Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23, 000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded_??_The enzyme was completely inhibited by diisopropylphosphorofluoridate (10^-2M) or potato inhibitor (250μg/ml).

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Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41, 000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenylalanine amide, poly-L-lysine and poly-L, a-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.

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Neutral proteinase II (the second peak in DEAE-cellulose chromatography) in the culture fluid of Aspergillus oryzae was purified the Amberlite IRC-50 adsorbed fraction by DEAE-cellulose chromatography and two steps of gel filtration through Sephadex G-100. The purified enzyme showed neither aminopeptidase nor carboxypeptidase activity. The molecular weight of the enzyme was estimated to be about 19, 300 by gel filtration method. The enzyme has an optimum pH of 5.5 to 6.0 in acetate and phosphate buffers. More than 70% of the activity was remained after heating at 90°C for 10min. About 70% of original activity was reduced in the presence of 10^-2M EDTA (pH 6.0) and the activity with 18% NaCI was almost 50% of the activity without NaCl.

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Eight α-N-acyl colistin nonapeptide derivatives including three aliphatic, four aromatic and one alicyclic derivatives were synthesized by the reaction of colistin nonapeptide with corresponding acid chlorides. This acylation reaction was carried out under the condition kept restrictedly at pH 5.0 in order to introduce an acyl group only to α-amino group but not to γ-amino group existing in a colistin nonapeptide molecule. Synthetic method and several physico-chemical natures of these acyl colistin nonapeptide derivatives are given in this paper.
All of the acylated derivatives thus synthesized exhibited characteristic antimicrobial activities. Antimicrobial spectra were substantially based upon a gram-negative type and not so much altered by chemical structures of acyl groups which were considerably differentiated from each other such as cyclic or chain form. Thus, more possible response of carbon size than its structure to the antimicrobial effectiveness was inferred. In spite of almost no toxicity and feeble antimicrobial activity of colistin nonapeptide itself, these acylated colistin nonapeptide derivatives showed a toxicity against mice at a dose of 16.9_??_70mg/kg in LD₅₀, which, however, was inferior to the toxicity of colistin sulfate, possibly correspondent to their much weaker antimicrobial activities, as a whole. Hence, it seems likely that acyl part of these acylated colistin nonapeptide derivatives including that of colistin is seriously responsible for the biological activities.

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Substrate specificity of purified preparations of phytase from Asp. terreus was examined. The enzyme showed broad specificity. It was found that Asp. terreus produced only one kind of acid phosphatase and it had phytase activity.
Effective materials for the enzyme formation were examined. The formation of the enzyme occurred only during times that mycelia was in contact with inositol.
By differential centrifugation and electron-microscopic autoradiography, it was determined that inositol was incorporated into the mycelia and that it was located at almost the same point as where the active enzyme was located.

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A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s°_{20, w} was determined to be 2.6S. The molecular weight was estimated to be approximately 19, 000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.
The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1g-atom of calcium and about 2g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.

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The effect of glucose on the formation of uricase by a strain of Streptomyces sp. incubated under conditions of nitrogen limitation was investigated. Glucose stimulated uricase formation in the presence of potassium ion and inhibited it in the absence of the ion. Glucose metabolism by the organism was altered in the absence of the ion, and this appeared to cause the inhibition of the enzyme formation. The stimulatory effect of glucose in the presence of potassium ion was to shorten the lag period. Comparisons of the enzyme formation with and without urate in the presence and absence of glucose revealed that glucose promoted the utilization of exogenous urate as the inducer. The effect of glucose appeared to require protein synthesis, since it was prevented by chloramphenicol. Cyclic adenosine 3', 5'-monophosphate showed apparently no effect on uricase formation of this organism.

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A conduction type calorimeter has been designed to chase microbial growth in batch system. The calorimeter is of a twin structure having thermopile plates as a temperature sensor. The heat evolution during the microbial growth at a required temperature can be observed as an output-voltage generated at thermopile terminals with a sensitivity of 58.5mV K^-1
A stainless steel cell with a volume of 300cm³ serves as a culture cell which is capable of being autoclaved prior to the initiation of calorimetric run, taking out from the calorimeter body.
Because of the twin structure, the apparatus works with sufficient stability in detecting small heat evolution for long duration. Its operation has been demonstrated with the growth of Sacch. cerevisiae grown on liquid synthetic medium under anaerobic condition.

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Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentuin Moench. One of them called BN-II is composed of salicylamine and alto-4-hydroxy-L-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and L-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2'-hydroxybenzyl)-allo-4-hydroxy-L-glutamine and N⁵-(4'-hydroxybenzyl)-L-glutamine respectively from their chemical and spectrometric properties.

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Saccharogenic amylase from Rhizopus javanieus sp. 3-46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1:1:6:2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1:1:9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6:2:2:2:12.

