Bulletin of the Agricultural Chemical Society of Japan Vol. 24(1960) No. 7
Studies on the Biosynthesis of Lignin
Part II. Changes of the Contents of the Enzymes and Organic Acids concerning the Lignification in the Growth of “Yugao” Fruits (Lagenaria leucantha Rosby var. clavata Makino)
The higher activities were obtained at pH 4.8 and 5.4 in the crude enzyme of peroxidase and inhibited by potassium cyanide. β-glucosi-dase was present and its opt. pH was 4.8. Laccase was scarcely found in the fruits. Some organic acids were detected by paper chromato-graphy; succinic, malic, citric, tartaric, quinic and shikimic acids. Especially, both enzymes and shikimic acid were shown to decrease with proceeding of lignification.
Nutritional requirements of three strains of Ped. soyae1, 2), one strain of Ped. acidilactici (Kitahara's strain) were determined from a complete synthetic medium. Upon inspection by the single omission method, Ped. soyae required glycine-betaine3), uracil, riboflavine, pyridoxine or pyridoxal, pantothenic acid, nicotinic acid, leucovorin, biotin, glutamic acid, arginine, histidine, isoleucine, leucine, phenyl alanine, valine, tryptophane, methionine, cystine and serine with excep-tion of the P-factor4). In addition to the above mentioned nutrients, a representative strain of the organism required xanthine, folic acid, thiamine, aspartic acid and threonine for the minimum synthetic medium. Ped. acidilactici Kitahara's strain required riboflavine, pyridoxine, pantothenic acid, nicotinic acid, biotin and all components of the basal medium consisting of amino acids except methionine, but it did not require leucovorin, glycine-betaine and organic bases. Nutritional require-ments of Ped. penlosaceus, Kitahara's strains5, 6) was proved to be quite identical with Ped. cerevisiae Pederson7-9) (=Ped. pentosaceus Mees5, 10)).
The sunlight flavor occurred when humulone or lupulone was added to beer after main fermentation was completed, and among the other phenols used, only acetolupuphenone [1-acetyl-3, 3, 5-(γ, γ-dimethylallyl)-cyclohexadiene-(1, 5)-diol-(2, 6)-on(4)] was found to cause the sunlight flavor.
A new toxic substance, Teleocidin, was isolated from the methanol extracts of cultured mycellium of Streptomyces 2A 1563 resembling Streptomyces mediocidicus. Teleocidin has strong toxicity to mice and its chemical and physical properties were examined.
Biological activities of a new toxic substance, Teleocidin, produced by a Streptomyces were examined. Among the toxic antibiotics produced by Streptomyces, 9 antibiotics were also tested for their toxicity toward Oryzias latipes, Daphnia magna and Bugula neritina larvae, and Antimycin A was found to show equal or more severe toxicity than Teleocidin. Teleocidin has shown certain inhibitory actions to Ascaris suilla and Turbatrix aceti and also has severe toxicity and skin irritative effects on mammalians.
On the reduction of isopulegone with sodium in aqueous ammonia, it was found that the main product was isopulegol. This fact indicates that the double bond not in conjugation with carbonyl group is not attacked.
A series of studies on the crystalline molecular complexes of tetracycline groups have been carried out in this laboratory, and the present paper deals with the compositions of the oxalic acid complexes of various tetracycline acid salts, which were examined by X-ray powder diffraction methods, infrared spectra, and chemical analyses. Insertions of salt anions and crystal water in the crystal lattice of the complexes, and the important role of the latter for crystallization are indicated.
Examination of the volatile compounds from Japan hemp plant shows that the phenol fraction consists mainly of eugenol with small amounts of guajacol. Two unknown carbonyl compounds are separated as their 2, 4-dinitrophenylhydrazones by the silicic acid-celite column chromatography from the phenol fraction.
