We examined the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus by attempting to isolate tryptic peptides of the enzyme. By solubilization and precipitation in buffers, the peptides were first fractionated into three. The main fraction was fractionated by ion-exchange chromatography. Twenty-seven peptides were generated from this fraction. The fraction insoluble at neutral pH was fractionated by SP-Sephadex C-25. From this fraction three peptides were obtained. The other fraction insoluble at acidic pH was fractionated by Bio-Gel P-60. Four peptides were isolated from this fraction. In total, thirty-four peptides were generated from the tryptic digest of the α-amylase. The amino acid sequences of twenty-one out of thirty-four peptides were completely determined, while those of the other thirteen peptides were partially determined. The peptides derived from the N- and C-terminal ends of the α-amylase were identified.

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We examined the primary structure of α-amylase produced by Bacillus subtilis var. amylosacchariticus by isolation and characterization of CNBr fragments of the enzyme. By solubilization and precipitation in a buffer, the fragments were first fractionated into two. The soluble fraction was fractionated by Bio-Gel P-30, and three fragments were obtained. The insoluble fraction was fractionated by SP-Sephadex C-25 and further purified by Bio-Gels, and five fragments were isolated. Amino acid sequences near the N- and C-terminus were determined with the eight CNBr fragments. By matching the sequences with those of methionine-containing tryptic peptides, alignment of the eight CNBr fragments was determined.

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To determine the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus, we have reported the isolation of thirty-four tryptic peptides and eight CNBr fragments from the enzyme. Since the alignment of the eight CNBr fragments was made by matching with six methionine-containing tryptic peptides, the order of tryptic peptides within each CNBr fragment was determined. In the case of four small CNBr fragments, sequence analyses using an automated sequence analyzer established the peptide orders within these fragments. For larger fragments, further fragmentation was done using chymotrypsin or staphylococcal protease V8 and the resultant peptides were isolated and sequenced. Consequently, the peptide orders within three out of four large CNBr fragments were established.

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Effects of exogenous Bt₂cAMP and Bt₂cGMP on the changes in lipase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, and invertase activities were investigated during the germination of pollen from Pinus densiflora SIEB. et ZUCC. Both nucleotides cooperatively restored the lipase activity which was depressed by exogenous sucrose, but had no effect on the change in glucose-6-phosphate dehydrogenase activity. Either Bt₂cAMP or Bt₂cGMP partially restored the glutamate dehydrogenase activity which was depressed by exogenous sucrose. Bt₂cGMP lowered the invertase activity during germination, whereas Bt₂cAMP reversed this depression. Using inhibitors of RNA and protein synthesis, we suggested that Bt₂cAMP and Bt₂cGMP control the invertase activity at the transcriptional and translational levels, respectively.

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Cosmomycins A, B, C and D, new differentiation inducers of Friend cell F5-5, accumulated in the mycelium and culture fluid of a new strain, Streptomyces cosmosus nov. sp. Cosmomycin A and cosmomycin B contain a rhodinosyl-rhodinosyl-rhodosaminyl and rhodinosyl-2-deoxy-L-fucosylrhodosaminyl group at C-10 of γ-rhodomycinone, respectively. Cosmomycins C and D respectively possess the same sugar chains as Cosmomycins A and B at C-10 and the common rhodinosyl-2-deoxy-L-fucosyl-rhodosaminyl group at C-7 of β-rhodomycinone. Cosmomycin D is a new anthracycline compound and cosmomycin A is a new anthracycline compound of microbial origin, but Cosmomycins B and C seem to be identical to γ-rhodomycin Y and β-rhodomycin S-2, respectively. Approximately 20.0%, 15.4%, 16.4% and 14.2% of the F5-5 cells were induced to respectively biosynthesize hemoglobin by 1.25 μg/ml of cosmomycin A, 1.25 μg/ml of cosmomycin B, 7.81ng/ml of cosmomycin C and 15.6ng/ml of cosmomycin D. Other anthracyclines such as aclacinomycin A1, adriamycin and daunomycin had no effect on differentiation.

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A new differentiation screening system employing a human neuroblastoma cell line. NB-1, was used to isolate staurosporine as an inducer obtained from the culture broth of Streptomyces actuosus. Staurosporine at a concentration of 20 nM induced elongation of neurites and cell enlargement one hour after treatment of NB-1. In addition, the agent had a cytotoxic effect against NB-1 at a concentration of more than 0.21 μM.

