Streptomyces sp. No. 280 produced several kinds of amylase inhibitors (amylase inhibitor A, B,
B' and C). Two amylase inhibitors (designated as AI-A₁ and AI-A₂) were obtained from an amylase inhibitor A fraction by paper chromatography. AI-A₁ inhibited muscle phosphorylase a much more than AI-A₂ and was hydrolyzed by sweet potato β-amylase whereas AI-A₂ was not. Both amylase inhibitors had a carbohydrate and were hydrolyzed by some kinds of amylases or acids. They lost their inhibitory activity against phosphorylase a after treatment with acids or hog pancreatic α-amylase, but they showed increased inhibitory activity toward porcine small intestinal sucrase.
Both AI-A₁ and AI-A₂ were composed of glucose and a basic moiety which gave a positive ninhydrin reaction. The molecular weights of AI-A₁ and AI-A₂ were estimated to be approximately 1300-1500 by gel filtration on a Sephadex G-15 column. The nitrogen content of the amylase inhibitors was found to be about 1.3% by elementary analysis.

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The reduction in mutagenic products was studied using Salmonella typhimurium TA 98 in the presence of tannic acid and some polyphenols during pyrolysis of protein. Albumin was pyrolyzed with or without tannic acid at different temperatures under N₂ or air atmosphere. The addition of tannic acid reduced the mutagenic activity of the pyrolysis products, and the reduction was observed in the basic and neutral fractions. The mutagenic activity of the pyrolyzates of albumin in the presence of polyphenols (such as quercetin, rutin, catechin, catechin gallate, n-propyl gallate, chlorogenic acid, pyrocatechol or protocatechuic acid at the rate of 0.2 g per 1 g albumin) was from 20 to 76% of that of albumin without additives. The content of mutagen 2-amino-9H-pyrido[2, 3-b]indole in pyrolyzates decreased in the presence of polyphenols.

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Suitable cultural conditions for polyphenol formation were examined in the amacha cultured cells. Factors tested included; inoculum size, sucrose concentration, various carbon sources, extract (malt extract and yeast extract), kinetin, auxins, initial pH, light exposure and phosphate concentration. Sucrose concentration had marked effects on cell growth and polyphenol production, and phosphate concentration was significant in polyphenol formation, although other investigated factors had no effects on polyphenol productivity. The time course of polyphenol accumulation in cultured cells showed that polyphenol content decreased at the lag and early exponential phases of growth, and increased remarkably till the stationary phase. Cells at the stationary phase did not accumulate polyphenol and even lost these polyphenols to the external medium as the cells reached the declining phase. Large scale culture of amacha cells was carried out using a 15 liter jar fermentor. Investigations on polyphenol content changes with amacha leaves were also carried out during growth. Isocoumarins, especially phyllodulcin, were formed as the glycosides at an early stage, and these glycosides were hydrolyzed to their aglycones by enzymesat a later stage.

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From membrane fraction of Gluconobacter melanogenus IFO 3293, 2-keto-D-gluconate dehydrogenase was solubilized and purified to an electrophoretically and ultracentrifugally homogeneousstate of about 400-fold in high yields of 41%. Purification was achieved by solubilization with 2% cholate and 0.2 M KCl, subsequent precipitation with ammonium sulfate and polyethylene glycol 6000, and chromatographies on CM-cellulose and hydroxyapatite columns in the presence of Triton X-100. The purified enzyme was tightly bound to a c-type cytochrome existing as a dehydrogenase-cytochrome complex. The molecular weight of the enzyme was determined to be about 133, 000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 61, 000 (flavoprotein), 47, 000 (cytochrome c) and 25, 000. The dehydrogenase was found to be a flavoprotein having a covalently bound flavin. Only 2-keto-D-gluconate was readily oxidized by the enzyme in the presence of dyes, such as ferricyanide, 2, 6-dichlorophenolindophenol or phenazine methosulfate. NAD, NADP and oxygen did not function as electron acceptors. Optimum pH for enzyme activity was 4.0, and optimum temperature was 39°C. The enzyme activity was not inhibited by sulfhydryl reagents or metal chelators.

