The specificity of the serine carboxypeptidase from Aspergillus saitoi (EC 3.4.16.1) was investigated on carboxyterminal amidated peptides such as benzyloxycarbonyl(Z)-Ala-Phe-NH₂, gastrin-related peptide, molluscan cardioexcitatory neuropeptide, eledoisin-related peptide, and (D-Ala², Met⁵)-enkephalinamide. The enzyme did not hydrolyze Z-Ala-Phe-NH₂. It acted only as a carboxyamidase for the carboxyterminal peptide bond of gastrin-related peptide and as an amidase for enkephalinamide. The enzyme had both carboxyamidase activity and amidase activity for molluscan cardioexcitatory neuropeptide and eledoisin-related peptide.
Whether the enzyme acts as an amidase or carboxyamidase, the hydrophobicity of P₃ and P₄ positions of the substrate may be important. After removal of the carboxyterminal amino acid amide and/or ammonia, the enzyme catalyzed the sequential liberation of the amino acid from the carboxyterminus of the substrate.

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An extract of Japanese green tea, one of the most popular drinks in Japan, was an inhibitor of the growth of Streptococcus mutans, a bacterium responsible for causing dental caries. The analysis of the extract revealed that the main antibacterial components of the extract were several polv phenolic compounds, especially gallocatechin (GC), epigallocatechin (EGC), and epigallocatechin gallate (EGCg). GC was the most active component and its minimum inhibitory concentration against the bacterium was around 250 μg per ml.

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Improvement of the α-tocopherol productivity of Euglena gracilis Z was achieved by derivation of an analog-resistant strain. Among various compounds tested, β-2-thienylalanine was selected as the best inhibitor for derivation of high α-tocopherol-producing strains.
A number of β-2-thienylalanine-resistant strains were obtained on UV irradiation with or without streptomycin treatment, and also on spontaneous mutation. One strain, S-T1, produced 4 times as much α-tocopherol as the wild type strain.
As the result of optimization of the culture conditions, strain S-T1 produced 48.4 mg per 1 of culture broth or 1.8 mg per gram of dry cell weight (DCW) of α-tocopherol intracellularly. The addition of homogentisate and ethanol increased the production to 130.3 mg per 1 or 4.8 mg per gram of DCW. Finally, a high amount of α-tocopherol, 180.4mg per 1 of 6.3 mg per gram of DCW, was obtained with the feeding of glucose during the cultivation.

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(1S)-[1-²H]-sn-Glycerol (1a) was obtained in three steps from (6S)-[6-²H]-1, 6-anhydro-β-D-galactopyranose and converted to the (1R)-isomer (1b) via an S_N2 reaction of a 1-O-methanesulfonylated derivative. The ¹H and ¹³C-NMR spectra well characterized their structure and stereo-chemistry.

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A general method to determine the absolute configuration of the glycerol moiety in glycopyranosyl glycerols is presented, which involves per-O-benzylation and acid hydrolysis of the glycosyl glycerol to give optically active 1, 2- or 2, 3-di-O-benzylated sn-glycerol (III). ORD and CD measurements of III and its benzoylated derivatives gave intensive optical rotations or Cotton effects to determine the absolute configuration at C2.

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We have cloned the β-glucanase gene from a β-glucanase producing strain, Bacillus subtilis Y-25, to construct a β-glucanase hyperproducing strain. The cloned 1.9 Kb EcoRI-HpaI fragment containing the entire β-glucanase gene was inserted into the EcoRI and PvuII sites of a multi-copy vector plasmid, pUB110. The resulting plasmid, named pLB100, was introduced into B. subtilis Y-25 to construct B. subtilis HL-25. HL-25 produced 347 units/ml of β-glucanase in a culture supernatant, which was about 19-fold higher than the amount produced by the original strain, Y-25, and corresponded to approximately 2 g of β-glucanase per 1. Furthermore, pLB100 is so stable in HL-25 that the proportion of cells carrying pLB100 was almost 100% after cultivation for 100 generations without a selective antibiotic.