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Perisperm starch granules of the dicotyledonous plant Amaranthus hypochondriacus L. were prepared from two homozygous lines (WxWx and wxwx) and their hybrid (Wxwx). The hybrid line was obtained by natural hybridization. By Sephadex G-75 column chromatography of isoamylase-debranched starches, the amylose content of WxWx starch was 16.9%, that of Wxwx was 10.7, and wxwx was zero. SDS-polyacrylamide gel electrophoresis showed that starch granules from two genotypes (WxWx and Wxwx) contained a Wx protein (MW=68, 000) which was supposed to be a starch granule-bound starch synthase and was associated with amylose synthesis, as observed in nonwaxy maize. The intensities of the stained protein bands were apparently correlated with the number of the Wx alleles. The Wx protein was not detected in the wxwx starch. These findings suggest that the Wx allele produces the Wx protein and amylose in the perisperm of A. hypochondriacus, with incomplete dominance over the wx allele. The Wx allele did not affect the fine structure of amylopectin and had little if any effect on susceptibility to glucoamylase and pasting properties of starch granules from these genotypes.

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The developmental changes in the structure and properties of endosperm starches were investigated using the near-isogenic lines for wx alleles of rice. The amylose content in nonwaxy starch was increased during the development of rice grains. Because the accumulation of amylose in endosperm stopped earlier than that of amylopectin during development, the percentages of amylose reached a maximum at the 17th day after flowering in nonwaxy endosperm. Since the distributions of the unit-chain length of amylopectin in waxy and nonwaxy starches were unchanged with the development of the grains, these amylopectins would be synthesized in a similar manner through development. The structure and properties of endosperm starches were reconfirmed to be conspicuously affected by the temperature at the early developmental stages of the grain-filling period, namely, they appeared to be characterized by the temperature at which the starch was accumulated in the endosperm.

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Fumes from phospholipids pyrolyzed at 500-700°C did not themselves show any mutagenicity on Salmonella strains, but when the pyrolyzates were treated with a sodium chloride precipitate, active carbon, or an anionic exchange resin, the filtrates were found to be mutagenic on Salmonella TA 100. Tests confirmed that the phospholipid pyrolyzates contained both mutagenic and inactivating substances of this mutagenicity. Low level mutagenieity was produced on Salmonella TA 98, but there was no such activity on the other strains. Preincubation of the pyrolyzates with S-9 mix had no activating effect on mutagenicity. The inactivating substances of the mutagenicity were isolated and identified as long chain fatty acids.

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A heat stable trypsin inhibitor was found in the bran of soft-shelled job's-tears (Coix lacrymajobi L. var. Ma-yuen Stapf) seeds. This inhibitor seemed to be a simple protein, and the molecular weight was about 12, 000. Similar to other heat stable trypsin inhibitors, this inhibitor also contained many cysteine or cystine residues in the molecule. This inhibitor inhibited bovine trypsin at the molar ratio of 1 to 2, showing that it was double-headed. Its activity was stable against the change of pH at the range of 3 to 11 and high temperature of 100°C under certain conditions. However, the degree of heat stability of the inhibitory activity depended highly upon the kind of the solution in which this inhibitor was dissolved.

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Rhodococcus sp. BPM 1613, a pristane oxidizing microorganism, grows on isoprenoid hydrocarbons such as phytane (2, 6, 10, 14-tetramethylhexadecane), norpristane (2, 6, 10-trimethylpentadecane) and farnesane (2, 6, 10-trimethyldodecane) as the sole carbon source, resulting in accumulation of oxidation products in the culture broth. The oxidation products of phytane, norpristane and farnesane in the respective culture broth were isolated and purified by the use of silica gel column chromatography. Their chemical structures were determined by instrumental analyses such as IR, NMR and mass spectrometry. The oxidation products of phytane were identified as 2, 6, 10, 14-tetramethyl-1-hexadecanol and 2, 6, 10, 14-tetramethylhexadecanoic acid, the product of norpristane as 2, 6, 10-trimethyl-l-pentadecanol, and that of farnesane as 2, 6, 10-trimethyl-1-dodecanol. All these oxidation products were either monoalcohols or monocarboxylic acids derived through oxidation of the isopropyl terminus of each alkane.
In addition, the relationship between the terminal structure of isoprenoid hydrocarbons and microbial oxidation was explored on the basis of these results.

