, respectively. Thus, the two saccharases showed merked differences in adsorption to and release from the wall. Adsorption of the cytoplasmic enzyme to the wall was reversible with changes in pH of the medium.
The results suggested that the wall-bound saccharase was essentially different from the cytoplasmic enzyme and consequently not an artifact.

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Association of the cell wall-bound saccharase of sugar beet seedlings with structural polysaccharides of the wall was investigated.
After sequential extraction with boiling water, 0.5% EDTA and 24% KOH from the wall of sugar beet, the polysaccharides were prepared as pectic substance I and II, and hemicellulose.Solubilization of the enzyme in the presence of the polysaccharides and the profiles on gel filtration of the enzyme with the polysaccharide suggested the formation of an enzyme-polysaccharide complex. The complex formation completely occurred at NaCl concentration below 0.2M, partly at 0.3M and only little at 0.4M. Among three types of wall polysaccharides used, hemicellulose had the highest affinity with the enzyme, while uronic acid content was the lowest. The reverse relation was observed with pectic substance I.
It was also observed by gel filtration that the polysaccharides used were associated in aqueous solution and dissociated in salt solution depending on its concentration. The enzyme was considered to form a complex with the associated molecule of the polysaccharide.

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A convenient procedure to assay the repellent activities of organic and inorganic substances against a sea snail Monodonta neritoides has been developed. By the use of this procedure, 8 authentic compounds and the benzene extracts of 11 species of plants were found to show such repellent activities.

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The 5/7 membered ring system of 6-dehydro derivatives (2, 3, 5 and 6) of grayanotoxin-II (1) was converted to a 6/6 membered ring system (8a and 8b) in consequence of an acyloin rearrangement in alkaline media. The configurations of C3 and C6 of the products (8a and 8b) were determined.

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The effect of L-leucine on L-lysine production was investigated in Brevibacterium lacto-fermentum.
L-Lysine production was inhibited by L-leucine specifically, and this inhibition was recovered completely by L-valine. The present study indicated that the inhibition of L-lysine production by the addition of excess L-leucine was accompanied with the repression of dihydrodipicolinate (DDP) synthetase. DDP synthetase was the first specific enzyme in lysine biosynthesis.
On the other hand, α-isopropylmalate (IPM) synthetase, which was the key enzyme and the first specific enzyme in leucine biosynthesis was inhibited by L-leucine, while L-lysine caused the complete reversion from its inhibition. From the above results, the possible physiological meanings of this coregulation mechanism of lysine and branched-chain amino acids biosynthesis in B. lactofermentum was discussed.

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The synthesis of dl-7-Methyldemethylvariotin with a less antibiotic potency than variotin is described.

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Chemical structure I was proposed for the tannic substance chestanin (MP-3) isolated from chestnut galls, on the basis of spectral data and enzymatic hydrolysis products.

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Five alkaloids were isolated from ripe fruits of Evodia rutaecarpa HOOK. f. et THOMS. Four of these were found to be indoloquinazoline type alkaloids: rutecarpine (I), evodiamine (II) and two new alkaloids, dihydrorutecarpine (IV) and its 14-formyl derivative (III). The remaining one was a new quinolone alkaloid, 1-methyl-2-nonyl-4(IH)-quinolone (V). The synthesis of II, III and IV was also achieved.

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Optical resolution of DL p-hydroxyphenylglycine was studied in order to develop a practi-cal method for the production of D-p-hydroxyphenylglycine useful as a starting material for preparing semisynthetic penicillin and cephalosporins. Among the various aromatic sul-fonates of DL p-hydroxyphenylglycine, the benzenesulfonate, the o-toluenesulfonate, the p-toluenesulfonate, the p-ethylbenzenesulfonate, the sulfosalicylate, and the 2-naphthol-6-sulfonate were easily resolved by a preferential crystallization procedure. The optically pure sulfonates of D-p-hydroxyphenylglycine were obtained in good yield and were easily con-verted to optically pure D-p-hydroxyphenylglycine without racemization. The undesired sulfonates of the L-isomer were readily racemized and could be reused for the resolution step. Industrial production of D-p-hydroxyphenylglycine by the present simple resolution method is considered to be very promising.

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Dichloromethane extracts of lamina and midrib cigarette smoke condensates were sub-jected to glass capillary column gas chromatography. Most peaks in the gas chromatogram were identified and the major components were quantitatively determined. The compositional differences between the two types of smoke were then compared. In midrib cigarette smoke the major components appear to be derived from cell wall substances by pyrolysis while lamina cigarette smoke consists of many sugar degradation products and essential oil com-ponents which would be directly transferred from tobacco.