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Some physicochemical properties of purified glucose isomerase from Streptomyces griseofuscus, S-41 were examined. The extinction coefficient (E^1%_1cm) and isoelectric point (p¹) were determined to be 11.4 and 4.0, respectively. The values for sedimentation coefficient (S^O_20w), partial specific volume (v), diffusion coefficient (D_20w) and intrinsic viscosity ([η]) were 8.50 S, 0.73 cm³/g, 4.6×10^-7 cm²/sec and 3.7 ml/g, respectively. The molecular weight was estimated to be 180, 000 by the sedimentation equilibrium method and 1 85, 000 by the gel electrophoretical method. The enzyme was found to contain four cobalt ions per molecule in atomic absorption spectrophotometrical analysis. The CD spectrum in the far ultraviolet region showed a minimum at 280 nm and a maximum at 198 nm. Mean residue ellipticity ([θ]) at 220 nm was estimated to be -11, 200 deg•cm²/d mol. The computed values for the contents of the secondary structure were as follows: α-helix, 40%; β-form, 36%; and random coil, 24%. The enzyme showed a visible CD spectrum having a negative peaks at 530 nm and 430 nm. This suggested the formation of a coordinated metal compound, Co-amino acid complex. The molecular dimension of the enzyme was examined by measuring the hydrodynamic parameters. The frictional ratio (a/b) was 5.0 for prolate ellipsoid and 0.2 for oblate ellipsoid. Stokes' radius was calculated to be 47A for a hydrated hypothetical sphere.

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The subunit structure of glucose isomerase isolated from Streptomyces griseofuscus, S-41 was studied using denaturants. The enzyme was quite stable to SDS under mild conditions, and dissociation into smaller subunits was dependent on pH and temperature. At acidic pH and high temperature, the enzyme showed rapid dissociation and inactivation. The dissociation into constituent subunits paralleled the loss of enzymatic activity. The enzyme was also dissociated into subunits by 6m guanidine hydrochloride. A single symmetrical elution peak was observed in Sepharose 6B column chromatography in the presence of 6 m guanidine hydrochloride, and the eluate showed no activity. Molecular weight estimations of the subunit, using both SDS polyacrylamide gel electrophoresis and gel nitration, were the same (43, 000). The N-terminal amino acid was identified as serine, and the yield of serine was estimated to be 43 percent from analyses of PTH and DNP derivatives. The sequence from N-terminal to the third amino acid was found to be Ser-Asp-Gin•••. Considering the molecular weight of the native enzyme as 180, 000 and the results obtained in the experiments here, it was concluded that native glucose isomerase was composedof four identical or very similar subunits with equal molecular weights and dimensions. The amounts of total and free thiols in the enzyme were estimated to be 6 mol and 2 mol, respectively, per mol of enzymeusing the titration method with DTNB. It was therefore considered that the enzyme contains two disulfide linkages.

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The influence of fasting and protein starvation on the profiles of liver and kidney lipids of rats was investigated. Rats on protein-free regimen were maintained on isocaloric diet up to 11 days without appreciable fall in food-intake by the spaced-feeding procedure, while rats on caloric depletion were starved for 5 days. Both types of dietary restrictions resulted in losses of body and organ weights of animals. The influence of stress conditions however, varied with respect to the individual lipid constituents as well as the organs studied. Fasting as well as protein-starvation caused an increase in liver total lipids, mainly in the form of triglycerides and a loss in hepatic cholesterol. In the kidney, fasting resulted in the loss of only free cholesterol. However, a preferential loss of esterified cholesterol occurred in protein starved rats leading to a 20-fold increase in the ratio of free to esterified forms. A significant loss (p<0.01) in phospholipid levels was apparent in the liver of fasted rats, while no such change was observed in the protein-starved group. The body and organ weights as well as the lipid profiles returned to normal levels as a result of realimentation of the experimental animals, suggesting recovery from nutritional stress.

