The effect of Streptomyces-pepsin inhibitor (S-PI) on fruit-body formation of Lentinus edodes (Berk.) Sing. was studied. The addition of S-PI to the culture medium(510 μg/ml) shortened the time required for mature fruit-bodies, and increased the fruiting-percentage and the overall yield 3.4 times compared to the control.
The intracellular proteinase in the mycelium was investigated. Proteinases having an optimal pH of 2.7 and 7.0 were found in the vegetative mycelial extract. When S-PI was added to the culture medium, their activities were strikingly changed; the carboxyl proteinase activity was remarkably decreased, and, in the contrary, the metal proteinase activity was increased to 1.5 times that of the control.
The carboxyl proteinase was purified. This enzyme was strongly inhibited by S-PI and synthetic pepsin inhibitors such as DAN and EPNP. The molecular weight and isoelectric point were 43, 000 and pH 3.4, respectively.

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A Streptomyces-pepsin inhibitor (S-PI or Pepstatin Ac), and DAN-insensitive carboxyl proteinase was found in a still culture filtrate of Lentinus edodes. The new carboxyl proteinase was purified, and about 9 mg purified enzyme was obtained from 19 liters of culture filtrate, with 11 % recovery. The enzyme showed a single band on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 40, 000 and pH 4.2, respectively. The enzyme did not contain histidine and was composed of 387 amino acid residues. The enzyme was most active between pH 2.72.9, and stable over a pH of 3.2-5.2 for 3 hr at 37°C. The enzyme was not inhibited by S-PI or synthetic pepsin inhibitors such as DAN and EPNP. The physicochemical and enzymological properties were very similar to those of Scytalidium lignicolum carboxyl proteinase A, which was reported to be an S-PI-, and DAN-insensitive carboxyl proteinase.

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The effects of salts were investigated on thermo-coagulation of bovine serum albumin (BSA). Salts affected the formation of coagulum, and a "minimum" (a col) on the turbidity-pH curve was observed at about pH 7.5. When the time course of turbidity was measured at more acidic pH (6.5) or alkaline pH (9.5), an induction period was observed at the more alkaline pH (9.5). The turbidity-pH curve for carboxyamidomethylated BSA was similar to that of original BSA. When salts were added to reduced, carboxyamidomethylated BSA solution, no "minimum" appeared on the turbidity-pH curve. The amount of free sulfhydryl groups, either in the presence or absence of salts, increased with heating time at more alkaline pH (pH 9.5). Intermolecular protein interactions occurred easily when salts were present. The endothermic peak on differential scanning calorimetry thermograms did not appear when salts were absent, but appeared when they were present.

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The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4, 4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.

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Three forms of alkaline phosphatase (Al-PMase) (EC. 3.1.3.1)were partially purified from Aspergillus oryzae grown on a bread medium which was in a phosphate-restricted condition. The purified enzymes were named Al-PMase I, II, and III, with pH optima at about pH 8, 99.5, and 10, respectively. Al-PMase II seems to be a major component in the Al-PMases of this fungus. The enzymes were satisfactorily stable at pH 710 at 30°C. Al-PMase I, II, and III were subjected to competitive inhibition with phosphate and commonly inhibited with Hg²⁺ and Mn²⁺. Al-PMase I and II were inhibited completely with EDTA, but Al-PMase III was insensitive to EDTA. Al-PMase II and III seemed to be a non-specific alkaline phosphatase. Al-PMase I showed less affinity to 5'-nucleic acids than 2'(3')-nucleic acids, and did not hydrolyze α-naphthyl phosphate, α-glycerophosphate, O-phosphoethanolamine, and phosphorylcholine.

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Barley was made into a normal and an over-modified malt, and the loss in starch was 14.6 % and 67.7 %, respectively. Starch granules, isolated from the barley and malts, were observed by scanning electron and light microscopes. In normal malt, 14 % of the large granules were eroded and the small granules remained almost intact. In the case of over-modified malt, 38 % of the large granules were eroded, and a marked reduction was found in the population of the small granules. Iodine affinities and blue values of the starches increased as malting proceeded. The malting of barley resulted in an apparent increase in the amylose component of the starch but hardly affected its molecular size distribution when examined by Bio-Gel A-50m column chromatography. The fine structures of the barley and malt amylopectins were compared by Shephadex G-50 and Bio-Gel P-2 column chromatographies after debranching with pullulanase. No change was observed during malting in spite of a significant reduction in the amylopectin component of the starch.