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A bacterial strain, SH-548, that produces a lytic enzyme toward intact cells of anilineassimilating Rhodococcus erythropolis AN-13, was isolated from soil. The isolated bacterium was identified as a Flavobacterium species. The growth conditions for the enzyme production by Flavobacterium sp. SH-548 were examined; organic nitrogen compounds, such as meat extract and Polypepton, were effective for its production. The lytic enzyme of this strain lysed intact cells of Rhodococcus, Bacillus, Nocardia, Corynebacterium, Brevibacterium, Streptococcus, Micrococcus, Cellulomonas and DAB (diaminobutyric acid)-type coryneform bacterial strains. However, it did not act on those of Staphylococcus aureus or gram-negative bacteria, Enterobacter, Escherichia, Klebsiella, Proteus or Pseudomonas strains. Bacterial strains having cell walls of the glycolyl type were readily lysed by this enzyme.

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To confirm the potential usefulness of amino acid residues as the protecting group of sugar hydroxyls, N-(benzyloxycarbonyl) and -N-(t-butoxycarbonyl) amino acids were condensed with methyl 4, 6-O-benzylidene-α-D-glucopyranoside (1) and methyl 2, 3-di-O-methyl-α-D-glucopyranoside (2), and the conditions were studied for the removal of aminoacyl groups from sugar moieties. The aminoacyl groups were easily removed by enzymatic hydrolysis using pronase E, trypsin and chymotrypsin, as well as alkaline treatment as with conventional acyls. We determined the utility of aminoacyl sugars as new protecting groups having some useful characteristics which never exist in normal circumstances.

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A 1.4-megadalton EcoRI restriction fragment carrying Bacillus subtilis speculation gene spoOB was cloned from the specialized transducing phage, ø105spoOB, into a unique EcoRI site of plasmid vector pUBHO, and four plasmids having a deletion in the 1.4-megadalton EcoRI fragment were constructed. Analysis of the polypeptides synthesized in B. subtilis minicells harboring these plasmids and the sporulation ability of strain UOT0436 (spoOB136 recE4) harboring these plasmids showed that the spoOB gene product is a polypeptide of 24, 000 daltons. Two-dimensional polyacrylamide gel analysis showed that the isoelectric point of this protein is almost neutral.

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Xylanases from alkalophilic thermophilic Bacillus spp. W1 and W2 were purified and characterized. The xylanases from the two strains were fractionated into two active components (I and II) by DEAE-Toyopearl 650M chromatography. Components I from the two strains had similar properties: optimum pH, 6.0; optimum temperature, 65°C; isoelectric point, pH 8.5 and 8.3; molecular weight, 21, 500 and 22, 500; and Michaelis constant, 4.5 and 4.0mg-xylan/ml. Components II from the two strains also had similar properties: optimum pH, 7.0-9.0 and 7.0-9.5; optimum temperature, 70°C; isoelectric point, pH 3.6 and 3.7; molecular weight, 49, 500 and 50, 000; and Michaelis constant, 0.95 and 0.57mg-xylan/ml. The activities of components I and II were inhibited by Hg⁺⁺ and Cu⁺⁺. Components I hydrolyzed xylan to yield xylobiose and higher oligomers, but components II produced xylose other than xylobiose and xylooligomers.

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The distribution of molasses pigment (melanoidin) decolorizing activity (MDA) was investigated in various Basidiomycetes. MDA was only found in some genera of the white-rot-fungi group of which Coriolus versicolor Ps4a showed high activity, a decolorization yield of approximately 80% under the optimal conditions. Production of MDA by C. versicolor was almost completely coincident with the growth of mycelia. The main MDA was due to intracellular enzymes and induced by the molasses pigment. The induced enzyme consisted of two types, namely a sugar dependent enzyme and a sugar independent enzyme. The decolorization by C. versicolor was due to the decomposition of the molasses pigment.

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Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48, 400-50, 000, an optimum pH of 5.5 and an optimum temperature of 30-35°C. P-III required glucose and O₂ for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.
On the other hand, P-IV had a molecular weight of 43, 800-45, 000, an optimum pH of 4.0-4.5 and an optimum temperature of 30-35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CME and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.
Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.