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Two enzymes, named E-1 and E-2, were purified from the culture supernatant of a β-glucanase (1, 3;1, 4-β-D-glucan 4-glucanohydrolase; EC 3.2.1.73) hyperproducing strain, Bacillus subtilis HL-25. Both purified enzymes were found to be homogeneous on SDS-polyacrylamide gel electrophones!s and to have an identical molecular weight of 24, 000. E-1 and E-2 have similar amino acid compositions, but their isoelectric points are pH 8.55 and 8.75, respectively. N-Terminal amino acid analyses showed that the N-terminal amino acid of E-2 is glutamine, which might be converted to pyroglutamic acid through spontaneous cyclization to yield E-1. Comparison of the amino acid sequence with that determined on DNA sequence analysis indicated that β-glucanase was produced as a precursor composed of 242 amino acid residues, including a signal peptide part consisting of 28 amino acids.
Detailed investigation of the crude enzyme preparation from the host strain, B. subtilis Y-25, also indicated the presence of two enzymes.

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A continuous-flow sensor, in which sulfite oxidase (SOD) was immobilized on CNBr-activated Sepharose, was developed for the determination of sulfite in white wine. Hydrogen peroxide produced by the enzyme reaction was monitored with a platinum electrode, which was covered with a dialysis membrane. The response of the sensor was linear in the range of 1 - 10 ppm sulfite, with a correlation coefficent of 0.999. The relative standard deviation for 10 injections was 2.3 % at the 5 ppm sulfite level. The gelatin coagulation procedure was introduced for the removal of interfering substances in wine such as polyphenol compounds. The method was applied to automatic sample pretreatment.

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An isooctane-addition agitated method is newly proposed for the assay of the hydrolytic activity of lipase. The hydrolytic activities of six kinds of microbial lipase were measured with this method at a high lipase concentration range and using olive oil, palm oil, and beef tallow as substrates. The newly proposed method gave equivalent or higher values than the previously proposed methods. In addition, the activity values calculated from the initial slopes of the progress curves gave a good proportional relation between enzyme concentration and activity in a wide range, although those calculated from the data obtained only at a fixed time (20 min) were out of proportion to the enzyme concentration by all methods tested.

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The distribution and the relaxation times of water in tomato fruits were measured by ¹H-NMR imaging with a resolution of 0.2 × 0.2 mm² area and 1 mm thickness. Water with a long relaxation time was preferentially accumulated in seeds and seed envelopes in immature green fruit. The total amount of water increased in mature red fruit, where water with a long relaxation time was localized in outer walls of the pericarp and water with a shorter relaxation time was distributed throughout all tissues except for seeds and seed envelopes. ¹H-NMR imaging apparently distinguished the physiological variations among different types of tissues and the physiological changes during maturation of tomato fruit.

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The possibility of producing amino acids under conditions of denitrification, i.e., with nitrate respiration as an energy-supplying system, was investigated using denitrifying bacteria. We found that Bacillus licheniformis A35 accumulated γ-polyglutamic acid (PGA) to a concentration of 8 mg per ml in a medium containing glucose and ammonium chloride or glutamic acid under denitrifying conditions. The bacterium also produced PGA aerobically. Thus, we found the de novo synthesis of PGA with B. licheniformis A35. The optimum conditions for PGA production were determined.
The purified PGA was found to be composed solely of glutamic acid; both D- and L -glutamic acid, in a molar ratio of 4:1. The molecular weight of PGA was about 3 × 10⁵.
Other bacilli tested produced some free amino acids but not under nitrate-respiration conditions, although some of them did produce PGA aerobically from L-glutamic acid.

