Four cyclic peptides were isolated from young unshiu (unripe fruit), orange and amanatsu peelings, and their structures were established on the basis of FAB-MS (CID method) and 2D-NMR spectroscopic data, and by chemical evidence. They were each found to consist of seven or eight amino acids. In the present study, compounds 1-4 are shown to be new cyclic Peptides.
The effects of reaction time and temperature on the volatile compounds formed from L-cysteine and propanal were studied in model systems heated in triglyceride-water (75:25). The volatile compounds, flavor profiles and yields formed in these model systems varied according to the reaction conditions. At 90 and 110°C, 2-methyl-2-pentenal was the most predominant component formed. Its maximum proportion occurred after heating for 1.5hr at 110°C, and markedly decreased with temperatures over 110°C. 2-Ethylthiazolidine was the major product in the system heated at 140°C for 1.5 hr. 2-Ethylthiazolidino[3, 4-b]thiazolidine could be detected in the system heated at 90°C for 1.5hr. At 170°C, the profile of the volatile products was very complex, higher temperatures favoring the formation of nitrogen- and sulfur-containing compounds, including pyrrole, pyridines, thiophenes, thiazolidines, 1, 2, 4, -trithiolanes and 1, 2, 4-trithianes.
Natural (+)-(1R, 2S, 3S)-methyl cucurbate (1b) and the (-)-δ-lactone of 3-epi-cucurbic acid (16) were synthesized from (+)-(1R, 6S, 7R)-bicyclo[4.3.0]non-3-en-7-ol (5). Asymmetric hydrolysis of the acetate (8) of (±)-5 with pancreatin gave optically pure the (+)-(7R)-alcohol (5) and (-)-(7S)-acetate (8). An ozonolysis product of (+)-5 was transformed to (-)-16 and (+)-(3S)-lb with inversion of the (7R)-hydroxyl group. Similarly, unnatural (-)-lb and (+)-16 were prepared from optically pure (-)-5. The growth inhibitory activities of these synthesized chiral compounds toward lettuce seedlings were examined.
We have recently reported that the conversion ratio of tryptophan to nicotinamide [urinary excretion of (nicotinamide + N1-methylnicotinamide (MNA)+ N1-methyl-l-pyridone-S-carboxamide (2-Py) + N1-methyM-pyridone-S-carboxamide (4-Py) (μmol/day)/tryptophan intake during urine collection (μmol/day)] was changeable according to the kind of dietary nitrogen sources. In these experiments, we investigated whether the fate of tryprophan is changeable according to nitrogen sources or not. Weanling rats were fed with a diet low in nitrogen (1.1311% nitrogen) containing a suitable amount of nicotinic acid (6mg/100g of diet) for 17 days. Egg white (EW), egg white proteolysate-5 (EWP-5), and mixture of amino acids simulating the amino acid pattern of EWP-5 were used as dietary nitrogen sources. The urinary excretion of 5-hydroxyindole-3-acetic acid and the sum of nicotinamide and its metabolites in the group fed with the EWP-5 diet was the lowest, but, the excretion ratio of (2-Py + 4-Py)/MNA, which has been reported to be an index of amino acid adequacy, in the group fed with the EWP-5 diet was the highest. From these findings, it was suggested that the tryptophan in EWP-5 (mixtures of small peptides) entered the degradation pathway of tryptophan such as serotonin and nicotinamide biosynthesis with difficulty, but that it was easy to enter the protein biosynthesis, compared with tryptophan in EW (protein and mixtures of amino acids).
Antisera against native yeast of 6 species, Candida intermedia, C. parapsilosis, C. guilliermondii, C. lambica, Crytococcus laurentii, and Rhodotomla rubra, were prepared for use as probes in an enzyme-linked immunosorbent assay (ELISA). These antisera reacted with yeast cell surface antigens that are thought to consist of protein moieties. The cross-reactivities between the antisera and yeast of 11 species were investigated by immunofluorescent and immunoblotting methods. It was shown that ELISA using the antiserum against C. intermedia or C. parapsilosis, is a valuable means of detecting yeast in orange juice. Yeast of more than 103 were capable of being detected by ELISA.