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An organism producing extracellular polysaccharide was isolated from soil and identified as Aeromonas hydrophila (Chester) Stanier. The effects of medium components and cultural conditions on production of the polysaccharide were studied. The optimal concentrations of carbon and nitrogen sources were 5 % and 0.3 %, respectively, for production of the polysaccharide. The optimal initial pH was 79. The maximum polysaccharide yield was obtained at 4-8 days of fermentation. From sucrose and raffinose as carbon source, the organism produced levan and acidic polysaccharide in the ratio of 7:3 and 4:6, respectively. From glucose, galactose, fructose, mannose, maltose and lactose, mainly acidic polysaccharide was produced. The acidic polysaccharide was found to contain galactose, mannose and glucuronic acid in a ratio of 5:4:2. The acidic polysaccharides obtained from sucrose and lactose seemed to be the same polysaccharide.

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A variety of bacteria and yeasts were examined for activities of biotin biosynthetic enzymes, including pimelyl-CoA synthetase, 7-keto-8-aminopelargonic acid (KAPA) synthetase, 7, 8-diaminopelargonic acid (DAPA) aminotransferase and dethiobiotin (DTB) synthetase. Among the strains tested, only Bacillus sphaericus, a DTB producer, showed significant activities for all four enzymes. The bacterium also exhibited high activity of biotin synthesis from DTB in an intact cell system. Using cell-free extract and intact cells, some properties of DAPA aminotransferase, DTB synthetase and biotin synthesizing reaction were examined.
Based on these results of enzyme activities DTB productivity of B. sphaericus was discussed.

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¹⁴C-DAEP [S-(2-acetylaminoethyl) dimethyl phosphorodithioate] was subjected to four different oxidative conditions, and the products were identified. On peracid oxidation in dichloromethane, DAEP gave the oxon (1) predominantly, and 2-acetylaminoethyl dimethoxyphosphinyl disulfide (3), N-acetylcysteamine (10), its oxidized dimer (11) and a further oxidation product of compound 11. This indicates that an unstable phosphorus oxythionate was initially formed, which lost sulfur, was rearranged, and hydrolyzed to give these products. Under other conditions, phosphinyl disulfide 3 was not found. DAEP was metabolized in vitro with a rat liver microsome-NADPH system via oxidation. The aqueous reaction condition prevented the formation of compound 3 from the intermediate, which predominated as well as the oxon formation under anhydrous or close conditions. The formation of various products with sunlight irradiation on glass plates or on bean leaves could be interpreted by oxidation at P=S, C₁ and C₂ positions, demethylation, and deacetylation, followed by further transformation. The initial formation of phosphorus oxythionate seems to play an important role in the oxidation of the organothionophosphorus compound.

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Gamma-radiolysis of fructose in aqueous solution under aerated conditions formed various oxidized products, such as dicarbonyl hexoses, lower molecular aldoses and aldonic acids. Among these radiolytic products, D-arabinohexosulose (1, G=2.2) and D-threo-2, 5-hexodiulose (2, G=1.5) were identified as major hexose derivatives, and D-threo-2, 3-hexodiulose (3) and D-lyxo-hexos-5-ulose (4) as minor products. The radiolytic processes were found to be derived through fructose radicals, similarly to anaerobic radiolysis of fructose. The mechanism of radiolysis was proposed to be initiated by hydrogen abstraction with hydroxyl radical, followed by formation and degradation of fructose hydroperoxy radicals.

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The regulation of enzyme synthesis has changed in Bacillus subtilis pleiotropic mutant lacking transketolase (tkt). The tkt mutant is hypersensitive to D-glucose repression of the synthesis of D-mannitol catabolic enzymes, such as D-mannitol-1-phosphate dehydrogenase and D-mannitol transport system. D-Gluconate, D-xylose and L-arabinose are also effectors for repression in the tkt mutant. In contrast, the synthesis of sorbitol catabolic enzymes, such as sorbitol permease and sorbitol dehydrogenase, are almost insensitive to D-glucose repression. These changes in the regulation of enzyme synthesis seem to be related to some defect in the cell surface structure of the tkt mutant by which other pleiotropic properties are also generated.

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An Hfr strain if Escherichia coli integratively suppressed by plasmid F showed high susceptibility to agents interacting with DNA at high temperature. Antitumor antibiotics, such as bleomycin, mitomycin C, netropsin, and streptonigrin, which interact with DNA, and intercalating dyes, such as acriflavine, 9-aminoacridine and ethidium bromide, were active in assays of preferential activity against the growth of the Hfr strain at 42°C. In the culture fluid of an actinomycete, dnacins, a new type of antibiotic interacting with DNA, were detected by this assay.

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A stereoselective synthesis of erythro-serricornin [(4RS, 6R, 7S)-4, 6-dimethyl-7-hydroxynonan-3-one] was completed starting from L-(+)-tartaric acid. The relative configuration of C(6)-methyl and C(7)-hydroxyl groups in naturally occurring serricornin was threo.