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The physico-chemical characteristics of purified arginine kinases from prawn and swimming crab were examined. The molecular weights of prawn and swimming crab enzymes were 40, 500 and 40, 000, respectively. Amino acid analysis indicated that there were some differences in the contents of proline, glycine, methionine, and lysine. The other amino acid compositions of these enzymes resembled each other.
Both enzymes were stable up to 20°C when they were treated for 10 min at various temperature levels. The enzymes lost their activities at temperatures higher than 25°C. They were more stable at pH 8.0 than pH 7.0. The optimum temperature for the enzyme of prawn was about 42°C and that for swimming crab was about 40°C. The pH optima for the activity of arginine kinase of prawn in the forward and in the reverse reactions were found to be 9.0 and 6.1, respectively. For the swimming crab, the similar optimum pHs at 9.2 in the forward reaction and 5.8 in the reverse reaction were observed. Both enzymes were activated most strongly with Mg²⁺ and Mn²⁺ followed by Ca²⁺, Co²⁺, and Fe²⁺. The enzymes were not activated by Sr²⁺, Cu²⁺, or Zn²⁺.
The optimum molar ratio of Mg²⁺: ATP in the forward reaction of prawn and swimming crab was found to be 1:1, and the molar ratio of Mg²⁺: ADP in the reverse reaction was 4:1 in both cases. Kinetic studies indicated that dissociation constants were rather different. In the prawn, dissociation constants for arginine, ATP, AP, and ADP were 0.19, 0.31, 0.67, and 0.29 mM, respectively, but in the swimming crab, they were 0.10, 0.18, 0.22, and 0.11 mM, respectively.

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Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000-9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.

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The effects of the injection of a large amount of N¹-methylnicotinamide (MNA) (500 mg per kg body weight) on the ratio of N¹-methyM-pyridone-S-carboxamide (4-py) to N¹-methyl-2-pyridone-5-carboxamide (2-py) excretion, and the activities of 2-py and 4-py forming MNA oxidases were investigated in rats. The injected MNA was excreted very rapidly into the urine; 46% of the dose was excreted from 0-3hr post-injection, 15% from 3-6hr, 6% from 6-9hr and 1.5% from 9-12hr. The ratio of 4-py to 2-py also decreased rapidly; the ratio being about 0.6, 0.4, 0.4 and 0.6 from 0-3hr, 3-6hr, 6-9hr and 9-12hr post-injection, respectively. This ratio then recovered rapidly; being about 2, 5.5, 8.5 and 9.7 from 12-24hr, 24-48hr, 48-72hr and 72-96hr post-injection, respectively. The normal range of 4-py to 2-py excretion ratio is 8-14. So, this ratio returned to a normal level by day 3 post-injection. The rats were killed 5 hr after the MNA injection. At this time (the lowest ratio was observed around this time), the activities of 2-py and 4-py forming MNA oxidases in the injected group were 59% and 11% of the normal levels, respectively. Therefore, it was found that the decreased ratio of 4-py to 2-py excretion with the MNA injection was mainly due to the higher inhibition of the 4-py forming MNA oxidase than of the 2-py forming MNA oxidase by the MNA injection.

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The effect of the addition of 0.26 % free tryptophan (Trp) to a 20 % casein diet containing 6 mg of nicotinic acid per 100 g of diet on the ratio of N¹-methyl-2-pyridone-5-carboxamide (2-py) plus N¹-methyl-4-pyridone-3-carboxamide (4-py) to N¹-methylnicotinamide (MNA) excretion was investigated in rats. The urinary excretion of MNA, 2-py and 4-py, respectively, increased statistically significantly with the feeding of a 0.26% Trp (the same as the content of the 20% casein diet) supplemented 20 % casein diet, although it did not increase with the feeding of a 40 % casein diet, compared with in the case of the 20 % casein diet [Agric. Biol. Chem., 52, 1765 (1988)]. So, the total urinary excretion of Nam and its metabolites was 1.8 times higher in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet. However, the ratio of 2-py plus 4-py to MNA excretion was much lower in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet (13.16 ± 3.75→5.49 ± 2.25). This decreased ratio was considered to be partially due to a decrease in the 4-py forming MNA oxidase, which decreased significantly with the feeding of the Trp supplemented diet. Furthermore, the metabolic fate of Trp was greatly affected by the form of Trp, free or bound.