For the purpose of improving sweetness and a further study on the structure-sweetness relationship of steviol glycosides, transglycosylation of stevioside by a variety of commercial glucosidases was investigated. It was revealed that two α-glucosidases gave glucosylated products. Transglucosylation of stevioside by Pullulanase and pullulan exclusively afforded three products, 13-O-[β-maltotriosyl(1→2)-β-glucosyl]-19-O-β-D-glucosyl-steviol (1), 13-O-β-maltosyl-(1→2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol (2) and 13-O-β-sophorosyl-19-O-β-maltotriosyl-steviol (3). All of these products have already been obtained by trans-α-1, 4-glucosylation of stevioside by the cyclodextrin glucano-transferase starch system, and 1 and 2 have been proven to be tasty and potent sweeteners. Transglucosylation of stevioside by Biozyme L and maltose afforded three new products, 4, 5 and 6, the structures of these compounds being elucidated as 13-O-β-sophorosyl-19-O-β-isomaltosyl-steviol(4), 13-O-β-isomaltosyl(1→2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol (5) and 13-O-β-nigerosyl(1→2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol (6). A significantly high quality of taste was evaluated for 4.
We attempted to find compounds that suppress the DNA impairment caused by D-glucose-6-phosphate (Glc-6-P) by observing the loss of ability of pBR322 to transform Escherichia coli, and found that APM (magnesium L-ascorbyl-2-phosphate) suppressed the loss of transformability to less than 1/100 of that in the absence of APM. When 2'-deoxyguanosine 5'-monophosphate (dGMP) was incubated in the dark with Glc-6-P and APM, changes in their absorbance patterns were observed, indicating possible suppression by APM of the interactions between dGMP and Glc-6-P. APM weakly suppressed the Maillard reaction.
To understand the mechanism by which γ-polyglutamic acid (γ-PGA) in the sticky material of natto was synthesized, we purified the γ-glutamyltranspeptidase (γ-GTP) (EC 184.108.40.206) from the culture broth of Bacillus subtilis (natto) to homogeneity. γ-GTP was composed of two subunits with molecular weight of 45, 000 and 22, 000. The N-terminal amino acid sequence of light subunit was homologous with that of γ-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60°C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55°C, but little loss of the activity was detected at 40°C. γ-GTP used glutamine as a γ-glutamyl donor and acceptor for γ-PGA synthesis. Dipeptides were better γ-glutamyl acceptors than free amino acids.
Growing rats meal-fed for 4 weeks with a 20% or 40% casein diet in the morning (9:00-11:00) and a non-protein diet in the evening (19:00-21:00), or vice versa, were examined for growth and metabolic changes. A pair of groups given the 40% casein diet at one meal and the protein-free diet at the other meal, although becoming a little different from each other in growth, did not significantly differ from the control given only the 20% casein diet at the two meals. A pair of groups alternately given the 20% casein and protein-free diets, although excelling in protein efficiency ratio, were far inferior in growth to the groups given the 40% casein diet at either of the two meals. In any case, the rats with alternation of the diets sufficient and deficient in protein preferred the 20% or 40% casein diet to the protein-free one at whichever feeding time, and had a higher body weight gain when the casein diet was administered in the evening. The differences among these groups in protein intake throughout the experimental period were roughly reflected in their growth curves. A similar tendency was also observed for the blood urea level and hepatic serine dehydratase activity. As to the individual free amino acids in the plasma, however, there was no significant difference between the control and other groups except for a few amino acids (Ser, Gly and Phe).
A novel mitogen, E-15, which induced blastogenesis of mouse splenocytes, was purified from the culture filtrate of an actinomycete through ethanol precipitation, anion and cation exchange column chromatography, and gel filtration. The producing organism was identified as Nocardia asteroides. Spectronic study demonstrated that it was a polysaccharide. Acid hydrolysis of E-15 yielded glucose and glucosamine. The active substance was eluted at the molecular mass between 90 kDa and 750 kDa on gel filtration. E-15 induced a mitogenic response of mouse splenocytes above 1 μg/ml and its potency of induction was superior to a lipopolysaccharide that is known to be a polyclonal B cell-specific mitogen. It showed no toxicity up to 100μg/ml. The cell surface phenotypes of the blast cells induced by E-15 were analyzed by flow cytometry; they had surface immunoglobulins but no Lyt-2 antigen. Thus it was suggested that E-15 was a B-cell-specific mitogen.