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Fatty acid contents were estimated in the cell wall of Saccharomyces. The fatty acids responsible for cell wall hydrophobicity were classified by ease of extraction to 'readily extractable' and 'bound' acids. The readily extractable fatty acids were easily extracted with pentane and chloroform-methanol. The fatty acids extracted with chloroform-methanol were quite effective for cell wall hydrophobicity, but the fatty acids extracted with pentane were not. The bound fatty acids comprised in the phospholipids phosphatidylethanolamine and phosphatidylserine, which were rigidly associated with the cell wall. These phospholipids were not extractable until they were released from the cell wall by pronase. Chloroform-methanol extraction caused a reduction in cell wall phospholipid content, particularly after treatment with pronase. The fatty acid content of the resultant cell wall was lowered to below 7% of initial content. Phospholipids contained more saturated fatty acid than readily extractable lipids. Phospholipids greatly contributed to cell wall hydrophobicity of various film strains of Saccharomyces.

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Bacillus sp. 11-1S, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56, 000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0-6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1, 4-glycosyl linkages in xylan and cellulose.

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Several 2-sulphanilamido-4-methyl-5-[5'-arylamino-1', 3', 4'-oxadiazolyrjthiazoles, 2-sulpha-nilamido-4-methyl-5-[5'-arylamino-1', 3'4'-thiadiazolyl]thiazoles, 2-sulphanilamido-4-methyl-5-[4'-aryl-5'-mercapto-1', 2', 4'-triazolyl]thiazoles and bis[4-aryl-5-(2'-sulphanilamido-4'-methyl-thiazol-5'-yl)-1, 2, 4-triazol-3-yl]disulphide have been synthesised with a view to study their biological properties. Some of the synthesised compounds checked fungal growth to a great extent.

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Gas chromatographic (GC) and high performance liquid chromatographic (HPLC) methods were developed for determination of a synthetic pyrethroid, 3-phenoxybenzyl d-cis, trans-chrysanthemate
(d-phenothrin, Sumithrin^®) in formulations, such as aerosol, dust, emulsifiable concentrate (EC), oil and premix, and also of coexisted other active ingredients. The active ingredients in formulations were determined by GC on a column of 2% DEGS using 2, 2'-biquinoline as internal standard, and by HPLC on a column of μBondapak Phenyl^® with acetonitrile-water (3 + 2) as a mobile phase. Optical isomers of d-phenothrin were hydrolyzed to corresponding chrysanthemic acids, which were derivatized into diastereomer esters with d-2- or l-2-octanol and determined by GC on a glass capillary column of silicone DC QF-1.

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An obligate and a facultative strains of iron-oxidizing bacterium, Thiobacillus ferrooxidans, were isolated and their properties were investigated. No difference was observed in the iron-oxidizing system of the two strains, while a distinct difference was observed in the D-glucose metabolizing system. Intact cells of the obligate strain, AP-19, did not have D-glucose oxidizing and ¹⁴C-glucose uptake activity, while they were both present in intact cells of iron grown, iron-glucose grown and glucose grown facultative strain, AP-44. Since cell-free extracts from both strains showed D-glucose oxidizing activity to the same degree, it was concluded that the obligate nature in AP-19 was due to lack of D-glucose uptake activity.

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Three isozymes that catalyze the NADPH-linked reduction of glyoxylate to glycolate were found in cell-free extracts of Neurospora crassa. They were designated glyoxylate reductase-1, 2 and 3 [EC 1.1.1.79]. Glyoxylate reductase-2 was purified about 5400-fold with an activity recovery of 11%, and the purified preparation was electrophoretically homogeneous. The molecular weight was estimated to be 110, 000 by the gel filtration method. The enzyme catalyzed the reduction of glyoxylate and hydroxypyruvate, and was about 4-fold more active with hydroxypyruvate than with glyoxylate at optimum substrate concentration. The enzyme was specific for NADPH as an electron donor, but showed slight affinity towards NADH, and the NADPH: NADH ratio of activity was about 7 : 1. The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH and NADH were found to be 5.7 mM, 85 μM, 3.1 μm and 0.23 mM, respectively. The other isozymes of glyoxylate reductase (GR-1 and 3) differed from GR-2 in molecular size and substrate specificity.