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Methylation analysis of five fractions of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 has shown that each fraction was a highly branched dextran with the branches being joined mainly through C-2. Detection of a small amount of 4-O-monomethyl-D-glucose has suggested that parts of the n-glucose residues were doubly branched at both C-2 and C-3. Detection of a larger amount of 2, 4, 6-tri-O-methyl-n-glucose in the hydrolyzates of the methylated products of the borate insoluble fractions has shown a greater percentage of linear α-1, 3-linked D-glucose residues in these fractions. It is suggested that the solubility of the dextran is closely related to the content of linear α-1, 3-linked D-glucose residues.

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The selective antagonists of blasticidin S elaborated by Streptomyces caespitosus var. detoxicus 7072 GC₁ showed several interesting biological activities to counteract the toxicity of the antibiotic against both plant and animal cells and was designated DX. C. This paper describes the production, isolation and some biological activities of DX. C.

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Separation of the active principles of DX.C was worked out into eight groups with the use of an ion exchange chromatography. Further purification of main components, the isolation and characterization of two main active principles were described.

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Studies on uptake, tissue distribution, and metabolsim of 3-(3', 5'-dichlorophenyl)-5, 5-dimethyloxazolidine-2, 4-dione (DDOD) by bean and grape plants, and degradation by soil and light were carried out using ¹⁴C- or ³H-DDOD. DDOD injected at stem or absorbed through roots of bean plants was transported mainly to leaf tissues. No downward movement of the label was observed. DDOD was decomposed in the nutrient solution to N-3, 5-dichlorophenyl-N-α-hydroxyisobutyryl carbamic acid (DHCA) and α-hydroxyisobutyryl3, 5-dichloroanilide (HDA). Metabolism of DDOD in bean plants or on grape berries themselves occurred to only a small extent if at all. When injected into grape trees, DDOD underwent some degree of metabolization to HDA and, probably, 3-(3', 5'-dichloro-4'-hydroxyphenyl)-5, 5-dimethyloxazolidine-2, 4-dione. In soil, DDOD broke down to DHCA, HDA, and 3, 5-dichloroaniline, but formation of tetrachloroazobenzene was not observed under the present experimental conditions. DDOD decomposed to some degree when irradiated with a xenon lamp.

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Olive oil hydrolyzing fraction (F-3) of Mucor lipase was separated into two fractions, F-3A and F-3B, by means of CM-Sephadex column chromatography. Both fractions were homogeneous and F-3A was crystallized. The pH optimum for olive oil hydrolysis of F-3A was at 9.0 and that of F-3B was at 8.0.
Chain specificities of the both enzymes were different with each other. Sedimentation coefficient (s_{20, w}) of F-3A was 2.8 S and that of F-3B was 3.1S.
Molecular weights calculated from sedimentation equilibrium data were 25, 400 for F-3A and 29, 000 for F-3B.

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Partially purified penicillin acylases (EC 3. 5. 1. 11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing D (-)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. inelanogenum contained high levels of penicillinase (EC 3. 5. 2. 6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.

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A newly described species of Streptomyces (named Sm. kagawaensis ATCC No. 21811) isolated from soil was found to produce a new antifungal antibiotic, prumycin, which belongs to amino sugar group. Prumycin was isolated from the fermentation broth by ion-exchange adsorption and gel-filtration methods. This antibiotic inhibited specifically the growth of Sclerotinia sp. and Botrytis sp. on flower pot test with kidney bean leaves and also was effective on the field test with various plants.

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Maltose phosphorylase (EC 2. 4. 1. 8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150, 000 and 80, 000, respectively. The enzyme does not contain pyridoxal-5'-phosphate as a cofactor. It can not act on maltitol, maltotriitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO₄, HgCl₂ and p-chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.

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Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s_{20, w} was 6.29S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1)×10, 000 from gel filtration with Sephadex G-200 and to be (9±1)×10, 000 from sedimentation equilibrium. These values were higher than that (6_??_7×10, 000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.

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L-Arginase (L-arginine amidinohydrolase, EC 3. 5. 3. 1) was purified in a crystalline fore from cells of Bacillus obtilis KY 3281 with an overall yield of 23.2% The crystalline enzyme had a specific activity of 858 i.u./mg-protein and was ultracentrifugally homogeneous. It was estimated to have a molecular weight of 115, 000±5000 by the method of Yphantis.
The enzyme highly specific for L-arginine showed the maximum activity at pH 10 with Mn²⁺ ion. The Km for L-arginine was 1.35_??_10^-2_M. The activity was competitively inhi bited by L-lysine, but not by L-ornithine and increased by the addition of Mn²⁺ or Co⁺² ions. The stable pH and temperature ranges became wider in the presence of Mn²⁺ ion and L- threonine.

