Kinichiro Sakaguchi

Study of alloisocitric acid fermentation

Abstract The occurrence of l s -alloisocitric acid in nature, as a new fungal product, has been demonstrated in the culture medium of P. purpurogenum var. rubrisclerotium No. 1148. The acid was obtained as its γ-lactone in a crystalline form. l s -Alloisocitric acid was the only detectable acid accumulated by this fungus under the given conditions, and the yield of the acid exceeded about 70% of glucose consumed. Two other species of Penicillium fungi were found to produce this acid from glucose. Tentative experiments concerning the mechanisms of this acid fermentation were undertaken using the resting-cell suspensions. An adaptive enzyme, DPN-specific l -alloisocitric dehydrogenase, was obtained from Achromobacter sp.

Study of alloisocitric acid fermentation. II. Tracer experiments on the fermentation mechanism

Abstract The mechanism of alloisocitric acid fermentation by the fungus, Penicillium purpurogenum var. rubrisclerotium No. 1148, was studied using C 14 O 2 and citric acid-1,5-C 14 . By the aerobic incubation with glucose in the presence of C 14 O 2 , C 14 O 2 was incorporated into carbons 1 and 6 of alloisocitric acid in equal amounts. The pathway was assumed including the carboxylation of the C 3 compound to the C 4 -dicarboxylic acid, and the condensation of C 2 with the C 4 -dicarboxylic acid, which was randomized completely through the dicarboxylic acid shuttle. Isocitritase type condensation of C 4 with C 2 , Thunberg reaction, and the glyoxylic acid bypath seemed unlikely to be operative. The incorporation experiments of citric acid-1,5-C 14 showed that the labeling patterns of the residual citric acid and alloisocitric acid formed were completely the same. It suggested the presence of some direct conversion pathway from citric acid to alloisocitric acid without splitting of the carbon skeleton. From these with some other data, it was presumed that alloisocitric acid might be formed immediately from citric acid which was recycling through the tricarboxylic acid cycle. The presence of citric condensing enzyme and a few other enzymes of the tricarboxylic acid cycle was shown.

Accumulation of allo-iso-citric Acid by Genus Penicillium. Part I

Pen. purpurogenumの一変種によってグルコースより大量に培地中に蓄積される有機酸が従来天然には認められて居なかったイソクエン酸のジアステレオマーであるLs-アロイソクエン酸である事を最終的に証明した. その蓄積の取量は消費グルコースに対して40%(重量)以上にのぼる事が認められた.

Studies on the Metabolism of D-amino acid in Microorganisms:Part. I. Degradation of D-glutamic acid in Genus Aerobacter

Several strains of bacteria belonging to genus Aerobacter were found to oxidize D-glutamate rapidly, while tbey show feeble oxidative activity toward the L-form even when they were grown in the medium containing DL-glutamate.The isolation of L-glutamate, a natural amino acid, from its DL-form was achieved by the degradation of D-glutamic acid using one of these strains.This may be the first observation on a natural amino acid obtained from the racemic one by the metabolic action of the organism.A new enzyme, D-glutamic acid oxidase, which is responsible for D-glutamate degradation in this organism and differs from Krebs’ D-amino acid oxidase, has been postulated.

Studies on the Natural Varitation of Koji-molds (Aspergillus oryzae and Asp. sojae)

Anastomosisに対する培養条件の影響をAsp. sojaeのcolor mutantを用いて, hanging drop culture及びmixed point inoculum plate cultureにより検討した. 1) 天然培地(麹汁,麦芽汁,米胚芽エキス,及び酵母エキス)では比較的低濃度がよい(通常の濃度の約1/10).即ち, anastomosisは培地の栄養状態の悪い時に起りやすい. 2) 合成培地(Czapek液)では1/2～1/20の濃度がよく,Czapek液中のK2HPO4, KCl, MgSO4の量の増減には関係がない. 3) Osmotic pressureのoptimumの濃度はNaCl 0.1mol., glucose 0.2mol., lactose 0.1～0.5mol.でこれらの濃度より高くても,低くても起りにくい. 4) C源とN源との関係はN源の絶対量が少く, C/N比の高い方がよい. 5) pHのoptimumの範囲はせまくpH 5.4～6.0であつて,酸性側では急に,アルカリ性側では比較的緩かに影響し,pH 4及びpH 8.8で完全に阻害される. 6) 前培養の培養期間は3ヵ月まではかわらないが, 7ヵ月になると一般に起りにくくなる. 7) 培養温度のoptimumは25～30°で繁殖の適温よりは幾分低い. 終りにのぞみ,有益な御助言に与つている和田教授及びAsp. sojaeのmutantを分譲された井口氏に謝意を表する.

