Bacillus circulans WL 12 was grown on cells of six species of yeasts as inducer substrates, and the courses of progress of β-glucanase production were compared. Also, the isoelectric focusing patterns of β-1, 3 and β-1, 6 glucanases were determined. Conspicuous differences were observed among the isoelectric focusing patterns of the crude enzymes produced with different yeast species as inducer substrates. Lyses of the cell walls of Pyricularia oryzae P₂ and several species of yeasts by the enzyme systems were compared. The lysis of the cell walls of Debaryomyces hansenii was stronger in the order of the complexity (number of major peaks etc.) of the β-1, 3 glucanases in isoelectric focusing patterns.

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A partially purified tannin was prepared by a K₂HPO₄-precipitation method from immature persimmon fruit (Diospyros kaki L., cv. “Hiratanenashi, ” astringent type). The kaki-tannin gave deiphinidin and cyanidin on acid hydrolysis. Its methylated derivative showed v_max 1720cm^-1 in IR spectrum and had the molecular weight of ca. 1.38×10⁴ daltons in Mw. From the results of toluene-α-thiol treatment, it is suggested that the kaki-tannin consists of catechin, catechin-3-gallate, gallocatechin, gallocatechin-3-gallate and an unknown terminal residue, and belongs to proanthocyanidin B group with a carbon-carbon interfiavan linkage from C-4 of one unit to C-6 or C-8 of another unit.

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For the purpose of making clear the macromolecular chemical properties of konjac gluco-mannan, the light scattering and viscosity measurements were carried out for the aqueous solutions of the partially methylated derivatives, and following results were obtained: 1) The weight-average molecular weight, Mw, and the root mean square of radius of gyration, <S²>^1/2 were 100×10⁴_??_120×10⁴ and 1100_??_1300 Å, respectively. 2) The <S²> versus Mw relationship was represented by the equation of <S²>=4.20×10^-1•Mw^1.08, and this fact suggested a random coil as a molecular form in the solution. 3) The intrinsic viscosity, [η], versus Mw relationship was represented by the equation of [η]=6.37×10^-4•Mw^0.74, which was considered to be useful for the purpose of estimating the molecular weight of konjac gluco-mannan by viscometric method.

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Eight oligosaccharides were isolated from an enzymatic hydrolyzate of cell walls of Rh. glutinis K-24 with red yeast cell wall lytic enzyme of Penicillium lilacinum. Two of these oligosaccharides were composed of mannose only and the others were composed of mannose and glucose. Linkage type between mannoses in the oligosaccharides was mainly β-1-4 bond. All of the linkages between glucose and mannose were β-1-6 bonds. The red yeast cell wall lytic enzyme hydrolyzed β-1-3 mannoside bond. Thus there might be a considerable amounts of β-1-3 mannoside bonds in cell walls. From these results, it might be concluded that main component of cell walls of red yeast was a β-glucomannan and the lytic enzyme from Pen. lilacinum was a glucomannase.

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Fibroin membrane was used as a support for immobilized β-glucosidase. The immobilized enzyme was prepared by drying fibroin-enzyme solution on a horizontal plate, followed by ethyl alcohol treatment which was an essential process for immobilization. The immobilized β-glucosidase was characterized enzymatically compared with soluble enzyme, using p-nitrophenyl-β-D-glucopyranoside as a substrate. The immobilized enzyme showed 47% of the activity of soluble enzyme and little decrease of the activity was observed both on re-use and storage. There were no significant differences in pH dependency between immobilized and soluble enzyme activity. Activation energy was slightly larger with immobilized enzyme than soluble enzyme. The enzyme was considerably enhanced by immobilization in stabilities against heating, electrodialysis and protease treatment. Apparent affinity for the substrate decreased as membrane thickness increased. Enzyme affinity for substrate in the membrane was discussed.