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The effects of neural blockers on the pancreatic enzyme secretion in response to an intraluminal infusion of soybean trypsin inhibitor and HCl were investigated. The stimulation of pancreatic enzyme secretion upon the intraluminal infusion of soybean trypsin inhibitor was not blocked by atropine, but was completely blocked by guanethidine. The intraluminal infusion of 0.08 N HCl, which is known as a potent secretagogue of secretin, caused a rapid augmentation of trypsin output, which was not blocked by atropine or guanethidine. Preinjection of CR-1392 (1.5mg/kg, i.p.), which is a strong cholecystokinin receptor antagonist, completely blocked the pancreatic response to soybean trypsin inhibitor, but not that to 0.08 N HCl. This inferred that guanethidine specifically suppressed the CCK-release from the small intestine.
These findings suggest that the pancreatic enzyme secretion in response to soybean trypsin inhibitor is mainly mediated by CCK, and that adrenergic modulation would be involved in the CCK-mediated pancreatic enzyme secretion in response to soybean trypsin inhibitor.

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The temperature dependencies of the optical rotation, light transmittance and viscosity of human serum albumin (HSA) solutions containing 2 M guanidine hydrochloride were investigated, in order to elucidate the gelation mechanism.
Unfolding of HSA molecules started at a certain temperature (T_d) and phase separation occurred at a higher temperature (T_s) than the T_d. The phase separation resulted in a heterogeneous, binary phase solution consisting of a fine coacervate particle phase and a dilute solution phase. At the phase separation point, there were more native state molecules than unfolded ones. No increase in viscosity was observed within the temperature range from the T_d to the T_s. An apparent increase in viscosity started at the T_s, showing that network formation proceeded from the T_s. These results suggested that the network formation was due to crosslinking among dispersed coacervate particles in the dilute solution phase.

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trans-3-Methylthioacrylamide (3-MTAA-NH₂) was isolated as colorless needles from the culture broth of Streptomyces sioyaensis, a siomycin-producer. This substance is considered to be not only a new metabolite from methionine but also a new substance. The isolation and identification of 3-MTAA-NH₂, as well as the cultural conditions for production, were investigated. A variety of other Streptomyces also produced 3-MTAA-NH₂ from methionine.

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We have attempted to develop an intraoral method which can measure the textural changes in foodstuffs during chewing by using electromyography (EMG). Forty-three foodstuffs with variable textural attributes were used.
Total chewing energy for these foodstuffs during chewing varied from 3 to 108 for the masseter muscle and 13 to 154 for the digastric muscle, respectively. Large differences in total chewing energy could be observed by EMG among the foodstuffs. The chewing energy for many foodstuffs revealed distinct differences throughout the chewing process. Foodstuffs could be categorized into six groups according to the changing patterns of chewing energy. EMG data and the number of strokes were influenced by masticatory index and salivary flow rate.

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Spheroplasts of auxotrophic mutants derived from Acetobacter aceti subsp. aceti No. 1023 were efficiently prepared by treatment with lysozyme, using sucrose as an osmotic stabilizer, and regenerated on an agar plate containing sorbitol and gelatin. In addition, spheroplast fusion between the several auxotrophic mutants was achieved in the presence of polyethylene glycol and CaCl₂. The frequency of fusion was found to be about 5 × 10^-5. Spheroplast fusion between A. aceti subsp. aceti No. 2 with the ability to grow at high temperature and A. aceti subsp. xylinum NBI1002 with high resistance to acetic acid was also achieved by the same method, with a frequency of 6.0 × 10^-6. The fusants showed various degrees of resistance to acetic acid and ability to grow at high temperature. One of the fusants, named No. 116, could produce acetic acid from ethanol continuously under conditions under which both parent strains were unable to grow. This suggests that spheroplast fusion is applicable to the breeding of strains for vinegar production.

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It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 μsec with an initial electric field strength of 7 kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16 kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.