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Corynebacterium sp. S-401 was isolated from soil using squalene as carbon source. Microbiological properties of this bacterium are described. The five metabolic products of this bacterium from squalene were identified as mono-, di-, tri-, tetra-, and pentahydrated squalene. The resting cells of the bacterium catalyzed the hydration of oleic acid to give (−)-10R- hydrbxyoctadecanoic acid and further oxidized product, 10-oxooctadecanoic acid, was also isolated from the reaction mixture.

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Two mutagenic compounds were isolated from the pyrolysate of soybean globulin: 2-amino-3-methyl-9H-pyrido[2, 3-b]indole (MeAC) and 2-amino-3-ethyl-9H-pyrido[2, 3-b]indole (EtAC). These two and other 3-substituted derivatives were synthesized by the condensation of 2-aminoindole with enaminonitriles, and tested for their mutagenic activity. The bulkiness of the alkyl group at C-3 position adjacent to the amino group was related to the mutagenic strength. 2-Amino-3-butyl-9H-pyrido[2, 3-b]indole and the bulkier alkyl derivatives were not mutagenic.

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A silver ion-resistant T. ferrooxidans which grew in the presence of 5 × 10^-4 M silver nitrate was obtained by subculturing cells in autotrophic medium supplemented with silver nitrate. The amount of silver ions released from the resistant-cells at pH 5 to pH 7 was larger than that from the sensitive-cells. Almost all silver ions in the cells were distributed in the cell-wall and cell-membrane. Since inhibition of cell growth and iron-oxidase by silver ions was decreased by the addition of the reduced form of glutathione (GSH) and a GSH dependent release of silver ions from the membrane was observed, it was proposed that GSH plays an important role in the resistance mechanism of the bacterium.

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A cyanide-tolerant strain was isolated from a soil sample, and identified as belonging to the genus Enterobacter. The microorganism accumulated a compound which produced a green color with ninhydrin, in culture fluid containing sodium cyanide and sodium fumarate. This compound was isolated from the culture fluid in crystalline form by using ion exchange resin column chromatography, and was identified as β-cyano-L-alanine (β-CNAla). The compound accumulated in the culture fluid with the growth of the microorganism, and the amount of β-CNAla accumulated depended on the amount of sodium cyanide added. Both intact cells and a cell-free extract of the microorganism catalyzed the formation of β-CNAla from O-acetyl-L-serine with sodium cyanide as substrates. The β-CNAla-forming enzyme was found to be localized in both the cytoplasm and cytoplasmic membrane of this microorganism.

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Concentrations of caffeoyl tartaric acid (CfT), p-coumaroyl tartaric acid (CmT), caffeic acid and p-coumaric acid in 50 samples of commercial white wines were determined by high-performance liquid chromatography. The average levels of CfT, CmT, caffeic acid and p-coumaric acid were 55, 21, 2.5 and 1.7 mg/liter, respectively. Most hydroxycinnamic acids were found to be in combined form with tartaric acid in wine.
The average amounts of CfT and CmT were much higher in Koshu wines (112, 53 mg/liter) than in Sémillon (23, 5 mg/liter), Chardonnay (29, 10 mg/liter) and Riesling wines (51, 13 mg/liter). The aqueous solutions of CfT and CmT were astringent and bitter, their threshold levels were about 50 and 25 mg/liter, respectively. The oxidative browning rates of phenolic cinnamates were much lower than in d-catechin, but CfT was shown to be better substrate for enzymatic browning. Therefore, the amounts of CfT and CmT in Koshu wine were considered to influence wine quality.

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Trioleoylglycerol (TO), trilinoleoylglycerol (TL), and trilinolenoylglycerol (TLN) were autoxidized in the dark at 37°C. Monohydroperoxides (MHP), the primary products, were isolated by preparative thin-layer chromatography (TLC). The isomeric compositions of their hydroperoxy fatty acid components were determined by gas chromatography-mass spectrometry (GC-MS) as follows-TO: the 8-, 9-, 10-, and 11-isomers; TL: the 9-, and 13-isomers; and TLN: the 9-, 12-, 13-, and 16-isomers. The proportions of isomers in each MHP did not vary with the oxidation time. The isomeric compositions of hydroperoxy fatty acid components obtained from autoxidized soybean and olive oils indicated that each unsaturated fatty acyl group of triacylglycerol (TG) in vegetable oils produced isomeric hydroperoxides during autoxidation in a way similar to the corresponding fatty acid methyl esters. The proportions of the isomers obtained from autoxidized oils changed with the level of oxidation. Isomers coming from linolenic acid in soybean oil and those from linoleic acid in olive oil decreased remarkably at a high level of oxidation.