The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned into Bacillus brevis. Under control of the strong cell wall protein promoter, the gene was efficiently expressed during the early stationary phase of growth, when cell densities were high. The expressed gene product was a soluble cytoplasmic protein and made up more than 20% of the total cellular protein. A simple heat treatment at 85°C for 10 min gave a virtually pure enzyme. Final isomerase yields were about 0.5 g isomerase per liter culture. The purified isomerase has an optimum temperature at 85°C, and an optimum pH around 7. The isomerase is stable at 85°C for several hours, opening possibilities for new uses.
1-IsobutyI-5-(4-phenoxyphenyl)imidazole (KK-98), an inhibitor of juvenile hormone (JH) biosynthesis in the cockroach, and related imidazole compounds were evaluated against silkworm, Bombyx mori, for their activity to induce precocious metamorphosis. KK-98 induced precocious metamorphosis in the 4th instar larvae at high doses. Replacement of the 4-phenoxy group by a 3-phenoxy or 3-benzyloxy group on the benzene ring increased the activity. Among this series of compounds, 5-(3-benzyloxyphenyl)-1-isopropylimidazole (8) showed the highest activity. The induction of precocious metamorphosis by compound 8 was rescued by the simultaneous application of methoprene, a JH minic. When newly molted 3rd instar larvae were treated with a high dose of compound 8, a few larvae formed larval-pupal intermediates in the 3rd instar stage, which has not been formed by treating of any other imidazoles so far.
To study the beneficial effects of traditional Indonesian foods on sugar and lipid metabolism, streptozotocin-induced diabetic rats were fed on purified diets containing 5% of either cellulose as a control or four kinds of Indonesian plants. One of them, Curcuma xanthorrhiza ROXB., improved the diabetic symptoms such as growth retardation, hyperphagia, polydipsia, elevation of glucose and triglyceride in the serum, and reduction of the ratio of arachidonate to linoleate in the liver phospholipids. C. xanthorrhiza specifically modified the amount and composition of fecal bile acids. Significance of these findings was discussed in the light of the improvement of several diabetic symptoms.
The hydrogenase reaction in Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, aerobic hydrogen-oxidizing bacterium, was studied in the membrane fraction, methionaquinone-depleted membrane, and purified membrane-bound hydrogenase. Both b and c type cytochromes were involved in the hydrogen oxidation. Methionaquinone mediated an electron transport between membrane-bound hydrogenase and cytochrome b560. Methionaquinone was reduced directly by purified hydrogenase. From these results, we conclude that methionaquinone is a direct natural electron acceptor for the membrane-bound hydrogenase in the strain.
During the screening program for atrial natriuretic peptide (ANP) receptor ligands of microbial origin, we isolated a novel nonpeptide ANP antagonist, HS-142-1, from a culture broth of Aureobasidium pullulans var. melanigenum. Structural analysis showed that HS-142-1 was composed of 20-30 kinds of β-1, 6-glucan esterified by caproyl groups; each component had an almost equal potency. HS-142-1 inhibited [125I]-rANP binding to its receptor in rabbit kidney cortex membranes with an IC50 of 0.3 μg/ml and antagonized ANP-induced cGMP production by bovine lung membranes in a dose-dependent fashion. The discovery of this nonpeptide ANP antagonist, HS-142-1, will provide a useful tool to study the physiological significance of natriuretic peptide system.
Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1. The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence. In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65°C and mature enzyme is detected. In the case of mutant genes containing partial deletions in the NH2-terminal pro-sequence, no proteolytic activity was detected and the precursor protein was found to be unstable in E. coli. These results indicate that the NH2-terminal pro-sequence is required to produce the active enzyme by stabilizing the precursor structure. Amino acid substitutions in the conserved sequence of the NH2-terminal pro-sequence found among subtilisin-type proteases made the processing faster compared with the wild type.
(±)-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrroiidine derivative (12), which was converted to 1 in a moderate yield.
The Interesterification of triacylglycerol with fatty acid was done to prepare triacylglycerol molecular species. Optimum operating conditions for the interesterification using a 1, 3-positional specific endocellular lipase from Rhizopus japonicus NR400 in a batch system were investigated. The reaction was done at 40°C for 5 hr in the following system: Trioleoylglycerol-palmitic acid=1:3.5 (mol/mol), 10ml n-hexane/g trioleoylglycerol, and 2500 units of enzyme/g trioleoylglycerol. Under these conditions, the content of palmitoyl groups in 1, 3-positions of triacylglycerol was about 60 mol%. Additional interesterification (2-cycle reaction) using palmitic acid and the novel triacylglycerol prepared by one-step interesterification (1-cycle reaction) resulted in a preparation of highly pure 1, 3-dipalmitoyl-2-oleoylglycerol.
