Deoxyribonucleic acid of amylase-producing bacteria : I. Trial of preparation from acetone-dried cells (1)

Concerning fresh and acetone-dried cells of amylase-producing bacteria, Bac. subtilis var. amyloliquefaciens Fukumoto, phosphorus distribution was determined by Schmidt-Thanhauser method. It was estimated that DNA-P is very low in content amounting to about 0.02 % of fresh cell or about 0.19 % of dried cell, while much more RNA-P is contained by 10 to 15 times of DNA-P. Therefore, it was suggested that this bacterial cell can not be suitable to DNA source in general, even if cells were multiplied manufacturally and discarded without any utilization. Using acetone-dried cells, several procedures for preparation of bacterial DNA were discussed by testing them. Firstly, cells were disrupted by grinding with some aid in mortar and extracted preliminarily with diluted NaC1 solution or 0.2 % citric acid to remove non-DNA substances as far as possible. From cell mass remained, DNA was extracted with 2 M NaCl and purified by several steps of treatment. These are mainly consist of Sevag\u27s method to remove proteins by chloroform-protein gel formation in concentrated NaC1 solution, and of Chargaff\u27s procedure to separate DNA from RNA by fractionation with alcohol in 10 % CaCl_2 solution. DNA was finally precipitated by adding it into 2 volumes of alcohol, removed moisture by stepwise treatment with alcohol of increasing concentration, washed with ether and dried in a vacuum desiccator, becoming white powder. However, it was found that this procedure is not satisfactory to preparation of DNA, regarding both purity and yield. Secondly, Dounce\u27s method using detergent was investigat e d. In this case, Japanese product, Emal OD, was used instead of original one, Duponol. However, DNA preparation also has much inpurities and its yield was very low. Finally, DNA was extracted with 0.5 % phenol according to Braun except that preliminary treatment with chloroform freezing cells had been omitted. From the extract, white powder of DNA was obtained after similar purification. The method was seemed to fit more or less for the purpose. However, the combination of phenol extraction with grinding in mortar was most suitable among all procedures tested. In connection with these trials, some properties of bacterial nucleoprotein as well as nucleic acid were observed. Especially, it may be noticed that Sevag\u27s method of removing protein is not complete. However, remaining protein can be eliminated by further treatment with chloroform in concentrated NaCl solution, when DNA precipitate had been fractionated with alcohol in CaC12 solution by Chargaff\u27s method after completion of Sevag\u27s procedure. Furthermore, it must be remembered that, in the procedure of Chargaff, RNA is precipitated together with DNA in CaC12 solution by adding 0.3 volumes of alcohol, in contrast from those of yeast. At any rate, DNA preparation from amylase-producing bacteria is very difficult and more suitable procedure must be investigated in respect of both purity and yield.アミラーゼ生産菌Bacillus subtilis var.amyloliquefaciens FukumotoK-49はアミラーゼを強力に生産する優良菌株であつて工業的アミラーゼ生産に広く利用されているが,液体培養に於けるファージの被害は特に解決を要する重要な問題である.一般に細菌バイラスの増殖に於て最も重要な役割をなすものは遺伝現象等に於けると同様にデオキシリボ核酸であつて,その性質の解明はファージ対策に不可欠な要素の1つである.我々の研究室では同じDNAバイラスである家蚕の多角体バイラスについて多年に亘つて研究が続けられ,本核酸の代謝系との関係も追及されて多くの興味ある知見が得られ,これらを含めた生成に関する機構が提案されている.而して家蚕バイラスで明らかにされたバイラス知見は細菌バイラスの場合にも有効な示唆を与えるものであつて,これらの関連性を考慮して先ずDNaseとの関係については既に検討を開始した.しかし核酸は複雑なものであり各生物に特異的な性質をもつため,唯単に酵素の面からの追及だけでは十分でなく核酸自体の研究と相侯つて初めて完全なものと云うことが出来る.一般に細菌のDNA含量は極めて低く,さらに分離操作中に細菌自体の酵素の作用を受けて部分的な変化を被り易いために,細胞内のままの完全なDNAを調製する事は甚だ難かしい.特にその最初の抽出操作は従来各種細菌についてそれぞれ多種多様の試みがなされたが,菌の種類によつて方法の有効性はそれぞれ異つており,従つてアミラーゼ生産菌についても多くの予備試験を要する事は当然である.他方,酒その他の味の成分として核酸乃至はその構成分の価値も報告され,とくにイノシン酸は新しい呈味物質として最近脚光を浴びつつある.またDNA原料としては含有量の高い胸腺や脾臓が最も優れている事は論を侯たないがその収集に制限があるので,若しアミラーゼ製造工場で酵素の生産に伴つて多量に培養増殖され然も現在利用される事なく廃棄されている菌体を利用し得るならば好都合であるのは当然であるので,その可能性を検討する事も無意味ではないと思われる