1. The presence of parasexual life cycle in Aspergillus oryzae was identified again. 2. The frequency of diploidization was increased from less than 5x10-4 to a few percent of the survivors by irradiating heterocaryotic conidia with ultraviolet light. The value is sufficiently high to utilize the process for breeding purpose. 3. Comparing the survival curves of two haploids and the heterocaryon with that of the heterozygous diploid, the diploid conidia were considered to have no selective advantage in survival during the irradiation. 4. As the first order somatic segregants of a diploid, ten out of the twelve possible phenotypes which were expected to come from the combination of white leucineless and yellow lysineless were really recovered. Some of them were proved to be still diploids. 5. A few supposed triploid and tetraploid strains were produced from the cross between a haploid and a diploid and between two diploids. 6. Ultraviolet light was demonstrated to be effective upon the segregation of a diploid as well as on the formation of a diploid. The effect on the former depends on the strain. 7. An attempt which has been carried out to improve industrial strains through this process will be published later.
Eleven bacteria, which were newly isolated from soils and sewage and are capable of producing α-ketoglutaric acid with higher yields such as 40-60% to glucose supplied in aerobic fermentation, were studied from the taxonomic point of view. Precise descriptions of morphological, cultural and physiological characteristics were performed. As a result of these descriptions two bacteria were identified as Escherichia coli, three as Escherichia freundii, one as Aerobacter cloacae. However the remaining five bacteria possessed polar flagella and fermented glucose, lactose and other sugars vigorously with the production of acid and gas. After exhaustive discussion we reached the conclusion that they should be assigned to a new genus in the tribe Pseudomonadeae. Consequently we proposed a new genus Kluyvera to which they should be assigned. A description of the new genus and descriptions of two new species of the genus Kluyvera are presented.
1. Searches for the microorganism which are able to grow in the medium, containing α-ketoglutaric acid and ammonium chloride, and do accumulate L-glutamic acid in the medium were carried out. The accumulation of L-glutamic acid was detected in the cultures of the microorganisms belonging to the following genera; Agrobacterium, Aerobacter, Aeromonas, Bacillus, Bacterium, Erwinia, Escherichia, Micrococcus, Serratia, Pseudomonas, Xanthomonas; Debaryomyces, Hansenula, Mycotorula, Pseudosaccharomyces, Saccharomyces, Willia, Aspergillus, Penicillium and Rhizopus. A strain of Pseudomonas ovalis showed the highest level of L-glutamic acid accumulation. A description of the morphological and physiological properties of this strain were presented. This strain was used in the subsequent experiments. 2. A test of the effects of nitrogen sources on L-glutamic acid formation revealed that ammonium chloride was the best nitrogen source for the L-glutamic acid formation. 3. The aerobic condition was favorable for the growth of this organism, but not for the accumulation of L-glutamic acid. 4. The accumulation process of L-glutamic acid was separated from the growth phase of the organism. After the organism was grown in the medium A3 on the shaker at 30°C for 17 to 18hrs., the cells were harvested by centrifugation and washed thrice. The washed cells were added to the fresh medium A3, and incubated at 30°C. The initial concentration of the cells were 30mg cells (wet weight) per ml. The highest yield of L-glutamic acid was obtained when the pH of the medium was maintained neutral or slightly alkaline and the incubation was carried out anaerobically. Within 24 to 30hrs., about 98% of the added α-ketoglutaric acid was consumed and 30μM L-glutamic acid per ml, an amount corresponding to 60% of the consumed α-ketoglutaric acid, were accumulated in the medium. 5. When the cell suspension was incubated aerobically or the pH of the medium was not controlled, the accumulation of L-glutamic acid remained at a lower level. 6. The enzymological studies suggested that L-glutamic acid was formed through either one, or both, of the following pathways; 1. α-ketoglutarate+TPNH2+NH3-L-glutamic dehydrogenase→ L-glutamate+TPN+H2O 2. α-ketoglutarate-TCA cycle→fumarate fumarate+NH3-aspartate→L-aspartate L-aspartate+α-ketoglutarate-aspartic-α-ketoglutaric transaminase →oxalacetate+L-glutamate The pathway 1 seemed to be mainly working in our organism used. 7. The problems derived from this work and left in the future studies were pointed out.
The principal pathways in the enzymic degradation of yeast ribonucleic acid by Aspergillus oryzae var. No. 13 have been established as shown in Figure 14, mainly by means of characterization of enzymic degradation products. Paper electrophoresis, paper chromatography, and ultraviolet spectrophotometry were employed for characterization. Ribonucleodepolymerase degrades yeast ribonucleic acid into 3′ (or 2′)- adenylic, 3′ (or 2′)-guanylic, 3′ (or 2′)-cytidylic, and 3′ (or 2′)-uridylic acids without liberation of inorganic phosphate. It does not degrade sperm deoxyribonucleic acid. The activity is still preserved after dialysis and contact with weak-base anion exchange resin (Amberlite IR-4B) or Japanese acid clay. This enzyme is remarkably thermostable, especially in the range of pH from 5.5 to 6.5. In 0.05N sodium acetate solution 94% of the activity is retained after heating for 10 minutes at 100°C. The optimum conditions for the activity are at about 60°C and pH 4.0. This enzyme is not influenced by ninhydrin, monoiodoacetate, potassium cyanide, as well as sodium fluoride. Mononucleotide phosphatase is thermolabile, and readily inactivated by physical or chemical treatments. Evidence suggests that this enzyme has properties similar to those of the general acidic phosphomonoesterase of plant origin. A brief procedure has been devised for the simultaneous detection of ribonucleodepolymerase and mononucleotide phosphatase on an agar plate. The splitting of inosine and guanosine is carried out by nonphosphorolytic hydrolase. Adenosine, cytidine, and uridine are not split by the Aspergillus oryzae enzyme. The direct liberation of hypoxanthine from 5′-inosinic acid by hydrolysis was first recognized. The enzyme responsible for this reaction is a nonphosphorolytic hydrolase and attacks 5′-inosinic acid specifically. Thus it seems that "5′-inosinate-N-ribosidase" might be an appropriate name for this enzyme.
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