In order to isolate and identify the unpleasa odor of wild hemp leaves, tops of the plant its flowering time was extracted with petroleu ether at first and the residue was extracted wi 90% ethanol. In this ethanol soluble fractic piperidine contained in free and combined for, in hemp was isolated and identified by meal of the paper chromatography analysis and i_??_frared spectra. The melting point of the picrolonate derive from wild hempen piperidine was 242_??_245°C and that of authentic piperidine was 242_??_244°C The infrared spectra of both picrolonates wei found to be coincident and Anal., found:_??_20.08, calcd. for C15H19N5O5: N, 20.06%.
(1) The survival-ratios of E. coli irradiated in each of the various types of meats and fish meats differed from each one another. (2) Small-neck clam, oyster and shrimp meats belonged to the group having high protective effect on the survivals of the strain. (3) Halibut and salmon meats belonged to the group having low protective effect. (4) In case of the same meat and the same concentration, the survivals in a raw meat were higher than those in a heated meat. (5) There could be observed some correlation between this protective effect and the substances analysed as the amino-type nitrogen by means of the Van Slyke method.
In case of using a nutrient agar medium, all three conditions resulted in protective upon three strains same as in case of E. coli. However, in case of using a pure agar, the conditions which show higher survivals of E. coli in a nutrient medium were divided into three conditions. The first showed the protective effect same as in case of nutrient medium. The second showed no influence, and the last showed, on the con-trary, lower survivals of the strain than those from the control. The results of this experiment are summarized as follows: (1) Three conditions which were the addition of each of succinic acid, thio-urea and amino-acid in a nutrient agar medium showed the same protective effect on the survivals of the above three strains as in case of E. coli. (2) In case of using a pure agar medium for irradiation, the effects of these conditions differed from each other. Higher survivals, similar survivals and lower survivals of the strains were observed. (3) The addition of succinic acid in a pure agar resulted in higher survivals of the straines. (4) The addition of thio-urea in a pure agar caused lower survivals. (5) The addition of amino-acid in a pure agar brought no influence on the survivals. (6) In case of E. coli in a pure agar containing thio-urea, the higher concentration resulted in higher survivals than the control, however, the lower concentration than a certain limit gave lower survivals than those from the control.
Glucomannan was isolated from holocellulose pulp of pine (Pines densiflora Sieb. et Zucc.) using dimethylsulfoxide and aqueous alkali. It was found that there were present also, in the pulp, separate two glucomannans as the possible contaminants. Purification of the glucomannan by extraction with dimethylsulfoxide, the following acetylation and fractionation resulted essentially homogeneous glucomannan triacetate [α]D -30.7° contained mannose and glucose only in the ratio 36:10. Measurements of intrinsic viscosity in the chloroform solution gave [η] 0.776 and 0.291 for high and low molecular parts of the acetate respectively. The glucomannan derived its acetate showed [α]D-36.5° and 78.5% mannose content
The chemical nature of fatty acids was studied on sphingomyelin which was prepared from horse spinal cord. Nervonic, palmitic, stearic and behenic acids were isolated from the hydrolysate of the sphingomyelin preparation. The presence, of behenic acid in sphingomyelin has in this paper been reported for the first time.
(1) The velocity of the phosphorylation of α-D-glucose, catalyzed by the purified yeast hexokinase, seems to be greater than that of β-D-glucose. The relative rate of phosphorylation of β-D-glucose is observed to be 60-70 (α-D-glucose=100). (2) The average Michaelis constants of yeast hexokinase are found to be 1.8×10-4 and 2.4×10-4 when it acted on α-D-glucose and β-D-glucose respectively.
By using the multiple ascent technique, the authors have resolved the first several oligosaccharides of the product of initial stage of potato phosphorylase action in both the absence and the presence of beet-saponin with maltotriose as primer. The resolved chromatogram was sprayed with a mixture of G-l-P and phosphorylase, followed by spraying with iodine solution to locate the spots in which starch synthesis occurred. Multi-chain mechanism of amylose formation from maltotriose and the suppression of length-ening of amylose chain by beet-saponin in the lag stage of enzyme action could be shown on paper chromatogram. No saccharides other than amylose series were recognized in the case of phosphorylase inhibition by beet-saponin.
