Studies on the Crystalline Substance Mainly Composed of MgNH_4PO_4・6H_2O Produced by Collybia velutipes (II)

1. Collybia velutipes (the parent strain) has lost the faculty of mushroom development, when cultured on the malt extract medium for several years. Studying the difference in inorganic component between the mycelia of the parent strain and the sterile descendant, the author observed a distinctive periodical fluctuation on phosphorus and magnesium components of the sterile descendant. Crystalline components were found in the old mycelium. They seemed to be a triclinic system (2×3mm.). Its volume was diminished by about one-third by heating five minutes at 150° in air, and its glassy surface turned dull by heating five minutes at 100° in water. By qualitative analysis, there was found much phosphate, magnesium and ammonium, with a little calcium and oxalate, and a trace of silicon. Its chemical formula was identified to be MgNH_4PO_4・6H_2O, contaminated with one percent of calcium oxalate. The crystalline substance is a product of the autolysis of the mycelium. 2. The author cultured mycelia on the malt extract medium prepared by the general method. Inorganic components were analysed at intervals, in the mycelia and the medium. In the normal mycelium (the parent strain), there was a little mineral component exuded from the mycelium into the medium, even after 70 days culture at 24°. When they were incubated at cool temperature soon the mushroom developed. But in the varied mycelium (the sterile descendant), when they were incubated for about 40 days, such mineral components as phosphorus, magnesium and calcium exuded into the medium. After 50 days incubation, these phosphorous compounds accumulated into the mycelium with the magnesium salt which was in the medium. The PO_4/Mg ratio increased in the mycelium was slightly a less than the PO_4/Mg ratio of MgNH_4PO_4. 3. The author incubated mycelia on a malt extract medium (Ballg. 7°, pH 6.0), and periodically analysed quantitatively the mycelial phosphorous compounds as acid soluble-P (inorganic-P, ⊿7-P, and residual organic-P), phospholipid-P, nucleic acid-P and phosphoprotein-P. In the varied mycelium or sterile descendants, there were distinctive fluctuations in the acid soluble-P and the nucleic acid-P by the culture days. That is, in the early culture, almost all the phosphorous compounds in the medium were absorbed by the mycelium. There were relatively abundant inorganic compounds in the young mycelium. When it came to the maximum growth, inorganic compounds reached a minimum, and ⊿7-P and other organic-P came to the maximum. After the maximum growth, organic phosphorous compounds decreased gradually and inorganic phosphorous compounds increased in the mycelium. Then they exuded out from it into the medium. In the old culture, some of them returned again into the mycelium (mycelial mass) producing a crystalline substance. 4. Grinding the defatted mycelium in a mill, extracted the ribonucleic acid (RNA) by 10 percent NaCl, and purified by barium acetate solution. It was N 13.93%, P 7.63%, N/P 1.83. Applying the RNA of the sterile descendant, the decomposition by the nuclease system in the old culture filtrate was investigated synthetically. In the old culture filtrate, there was much inorganic-P. So the dialysed filtrate was prepared as an enzyme solution. By this treatment, inorganic-P releasable activity remarkably decreased. Its optimum pH was 5.8 interfered by NaF. By the treatment of dialysis, and sensitivity for heating or NaF, the enzyme activity of the culture filtrate was divided into two groups. The one was RNA degradable, stable to heat and was not interfered with dialysis and NaF. The other was inorganic-P splitable, feeble to heat and was interfered by dialysis and NaF. The optimum temperature of them were 50°. 5. Collybia velutipes (the parent strain) and its sterile descendant were cultured on the malt extract medium and were analysed periodically its carbohydrate fractions as 10% trichloro acetic acid (TCA) extractable, 30% KOH extractable, Fehling's solution precipitable, 2N acetic acid extractable, and insoluble carbohydrate. In the normal mycelium or the parent, there was not any distinctive differences in the young and old mycelia. But in the varied mycelium or sterile descendant, there were distinctive differences in them. Especially, TCA extractable fraction, was 39.73% in the young, and 13.15% in the old mycelium. In the insoluble fraction, there was chitin or chitin-type, about 3.8% of the mycelium. It was negative to carbohydrate reaction by anthrone reagent, or to protein by nynhydrin, and reduced Fehling's solution, N 4.8%, after being treated 3 hours by cone. HCl yielded a crystalline substance. There was a little cellulose or cellulose-type in the insoluble fraction. It was insoluble in 10% hot H_2SO_4, and readily soluble in 50% hot H_2SO_4. It reduced Fehling's solution. In the hydrolysed carbohydrates of mycelia, much glucose and a little mannose or mannose-type was observed by paper partition chromatography. 6. The author cultured mycelia of sterile descendant on the malt extract medium, and fractionized polysaccharides in it with ethanol. Though there were some polysaccharides in the medium before the incubation, when a mycelium grew on it, they decreased, and other polysaccharides were produced in it. When they were incubated normally on the medium, polysaccharides which were precipitated in 50% or 60% ethanol were produced in abundance. When they were incubated in the medium anaerobically, "water-hardly-soluble fractions" were produced in abundance. In this fractions there was nitrous substance, N 6.17%, it did not show any protein or carbohydrate reaction. Perhaps it will be chitin or its derivative. 7. a). Oxalic acid and ammonium production, and variation of pH in the medium were tested periodically for 75 days. b). Condition of MgNH_4PO_4 formation on the normal temperature were tested, for ammonium magnesium phosphate may exist in different forms, according to the composition of the mother liquid at the time of precipitation. Precipitates of ammonium magnesium phosphate were obtained by adding NH_4OH to the mixed solution of KH_2PO_4, MgCl_2 and (NH_4)_2SO_4 or C_2O_4(NH_4)_2. When (NH_4)_2SO_4 was used, precipitation began pH 5.10, and C_2O_4(NH_4)_2, pH 5.78. The effect of potassium and ammonium oxalate were to decrease the weight of the precipitate for the normal. But the existence of oxalate was to produce a normal compound, MgNH_4PO_4・6H_2O, unless excessive MgCl_2 existed. In conclusion the author wishes to express his cordial thanks to Prof. S. FUNAHASHI of the University of Tokyo, the late Prof. K. TOGASHI, Prof. S. IMAI, Mr. K. NARITA of our university and Dr. H. E. GRUEN of Harvard University for the kind advices and helps which they have given to him in the course of writing this thesis