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The effect of pale, soft and exudative (PSE) porcine muscles on the color of processed meat products was investigated in sausages prepared in the laboratory. Eighteen muscle samples obtained at 24hr postmortem were tested. CFR (color forming ratio) tended to decrease with decreasing pH value. Muscle samples with extreme PSE characteristics produced low CFR sausages. A significant difference was not observed in the amount of total heme pigments in normal and PSE muscle. The effect of pH (4.926.23) on laboratory sausage from normal muscle showed that CFR gradually increased at lower pH values. The PSE conditions were also simulated by incubation of normal muscle adjusted to low pH (5.45.0) for 90 min at 40°C. Laboratory sausages were prepared from the PSE-like muscle after adjustment to pH 6.0. The PSE-like muscles had changes in CFR similar to those obtained with PSE muscles. The reasons for the decrease in CFR are discussed.

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Escherichia coli OmpF protein of the outer membrane behaved essentially the same as the OmpC protein on reconstitution of the cell surface. An ordered hexagonal lattice structure was reconstituted on the entire surface of the lipoprotein-bearing peptidoglycan sacculus from the OmpF protein and lipopolysaccharide. With the lipoprotein-free peptidoglycan sacculus, the hexagonal lattice structure was formed independently of the sacculus. In the absence of the peptidoglycan, the OmpF protein and lipopolysaccharide were assembled into a vesicle with an ordered hexagonal lattice. The role of lipopolysaccharide in lattice formation was taken over by lipid A or even by fatty acid. However, the lattice constant with these lipopolysaccharide derivatives was appreciably smaller than that with the wild-type lipopolysaccharide. The importance of the lipoprotein in the interaction between the outer membrane and the peptidoglycan layer was also shown by using a lipoprotein-negative mutant.

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Three polymerizable ATP derivatives, N⁶-[N-(6-methacrylamidohexyl)carbamoylmethyl]-, N⁶-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl]carbamoylmethyl]-, and N⁶-[N-[N-(2-hydroxy-3-methacrylamidopropyl)carbamoylmethyl]carbamoylmethyl]-ATP, were synthesized and radically copolymerized with comonomers [acrylamide, N-(2-hydroxyethyl)-, N-ethyl-, N, N-diethylacrylamide, acrylic acid, and 6-methacrylamido hexylammonium chloride] to obtain 18 new polymer derivatives of ATP. The molecular weight distributions were controlled by appropriate initiator concentrations. The monomeric and polymeric ATP derivatives were all coenzymically active against both hexokinase and glycerol kinase. The observed coenzymic activities (Km and V_max) are discussed in connection with the structures of the derivatives.

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An extracellular proteolytic enzyme was produced by Conidiobolus lamprauges accompanied with chitin-binding hemagglutinin. The protease was partially purified by ammonium sulfate precipitation and CM Sephadex, concanavalin A-Sepharose, and Sephadex G-75 chromatographies. The molecular weight was estimated to be about 20, 000 by gel filtration.
Agglutination of human erythrocytes by C. lamprauges agglutinin, wheat germ agglutinin, soy bean agglutinin and concanavaline A was accelerated by treating erythrocytes with this enzyme, whereas agglutination by Streptomyces L-fucose-specific lectin was not affected. Agglutination by Vibrio was repressed.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the erythrocyte membrane preparations from digested cells showed that this protease and pronase hydrolyzed "Band 3" and sialoglycoproteins [Fairbanks et al., Biochemistry, 10, 2606 (1971)] whereas papain and trypsin hydrolyzed only the latter. The relationships between these membrane proteins and the receptors for hemagglutinins were discussed.

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The host-specific pathotoxin complex isolated from the culture filtrate and mycelial mat of Bipolaris (Helminthosporium) maydis, race T, was reduced to hydrocarbons. The hydrocarbons were separated by molecular sieves and analyzed by GC-MS. The MS gave typical spectra of normal, odd carbon numbered hydrocarbons from C₃₅H₇₂ to C₄₉H₁₀₀. Compounds differing from hydrocarbons only in the presence of an internal tetrahydropyran ring were obtained as byproducts from the toxin-complex reductions. These compounds were analyzed by CI (chemical ionization)-GC-MS and HR (high resolution)-GC-MS. The MS data supported the hypothesis that the C₃₅- to C₄₉- toxins are analogous compounds with many common partial structures. The configurations at both the C-6 and C-8 carbons of the major components appeared to be R.

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Amino-thiol compounds were found to show mutagenicity, when they were activated with S-9 Mix. Further, the mutagenicity also appeared when these amino-thiol compounds were activated with pepsin, catalase, D-amino acid oxidase and thermolysin. All of their activating effects remained even after they were heated in a boiling water bath for 10 min. On the other hand, pancreatic lipase, lysozyme, peroxidase, papain, bovine serum albumin, casein, hemoglobin, RNase, gelatin and ovoalbumin did not show any effect on the mutagenicity of these amino-thiol compounds.
Kinetic studies of the reaction between these aminothiol compounds and pepsin or catalase suggested that the activation seems to be caused by proteins but not enzymatically.

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