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The two low density lipoproteins (LDL₁ and LDL₂) in pig serum were isolated and their aqueous solutions extracted with ethyl ether. On ultracentrifugation four components of 13, 20, 24 and 30S were evident in both fractions, and occasionally a fifth of 10S could be detected. The 13S component represented about 70% of the extracted material and its protein moiety was estimated to be half that of the parent lipoprotein. This shows that both LDL fractions contain protein subunits that dissociate on lipid extraction, and these apparently reassociate to form the heavier components. Apart from lipid content the only difference between LDL₁ and LDL₂ was a small, but statistically significant, difference in the histidine content.

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The effects of several factors on the enzymatic production of 3, 4-dihydroxyphenyl-L-alanine (DOPA) and 3, 4-dimethoxyphenyl-L-alanine (DMPA) by transamination reaction were investigated using wet cells of Alcaligenes faecalis IAM 1015. In addition, some experiments for the cultural conditions for transaminase production were performed. DOPA and DMPA were obtained in 80 and 90% yields, respectively, using the mixture of L-glutamate and L-aspartate as amino donors. Accumulation of DMPA in the culture under the growing state of the bacteria was also confirmed.

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5' Phosphodiesterase (5'-PDase) which degrades RNA to nucleoside-5'-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5mg of 2, 4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5'-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.
The 5'-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5'-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5'-PDase activity as the mother plant on dry cell weight basis.

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Neutral constituents of volatiles in the ether extract of cultured broth of Sporobolomyces odorus AHU 3246 were analyzed by gas chromatography-mass spectrometry and other methods.
Identified compounds were as follows: Methyl, ethyl, isobutyl, n-butyl, isoamyl, n-amyl, benzyl, and β-phenylethyl alcohol; formaldehyde, acetaldehyde, benzaldehyde, phenylacetaldehyde, acetone, and methyl ethyl ketone; ethyl formate, ethyl acetate, and di-n-butyl phthalate; γ-decalactone (4-decanolide) and 4-hydroxy-cis-6-dodecenoic acid γ-lactone (cis-6-dodecen-4-olide). Di-n-butyl phthalate and parts of methyl, ethyl, and n-butyl alcohol and ethyl acetate were thought to be contaminants. γ-Lactones produced by the yeast were determined by GLC.
Although nine strains of six species of carotenoid pigment accumulating yeasts were cultured under the same conditions, neither flavorful smelling nor γ-lactone production detected in their cultured broths.

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Pantothenate kinase (ATP: pantothenate 4'- phosphotransferase, EC 2. 7. 1. 33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45, 000. The enzyme catalyzed the formation of pantothenic acid 4'-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^-5M for pantothenate, 3.5×10^-5M for ATP, and 10^-3M for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.

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2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel. The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110, 000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^-3M). Km value of the enzyme for GA was 1.3×10^-2M and for 2KGA was 6.6×10^-3M. Km values for NADP and NADPH₂ were 1.25×10^-5 and 1.52×10^-5M, respectively.

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Lipase activators were extracted with ethyl ether from the culture medium of Candida paralipolytica at pH 2 and two kinds of activators were separated by silica gel column chromatography. The main component (activator A) was an oily liquid, and showed an Rf value different from that of oleic acid on thin-layer chromatograms. One mole of activator A seemed to be necessary to activate one active site of the lipase and it lowered the optimum pH of the lipase from 8.2 to 7.0. In relation to this, it was found that urea, ethanol and sodium chloride had the ability to lower the optimum pH.

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In attempt to obtain an efficient mutant for the production of flavin-adenine dinucleotide (FAD) from flavin mononucleotide (FMN) and adenine, Sarcina lutea IAM 1099 was treated with N-methyl-N'-nitro-N-nitrosoguanidine, and a purine base-requiring and adenosine deaminaseless mutant was obtained as the favorable one. The mutant accumulated more than three fold as much as that by the parent strain.

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Various proteases were compared with each other for their ability of generating tastes from a fish protein concentrate (FPC), and Pronase was selected as an enzyme producing a large amount of brothy taste peptides. From the FPC hydrolysate obtained by treatment with this enzyme, an acidic fraction of mol. wt. lower than 1000 was prepared by ultrafiltration and subsequently by column treatments with activated charcoal and two different ionexchangers. The acidic fraction was rechromatographed on Amberlite CG-120 to obtain a fraction containing neither free aspartic acid nor free glutamic acid. The resultant acidic, oligopeptide fraction was found to taste considerably brothy and have a favorable after-taste effect.

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3-Isobutyroxy-β-ionone (III) is the proposed structure of quiesone, the naturally occurring inhibitor of the germination of Peronospora tabacina conidia. This was synthesized as a racemate and shown to possess qualitatively identical biological activity as quiesone itself. Employing an intermediate of this synthesis, dl-dehydrovomifoliol (VII) was also synthesized.

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The Rupe rearrangement of the appropriate acetylenic alcohols afforded dihydro-β-damascone (XII) and its 3-hydroxy derivative (I). The latter is an aroma component of Manila leaf tobacco.

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