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During the course of phosphorylation of adenosine and AMP by acetone dried cells of yeast, enzymes in related to glycolysis and adenosine kinase as well as adenylate kinase were found to be released into the supernatant of the reaction mixture. The supernatant effectively phosphorylated adenosine and AMP at high phosphate concentrations utilizing glucose as an energy source, and the ATP accumulated was maintained at a high level still after 24 hr of incubation. A continuous reaction apparatus for regeneration of ATP equipped with a semi-permeable membrane was designed and constructed taking into account the specific characteristic of the present enzyme reaction systems. The released enzymes were applied to the apparatus equipped with semi-permeable membranes, and continuous phosphorylation of adenosine to ATP was examined under different operational conditions. When the resi-dence time was 18 hr, 80% of the initial adenosine was continuously phosphorylated to ATP by this apparatus, in which the collodion membrane was used as a semi-permeable membrane.

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Streptomyces griseoincarnatus strain No. KTo-250 produced three kinds of inhibitors designated as API-2a, b and c. Among them, API-2b was purified previously.¹⁾ API-2c, produced rather in late phase of culture, was purified by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on Sephadex G-100.
From the result of amino acid analysis, it was demonstrated that API-2b and c have an isoleucine residue in their molecules. Furthermore, both inhibitors differed from other microbial alkaline protease inhibitors in chemical and physico-chemical properties.
API-2b was converted to c by washed mycelium of the strain, and from the examination of the terminal amino acid, some amino acid residues from the amino-terminal part in API-2b are thought to be removed to form API-2c.

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Two series of synthetic polymers (or oligomers), one is polyethylene and the other is nylon, were subjected to microbial degradation, and several assimilating bacteria of each substrate were obtained. In some cases, such as parafl'ine wax (PW) assimilating bacteria, lower homologues, i.e., PW, seemed to induce assimilating ability against higher homologues, i.e., polyethylene (PE), but not the case in polyethylene glycol (PEG) assimilating bacteria. PEG and 6-nylon oligomer assimilability of some strains were lost by four serial transfers on nutrient agar, but it was not ascertained that the assimilating ability was plasmid borne. Representative PEG-400 assimilating bacteria, which may fall into Alcaligenes or Pseudomonas, effectively and inducibly degraded PEG under aerobic condition. From the analysis of structures of two main degraded products derived from a model substrate, tri-ethylene glycol, it is supposed that PEG may be degraded oxidatively by splitting C₂ unit from an alcohol terminal which is at first oxidized to carboxyl.

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Complete isolation of Fraction I protein from alfalfa leaves was achieved by a combi-nation of ammonium sulfate fractionation and gel filtration. Analytical ultracentrifugation gave a s°₂₀, _w, W value of 18.0. Judging from the CD spectrum the protein contains a large amount of β-form as well as tobacco F-I protein. Electron micrographs showed closely similar appearances for the two F-I proteins. The F-1 protein (from alfalfa leaves) was separated to large subunits (53, 000 daltons) and small subunits (14, 000 daltons) on SDS gel electro-phoresis. Further the amino acid composition of the large subunit was found similar to those of tobacco and spinach, but considerably different from them in small subunits.

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A fluorometric manual technique has been demonstrated for determining the amino acid sequence of a polypeptide, by the combined use of fluoresceinisothiocyanate (FITC) and phenylisothiocyanate (PITC). This method permits continuous assignments of 10 residues with 5.6 nmoles of myoglobin or 23 residues with 45 nmoles.

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A reaction of α-isocyanoacetic acid amides (Ib-g) with acetaldehyde was carried out to develop a useful method for the synthesis of threonine. The reaction in the presence of potassium hydroxide in methanol afforded predominantly trans-5-methyl-4-N'-substituted-aminocarbonyl-2-oxazoline (IIIb-f), which was easily hydrolyzed to DL-threonine, in a high yield. This method was extended to the synthesis of threo-β-hydroxynorvaline and threo-β-hydroxyleucine in excellent yields.

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Two new gibberellins A₅₀ and A₅₂ were isolated from seeds of Lagenaria leucantha Rusby var. clavata Makino. Their structures were shown to be ent-2α, 3α, 10, 11α-tetrahydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone (1) and ent-2α, 3α, 11β, 20-tetrahydroxy-gibberell-16-ene-7, 19-dioic acid 19, 20-lactone (10), respectively.

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The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B-11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and co-lumn chromatographies on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (S₂₀, _w=3.2).

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α-Methylene γ- and δ-lactones can be prepared in fair yields by a two step procedure involving formation of the a-hydroxymethylene or α-ethoxyoxalyl sodio derivatives of γ- or δ-lactones followed by their condensation with formaldehyde.

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