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More detailed study on the reactivities of non-protein substances in a modified Lowry procedure using chloramine-T (CAT) has been performed. Many compounds, which hardly showed the color by the original procedure, became to react with the phenol reagent in the modification which used CAT in trichloroacetic acid (TCA) solution. However, the color yields obtained by this modification of certain of these compounds were markedly or considerably repressed when one of the sulfhydryl (SH) compounds coexisted in the reaction mixture. Moreover, the absorbances of phenols, tryptophan, tryptamine, several purine bases, and penicillin G which give the deep color by the original procedure were greatly or partially decreased by this modification.
On the other hand, in almost all the tested compounds, the absorbances obtained by the other modification which used CAT under the alkaline pH were not appreciably higher than those obtained by the original procedure.

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The formation of D-pantothenic acid-α-glucoside (PaA-α-G) was found from D-panto-thenic acid (PaA) and maltose in incubation mixtures of microorganisms, especially Saccharomyces yeasts and Sporobolomyces coralliformis IFO 1032. The reaction conditions were investigated for formation of PaA-α-G by resting cells of Spor. coralliformis. The formation of the compound increased with PaA concentration (3_??_20mg/ml). The yield was maximum at 5_??_10mg/ml of PaA. Cetyl trimethyl ammonium bromide (0.1%) promoted the formation of PaA-α-G. Sucrose was the optimal α-glucosyl donor. When 30mg/ml of sucrose was fed to the reaction mixture (initial sucrose, 100mg/ml; and PaA, 10mg/ml) at 12-hr intervals, 5.74mg/ml (3.30mg/ml as PaA) of PaA-α-G was formed in 48-hr incubation at 28°C with shaking. PaA-α-G was also formed by yeast α-glucosidase, mold maltase and the cellfree extract of Spar. coralliformis. The compound showed approximately 9_??_10% and 0.1_??_0.3% (molar ratio) of activity of PaA for Saccharomyces carlsbergensis ATCC 9080 and Lactobacillus plautarum ATCC 8014, respectively. The compound had the same microbiological activity as authentic 4'-O-(α-D-glucopyranosyl)-D-pantothenic acid.

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An α-amylase [α-1, 4-glucan 4-glucanohydrolase, EC 3.2.1.1.], found in the culture filtrate of a strain of Thermoactinomyces vulgaris, was purified by ammonium sulfate fractionation, and DEAE-cellulose and CM-cellulose chromatographies. The purified enzyme showed a single band on disc gel electrophoresis. The optimum reaction pH and temperature were determined to be around pH 5.0 and 70°C. The isoelectric point was determined to be pH 5.2. The α-amylase was stabilized by Ca²⁺.
The α-amylase was found to hydrolyze pullulan to panose. Therefore, the hydrolytic pattern of this enzyme is different from those of pullulanase and isopullulanase.

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The growth of Gluconobacter suboxydans was supported by addition of NH₄Cl as sole nitrogen source. Glutamine synthetase and glutamate synthase activities were revealed in the cell-free extracts of NH₄Cl-grown cells. The two enzymes were separated and partially purified about 120 and 200 fold, respectively.
The optimum pH of glutamine synthetase was 8.0. The Km values for L-glutamine, ammonia and ATP were 6.7mM, 3.3mM and 0.56mM, respectively. Mg²⁺ was indispensable for the activity. The enzyme activity was inhibited by glycine, L-alanine, L-hisdidine, ADP and AMP.
Although glutamate synthase was strikingly unstable, it was reactivated 2_??_3 fold by addition of dithiothreitol. The optimum pH was 8.5. Only L-glutamine, a-ketoglutarate (α-KGA) and NADPH were the specific substrates and coenzyme. Their Km values were 0.71mM, 2.2μM and 20μM, respectively. The enzyme activity was inhibited by various kinds of amino acids and α-keto acids or NADH.
The effects of culture conditions on the enzyme activities were also investigated.