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Lutein had novel spectroscopic properties in the visible region on the formation of complexes with several proteins [S. Takagi, M. Shiroishi and T. Takagi, Agric. Biol. Chem., 44, 2111 (1980)]. The effects of pH, molar ratio of lutein to protein, and the variety of protein on the phenomenon was studied. The phenomenon was insensitive to these parameters. Solubilization into micelles of deoxycholate was found to induce no optical activity in contrast to bilirubin by Perrin et al. [J. H. Perrin and M. Wilsey, Chem. Commun., 769 (1971)].
It is strongly suggested in this paper that the observed changes in spectroscopic properties including the novel one in circular dichroism come chiefly from mutual interactions between lutein molecules in the complexes. Changes in spectroscopic properties comparable to those for lutein were observed with β-cryptoxanthin but not with canthaxanthin or ethyl β-apo-8'-carotenoate, although the latter two formed complexes with ovalbumin. The presence of at least one asymmetric carbon atom in the ionone rings seems to be essential for the novel spectro-scopic changes to be observed. The possible correlation of the trans-cis conformational change in the conjugated double bond system was discussed. The optical activity was presumed to come from the intermolecular dipole-dipole coupling with the chiral spatial orientation.

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A cadmium and zinc-binding protein similar to metallothionein has been isolated from Tetrahymena pyriformis exposed to cadmium chloride. This protein contained 32.4% half-cystine, 23.7% acidic amino acids and 10.1% lysine. The amino acid composition was similar to that of copper-thionein of yeast. The metal-binding protein of Tetrahymena pyriformis contained 3.7 g atom cadmium, 0.7 g atom zinc, and 0.1 g atom copper per mol, and showed the spectral characteristics of cadmium-thionein, i.e., a broad shoulder at 250 nm and low residual absorption at 280 nm. The molecular weight of this protein was determined to be 11, 000 by gel filtration in 6m guanidine hydrochloride, although it moved like a protein with a molecular weight of 30, 000 on ordinary gel filtration.

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Phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) from Escherichia coli AHU1520 was purified until electrophoretically homogeneous by a single procedure, using a linear gradient elution of 2, 3-bisphosphoglycerate between 0 and 0.5 mM on a blue dextran-Sepharose 4B column chromatography. The phosphoglycerate kinase was inhibited by blue dextran competitively with respect to 3-phosphoglycerate, and noncompetitively with respect to
ATP. The inhibition of the kinase by Cibacron Blue was competitive with respect to 3-phosphoglycerate at both saturated and unsaturated levels of ATP; the inhibition by the dye toward ATP was shifted from a mixed-type at a saturated level of 3-phosphoglycerate to competitive-type at an unsaturated level. Further inhibition studies implied that two molecules of the dye were bound per molecule of phosphoglycerate kinase with an unsaturated level of ATP used as a fixed substrate. The shape of the difference spectrum of Cibacron Blue in the presence of the kinase was very similar to that of a difference spectrum generated upon addition of KCl, but was markedly different from the shape of the spectrum produced by the addition of glycerol.

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Theoretical and experimental investigations were conducted on growth kinetics and substrate consumption of hydrogen bacterium Alcaligenes hydrogenophilus. The kinetic models proposed were evaluated by continuous cultures of A. hydrogenophilus under various conditions. Thekinetic models closely simulated continuous cultures of A. hydrogenophilus.

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Soybean seedlings were grown at 28°C under dark or light conditions for 12 days. Non-polar lipids (NPL) were separated by silicic acid column chromatography from total lipids in epicotyl containing young leaves, hypocotyl and root. The glyceride (TG, DG, and MG), free fatty acid (FFA) and sterol lipid (SE) components in NPL were analyzed mainly by thin-layer and gas-liquid chromatographies (TLC and GLC).
During germination, the amounts of polar lipids (PL) markedly increased in the tissues of soybean seedlings, especially in light-grown seedlings, whereas these of NPL increased slightly or maintained constant values. The features of the compositions and changing patterns of NPL in the tissues were more clarified in light-grown seedlings than in dark-grown ones. The pattern of change in fatty acid composition was similar in TG and 1, 2-DG, which showed higher proportions of linoleic and linolenic acids, whereas FFA, 1, 3-DG or MG had high proportions of saturated fatty acids. These results indicate that the compositions and changing patterns of NPL and their fatty acids in the tissues depend on the differences under two germinating conditions tested.