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The enzyme system (culture filtrate) from Streptomyces sp. W19-1 formed gentiobiose from curdlan (β-1, 3-glucan). The mechanism of the formation of gentiobiose was investigated in this study.
Two kinds of enzymes, β-1, 3-glucanase and β-glucosidase (transglucosidase), were isolated from the culture filtrate of the strain by hydroxylapatite column chromatography. The β-1, 3-glucanase hydrolyzed curdlan to glucose and laminari-oligosaccharides, and the β-glucosidase formed gentiobiose by transglucosylation from the resultant laminari-oligosaccharides, especially laminaribiose. The two enzymes took part in the formation of gentiobiose from curdlan.

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A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412, 000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.

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We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.
We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6g dry wt/liter of the cells was inoculated.

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Methyl eicosapentaenoate (methyl 5, 8, 11, 14, 17-eicosapentaenoate) was subjected to autoxidation and methylene blue-sensitized photooxidation. The secondary oxidation products were separated, and characterized by gas chromatography-mass spectrometry. The autoxidation products included hydroperoxy endoperoxide isomers, prostaglandin-like hydroperoxy bicyclic endoperoxide isomers and 5, 18-dihydroperoxide. The photosensitized oxidation products included only dihydroperoxide isomers as the secondary products.

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Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.

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An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4, 000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca2+. The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 10⁵ transformants per μg plasmid DNA was achieved.

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Nitrate reductase activity, assayed either in vivo or in vitro was considerably higher in bean (Phaseolus vulgaris L.) leaves from 7-day-old light grown seedlings than those from dark grown, both in the absence as well as presence of nitrate. Cytochrome c reductase activity was however similar in both regimes, while peroxidase was lower in light than in dark. The light stimulated increase in nitrate reductase activity in leaf segments from dark grown seedlings was inhibited by cycloheximide, DNP, chloramphenicol, and sodium tungstate and was unaffected by lincomycin and DCMU. Under similar conditions, the increase in total chlorophyll was inhibited completely by cycloheximide and DNP, partially by chloramphenicol and lincomycin, and was unaffected by tungstate and DCMU. A supply of 1 - 5 mM reduced glutathione increased enzyme activity in the dark and also to some extent in light. The substrate induction of enzyme activity started after a lag of one hr in light or dark and continued for either 5 hr in the dark or 8 hr in light. Two proteinaceous inhibitors (Factors I and II) of nitrate reductase were isolated by ammonium sulfate precipitation and Sephadex gel filtration. The amount of Factor I was higher in the dark than in light. The amount and activity of Factor II was however, almost equal in light and dark. The inhibition of enzyme activity by these inhibitors increased with their concentration. It is proposed that light increases nitrate reductase activity by decreasing the amount of a nitrate reductase inhibitor.

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The mechanism and control of protein degradation in cells are quite mysterious. We investigated the change of protease activities in animals fed a vitamin E-deficient diet. The Ca²⁺- activated protease activity was not significantly changed in vitamin E-deficient rats during the 45 weeks of the experiment. The cathepsin B activity was increased in those animals. Electron microscopic observation on the muscle of the vitamin E-deficient rats showed destruction of myofibrils at the Z-line, narrowness of myofibrils, and dispersed myofibrils. The M-line, which is known to disappear with cathepsin L treatment, was clearly observed. The phagocytosis of muscle cells by macrophages was also observed. These results show that the abnormal myofibril protein degradation in muscle tissue of vitamin E-deficient rats is not only due to the activation of macrophages and the increment of lysosomes in muscle cells, but also due to the protease which can destroy the myofibril at the Z-line. It may be a Ca²⁺-activated protease.

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Synthesis of trehalose from maltose by a coupled enzyme system with trehalose phosphorylase and maltose phosphorylase has been studied. Trehalose phosphorylase was partially purified from Euglena gracilis and maltose phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5-7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.
Immobilized maltose and trehalose phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.

