Invertase (β-D-fructofuranoside fructohydrolase, 3. 2. 1. 26) of Candida utilis was obtained not only from the autolysate of the cells, but also from the culture filtrate and the supernatant of the aerobically incubated cell suspensions. The invertase formed in the yeast cells was not influenced by the kind of sugar used as a carbon source for culture, and the release of the enzyme from the cells occurred under aerobic condition. The addition of sugar to the aerobically incubated cell suspension prevented the enzyme release. Liberation of invertase from the yeast cells was also found to occur under another condition, and the procedure designated as the salt-steeping method was described in the present paper.
Among the invertase preparations by the above methods, however, the preparation from the aerobically incubated cell suspension was most pure and obtained easily in a highly purified state by treating with columns of DEAE-cellulose and Sephadex G-200.

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A variety of microorganisms were tested for their extracellular L-asparaginase productivity and it was found that many bacteria, fungi and yeasts are positive for it. Especially some strains in the genera Pseudomonas, Candida and Rhodotorula were able to produce a large amounts of the enzyme. Escherichia coli, however, that contained intracellular enzyme was unable to produce extracellular one. In enzymologieal properties some differences were noted among these extracellular enzymes. Pseudomonas asparaginase showed glutaminase activity too, but the asparaginases of Candida and Rhodotorula were unable to hydrolyze glutamine. Candida L-asparaginase was most stable to heat-treatment.

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Oxidation of methyl 2-trans-β-ionylideneacetate with N-bromosuccinimide afforded methyl 2-cis and trans-3'-hydroxy-β-ionylideneacetates. NaBH₄ reduction of methyl 2-cis-3'-keto-β-ionylideneacetate and ethyl 4'-keto- α-ionylideneacetate gave methyl 2-cis-3'-hydrxy-β-ionylideneacetate and ethyl 4'-hydroxy-α-ionylideneacetate respectively. Further, methyl 4'-methoxy-epoxy-β-ionylideneacetate was prepared by epoxidation of methyl 4'-methoxy-β-ionylideneacetate. And then methyl 4'-hydroxy-1', 2'-dihydro-β-ionylideneacetate was synthesized from ethyl 4-keto-α-cyclogeranate. Growth inhibitory activities of the above compounds on rich seedlings were examined.

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Several conditions for the formation of coenzyme A from pantothenic acid and cysteine in the presence of ATP were investigated using Brevibacterium ammoniagenes IFO 12071 (ATCC 6871). The higher activity of the formation of coenzyme A was observed with the cells grown on acetate-medium. The effects of the substrate concentration, phosphate buffer concentration, pH and cell concentration on the formation of coenzyme A were examined and the amount of coenzyme A formed reached a maximum value of 1.2mg per ml of the reaction mixture. Several surfactants belonging to cationics and anionics brought about 1.8-fold stimulation of the formation of coenzyme A.
The considerable accumulation of coenzyme A in the culture broth during the growth was also observed.

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Changes of salt-soluble and salt-insoluble zymogen β-amylase (Z-β-A), papain-soluble and papain-insoluble Z-β-A were pursued during germination of barley. These four types of Z-β-A decreased as germination progressed and correspondingly the salt-soluble active β-amylase. (A-β-A) increased. The gibberellic acid (GA)-treatment during germination c-celerated the activation of Z-β-A, but had no effect on the amount of total β-amylase.
The incubation of isolated aleurone layers with 0.5 μM GA at 25°C for 5 days resulted in a remarkable increase in the total saccharogenic activity, to which the contribution of β-amylase was found to be much smaller than that of α-amylase.
From these results it may be reasonable to conclude that the increase in A-β-A during germination is ascribable to the activation of Z-β-A and that the amount of A-β-A synthesized during germination, if any, is negligibly small in comparison with that of A-β-A activated from Z-β-A.

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Many amino acids were liberated by acid hydrolysis of the non-cationic fraction of pea seedling extract, suggesting the existence of ninhydrin-negative conjugated amino acids. The non-cationic fraction was subjected to cellulose column chromatography and gel-filtration with Sephadex G-15 and was separated into 20 fractions which gave ninhydrin color after alkaline hydrolysis. Alanine and glutamic acid, the most predominant amino acids in the hydrolyzate of the non-cationic fraction, occurred in many fractions. These results indicated that alanine and glutamic acid existed in various conjugated forms. In one of the fractions, there was evidence suggesting the presence of a peptide containing equimolar amounts of alanine and glutamic acid.

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The browning reaction in soy sauce was compared with that in the model system containing sugars (glucose 4%, xylose l%) and amino acids (glycine 5%, glutamic acid 5%). In both cases, browning was developed on heating or static oxidation (spontaneous oxidation). On shaking oxidation (obliged oxidation), on the contrary, browning was observed only in soy sauce, and in the model system it was rather repressed by this treatment. The brown pigments in the model system were fractionated into two components, designated as F-A and F-B, by gel filtration with Sephadex G-25. The amount of F-A was increased on oxidation, and that of F-B was increased on heating but decreased on oxidation. The color of F-A was dark tone and that of F-B was light tone. F-A and F-B were suggested to be analogous to brown pigment P-I and P-III in soy sauce, respectively, judging from the results of gel filtration, characteristics of the color and UV absorption spectra.
Amino acid solutions (hydrolyzates of soybean) resembled soy sauce more closely than the model system in the browning behaviors on shaking oxidation and in chromatographic patterns of brown pigments on Sephadex G-25.
In addition, not only glycine and glutamic acid but also many kinds of amino acids were found to be lost considerably during the browning of soy sauce.