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The protein which is antigenically equivalent to the soluble glycoprotein (SGP) isolated from milk fat globule membrane was immunologically found to be contained in whey protein. The identical antigeneicity between the SGP and an unknown protein component of whey was certified by double immunodiffusion and immunoelectrophoretic assays, using the antiserum of the SGP and its antiserum absorbed with whey protein, and using the whey protein fractions fractionated by gel filtration or ion exchange chromatography.
The concentration of this protein in whey (anti-SGP reacting protein) was estimated to be about 15% of total whey protein by simple radial immunodiffusion assay.
The major whey protein components, such as β-lactoglobulin, α-lactalbumin, bovine serum albumin, and IgG-immunoglobulin were confirmed to be not the anti-SGP reacting protein by the indirect elimination method. Anti-SGP reacting protein was found to be rich in the heat-stable protein fraction which corresponds to the so-called proteose-peptone fraction.

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Glucoamylase (from Rhizopus niveus) was immobilized to SP-Sephadex C-25 and C-50 by ionic linkage. The kinetic and physical properties of the immobilized glucoamylase were quantitatively investigated using maltose as substrate. The change of kinetic parameters, k₀, and Km, due to immobilization was explained in terms of decrease of pH in the ion exchanger, which was estimated from Donnan's equilibrium. Binding equilibrium between the enzyme and the ion exchanger was investigated. A linear relationship between the concentrations of enzymes in the ion exchanger and in the outer solution phase was observed over a wide range of enzyme concentrations. Distribution coefficients were obtained, by which the amount of the enzyme immobilized can be calculated. Diffusion coefficients of the enzyme in the ion exchanges were also estimated from analysis of the adsorption process. A theoreti-cal equation to predict the half-life of the activity of immobilized enzymes caused by desorption of the enzymes was proposed in terms of the distribution coefficient, the diffusion coefficient and the operational variables of a continuous reaction.

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The steam-volatile constituents of fresh radish of Japanese and Kenyan origin have been studied by GC-MS technique. The over-all pattern of compounds in the two materials was similar. Ten mustard oils, of which pentyl, hexyl, and 4-methylpentyl isothiocyanate have not previously been reported as products of natural derivation, two related nitriles, dimethyl disulfide, methyl methanethiolsulfinate and 1-methylthio-3-pentanone, a novel sulfide-ketone, together constitute the major volatile sulfur products in the two radish materials. A few nonsulfur volatiles have also been identified. The diversity in chemical structure of the sulfur constituents becomes less surprising when regarded in terms of their biogenetic origin.

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A number of new 5-/6-fluoro-2-substitutedaryl-3-indolylglyoxamides and their corresponding tryptamines have been synthesized. 5-/6-Fluoro-2-arylindoles, prepared by Fischer indole synthesis on treatment with oxalyl chloride and subsequent reaction with secondary amines, gave 5-/6-fluoro-2-aryl-3-indolylglyoxamides. The indolylglyoxamides were reduced with lithium aluminium hydride to yield corresponding tryptamines. All the synthesized compounds have been characterized by IR, PMR and ¹⁹F NMR spectral studies. CNS activity of some representative compounds has also been evaluated.

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Amadori compounds, one of important factors contributing to oxidative browning of soy sauce, were isolated and identified from miso, white wine and Saké by ion exchange chro-matography, gel filtration and partition chromatography. Fructose-leucine, fructose-isoleucine, fructose-valine and fructose-alanine were present in miso, white wine and Saké. Fructose-glycine was identified in miso and Saké, and fructose-proline was found in white wine. Amino-carbonyl reaction was suggested to be involved in oxidative browning of miso, white wine and Saké.