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Surface substances were isolated by sonication from the germinated spores of various strains of Ceratocystisfimbriata and characterized in relation to host-parasite specificity. The substances from the sweet potato strain, compatible with sweet potato, potently inhibited the spore agglutination of various strains by spore-agglutinating factor from sweet potato roots, while the substances from incompatible strains, that is, coffee, taro, and almond strains, weakly inhibited this agglutination. The substances from the sweet potato strain increased ethylene production from sweet potato roots infected by all strains tested, sweet potato, coffee, taro, and almond strains, which was possibly an index of pathogenicity. On the other hand, the substances from incompatible strains, coffee, taro, and almond strains, suppressed the ethylene production from the tissue infected by all four strains except the substances from almond strains on almond strain. Heat and trypsin treatments inactivated the spore agglutination inhibitory activity of the surface substances. Coincidently, these treatments extinguished the effect of the surface substances on pathogenicity of C. fimbriata on sweet potato roots.

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The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the lowaffinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.
By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.
From these results, it was inferred that the binding site abolished by the iodination is the LAbinding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.

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β-Galactosidase and glucose oxidase were immobilized with bovine serum albumin using glutaraldehyde on to a glassy carbon electrode silanized with 3-aminopropyltriethoxysilane. The laboratory-constructed lactose electrode was used for flow injection analysis to determine the lactose content in milk. Electrochemical interference could be detected by a non-enzymatic electrode (W₂) and the current was subtracted from that of the enzymatic electrode (W₁), providing an accurate measurement of the hydrogen peroxide that was enzymatically generated. The peak current was linearly related to the lactose concentration in the range 10^-4-1.5 × 10^-3M (original concentration) and 40 samples/hr could be analyzed. The relative standard deviation for 10 assays was less than 2%. The proposed method was compared with the chloramine T method and the values determined by both methods were in good agreement.

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Fruiting bodies were formed around a Penicillium colony which appeared as a contaminant in a culture of Schizophyllum commune, and this phenomenon was reproduced with a synthesized system consisting of S. commune IAM 9006 and P. funiculosum A-1. The active substances were recovered in an acetone extract of the mycelia of P. funiculosum, purified by silica gel column chromatography and reverse-phase high-performance liquid chromatography, and characterized by infrared spectroscopy, gas-liquid chromatography, gas-liquid chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy. They were ceramides and cerebrosides having nonadecasphingadienine and 2-hydroxy fatty acid moieties in common. The major component was identified as (4E, 8E)-N-2'-hydroxy-(E)-3'-octadecenoyl-1-O-β-glucopyranosyl-9-methyl-4, 8-sphingadienine.

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A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307, a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.
Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cellfree extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺. The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malolactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.

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A transglycosylation reaction with moranoline (1-deoxynojirimycin) was carried out with α-cyclodextrin as the glucose donor and Bacillus macerans amylase as cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products were hydrolyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus. The hydrolyzate (the transglycosylation product of the lowest molecular weight) was isolated and the structure was found by physico-chemical methods to be 4-O-α-D-glucopyranosyl-moranoline.

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A simple method for the synthesis of various purine arabinosides from purine bases and uracil arabinoside by microbial transarabinosylation is described. A wet cell paste of Enterobacter aerogenes AJ 11125 showed a wide substrate specificity range for purine bases. Not only naturally occurring purine bases such as adenine and hypoxanthine but also unnatural bases such as 6-thioguanine and 2-chlorohypoxanthine were catalyzed to give the corresponding purine arabinosides. The enzymatically synthesized purine arabinosides were isolated from the reaction mixtures and identified by physicochemical means. The biological activities of the compounds were investigated and it was found that thioguanine arabinoside and 2-methyladenine arabinoside have potent activity against Hela cells, and their ED₅₀ were 10.5 and 21.5μg/ml, respectively.

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The influence of nineteen flavonoids on cow's milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) was investigated. The enzyme activity was estimated by measuring the increase in absorbance at 290nm due to uricate formation as well as by a colorimetric method assaying hydrogen peroxide generated from uricate by uricase. Among the flavonoids tested, myricetin, kaempferol, quercetin, fisetin, quercitrin, and morin inhibited the enzyme strongly at 50μM; the concentrations which gave 50% inhibition (ID₅₀) were 2, 2, 3, 7, 15, and 19μM, respectively. The inhibition mode of the former three compounds was of mixed type and the kinetic parameters were determined. Chrysin and naringenin were moderately inhibitory, while other flavonoids showed weak to no inhibition.

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