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A compound, non-acetylatable and diazotizable Bratton-Marshall's reaction-positive compound, was accumulated in pea seedlings in a “folate-deficient” state induced by soaking the seeds in 2×10^-3M sulfonamide, 5-methyl-3-sulfanilamide isoxazole. The compound was isolated and characterized as 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICA-ribotide), known as an intermediate compound in the de novo biosynthesis of inosinic acid in animals and microorganisms. Characterization was based on its paper chromatographic behavior, ultraviolet absorption spectra, infrared absorption spectrum, and the molar ratio of its constituents.
Using the isolated AICA-ribotide as substrate, AICA-ribotide formyltransferase (EC 2. 1. 2. 3) was found in extracts of pea seedlings. The enzyme was purified about 40-fold and some of its properties were investigated. Optimum pH was 7.4, and optimum temperature was 37°C. In the enzyme reaction, AICA-ribotide and 10-formyltetrahydrofolate were specifically required as substrates with K⁺ as an essential component. Michaelis constants for AICA-ribotide and 10-formyltetrahydrofolate were 3.1×10^-5M and 2.0×10^-4M, respectively. Since the enzyme preparation contained inosinicase, the product formed in the enzyme reaction was shown to be inosinic acid by paper chromatography.

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A partially purified phosphopeptide fraction with low N/P ratio has been obtained from the small intestinal contents of rats, which was collected at the first one hour after 1.5hr spaced-feeding of the diet containing 20%, casein.
After precipitation of protein with trichloroacetic acid, acid-soluble fraction was chromatographed on the column of Sephadex G-25.
A fraction containing large peptide (s) having the N/P atomic ratio of about 7 to 9 and free from lower molecular substances was rechromatographed with Sephadex G-50.
This fraction, amounted nearly 30% of the total non-protein nitrogen of the intestinal contents, showed high calcium-binding activity as determined with Chelex-100.
These results suggest a special role of phosphopeptide stage on the stimulating calcium absorption in the intestinal tract during the digestion of casein.

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Changes of casein and lysozyme during roasting at various times and temperatures (100_??_300°C), especially those in amino acid composition and formation of some nonvolatile degradation products, were investigated.
Tryptophan, methionine, basic amino acids and β-hydroxy amino acids were easily decomposed as compared with acidic amino acids and other neutral amino acids in casein and lysozyme. In hot water extract of roasted casein were detected some free amino acids, peptides, organic acids such as α-ketoglutarie, tartaric and malic acids, and indole. It is considered that free amino acids are produced mainly through ionic cleavage of peptide bond with the water bound within casein.

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Strain M-1 which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω, ω'-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.
On the other hand, since the growth rate of strain M-1 on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-1 was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8g/liter was obtained ofter 3 days' cultivation.

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Crystalline nuclease O obtained from autolyzed Aspergillus oryzae hydrolyzed heatdenatured calf thymus DNA 19 times faster than native DNA. Digestion of the heatdenatured DNA with an exess of the enzyme produced mono-, di- and tri-nucleotides with 5'-terminal phosphate, which amounted 3.4, 58.3 and 38.2%, respectively, of total degradation products. Hydrolysis of the native DNA with a sufficient amount of nuclease O produced mono-, di-, tri- and tetra-nucleotides with 5'-terminal phosphate, which amounted 1.9, 47.9, 36.7 and 13.6%, respectively, of total degradation products. Although nuclease O showed no strict base specificity on the native and heat-denatured DNA, diand tri-nucleotides in the digests were resistant to further hydrolysis by nuclease O. Native DNA was hydrolyzed by nuclease O through the mechanism of single strand break, which was shown by neutral and alkaline sucrose density gradient centrifugations.

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Two new analogues of the Cecropia juvenile hormone lacking the methyl group at C-3 were synthesized and were shown to possess very high juvenile hormone activity.

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Glutamine and ornithine were found to stabilize effectively carbamoyl phosphate synthase (CPSase) partially purified from the larvae of Aldrichina grahami reared aseptically.
Glutamine, ATP, and Mg ion were required for the enzyme reaction. A high concentration of ammonia could replace the requirement of glutamine; N-acetylglutamate could not enhance the reaction. The apparent Km for ammonium ion, however, was much higher than that for glutamine. The concentration of ATP required for half maximal velocity was 1.0×10^-2M.
Various kinds of nucleotides of pyrimidines and purines inhibited the enzyme reaction. The reaction product in the assay system radioautographically coincided with citrulline.