A Study on the Durability of the “Kuro-Koji” Mould

It is widely recognized that “Tane-Koji”, or a starter for black Koji making, consisting chiefly, of conidia of black Aspergilli, keeps not so well as those of yellow green Aspergilli or Asp. oryzae and Asp. sojae. The present research has been carried out to find out resistant strains for preservation of “Tane-Koji” among Kuro Koji (black Asp.) moulds. About 14 years ago (from April .to July, 1935) one of the authors (K. SAKAGUCHI) collected 402 samples of Kuro-Koji or mouldy material for Awamori (a distilled wine from rice) and Imo-Shochtu (a distilled wine from sweet potato) making, of soils of the factories and of the fermenting mash, from the southern districts of Japan, such as Kagoshima and Miyazaki prefectures and Luchu and Hachijo islands, and isolated from them about three hundred different strains of black Aspergilli and classified themt(1). From those samples which have been preserved in tin cans for these 14 years, the authors isolated the black Aspergiilli and other moulds in May, 1949. (1) The isolated species, which have survived for so long a time, were A. Usamii (2 strains), A. Saitoi (28), A. Saitoi var. Kagoshimaensis (180) and A. awamori (40). While A. Inuii., A. aureus, and A. Nakazawai have not been found. (2) The amylolytic powers and the rates of acid productivity of the newly isolated strains were compared with those which had practically been used in the above fermentation industries. And several strains belonging to A. Usamii and A. Saitoi var. Kagoshimaensis were found to be effective for the purpose.

A Study on the Nature of the Conidial Walls of Asp. oryzae and Asp. sojae

As for the conidial walls of Asp. f laves-oryzae, THOM and CHURCH (1921)(1) pointed out that “the walls are so thickened as to leave circular, elongated, or winding pits giving a rough or echinulate effect when viewed with low magnification” One of the present authors(2), on his taxonomic studies upon Asp. oryzae strains, has found that some members of these yellow green Aspergilli, especially those used in Soy sauce or Tamari Soy sauce brewing, gave manifestly echinulate appearance in the surface of the conidial walls under ordinary or low magnification. and the walls of I conidiophores were seen to be quite smooth or clear against so-called “pitted” nature which are prevalent among ordinary members of this group. Accordingly SAKAGUCHi and YAMADA (1944)(3) hava proposed a new classification, dividing these strains into two main groups; i.e. (1) Asp. oryzae s. str. SAKAGUCHI and YAMADA or Asp. flavus-oryzae, TH. & CH. and (2) Asp. sojaae nov. sp. S. & Y. The present investigation has been intended to clarify the problem using an electron microscope. As shown in the figures, it has been found out, contrary to the authors expectation, that the both species gave equally echinulate appearance of the, same nature on their conidial walls, except that those of Asp. sojae have somewhat higher echinulation than the other in the extent of 0.2μ or higher which is within the limit of the resolution of ordinary microscope.

Studies on the Acid Fermentation by Rhizopus Species. (Part III)

1) フマール酸生産種(Rh. G. 34),乳酸生産種(Rh. G. 36)及び中間種(Rh. No. 7)を用ひ,培養基の各種水素イオン濃度に於ける葡萄糖からの生産物の量的關係を檢した. 2) 生理的條件に於て乳酸を生産する種類即ち乳酸生産種及び中間種は培養基のpH價の増大と共にフマール酸量:乳酸量の比を増し,乳酸のみを生産する種類(乳酸生産種)でも, pH=7を超えればフマール酸を伴つて生産する様になる. 3) フマール酸のみを生産する種類にあつても,上記の比は培養基の水素イオン濃度によつて影響され,中性乃至微アルカリ性に於て最低點が認められる. 4) 不揮發性酸,即ちフマール酸及び乳酸の生産の最適水素イオン濃度はpH=7の附近にある. 5) 揮發性酸の生産はpH=7附近から著しくなり, pH價と伴つて増加する.この事實は第2報の要旨に述べた様な分解方法の増強,又は中間生成物としての揮發酸の捕獲による集積の何れかの原因によることを意味すべきものとして興味が深い. 終に臨み高橋偵造先生並びに本實驗の一部を分擔された木下祝郎農學士に深厚な謝意を表する.

On Colour Reactions of Dibasic Acids of Fatty Series, Especially of Fumaric Acid

As the reagents required are resorcinol and sulphuric acid, the procedure is already known in the case of succinic A piece of fumaric acid of the size of a pins head is placed in a test tube with about ten times its quantity of powdered resorcinol and 1-1.5 c.c. of concentrated sulphuric acid then heated gently in the beginning and later briskly until it attains nearly 190-195•Ž. Whereby the liquid becomes red with a green fluorescence. After cooling add some water and heat again. Cool well and a few drops of the reaction products are introduced drop by drop into ammonia, which colours red with a green fluorescence. This reaction is to be given by the dilute solution of fumaric acid, thus :A few drops of a 0.1 % solution of fumaric acid placed in a test tube with equal drops of a 0.1 per cent solution of resorcinol and 1-1.5 c.c. of concentrated sulphuric acid gives a liquid which on treating as mentioned above shows a yellow colour with green fluorescence. Anhydride : i. e. maleinic anhydride gives quite the same colouration as met with by crystals of fumaric acid. Succinic acid gives a yellow colouration in the first part of the reaction and when introduced into ammonia it changes to a green fluorescent liquid. Malic acid on treating as above gives a liquid which on heating becomes at first yellow and then crimson-red even after the addition of water. A blueish violet colour is produced by adding 2 or 3 drops of this crimson-red colour fluid into ammonia. This colouration is quite specific to malic acid and may distinguish this