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Nicotine biosynthesis in tobacco callus tissues was investigated by comparing high nicotine producing strain OMT-53 with scarcely producing strain NT-5. NT-5 strain which had been subcultured for about 5 years with a medium containing 1.0 ppm 2, 4-D, produced very little amount of nicotine even after more than 47 subcultures on OT-23 medium containing 0.15ppm α-NAA. On the other hand, OMT-53 strain maintained a constant high level nicotine producing (ca. 2% or more on D. W. basis) capacity over 50 successive cultures on the same medium. The growth response of NT-S callus tissues to α-NAA concentration was quite different from that of OMT-53. Nicotine production assumed more distinctive patterns with these two strains. NT-5 strain never produced nicotine at any concentrations of α-NAA examined, whilst OMT-53 strain produced abundant amount of nicotine within a narrow range of α-NAA concentration having the optimal value of 0.15ppm. Nicotine supplemented to culture medium could not be decomposed either with NT-S or with OMT-53 callus tissues. The nicotine production by OMT-53 was not affected at moderate level (1mM) of supplemented nicotine, but at a very high level (5mM) of the supplemented nicotine, the nicotine production seemed to have been affected considerably. Some precursors supplemented to NT-5 callus cultures did not stimulate the nicotine biosynthesis at all, even though slightly positive effect was recorded with OMT-53 callus cultures. NT-5 callus tissues had much smaller pool size of total free amino acids than OMT-53. The ratio of each amino acid in total free amino acids was not so different between both callus tissues. On the basis of these results, some discussions were made on nicotine biosynthesis in tobacco tissue cultures.

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Leucine dehydrogenase was inhibited by p-chloromercuribenzoate and HgCl₂, but not by 5, 5'-dithiobis (2-nitrobenzoic acid), 4, 4'-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl₂ was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12sulfhydryl groups were titrated per molecule in the presence of 8M urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.
These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group.

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Reactivation and aging of α-chymotrypsin inhibited by organophosphorus compounds including delayed-neurotoxic and non-neurotoxic agents were studied. Delayed-neurotoxic o-tolyloxy and phenyl analogs of salioxon, and (-)-EPN-oxon reacted stoichiometrically with α-chymotrypsin to inhibit its esterase activity, whereas non-neurotoxic salioxon and (+)-EPN-oxon inhibited the enzyme less specifically as compared with the former three compounds.
The enzyme inhibited by EPN-oxon isomers was slowly reactivated and completely dephosphorylated with about 300 molar equivalents of PAM to regenerate the active enzyme. On the other hand, the enzyme inhibited by saligenin cyclic phosphoryl compounds was not reactivated even by the action of PAM. The phosphorylated enzyme underwent rapid cleavage of the P-O-salicyl linkage in processes referred to as aging, yielding an inactive enzyme with an ionized acidic phosphorus group. There seems to be no differences in reactivation and aging processes of the inhibited α-chymotrypsin between delayed-neurotoxic and non-neurotoxic compounds.

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Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyl peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsin-inhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G-50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.
Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.

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A peptide fraction with a delicious taste was isolated from the gravy of beef meat. By successive application of gel filtration with Sephadex G-25, chromatography on an ion exchange resin (Dowex 50×4), and paper electrophoresis, the peptide was purified. The amino acid sequence of the purified peptide was determined by Edman degradation for N-terminal and Cpase A method for C-terminal sequence. As a result, the primary structure of the peptide was proposed as follows; H-Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala-OH.

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A variety of 2, 3-dihydrobenzo [b] furan-3-yl and 2, 3-dihydrobenzo[b]thiophen-3-yl chrysanthemates were prepared and their insecticidal activity was tested on the American cockroach. Of these chrysanthemates, 7-methyl, 7-chloro and 5-chloro-4, 6-dimethyl-2, 3-dihydrobenzo-furanyl esters (9c, 11c and 18c) were much more potent than allethrin. Substituents on the 7-position of either the benzofuran or benzothiophen nucleus generally enhanced the activity compared with those on other positions.

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Relationships between L-lysine productivity and the formation of alanine in Brevibacterium lactofermentum were investigated.
Alanine can be formed from pyruvate by transaminase with L-amino acid, and directly from aspartate in the presence of α-ketoglutarate and L-leucine. The later suggests that aspartate β-decarboxylase may contribute to the formation of L-alanine.
However, this activity is approximately 1/20_??_1/50 of transaminase activity. Productivity of L-lysine was inversely as the level of pyruvate-L-amino acid transaminase. It was cofirmed by the derivation of alanine auxotrophs from AJ3445 (AEC^r). The best L-lysine producer, AJ3799, accumulated 39mg/ml of L-lysine and lacked pyruvate-L-amino acid transaminase.

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