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Microbial transformation of the nucleoside analogue antibiotic showdomycin was performed using some Streptomyces species. Both the growing culture and the resting cells of Streptomyces sp. No. 383 arrested the antibacterial activity of showdomycin. The inactivated showdomycin was isolated from the reaction mixture by carbon chromatography and was identified with an isoshowdomycin sample which has been chemically derived from showdomycin. It is conjectured that the conversion of showdomycin to isoshowdomycin results from isomerization by an enzyme of Streptomyces sp. No. 383.

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In the course of the study on glutamic acid fermentation by Arthrobacter paraffineus, in which n-paraffin was used as the sole source of carbon, it was observed that fatty alcohollike lipid was accumulated in the paraffin layer of culture medium. This was isolated and identified as the primary fatty alcohol having the corresponding carbon skeleton to that of n-paraffin used as the carbon source.
The accumulation of fatty alcohol occurred rapidly in the early-log phase. The maximum amount of the accumulation was approximately 0.5mg/ml after 6 hr incubation. Incontrast with the production of glutamic acid and trehalose, the addition of penicillin gave no effect on the accumulation of fatty alcohol.
Acetone-treated cells of the n-paraffin-grown bacterium still had the oxidative activity of n-paraffin, and the formation of fatty alcohol from n-paraffin was observed only by the reaction in alkaline pH.

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Brevibacterium ammoniagenes ATCC 6872 was previously reported to accumulate large amounts of IMP, AMP, ADP, ATP, GMP, GDP and GTP from the corresponding purine bases. Using the similar process, ribotidation of some purine analogues was attempted, and it was found that Br. ammoniagenes ATCC 6872 was able to produce remarkable amounts of 6-thio-IMP, 6-thio-GMP, 6-thio-XMP, 6-methylpurine riboside 5'-monophosphate (6-methyl-Pu-RP), 2-methyl-IMP, 6-methylamino-Pu-RP, 8-aza-IMP and AICAR from the corresponding base analogues. When 2, 6-diamino-Pu, 6-hydroxylamino-Pu, 2-fluoroadenine, 2-methyladenine, 8-azaadenine or 8-azaguanine was added to the culture, 5'-diphosphate and 5'-triphosphate in addition to 5'-monophosphate of the corresponding riboside were produced. When the organisms was incubated in the presence of 8-aza-guanine, 8-aza-2' (or 3'), 5'-GDP was accumulated together with 8-aza-GMP and 8-aza-GDP in the culture medium.
It was also found that IMP was accumulated with 6-thio-IMP in the culture medium of the organism incubated with 6-mercapto-Pu.
The crystals of sodium salt of 6-thio-IMP was isolated from the culture medium of the organism incubated with 6-mercapto-Pu. It was recognized that 6-thio-IMP had _??_ IMP-like taste.

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Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with L-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259, 000. The crystalline enzyme catalyzed the conversion of L-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from D-tyrosine, Smethyl-L-cysteine, 3, 4-dihydroxyphenyl-L-alanine, L- and D-serine, and L- and D-cysteine, but at lower rates than from L-tyrosine. L-Phenyl-alanine, L-alanine, phenol and pyrocatechol inhibited pyruvate formation from L-tyrosine.
Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, L-tyrosine, or the competitive inhibitors, L-alanine and L-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.

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α-Glucosidase (α-D-glucoside glucohydrolasc; EC 3. 2. 1. 20) has been extracted from bovine spleen and separated into two fractions (Fractions A and B) by gel filtration on Sephadex G-200. Fraction B which was retarded on Sephadex columns contained only an acid α-glucosidase. This enzyme catalyzed hydrolysis of maltose and glycogen at comparable rates. Glycogen was hydrolyzed almost completely to glucose. The results of heattreatment and of inhibition by turanose demonstrated that Fraction A contained both acid and neutral α-glueosidases. The neutral enzyme in Fraction A was further separated into four different components on preparative polyacrylamide gel electrophoresis. All of these neutral enzymes showed similar catalytic properties each other, and hydrolyzed maltose much more rapidly than glycogen. The acid enzyme in Fraction A was inactivated on the electrophoresis and was not further characterized.

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To establish a novel process for the production of L-glutamic acid from n-paraffins, a glycerol auxotroph GL-21, a new type mutant, was successfully obtained from Corynebacterium alkanolyticum No. 314 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This auxotroph required glycerol for its growth regardless of the carbon source used.
At 72 hr, this mutant GL-21 produced about 40mg/ml of L-glutamic acid from n-paraffins in the culture broth at 0.01 per cent addition of glycerol in the absence of penicillin.
A thiamine auxotroph, a biotin auxotroph and an oleic acid auxotroph were also obtained by a similar technique, but these auxotrophs were found to be inapplicable for theproduction of L-glutamic acid from n-paraffins.

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Application of thin-layer chromatographic techniques to the analysis and preparation of triose reductone from naturally occuring reductone compounds has become an important tool in reductone chemistry. A satisfactory method for the separation of triose reductone and related compounds by thin-layer chromatography, using silica gel plate and various solvents as developer, is described. Seven reductones were separated from each other by two-dimensional chromatography.

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