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An exo-β-1, 3-glucanase was purified from the commercial enzyme preparation "Kitalase" which is a yeast cell wall lytic enzyme preparation. The purification procedures consisted of following steps: ammonium sulfate fraction, SP-Sephadex C-50 and CM-Cellulose C-32 column chromatography, and Sephadex G-100 gel filtration. The optimum pH value was 5.8, and the optimum temperature was 55°C. The enzyme was stable in the pH range of 5.1 to 9.8 and at temperatures below 53°C. The isoelectric point and the molecular weight were estimated to be pH 9.3 and 73000, respectively. The enzyme was shown to bypass β-1, 6-linkaged branches to cleave β-1, 3-linkages when scleroglucan was used as substrate. The Km values for laminarin, laminaripentaose, laminaritetraose and laminaritriose were 0.16, 2.01, 2.24 and 1.34 mM, respectively.

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The crude enzyme fraction of precipitates resulting from the addition of 70% alcohol to the culture filtrate of A. lunatus was separated by CM-Sephadexand Sephadex G-75 chromatography into 13 fractions having lytic activity for M. radiodurans, M. lysodeikticus and P. radiora. Five of the fractions showed similar lytic activity spectra, but the other fractions were separated by the specificities of their lytic activities. This result indicates that the wide lytic spectrum of the crude enzyme against microorganisms is attributable to the action of many lytic enzymes. All fractions, except for P2-2 fraction (designated as the P2-2. enzyme), contained at least two proteins as determined by disc gel electrophoresis. The P2-2 enzyme was purified 34-fold by rechromatography on Sephadex G-75, and appeared to be homogeneous on disc gel electrophoresis. The enzyme was able to lyse intact cells of M. radiodurans and M. lysodeikticus without detergent, and those of P. radiora with detergent, but was not able to digest casein.

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The enzymatic properties of P2-2 enzyme were determined by using of M.radiodurans. The enzymewas: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16, 000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.

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An antagonistic fungus to Valsa ceratosperma was isolated from soil, and identified as Fusarium solani. The fungus was found to produce at least two antifungal substances in stationary culture. These two substances were isolated from their culture filtrate as chromatographically homogeneous, amorphous solids. Their examined physico-chemical properties appeared to be identical with cyclosporin A and C, obtained from the fermentation broth of Trichoderma polysporum, and they showed a pronounced inhibitory effect on growth of V. ceratosperma.

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Partially purified, cell-free extracts of Clostridium rectum effectively degraded lindane (y-BHC) and related polychlorocyclohexenes, in the presence of dithiothreitol. The first step of the reaction was proved to be reductive dechlorination. These compoundswere also reductively dechlorinated by polarography. The half-wave potentials had a linear relationship to the logarithmic values of the biodegradation rates.

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A glutamine production process was established by combining alcoholic fermentation of baker's yeast cells with glutamine synthetase from the bacterium Gluconobacter suboxydans. The maximumamount of glutamine formed under optimum conditions was about 20mMin 3 hr with 80% yield based on glutamate, substrate. The fermentation proceeded in two steps: the accumulation of energy in a form of fructose 1, 6-diphosphate (FDP) by yeast fermentation of sugar based on the Harden-Young effect and the fermentation of FDPcoupled with glutamine synthetase reaction (an endergonic reaction) through an ATP-ADPsystem. The following factors were found to be important: (a) the ratio of the activities of yeast fermentation of sugar and glutamine synthetase, (b) effect of contaminating enzyme(s) in glutamine synthetase preparation, and (c) enzymatic properties of glutamine synthetase.

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Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20mMsodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 msodium chloride and 7niM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.
On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.

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Gibberellin A₅₉ has been isolated from immature seeds of Canavalia gladiata (sword bean) and its structure was determined to be Δ²-gibberellin A₂₁ (1).

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