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The performance of a bioreactor with a microfiltration module for the production of an intracellular enzyme, superoxide dismutase (SOD), by Streptococcus lactis is described. The fermentation system involving the bioreactor enables the continuous removal of metabolites inhibitory for cell growth and the complete recycling of the cells to the bioreactor. In a fed-batch (FB) culture with filtration, in which the main metabolite, lactic acid, in the culture broth was maintained at a low concentration, S. lactis was cultivated to the high concentration of 15.5 g-dry cells/1. The SOD content of the cells remained at almost a constant level throughout the cultivation and the productivity of SOD as well as cells per unit time was 4.3-fold as high as that in the case of a conventional batch culture without filtration. Repeating the FB culture with filtration enhanced the productivities of SOD and cells further, as compared with those in the case of the FB culture with filtration.

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Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkei Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/1) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co²⁺, Cu²⁺, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Eschevichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.

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In order to obtain further information on the changes in liver lipids, either a basal or a lysineexcess diet was refed to previously starved rats or fed to previously non-starved rats. Liver lipid accumulation was observed in previously starved rats refed the lysine-excess diet for 7 days, but not in rats without previous starvation. The liver lipid did not accumulate with another 8 days' feeding (15 days' refeeding). The addition of methionine alone or in combination with threonine to the lysineexcess diet had no effect on the liver lipid level. The decrease in serum triacylglycerol in rats refed the lysine-excess diet was preceded by lipid accumulation in the liver. Urinary potassium during the initial two days increased with refeeding and feeding. Marked excretion of orotate was observed for 2 days from the initiation of refeeding of the lysine-excess diet and it then decreased. Thus, such a marked increase in the urinary excretion of orotate might be associated with the stimulation of orotate biosynthesis and with lipid accumulation in the liver.

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The supplementation of egg yolk phospholipid (PL) containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to a cholesterol-free purified diet causes a reduction in the serum cholesterol level in rats [J. Nutr., 112, 1805 (1982)]. The present study was carried out to determine if dietary egg yolk PL also exerts this hypocholesterolemic action in rats given a high cholesterol diet and if this action is influenced by the constituent fatty acids. Egg yolk PL suppressed the elevation of serum cholesterol irrespective of its fatty acid composition, while purified PC had no effect, suggesting that the ethanolamine portion is responsible for this hypocholesterolemic effect. Egg yolk PL and PC containing longer-chain polyunsaturated fatty acids (arachidonic and docosahexaenoic acids) lowered the serum triglyceride level, while their hydrogenated forms did not. The present results, therefore, indicate that the hypolidemic effect of dietary egg yolk PL can be modulated by the combination of the constituent fatty acids as well as the base moieties. This hypolipidemic effect, however, appeared not to be related to the activities of adipocyte lipoprotein lipase and serum lecithin: cholesterol acyltransferase.

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Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in Osmoregulation in the bacterium.

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An enzyme that catalyzes the synthesis of S-carboxymethyl-L-cysteine from 3-chloro-L-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S-carboxymethyl-L-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84, 000 and gave one band corresponding to a molecular weight of 37, 000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-Ala and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4mM. The enzyme showed very low activity as to the α, β-elimination reaction with 3-Cl-Ala and L-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.

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This study was undertaken to investigate the response of sucrase (SA), alkaline phosphatase (ALP), and leucine aminopeptidase (LAP) activities localized in the brush border membrane of the small intestine to ingested nutrients. In rats previously meal-fed on carbohydrate-free diets, the maximal increase in SA activity after the administration of a 18 % casein and high sucrose diet (HSD) occurred 24 hr after the beginning of the 12-hr period of HSD-feeding, but ALP and LAP activities reached the maximal levels 12hr after the HSD administration and then rapidly declined. These changes in three enzyme activities were similar to those after HSD-feeding following a 2-day fast, but those were not observed after giving a carbohydrate-free and high fat diet. The increases in these enzyme activities after giving HSD were not observed entirely after giving HSD containing 0.5% concanavalin A, which was undigestible and preferentially bound mitotically active cells of the small intestine. From these findings, it can be suggested that the adaptive response of these enzyme activities to ingested nutrients, as sucrose and casein, was produced on the luminal surface of the small intestine, especially on the immature cells.

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