A novel ethanol dehydrogenase with high activity against dulcitol 1-phosphate (D1P-EDH) was purified from Salmonella typhimurium IFO 12529 grown in a medium containing dulcitol as a carbon source. D1P-EDH was purified from a crude extract of S. typhimurium cells by (NH4)2SO4 precipitation and column chromatographies on Blue-Cellulofine, Sephacryl S-300, and Zorbax GF-250. D1P-EDH was purified 277-fold with an activity yield of 21.3%. The purified preparation gave a single band on an electrophoregram. The activity staining of the electrophoregram of the (NH4)2SO4 precipitate indicated that there was no isozyme of D1P-EDH in the extract. The molecular weight of D1P-EDH was estimated to be 158, 000 by gel filtration and 40, 000 by SDS-polyacrylamide gel electrophoresis. D1P-EDH showed its maximal activity in a pH range from 9.0 to 9.5. D1P-EDH was stable in a pH range from 6.0 to 10.0 and was also stable at 30°C for 120 min. The purified preparation oxidized fructose 6-phosphate and galactose 6-phosphate to the same extent as DIP and oxidized much more ethanol than DIP. D1P-EDH activity was strongly inhibited by p-chloromercuribenzoic acid and NaN3 though it was activated by A13+, Ba2+, Ca2+, andFe2+.
A transformation system with efficiencies between 6 and 50 stable transformants per μg of DNA was developed for Penidllium urticae Jl (ATCC48163) using hygromycin B-resistant pi asm ids containing or not containing fragments of the P. urticae genome. The tandem repeated integration and/or random integration of vector DNA were observed. Although P. urticae was unable to grow in the presence of 200 μg/ml hygromycin B, the transformants were resistant to more than 5 mg/ml 8of hygromycin B.
A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thermophilum strain T, which is obligately symbiotic with the thermophilic Bacillus strain S. The enzyme was purified 21-fold to homogeneity with 19% recovery by a series of chromatographies using anion-exchange, hydroxylapatite, hydrophobic interaction, and MonoQ anion-exchange columns. The molecular weight of the purified enzyme was estimated to be approximately 210, 000 by gel filtration, while the molecular weight of its subunit was 46, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The isoelectric point of the enzyme was 4;9. The tryptophanase was stable to heating at 65°C for 20 min and the optimum temperature for the enzyme activity for 20 min reaction was 70°C. The optimum pH was 7.0. The NH2-terminal amino acid sequence of this tryptophanase shows similarity to that of Escherichia coli K-12, despite a great difference in the thermostability of these two enzymes. The purified enzyme catalyzed the degradation (α, β-elimination) of L-tryptophan into indole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate. The Km value for L-tryptophan was 1.47 mM. 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, and L-serine were also used as substrates and converted to pyruvate. The reverse reaction of α, β-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate.
The regioselectivity of six serine proteases for the amino groups of lysine was investigated. α-Chymotrypsin showed a preference for the α-amino group, although the selectivity can be varied 10-fold depending on the reaction medium. Subtilisin Carlsberg and other bacterial proteases were highly specific for the ε-amino group, regardless of the reaction medium: they were used as catalysts for the preparative synthesis of isopeptides in anhydrous tert-amyl alcohol.
Several analogs modifying the 6-methoxy-2-methoxymethyl-3-(3, 4-methylenedioxyphenyl)-1, 4-benzodioxan-7-yl group of haedoxans were synthesized and their insecticidal activity was examined. 2-(2, 6-Dimethoxyphenoxy)-1-hydroxy-6-(2-methoxy-5-methoxyethoxyphenyl)-3, 7-dioxabicyclo[3.3.0]octane, which lacked the 3-(3, 4-methylenedioxybenzyloxy) moiety of the benzodioxanyl group, was not insecticidal, but caused prolonged paralysis of the housefly. A compound replacing the 6-(2-methoxy-5-methoxyethoxyphenyl) by 6-(5-butoxy-2-methoxyphenyl) exhibited insecticidal activity comparable to one thirty-thousandth of that of haedosan A. It became evident that the 1, 4-benzodioxane framework charging the 3-(3, 4-methylenedioxy)phenyl group is important for the insecticidal activity of haedoxans.