Fates of N′-Methylnicotinamide and Nicotinamide N-Oxide in Mice

This experiment was performed to investigate the possibility that N′ -methylnicotinamide (N′-methyl-3-pyridinecarboxamide) and nicotinamide N-oxide have niacin activity or not in animals. When 20 mg N′-methylnicotinamide per mouse was administered, urinary excretion of nicotinamide, N¹-methylnicotinamide (MNA), N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) increased 24-, 3-, 3-, and 3-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were almost the same as those when 20 mg nicotinamide was administered. Therefore, the relative activity of N′-methylnicotinamide to nicotinamide as niacin was considered to be about 1. When 20 mg nicotinamide N-oxide per mouse was administered, urinary excretion of nicotinamide, MNA, 2-Py, and 4-Py increased 6.4-, 1.8-, 1.6-, and 1.7-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were about 1/2 of those when 20 mg nicotinamide was administered, so the relative activity of nicotinamide N-oxide to nicotinamide as niacin is considered to be about 1/2. In conclusion, it was found the possibility that the reactions N′-methylnicotinamide → nicotinamide and nicotinamide N-oxide → nicotinamide occur, at least in mice, and that therefore N′-methylnicotinamide and nicotinamide N-oxide have niacin activity.     

Serratia Protease

The substrate specificity of Serratia protease was determined using various synthetic substrates. The enzyme did not participate in the hydrolysis of di- and tri-peptides except benzoylglycylleucinamide which was split at a limited rate into hippuric acid and leucinamide. The enzyme action on larger peptides was also studied. The enzyme cleaved the gly-leu bond in eledoisin related peptide and the gly-phe bond in bradykinin. The enzyme split oxidized insulin B-chain at twelve different peptide bonds.     

Serratia Protease

Protease from a strain of Serratia contained one gram atom of zinc ion per mole and the zinc ion was essential for the activity. Also zinc-free apoenzyme was isolated as a crystalline form from the native-enzyme. Several metalloenzymes were prepared by the addition of corresponding metal ions to the apoenzyme. Studies on activities toward the hydrolysis of casein showed that relative activities of native- (zinc), cobalt- and manganese-enzyme were 1.0, 1.2 and 0.8, respectively. Toward the hydrolysis of hippurylleucinamide, however, specific activity of cobalt-enzyme was about 10 times that of the native- (zinc-) enzyme. Spectroscopic studies did not reveal any significant differences in conformations among native-enzyme, apoenzyme and the other metalloenzymes.     

Acid Protease Produced by Trametes sanguinea,a Wood-destroying Fungus

A wood-destroying fungus, Trametes sanguinea, produced a potent acid protease in a submerged culture. Maximum proteolytic activity of the culture was attained after 140-hours cultivation in a medium containing dextrin and corn steep liquor. The acid protease was obtained in crystalline form from the mycelium-free culture filtrate by the following successive treatments: acetone precipitation, ionexchange column chromatography, ammonium sulfate fractionation, dialysis, and crystallization by acetone. Throughout the over-all process, the acid protease was purified approximately 30-fold with about 8% recovery of the original activity.     

Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

Studies on Erythorbic Acid Production by Fermentation

Screening was carried out for erythorbic acid (EA)-producing strains from about 5,000 newly isolated fungi and bacteria. Penicillium notatum FY 115 was screened out as most powerful EA producer. Only Penicillium, but no other genera, was obtained as EA producers from our screening program. Monospore selections and mutagenic treatments succeeded to elevate the yield of EA over 40% to glucose supplied. Various cultural conditions were studied, and pH change during fermentation process was proved to be most important for favorable EA production. Over 80% yield could be obtained when washed mycelium was used in dilute glucose solution.

Abundant accumulation of EA by the strain FY 115, Penicillium sp., in fermentation broth was studied, and EA, both free and Na-salt, was obtained as crystal in the yield of about 45% to glucose supplied, in the media of 8% glucose by jar fermentor, in considering the inhibitory effect of some metal ion.

Extraction processes were improved to elevate the yield and was developed the continuous multi-bed extraction system of anion-exchange resin, which resulted in the yield of 90.9% of EA from fermentation broth in sum total.