Comparison of Mineral Balances in Germfree and Conventional Mice When Sodium Phytate is Added to Purified Diet

A strain of Alcaligenes isolated from soil was a good producer of β-glucuronidase, and the enzyme was purified from the cell-free extract by sequential column chromatography on DEAE-Toyopearl, Toyopearl HW-55F, and Phenyl-Sepharose CL-4B. By these procedures, two β-glucuronidases designated as β-glucuronidases I and II were purified 240- and 508-fold, respectively. β-Glucuronidase I, with a molecular weight of 75,000, had an optimum pH at 7.5 and the enzyme II, with a molecular weight of 300,000, had maximum activity at pH 6.0. Both enzymes were strongly inhibited by saccharo-1,4-lactone, glucaro-δ-lactam, p-chloromercuribenzoate, Hg²⁺, and N-bromosuccinimide. β-Glucuronidase I was active toward estrogen-3-β-glucuronides and inert toward β-glucuronide conjugates of menthol, estrogen-17β-, estrogen-16α-, androsterone-3α-, testosterone-17β-, cortisol-17α-. β-Glucuronidase II hydrolyzed all of these substrates. β-Glucuronidase I was inhibited by phenolphthalein and its glucuronide.     

Studies on the ɛ-Lysine Acylase

Since Achromobacter pestifer EA isolated from soils shows markedly high ?-Iysine acylase activity compared with those of the other microorganisms ever tested, cultural conditions for the production of this enzyme were investigated.

As a result, it was confirmed that simple medium containing 1% peptone, 5% glucose and some inorganic salts is most suitable for the enzyme production and that much more ?-Iysine acylase is produced by shaken culture or submerged culture in jar fermentor than by stationary culture. α-Amino acylase activity in this organism was also studied.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

N-Acetylornithine-δ-aminotransferase-deficient and N-Acetylglutamokinase-deficient Arginine Auxotrophs of Corynebacterium glutamicum

An N-acetylglutamokinase-deficient arginine-requiring mutant, KY9390 and an N-acetylornithine aminotransferase-deficient arginine-requiring mutant, AA-7 were derived from a wild-type strain and an l-arginine-producing mutant of Corynebacterium glutamicum, respectively. KY 9390 accumulated 7.5 mg per ml of N-acetylglutamic acid and AA-7 accumulated 2 mg per ml of N-acetylglutamate-γ-semialdehyde, intermediates of arginine biosynthesis, in a culture medium.

The production of N-acetylglutamate-γ-semialdehyde by AA-7 was not affected by the concentration of l-arginine in the medium, whereas that of N-acetylglutamic acid by KY 9390 was inhibited by the addition of l-arginine in the medium.     

Isolation and Characterization of Two Plasmids in Bacillus subtilis var. amylosacchariticus

Five bufadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana×tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC₅₀=0.4 μM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Identification of d-Erythro-Dihydroneopterin Triphosphate as a Product of Guanosine Triphosphate Cyclohydrolase from Serratia indica

Guanosine triphosphate cyclohydrolase (EC 3.5.4.16) was previously shown to exist in two forms (GTP cyclohydrolase D-I and D-II) in Serratia indica IFO 3759, and they were homogeneously isolated. The present study deals with the characterization of their reaction products. A fluorescent product formed from guanosine triphosphate by GTP cyclohydrolase D-II was identified as 7,8-dihydroneopterin triphosphate by its absorption spectra, phosphate analysis and gas chromatography-mass spectrometry of the dephosphorylated trimethylsilyl derivative. After oxidation and dephosphorylation, the d-erythro configuration of the side chain was made clear by the elution profile on ECTEOLA-cellulose chromatography, Rf values on thin-layer chromatography and by biological activity to Crithidia fasciculata ATCC 12857. The fluorescent products from GTP cyclohydrolase D-I and D-II were indistinguishable.     

Isolation and Restriction Mapping of a Pseudomonas Plasmid

The effects of the injection of a large amount of N¹ -methylnicotinamide (MNA) (500 mg per kg body weight) on the ratio of N¹ -methyl-4-pyridone-3-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 MN A 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 12~24 hr, 24 ~48 hr, 48~72 hr and 72 ~96 hr 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.     

Production and Properties of Lipase from a Newly Isolated Chromobacterium

Out of some 750 strains of microorganisms, a potent bacterium for lipase production was isolated from soil and was identified as Chromobacterium viscosum.

The bacterium accumulates lipase in culture fluid when grown aerobically at 26°C for 3 days in a medium composed of soluble starch, soy bean meal, lard and inorganic salts.

Chromobacterium lipase had an optimum pH of 7.0 for activity at 37°C, and an optimal temperature of 65°C at pH 7.0. The enzyme retained 80% of the activity when heated for 10 min at 70°C. This lipase was capable of hydrolyzing a variety of natural fats and oils, and it was more active on lard and butter than on olive oil. The activity was stimulated by Ca²⁺, Mg²⁺, Mn²⁺ and inhibited by Cu²⁺, Hg²⁺ and Sn²⁺. It was not diminished but rather stimulated by a high concentration of bile-salts.     

Reductive Dechlorination of 1,2,4-Trichlorobenzene by Staphylococcus epidermidis Isolated from Intestinal Contents of Rats

Twelve bacterial strains which were concerned with dechlorination of 1,2,4-trichlorobenzene (TCB) were isolated from the intestinal contents of rats and it was found that they belonged to Staphylococcus epidermidis (strain A-F), Staphylococcus saprophyticus (strain G), Streptococcus sp. (strains H and I), Bacillus sp. (strain J), Gram negative rod (strain K) and Lactobacillus sp. (strain L).

In Staphylococcus epidermidis (Strain A), TCB was mainly converted to o-dichlorobenzene and the latter was preferentially converted to monochlorobenzene (MCB) among dichlorobenzenes (DCBs). These conversions proceeded only under a gas phase of hydrogen. Furthermore, dry and broken cells of intact bacteria also maintained the dechlorinating activities, which were stimulated by the addition of NADPH.

Therefore, it was supposed that the conversion of TCB to MCB via DCBs was reductively carried out by enzymes originating from the isolated bacteria.