In relation to the elucidation of the mechanism of the browning reaction, isolation in crystalline form, structure and characterization of the intermediate compounds are studied. Glycine-isoglucos-amine (1-deoxy-l-glycino-D-fructose) was prepared in crystalline form by conversions of glycine-N-D-glucoside and -N-D-mannoside (both Na salt) and also from heated solLion of glycine and D-glucose. Similarly, crystalline glycine-isogalactosamine (I-deoxy-l-glycino-D-tagatose) (m.p. 165_??_168°C) was obtained by rearrangement of glycine-N-D-galactoside (Na salt) and from the browning mixture of glycine and D-galactose. Infrared studies indicated that the absorption near 3, 500 cm -1 is characteristic to isoglucosamines but not to isogalactsamine. On the infrared and periodate oxidation data, the structure of the amino acid-isoglycosamines is considered.
Substituted β-benzyl-γ-butyrolactones were synthesized. Hydrolysis and decarboxylation of ethyl α-allyl-α-benzyl malonate gave α-allyl-α-benzyl acetic acid. Esterification of the acid and subsequent reduction with lithium aluminum hydride afforded β-benzyl-β-allyl ethanol. Oxidation of the double bonds with potassium permanganate followed by periodate treatment, silver oxide oxidation and lactonization yielded β-benzyl butyrolactones. Thus β-benzyl-, β-piperonly-, β-3, 4, 5-trimethoxybenzyl- butyrolactone were prepared. Condensation of piperonal and β-piperonyl-γ-butyrolactone with sodium ethylate afforded (±) hibalactone
Nicotine-N';-oxide (II) was purified to give a crystalline form which has m. p. 170_??_171°C, and [α]_??_=+65.3°C. The reaction of nicotine-N';-oxide with acetic anhydride afforded a good yield of l';-(3-pyridyl)-4';-(N';-acetylmethylamino)-l';-propanone (V) which was hydrolyzed to give pseudoöxynicotine (III). Nicotine-N';-oxide (II) either with acetyl chloride or with benzoyl chloride, under similar conditions, furnished pseudoöxynicotine (III) without giving the corresponding acyl compound as an intermediate. 2';-Methyl-6';-(3-pyridyl)-tetrahydro-l';, 2';-oxazine (XII) rearranged from nicotine-N';-oxide reacted neither with acetic anhydride nor acetyl chloride. Reduction of the oxime (IV) of pseudooxynicotine dihydrochloride gave dl-l';-amino-1';-(3-pyridyl)-4';-methyl-aminobutane (VII) as a main product. The hydrazone (VIII) of pseudoöxynicotine dihydrochloride subjected to a modification of the Wolf-Kischner reaction, was reduced to yield dihydrometanicotine (IX). The pyrolysis of N';-methylmyosmine (IV) gave N';-methylnicotinamide (XI) and nicotyrine (X) in low yields. The presence of N';-methylmyosmine in the autoxidation mixture of nicotine was also established. Oxidation of nornicotine (XIV) with hydrogen peroxide furnished myosmine-N';-oxide (XV) whose identity was established by its chemical and physical properties. This oxide, on pyrolysis, gave nornicotyrine (XVII) and myosmine (XVI).
Enzymatic Degradation of Lignin
Part I. Paperchromatographical Separation of Intermediate Degradation Products of Lignin by the Wood-rotting Fungns Poria subacida (Peck) Sacc
1. It was confirmed that p-hydroxy-m-meth-oxyphenylpyruvic acid occurred in the culture filtrate of Poria subacida, in the medium of which native lignin, xylose and asparagine were contained. 2. Guaiacylglycerol-β-coniferylether and va-nillic acid-like compound were recognized in the extracts of the decayed spruce wood-meal attacked Poria subacida. 3. The decayed lignin extracted from the decayed wood-meal showed the remarkable variation and the existence of the more pre-dominant amount of carbonyl groups, compared with native lignin on their ultraviolet and infrared absorption spectra, and gave no purple-red colour reaction with phloroglucin-hydro-chloric acid reagent. 4. Depending upon the above results, the scheme of degradation of coniferous lignin by the function of the wood-rotting fungus Poria subacida was presumed and discussed.