In our institute, Bac. subtilis var. amyloliquefaciens FUKUMOTO K 49 which produces amylase in culture media had been employed for the studies on DNA, DNase and bacteriophages. In connection with these studies, some genetic properties of this strain K 49 was investigated. One resistant strain was obtained among approximate 10^6 K 49 cells which had been treated with 100 to 200 units/ml penicillin G in culture medium, while about 1 per 10^8 with 200 to 500 μg/ml dihydrostreptomysin. Phage-resistent strain was also isolated with rate of 1 per 12,000 to 13,000 cells after treating them with excess of phage. On the other hand, they survived nearly proportionally to time exposed to UV light 50 cm under 10 W UV lamp. M I medium prepared mainly from soybean extract (note of Fig. 2) is the most suitable one for K 49 and has been employed in the industrial production of amylase, whereas K 49 does not grow in some synthetic media such as FY (note of Fig. 4) and Anag (note of Table 7). However, when yeast extract or casamino acid had been supplemented to them, K 49 can grow, suggesting the presence of certain nutritional factor necessary for growth of K 49. Through paper chromatography of yeast extract followed by auxanographic test using FY as minimum medium, Rf values of the active substance were calculated to be 0.62 with butyl alcohol : acetic acid : H^2O (4:1:2) or 0.87 with 80% phenol. Further auxanographic examination with pieces of crystal of various amino acids revealed that only leucine is effective for growth of K 49, whereas no ability was observed with valine, isoleucine, threonine, arginine, glutamic acid, phenylalanine, tyrosine and methionine. Maximum growth was attained by addition of about 200 μg/ml leucine to FY medium. However, growth and amylase production were more or less delayed in the supplemented medium. Considering the biosynthetic pathway of leucine, effect of α-ketoisovalerate and α-ketoisocaproate on the growth of K 49 was also examined. The auxanography indicated that the latter is available for K 49 in place of leucine, while the former is not. Thus, it was demonstrated that the strain K 49 requires leucine or α-ketoisocaproate for growth as the indispensable nutritional element, because biosynthesis of leucine by K 49 is blocked between α-ketoisocaproate and α-ketoisovalerate. When spores or extremely low amount of K 49 cells had been inoculated to leucine-supplemented FY medium, no growth was observed, while growth was attained in M I complete medium after the same inoculation. However, growth was established by additional supply of some extract of K 49 cells, yeast or soybeans to leucine-FY medium. This probably suggests that another nutritional factor than leucine must be present in media for growth of K 49 from spore stage. By auxanographic surveys with amino acids, vitamins, nucleotides and others, the factor could not be identified. However, for isolating it, solubility was preliminary examined and illustrated. Namely the factor is typically soluble in water, acetic acid and methyl alcohol, whereas it is insoluble in ethyl alcohol and acetone of high concentration or ether. Transformability of K 49 was comparatively examined with that of Bac. subtilis Marburg. Transformation could not be observed for K 49 concerning leucine requirement and streptomycin resistivity, while that as to tryptophan and histidine requirement was attested with high rate for Marburg strain. Thus, it was demonstrated that transformability of K 49 was not or very hardly observed.
アミラーゼ生産菌Bac. subtilis var amyloliquefaciens FUKUMOTO K 49について耐性獲得能,栄養要求性,型質転換能など二,三の遺伝的性質を検討した. (1) ペニシリン耐性菌は培地1mlあたり100ないし200単位添加培養することによつて概略10^6あたり1個の割合で得られた.またデヒドロストレプトマイシンでは200μgないし500μgで10^8あたり1個の割合であつた.一方ファージ耐性菌は過剰のファージ処理によつてほぼ12,000ないし13,000に対し1個の比較的に高い割合で出現した.しかし紫外線照射に対しては少なくも用いた条件(10W,50cm)では照射時間に応じて菌の生存数が低下した. (2) Auxanographyによつて本菌の生育にはロイシンが必要であるがバリンやイソロイシンは必要でないこと,およびこれはロイシン合成経路のうちα-ケトイソバレリアン酸とα-ケトイソカプロン酸との間が菌に欠けているためであることが明らかになつた.しかし胞子から生育する場合にはさらに別の因子も必要であつて菌体内のほか大豆粕および酵母抽出液にも含まれていた.しかも26種類のアミノ酸,ポリペプトンないしカザミノ酸,18種類のビタミン類を初めヌクレオチド,有機酸その他いずれも無効であつた.さらにこの有効因子の種々の溶媒による溶解性も検討された. (3) 形質転換能をMarburg株のものと比較検討し,少なくもMarburg株で可能な条件では出来ないことを示した.